Elevated C‐peptide has been suggested as a risk factor for coronary artery disease (CAD). Elevated urinary C‐peptide to creatinine ratio (UCPCR) as an alternative measurement is shown to be related to insulin secretion dysfunction; however, data regarding UCPCR predictive value for CAD in diabetes mellitus (DM) are scarce. Therefore, we aimed to assess the UCPCR association with CAD in type 1 DM (T1DM) patients.n = 84) and without‐CAD (n = 195). Furthermore, each group was divided into obese (body mass index (BMI) ≥ 30) and non‐obese (BMI < 30) groups. Four models utilizing the binary logistic regression were designed to evaluate the role of UCPCR in CAD adjusted for well‐known risk factors and mediators.279 patients previously diagnosed with T1DM included and categorized into two groups of CAD . Also, the well‐acknowledged risk factors including being active smoker, hypertension, duration of diabetes, and body mass index (BMI) as well as higher levels of haemoglobin A1C (HbA1C), total cholesterol (TC), low‐density lipoprotein (LDL) and estimated glomeruli filtration rate (e‐GFR) had more significant pervasiveness in CAD patients. Based on multiple adjustments by logistic regression, UCPCR was a strong risk factor of CAD among T1DM patients independent of hypertension, demographic variables , diabetes‐related factors , lipid profile  and renal‐related indicators  in both patients with BMI≥30 and BMI < 30.UCPCR is associated with clinical CAD, independent of CAD classic risk factors, glycaemic control, insulin resistance and BMI in type 1 DM patients. Urinary C‐peptide to creatinine ratio (UCPCR) is associated with both insulin secretion disturbance and insulin resistance. Among type 1 diabetes mellitus (T1DM) patients, the mean UCPCR is significantly higher in CAD group compared to non‐CAD group. UCPCR is independently associated with CAD. UCPCR leads to a similar risk of CAD in both obese (>30 kg/m2) and non‐obese (<30 kg/m2) patients with T1DM. Finally, a total of 279 subjects (84 patients with CAD and 195 patients without CAD) have been permitted for evaluation in the present study. Written informed consent has been acquired from patients, and the study has been given approval by the ethics review board of the Shahid Beheshti University of Medical Sciences .The present research has enrolled 279 patients with a T1DM diagnosis at Shahid Modarres Hospital affiliated to Shahid Beheshti University of Medical Sciences in Tehran, Iran, from November 2020 to November 2021  was calculated by the weight divided by the square of the height (kg/m2). Blood pressure (BP) has been recorded three times consecutively, on the left arm following the seated condition of patient lasting for at least 5 min. Consequently, the average of three recorded BPs was employed for evaluations. Blood samples have been taken after 8‐hour fasting and before the breakfast. Fasting glucose, low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), triglycerides (TGs), total cholesterol (TC), creatinine (Cr), and uric acid (UA) were quantified. High‐performance liquid chromatography was used to determine HbA1C levels. A chemiluminescence immunoassay analyser has been used to detect C‐peptide and blood insulin levels. An immunoturbidimetric test analyser has been employed to detect albuminuria, and the e‐GFR has been determined by the Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) equation and creatinine levels. Patients collected urine specimen at the time of blood sampling and the urinary C‐peptide and creatinine were analysed using electrochemiluminescence immunoassay and Roche P800 platform, respectively, to calculate UCPCR (nmol/mmol).2.32 has been described as obesity, based on the criteria of the World Health Organization (WHO) obesity classification2) and obese (BMI ≥30 kg/m2). Diabetic cases who had already been identified were discovered by an examination of their health records using the WHO criteria from 1999: random blood glucose levels of more than 200 mg/dL (11.1 mmol/L), Fasting blood glucose levels of more than 126 mg/dL (7.0 mmol/L), with clinical manifestations , or an abnormal oral glucose tolerance test result. Abnormal glycaemia must be presented on two separate occasions if the patient had absence of symptoms. The current use of antihypertensive therapy or a blood pressure of 140/90 mmHg or was considered hypertension. The following criteria were used to diagnose CAD in the Department of Cardiology at Shahid Modarres Hospital in Tehran, Iran: (1) ischemia in the ECG, (2) Angina and (3) hard CAD. Angiography‐shown coronary artery blockage of 50% or more, coronary revascularization, or a history of myocardial infarction (MI) verified by Q waves evident in the ECG or medical records was considered hard CAD.A BMI ≥30 kg/m2.4t test were utilized to evaluate differences between the two groups, while one‐way ANOVA has been utilized for three groups or more. To compare continuous data, the Mann–Whitney U‐test was utilized. In T1DM patients, the risk variables for CAD were determined using binary logistic regression in 4 designed models: Model. A: Not adjusted for variables; Model. B: Adjusted for age, sex, and BMI; Model. C: further adjustment for diabetes‐related variables ; and Model. D was adjusted for model C variables as well as active smoking, alcohol consumption, hypertension status, TC, HLD, LDL, TG, eGFR, uric acid, albumin, creatinine and albuminuria. Statistical significance has been defined as a two‐sided alpha less than 0.05. SPSS version 26.0 has been employed for all statistical evaluations (SPSS).Categorical variables and continuous data having a normal distribution have been presented as percentages (%) and the mean ± standard deviation (SD), respectively. The chi‐squared test and Student's 33.1p = .12). The BMI (p = .01) and history of smoking (p = .04) were also greater in the CAD group, while the history of alcohol consumption did not show a contrast between the two groups. Regarding the clinical history, prevalent hypertension (p = .01) and the duration of diabetes  were grater among CAD patient. To compare the laboratory data, the significant characteristics were as follows: The levels of HDL and e‐GFR had a statistically lower mean in the CAD group, whereas the levels of HbA1C, TC, LDL, albuminuria and more importantly UCPCR all had a statistically higher mean in the CAD group (All with p < .05).Table.For a more precise assessment of the patients, we decided to evaluate and compare them separately based on whether or not they had BMI exceeding 30. Accordingly, as shown in Table.3.2p‐value = .000); in the case of hypertension, it was discovered that patients who had a history of hypertension were 2.1 times more likely to have developed CAD ; in the case of HbA1C, it was found that elevated levels of HbA1C could cause a 30% increase in the risk of CAD ; and finally in the case of UCPCR, it was indicated that increased levels of UCPCR could elevate the risk of CAD for 50% .Table.As the focal point of our study is to assess the effect of UCPCR, Table.4In the present study, we have attempted to assess UCPCR levels in CAD patients and figure out whether CAD could be predicted in patients based on their UCPCR levels. Our analysis showed that UCPCR levels were significantly greater in CAD patients as a whole, and as sub‐grouped based on having a BMI ≥30. Also, further modellings conducted on the patients indicated that CAD cases, when adjusted for a variety of different variables, still had remarkably greater levels of UCPCR, regardless of their BMI.Aggravation of systematic inflammation,Moreover, clinically, it was revealed that C‐peptide is related to a decrease in glomerular hyperfiltration and proteinuria, leading to increased risk of nephropathy. Also, low C‐peptide levels were correlated with greater glucose level variability, increased HbA1c, poor glycaemic control lower and as a result increased risk of retinopathy, neuropathy, foot ulcers and mortality.In this study, we compared CAD and non‐CAD patients based on the clinical and laboratorial characteristics and also categorized by BMI. We also considered well‐acknowledged risk factors of CAD including hypertension, dyslipidaemia, treatment with statins, mild to moderate reduced eGFR, diabetes‐related factors and sociodemographic properties. Similar to previous studies, those with CAD had significantly higher incidence of hypertension, albuminuria, TC, LDL, HbA1C, longer T1DM duration, as well as declined levels of eGFR and HDL. It is noteworthy that treatment with statins was generally high (>85% in both groups) in the participants of this study; thus, its effect on lowering the risk of coronary artery disease may have been diluted. This might explain why there is not a clear link between statins and coronary artery disease in this study.A considerable number of diabetic individuals are obese with elevated BMI as the independent risk factor of CAD. Moreover, urinary C‐peptide, levels of C‐peptide and UCPCR could be influenced by weight. Therefore, following investigating the role of UCPCR on clinical CAD, we decided to analyse whether UCPCR have different effect in different BMI groups of T1DM patients. We obtained that among obese (BMI≥30) as well as the non‐obese (BMI < 30) group, UCPCR was related to CAD regardless of conventional CAD risk factors. These findings imply that UCPCR may be linked to or implicated in the production of atherosclerotic plaques within the coronary arteries of T1DM patients, both obese and non‐obese. Herein, UCPCR was suggested as a risk factor of CAD with the adjusted OR of more than 1, greater in non‐obese patients in comparison with obese patients. This may suggest the greater implication of UCPCR measurement in non‐obese patients.One of the reasons for the importance of this study is that measuring UCPCR is simple, cost‐effective and practical in comparison with serum assays of C‐peptide and insulin.In conclusion, this is a novel topic regarding the predictive role of UCPCR in patients with T1DM with lack of previous research studies. UCPCR is a practical and non‐invasive biomarker that has shown its effectiveness in differentiating T1DM from other DM groups, guiding the individual's treatment regarding poor‐ or well‐controlled DM, and as we showed, the subsequent CV risk prediction. We discovered that in T1DM patients, whether obese or not, UCPCR is associated with clinical CAD, independent of established CAD risk variables such as insulin resistance and glycaemic control. Meanwhile, further studies are required to evaluate the expansion of clinical implications and cost‐effectiveness of this marker in both fields of medical management and biomedicine.Written informed consent has been acquired from patients to publish this study according to the journal's patient consent policy. Moreover, the authors all declare that patients' confidentiality has been respected. (Ethics code: IR.SBMU.MSP.REC.1400.083).Farzad Esmaeili Tarki: Data curation ; methodology ; project administration ; resources . Fatemehsadat Rahimi: Data curation ; methodology ; writing – original draft . Nasser Malekpour Alamadari: Conceptualization ; project administration ; supervision .There is no funding to present study.The authors declare no conflict of interest.The authors all declare that this manuscript is not published elsewhere.


Women with breast cancer and improved survival often experience treatment‐related impairments. High‐intensity interval training (HIIT) has emerged as a promising exercise therapy modality for adult cancer patients. However, the overall effects of HIIT in breast cancer patients remain scarce and controversial. Therefore, we conducted a systematic review and meta‐analysis to comprehensively evaluate the impact of HIIT on health‐related outcomes in breast cancer patients.We searched the PubMed, Embase, and Web of Science from inception to November 7, 2022. Eligible studies included randomized controlled trials that compared HIIT interventions with usual care (UC) or MICT in breast cancer patients. The primary outcome assessed was physical fitness, and exploratory outcomes included body composition, blood‐borne biomarkers, and patient‐reported outcomes. Summary data were extracted, and standardized mean differences (SMD) were calculated for meta‐analysis. For outcomes that could not be pooled, a systematic review was conducted.2peak). The SMD for VO2peak (L/min) and VO2peak (mL/kg/min) was 0.79  and 0.59 , respectively. No significant differences in VO2peak were found between the HIIT and MICT groups. Meta‐analyses on body composition and blood‐borne biomarkers showed no significant differences between HIIT and UC. Systematic review indicated favorable effects of HIIT on muscle strength, fatigue, and emotional well‐being.Our analysis included 19 articles from 10 studies, encompassing 532 participants who met the inclusion criteria. Pooled results demonstrated that HIIT was superior to UC in improving peak oxygen uptake (VO2peak and may also enhance muscle strength and alleviate fatigue and emotional symptoms in breast cancer patients. HIIT should be considered as an important component of exercise prescription in breast cancer care. Further studies with larger cohorts are needed to determine the clinical significance of HIIT‐induced changes in terms of other outcomes in women with breast cancer.HIIT is a time‐efficient alternative to MICT for improving VO The complete list of synonyms and the detailed search strategy are available in Table 2peak, peak heart rate (HRpeak), maximal heart rate (HRmax), or equivalent rating of perceived exertion (RPE) ≥16 on the BORG's 6–20 scaleFull‐text articles in the English language were included if they met the following criteria based on the PICOS principle: (1) participants were breast cancer patients or breast cancer survivors; (2) the intervention included HIIT with a clear description, and high intensities were defined a priori as ≥75% of VOTwo investigators (XC and XS) independently reviewed the search results. Irrelevant studies were excluded by reviewing the title and abstract according to the pre‐specified selection criteria, and potential articles were selected for full‐text screening. Any disagreements on eligibility were resolved through discussion between the two investigators. One study including participants with breast cancer and other categories of cancers was excluded after our discussion, because the result would be influenced by other cancers.2.2The quality of included articles was evaluated by two reviewers (XC and XS) separately, utilizing the Revised Cochrane risk‐of‐bias tool for randomized trials (RoB 2),2.32peak, and the secondary outcome was other cardiopulmonary fitness indices, muscle strength, body composition, blood‐borne biomarkers, and patient reported outcomes. The two investigators independently completed the data extraction and checked with each other to reach a consensus.Basic information from the included studies was extracted into a pre‐piloted Microsoft Excel sheet developed based on the PICOS principle by XC and XS after full‐text reading. The primary outcome was VOI2 statistics, and heterogeneity was considered significant if the p‐value was less than 0.10 or if I2 was greater than 50%. We presented all combined results as SMD with its corresponding 95% confidence interval (95% CI) in the table. An SMD that overlapped with zero indicated that there was no significant difference between the different groups. We utilized Review Manager 5.4 software to generate the forest plot. For results that were only available in figure form in the original articles, we extracted the relevant data using Engauge Digitizer software.We conducted meta‐analyses on outcomes that were reported in two or more studies, and for outcomes that were reported in only one study, they will be described in the result part. The meta‐analyses were performed using STATA 16.0, and we calculated the pooled estimate of SMD as all outcomes were continuous variables. We used Cohen's d to calculate the effect sizes of each endpoint, and we employed a random‐effects model. We evaluated the heterogeneity using the Chi‐squared test and 33.1A total of 429 articles were initially identified from three databases in our systematic literature search, as shown in Figure n = 532 participants (mean age 54.05 ± 9.36 years) were included. Specifically, n = 296 (52.27 ± 9.49 years), n = 40 (56.66 ± 7.49 years), and n = 196 (51.57 ± 9.19 years) received HIIT, MICT, and UC, respectively. The duration of HIIT intervention ranged from 6 to 16 weeks, with an average of 10.4 (3.07) weeks. And the average number of weekly HIIT sessions was 2.7 (0.46).Among the 10 studies included in our review, a total number of All participants were women with Stage I–III breast cancer without metastases, recurrence, or secondary cancers at the time of enrollment. Except for two studies that recruited patients undergoing chemotherapyIntervention compliance was primarily evaluated by the percentage of attendance at arranged training sessions, which was generally high for both HIIT and MICT groups, except for the study by Mijwel et al., in which the attendance rates of the RT‐HIIT and AT‐HIIT groups were 68% and 63%, respectively. Training adherence for HIIT ranged from 78.7% to 100%, and for MICT, the rate ranged from 79.4% to 98.6%. The overall dropout rate across all studies was 8.89% (53/596), with HIIT at 7.59% (24/316), MICT at 4.76% (2/42), and UC at 12.22% (27/221). The dropout rates of individual studies ranged from a minimum of 0% to a maximum of 14.17% (34/240).3.2The results of the risk of bias assessment for all included studies are presented in Figure 3.3The pooled estimates of health‐related outcomes between HIIT and MICT or UC are summarized in Table 3.3.12peak. Two studies compared post‐intervention VO2peak (L/min) in different randomized groups, and the pooled result was 0.78  compared to UC.2peak (mL/kg/min) between the HIIT group and the UC group.p = 0.04, Figure 2peak (mL/kg/min) between the HIIT group and the MICT group  .Several studies have investigated the impact of HIIT on VOp = 0.663). For the sit‐to‐stand (STS) test, the estimated pooled SMD was 0.23 . Additionally, Lee et al. utilized the timed up and go (TUG) and Margaria–Kalamen stair test, which indicated that no significant difference between groups was observed in the TUG test .p = 0.013).In terms of physical function, the six‐minute walk test (6MWT) was a commonly used endpoint for evaluating functional performance. Two articles compared the results of the 6MWT between the HIIT and control groups.p = 0.462). Two other studies compared strength measured by one‐repetition maximum (1RM) between the HIIT and control groups.I2 = 72.2%) was reported, and the pooled result overlapped zero . Another study assessed both lower body strength (LBS) and upper body strength (UBS), and found a significant increase in both HIIT and MICT groups.Regarding muscle strength, two studies compared handgrip strength between the HIIT and UC groups for the surgery side.p = 0.23). Additionally, peak workload, minute ventilation, and peak heart rate were also used to assess cardiovascular fitness, but no significant differences were observed either between or within groups.As for cardiovascular fitness, Northey et al. examined several indices like middle artery mean blood flow velocity (MCAvmean) and mean arterial pressure (MAP).p = 0.005), improved activity of oxphos complexes , and greater cross‐sectional area (HIIT vs. control: p = 0.02).Moreover, Mijwel et al. conducted a biopsy analysis to compare the skeletal muscle fiber characteristics between cancer patients undergoing HIIT and those in the UC group.3.3.2p = 0.108).p = 0.786).p = 0.031).Three studies investigating the effect of HIIT on body mass indicated no significant decrease compared to the UC group .p = 0.332).Two articles comparing post‐intervention fat mass between the HIIT and the UC group showed an estimated SMD of −0.27 .p = 0.66).In respect of lean mass, two articles compared the effect of HIIT and the UC group, and the pooled result showed no significant difference between the two groups .p = 0.376).The impact of HIIT on blood‐borne biomarkers has been investigated by several studies. Three articles reported on the effect of HIIT on IL‐6 levels compared to UC, with a SMD estimate of −0.72 , and the levels of CD40‐L, EGF, CCL17, and CASP‐8 attenuated significantly after UC, while the levels of TRAIL, DCN, ISOSLG, CSF‐1, FasL, and CXCL9 increased in the UC group. However, there was no significant difference between HIIT intervention and UC in terms of all inflammatory markers.Hiensch et al. compared a large cluster of blood‐borne biomarkers between and within groups.Moghadam et al. reported significant reductions in TNF‐α and leptin levels in both the HIIT and MICT groups, while adiponectin levels were significantly increased in both groups.p = 0.018; miR‐155: 1.3 [0.4]; p = 0.005; miR‐27a: 1.5 [0.02]; p = 0.037; miR‐10b: 1.95 [0.6]; p = 0.031), while the level of miR‐221 was not significantly influenced by HIIT . The study also found that the levels of several tumor suppressor miRs (TsmiRs) were upregulated after HIIT , except for miR‐9 .Alizadeh et al. monitored serum microRNA (miR) levels in patients undergoing hormone therapy.3.3.4p = 0.09).Several studies have used patient‐reported outcome measures to evaluate the effects of HIIT on cancer patients. Mijwel et al. compared cancer‐related fatigue (CRF) between HIIT and UC group using the Piper Fatigue Scale (PFS).HRQoL was measured by the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC‐QLQ‐C30), and Mijwel et al. found that emotional function was higher in the HIIT group, while negative physical function and pain scores were relatively lower.p < 0.05), and the score of “feeling irritable” in the emotional dimension was also lower in the HIIT group . No other significant differences were found.Symptoms and symptom burden were measured by the Memorial Symptom Assessment Scale (MSAS) after a 16‐week intervention of AT‐HIIT or RT‐HIIT in the Optitrain trial.Furthermore, Lee et al. used the Functional Assessment of Cancer Therapy‐Breast Cancer (FACT‐B), 15‐item Five‐Facet Mindfulness Questionnaire (FFMQ‐15), and Multidimensional Fatigue Inventory with 20 questions (MDFI‐20) to assess patient‐reported outcomes.42peak) compared to the UC group, but no significant differences were observed between the MICT group and HIIT. Moreover, no significant differences were found in other physical fitness indices, body composition, and blood‐borne biomarkers between the MICT and UC groups. Furthermore, based on our systematic review, it was observed that HIIT may also enhance muscle strength, reduce CRF, and alleviate emotional symptoms, although further empirical evidence is still required. In the subsequent sections, we will interpret and summarize the findings, as well as discuss future directions for investigating the effects of HIIT in breast cancer patients.The objective of this systematic review and meta‐analysis was to investigate the potential beneficial effects of HIIT on health‐related outcomes in breast cancer patients compared to MICT and UC groups. Our pooled analysis of 10 RCTs indicated that HIIT led to a significant improvement in cardiorespiratory fitness  can negatively impact health outcomes and have long‐lasting effects, it is important to differentiate the effect size of HIIT between these two conditions. Furthermore, exploring the effectiveness of HIIT across different cancer types and treatment time points should be considered as a future direction in the field of supportive medicine in cancer care.52peak as compared to UC. HIIT may also improve muscle strength and alleviate CRF and emotional symptoms. However, the evidence is limited regarding the effects of HIIT on other outcomes. We recommend that HIIT could be considered as a feasible exercise intervention for breast cancer patients during and after treatment. Further studies with larger cohorts are needed to determine the clinical significance of HIIT‐induced changes in body composition, cardiovascular fitness, and blood‐borne biomarkers in women with breast cancer.Our study suggests that HIIT is an effective alternative strategy to MICT, leading to significant improvements in VOXudong Chen: Conceptualization (lead); formal analysis ; investigation (lead); methodology ; project administration (lead); resources ; supervision ; validation ; writing – original draft (lead); writing – review and editing . Xuyuan Shi: Conceptualization (supporting); formal analysis ; investigation ; project administration ; resources ; validation ; writing – original draft ; writing – review and editing . Zhiruo Yu: Formal analysis (supporting); investigation (supporting); validation (supporting); writing – original draft ; writing – review and editing (supporting). Xuelei Ma: Conceptualization (supporting); investigation (supporting); project administration (supporting); validation ; writing – review and editing (supporting).This research received no external funding.The authors declare no conflict of interest. We are not using any copyrighted information, patient photographs, identifiers, or other protected health information in this paper. No text, text boxes, figures, or tables in this article have been previously published or owned by another party.Table SA1.Click here for additional data file.Table SA2.Click here for additional data file.


The energy restriction and early TRE (eTRE) subgroups resulted in greater weight loss. TRE (8/16) showed beneficial effects on the homeostatic model assessment of insulin resistance , but had no significant effect on other parameters of glucose metabolism and lipid profiles. In conclusion, TRE (8/16), especially eTRE, or in combination with caloric intake restriction, is a potential therapeutic strategy for weight control in overweight and obese adults. TRE (8/16) also reduced HOMA‐IR; therefore, it may have a positive effect on glucose metabolism.Time‐restricted eating (TRE) is a new therapeutic strategy for the management of weight loss and dysmetabolic diseases. At present, TRE  is the most common form of TRE. Therefore, this meta‐analysis included randomized controlled trials (RCTs) on TRE (8/16) in overweight and obese adults to determine its impact on body weight and metabolism. Articles reviewed from PubMed, Ovid MEDLINE, Embase, and Cochrane Central Register for the relevant RCTs that compared TRE (8/16) to non‐TRE in overweight and obese adults. Eight RCTs were included in this meta‐analysis. Participants following TRE (8/16) showed significant body weight reduction  and fat mass reduction . There was no significant difference in lean mass change with TRE intervention  is a potential therapeutic strategy for controlling weight in adults with overweight and obesity, especially the elderly and people who do not exercise regularly, and eTRE maybe the better choice for weight loss. TRE (8/16) also reduces HOMA‐IR, so maybe it has a positive effect on glucose metabolism. This review aimed to assess the time‐restricted eating (8/16) on body weight and metabolism in overweight and obese adults. A meta‐analysis and systematic review was conducted up to May 2022. The search strategy was based on database‐specific subject headings and keywords. Medical subject headings (MeSH) and free‐text search terms were used. Restrictions on human studies were also included. We also manually scanned the reference lists from trials, review articles, and reports to identify any other relevant data. Search terms included combinations of “time‐restricted feeding,”, “time‐restricted diet,” “time‐restricted meal,” “time‐restricted fasting,” or “time‐restricted eating” and “body mass index,” “adiposity,” “overweight,” or “obesity” (the details of the search strategy are presented in Appendix 2.22. (2) Intervention: at least one treatment arm with TRE (8/16) intervention. (3) Comparison: non‐TRE . (4) Study duration≥4 weeks. (5) Study type: RCT.The following were the inclusion criteria. (1) Population: adults aged 18 years or older; overweight or obese people with BMI ≥25 kg/m2.3Studies with ambiguous descriptions of the TRE regimen; conference abstracts without outcomes, reviews, case reports, or protocols; studies including participants with acute or chronic diseases, such as gastrointestinal diseases or cancer, that affect the outcomes; or non‐RCTs and nonrelevant studies were the exclusion criteria.2.4The primary outcome was the change in body weight between the TRE (8/16) and control groups. Secondary outcomes were the changes in body composition , waist and hip circumference, blood pressure, lipid profiles , and glucose metabolism  between the TRE (8/16) and control groups. The included studies were required to assess one of the primary or secondary outcomes.2.5Based on the inclusion and exclusion criteria, two authors (HL and CY) sequentially enrolled the trials and extracted data independently into a predesigned database. The following information was extracted: publication information (first author's name and publication year), participant characteristics , and primary and secondary outcomes. Discrepancies in eligibility between the two researchers were resolved through discussions. If necessary, a third researcher (XLZ) was involved.http://digitizer.sourceforge.net) were used to estimate or extract data.Continuous variables were expressed as mean ± standard deviation (SD). For the trials that merely reported the range/interquartile range or for which data were expressed with figures, the formula introduced by Wan et al.  and the 2.6The risk of bias was assessed according to the Cochrane tool for assessing the risk of bias in randomized clinical trials Table , includi2.7I2 value. I2 ≥ 50% was defined as high heterogeneity. The Hartung–Knapp–Sidik–Jonkman (HKSJ) method  were identified by the original screening Figure . The mai3.2The risk of bias of the studies is shown in Figure 3.33.3.1p = .006, I2 = 74%), indicating a significant weight loss using the TRE intervention , while the dTRE subgroup  and a limited number of studies (four studies) in the dTRE group, the HKSJ method was further conducted, but it did not alter the result  and delayed TRE (dTRE). Specifically, the food intake of eTRE started before 12:00 w et al.  could now et al.  reportedp = .002; I2 = 69%), while the <40 years old subgroup . Because there was only one study , while the nonenergy restriction subgroup  in the nonenergy restriction group, the HKSJ method was further conducted, but it did not alter the results . Because there was only one study , while the <40 years subgroup . Because there was only one study , while the <40 years group , indicating no significant difference in visceral fat mass change with TRE intervention  and limited number of studies (three studies), the HKSJ method was further conducted, but it did not alter the result , indicating no significant difference in hip circumference changes with TRE intervention  and a limited number of studies (four studies), the HKSJ method was further conducted, but it did not alter the results .Four studies , indicating no significant difference of LDL‐C change with TRE intervention , indicating no significant difference in TG change with TRE intervention , indicating no significant difference in HbA1c change with the TRE intervention , the weight reduction led to a greater fat mass loss  in participants ≥40 years old. Moon et al.  achieved significant weight loss and fat mass reduction in overweight and obese adults, but exerted no effect on the lean mass of the body. However, after subgroup analysis, we found that TRE (8/16) reduced body weight, fat mass, and lean mass simultaneously for people over 40 years of age compared to the control group, but it had no significant effect on body weight, fat mass, and lean mass in the <40 years old group. The potential health and metabolic benefits of weight loss are associated with minimal reduction in lean mass  intervention; however, there was no significant weight loss compared to the control group. Interestingly, in the energy‐restricted subgroup, the TRE group had more weight loss than the control group under the same energy‐restricted proportion in both groups. It is possible that TRE and energy restriction have a stack effect on weight control; however, the mechanism needs further study. In addition, TRE (8/16) intervention reduced fat mass without affecting lean mass in both the energy‐restricted and nonenergy‐restricted subgroups. Therefore, TRE could still be a healthy eating regimen.The pooled data showed that waist circumference decreased in the TRE (8/16) group, whereas the waist–hip ratio and visceral fat did not change significantly. This indicates that waist circumference alone does not reduce visceral fat. Previous studies have shown that the best predictor of visceral fat is the waist‐to‐hip ratio  intervention did significantly reduce HOMA‐IR, but there were no significant changes in blood glucose, insulin, and HbA1c levels. IR is now used as a screening indicator for the primary prevention of DM. IR commonly coexists with obesity  and control groups. This is inconsistent with the results of previous meta‐analyses , especially eTRE, or in combination with caloric intake restriction, is a potential therapeutic strategy for weight control in overweight and obese adults. TRE (8/16) also reduced HOMA‐IR; therefore, it may have a positive effect on glucose metabolism.The authors declare no conflicts of interest.This review used only published data sources. An ethical review by the Research Ethics Committee was not required.Appendix S1.Click here for additional data file.Appendix S2.Click here for additional data file.


Decreased blood‐tissue oxygenation at high altitude (HA) increases mitochondrial oxidant production and reduces exercise capacity. 5‐Hydroxymethylfurfural (5‐HMF) is an antioxidant that increases hemoglobin's binding affinity for oxygen. For these reasons, we hypothesized that 5‐HMF would improve muscle performance in rats exposed to a simulated HA of ~5500 m. A secondary objective was to measure mitochondrial activity and dynamic regulation of fission and fusion because they are linked processes impacted by HA. Fisher 344 rats received 5‐HMF (40 mg/kg/day) or vehicle during exposure to sea level or HA for 72 h. Right ankle plantarflexor muscle function was measured pre‐ and post‐exposure. Post‐exposure measurements included arterial blood gas and complete blood count, flexor digitorum brevis myofiber superoxide production and mitochondrial membrane potential (ΔΨm), and mitochondrial dynamic regulation in the soleus muscle. HA reduced blood oxygenation, increased superoxide levels and lowered ΔΨm, responses that were accompanied by decreased peak isometric torque and force production at frequencies >75 Hz. 5‐HMF increased isometric force production and lowered oxidant production at sea level. In HA exposed animals, 5‐HMF prevented a decline in isometric force production at 75–125 Hz, prevented an increase in superoxide levels, further decreased ΔΨm, and increased mitochondrial fusion 2 protein expression. These results suggest that 5‐HMF may prevent a decrease in hypoxic force production during submaximal isometric contractions by an antioxidant mechanism. High altitude (HA) exposure affects all phases in oxygen transport that increases mitochondrial oxidant production and negatively impacts exercise performance. 5‐Hydroxymethylfurfural (5‐HMF) is a natural compound that increases blood oxygenation and functions as an antioxidant. Here, 5‐HMF reduced skeletal muscle oxidant production and prevented a decline in isometric force production at 75–125 Hz after a 72 h exposure to a simulated HA of 5500 m. The next morning rats were anesthetized for implantation of osmotic pumps containing 0.9% NaCl or 5‐HMF (40 mg/kg/day). Within 30 min of recovering from anesthesia, animals were placed in chambers and exposed to sea level or a simulated HA of ~5500 m for 72 h. This level of hypobaric hypoxia was selected because it increases pulmonary arterial pressure and causes hypophagia in rats, resembling the effects of HA in humans  for cleaning, weighing of food, and replenishment of food and water. After 72 h rats were anesthetized and muscle function testing was repeated. Blood was collected via cardiac puncture for measurement of complete blood count and arterial blood gas. In the contralateral limb, the gastrocnemius, plantaris, and soleus muscles were collected, weighed, and frozen. An additional group of rats (n = 12) completed air and HA exposures only, and were used for live cell imaging experiments. For molecular analysis, the soleus muscle was selected because it contains mostly slow myosin heavy chain (MHC)  and is rich in mitochondria, thus highly susceptible to oxidant damage  between the ages of 10–12 weeks and weighing 200–225 g remained on a 12:12 light:dark cycle with free access to food and water. The experimental design is shown in Figure “The study protocol was reviewed and approved by the Walter Reed Army Institute of Research/Naval Medical Research Center Institutional Animal Care and Use Committee in compliance with all Federal regulations governing the protection of animals and research. The health status of animals was monitored daily, and the research was conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care‐International. Euthanasia was carried out in accordance with the recommendations and guidelines of the American Veterinary Medical Association.”2.22, 0.04% CO2, balance N2) to match the FiO2 at 5500 m. This was achieved by mixing air with pure N2 and confirmed by a gas analyzer. Total time for testing was 15–20 min per animal.Right ankle plantarflexor muscle function was measured using a rodent isokinetic dynamometer as previously described  or 0.9% NaCl (vehicle) were transferred to osmotic pumps (Alzet Model 2001) set to deliver 40 mg/kg body weight per day and placed in warm saline overnight for priming per manufacturer's instructions. The following day rats were anesthetized a second time with 2% isoflurane, and the area between the scapula was shaved and the skin was cleaned. Bupivacaine hydrochloride (4 mg/kg) was injected into the site and a small lateral incision was made along the center of the back. The pump was inserted into the subcutaneous pocket and the incision site was closed with staples. Buprenorphine (0.02–0.5 mg/kg) was administered subcutaneously for prophylactic analgesia and the animals recovered from anesthesia within 30 min.2.4Rats were placed individually in cylindrical Plexiglas chambers and decompressed to ~5500 m (300 m/min) or remained at sea level pressure for 72 h (26°C). Every 24 h the chambers were cleaned, the rat's food and body weight were recorded, and the food and water replenished (~40 min). HA rats were compressed to sea level at a rate of 300 m/min. Rats were placed in temporary chambers and breathed the same normoxic or hypoxic gas mixtures during this time.2.5i) and arterial blood gas (ABL90 Flex Plus Radiometer). The plantarflexors  were harvested, weighed, and flash frozen in liquid N2 and stored at −80°C. Plantarflexor muscle weights were used to measure the degree of muscle atrophy.Following exposures and muscle function testing, rats were removed from the force plate and transferred to a surgical table breathing the same normoxic or hypoxic gas mixtures with isoflurane  phosphate buffer saline. The po2 was estimated to match intramuscular po2 at ~5500 m. The samples were transported to the Uniformed Services University of the Health Sciences within 60 min of collection for isolation of myofibers as previously described  placed within the cell culture incubator maintained at 37°C. Superoxide was measured using 5 μM dihydroethidium (DHE)  (Ex: 500/Em: 588). Mitochondrial membrane potential was measured using 100 nM of tetramethylrhodamine ethyl ester (TMRE)  (Ex: 555/Em: 579)  were used: mouse anti‐total OXPHOS , rabbit anti‐dynamin‐related protein 1 , rabbit anti‐optic atrophy 1 , rabbit anti‐mitofusion 1 , rabbit anti‐mitofusion 2  and rabbit anti‐GAPDH . The following secondary antibodies were used : goat anti‐mouse  and goat anti‐rabbit . Membranes were imaged with a Bio‐Rad ChemiDoc MP Imaging System. Densitometry quantification was performed using ImageJ software.2.8Opa1 (Cat# 4331182), Mfn1 (Cat# 4331182), Mfn2 (Cat# 4331182), dynamin‐related protein 1 , mitochondrial fission 1  and GAPDH (Cat# 4351368). qPCR was completed using an iCyclerQ (Bio‐Rad). Gene cycle threshold (Ct) values were normalized to GAPDH and data were quantified by the 2CT−ΔΔ method.RNA was isolated from ~25 mg of soleus muscle using TRIzol® Reagent (ThermoFisher Scientific). One microgram of RNA was used for first strand cDNA synthesis using QuantiTect Reverse Transcription Kits  and amplified in a TaqMan PCR Master Mix (Cat# 201445) with the following Thermo Fisher Scientific probes and primers: 2.9t‐test was used for pairwise multiple comparisons. Data were log transformed if normality and variance homogeneity assumptions were not met. Data are presented as medians with quartiles, individual data points with medians and quartiles, and means ± SD or SE. Values of p < 0.05 were considered statistically significant. Data were analyzed using SigmaPlot version 14.0 (Systat Software).Statistical power and sample size were determined from mean peak isometric torque measurements obtained in our unpublished preliminary experiments. From these data it was determined that 9–10 rats/group were required to detect an approximate difference of 20% with an alpha of 0.05 and 80% power. Body weight, food intake, and muscle stimulation data were analyzed using a two‐factor repeated measures ANOVA. All other data were analyzed using a two‐factor ANOVA. A Bonferroni 33.1Exposing rats to a simulated HA of ~5500 m for 72 h led to a 58%–72% reduction in food intake Figure , a 9%–163.22 of 10.5%, arterial blood pH increased by 0.042 units and PaO2, SaO2, and PaCO2 fell by 20%, 23%, and 44%, respectively . Continuous delivery of 5‐HMF, however, did not improve SaOL and bH is slowed .No competing financial interests exist.“The views expressed in this journal article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. The study protocol was reviewed and approved by the Walter Reed Army Institute of Research/Naval Medical Research Center Institutional Animal Care and Use Committee in compliance with all applicable Federal regulations governing the protection of animals in research. The experiments reported herein were conducted in compliance with the Animal Welfare Act and per the principles set forth in the “Guide for Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 2011. The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of Uniformed Services University of the Health Sciences, The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., the Department of Defense (DoD), the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Some of the authors are military Service members or employees of the U.S. Government. This work was prepared as part of their official duties. Title 17, U.S.C., §105 provides that copyright protection under this title is not available for any work of the U.S. Government. Title 17, U.S.C., §101 defines a U.S. Government work as a work prepared by a military Service member or employee of the U.S. Government as part of that person's official duties”.


The emergency department assessment of chest pain score‐accelerated diagnostic protocol (EDACS‐ADP) are commonly used for risk stratification in undifferentiated patients with acute chest pain. This systematic review aimed to investigate EDACS‐ADP for risk stratification of emergency department (ED) patients with chest pain. The PubMed, Web of Science, Medline, Cochrane Library, China National Knowledge Infrastructure, and Wanfang databases were searched for related studies without restrictions on the publication year. The Quality Assessment of Diagnostic Accuracy Studies 2 tool was used to assess the risk of bias, and Stata 16.0 was used to determine the combined sensitivity, specificity, positive diagnostic likelihood ratio (DLR), and negative DLR. Twelve studies comprising 14 290 patients were identified. Of these, 7537 (52.74%) patients were considered low risk, and 67 (0.89%) had major adverse cardiovascular events (MACE), including myocardial infarction, stroke, and cardiovascular death within 30 days of the patients' ED presentation. EDACS‐ADP showed a combined sensitivity of 0.97 ; specificity, 0.58 (0.53−0.63); positive DLR, 2.34 (2.08−2.63); negative DLR, 0.04 (0.02−0.09); diagnostic odds ratio, 53.11 (26.45−106.63); and summary receiver operating characteristic area under the curve, 0.83 (0.79−0.86). Despite the large statistical heterogeneity of the results, EDACS‐ADP identified a considerable number of low‐risk patients for early discharge, with a specificity >50% and an incidence of MACE within 30‐days of patients' ED presentation <1%. Thus, it is a useful tool with a potential for clinical application. Emergency department assessment of chest pain score‐accelerated diagnostic protocol (EDACS‐ADP) identified a considerable number of low‐risk patients for early discharge, with sensitivity of 0.97, specificity of 0.58, and an incidence of major adverse cardiovascular events within 30‐days of patients' ED presentation 0.89%. It is a useful tool with a potential for clinical application. Conference abstracts were hand searched and included if they gave sufficient methodological detail to demonstrate they met the inclusion criteria. The following search strategies were implemented:English databases: #1: chest pains [MeSH Terms]; #2: (Chest Pain [Title/Abstract]) OR  OR  OR  OR  OR (Thoracodynia [Title/Abstract]); #3: (Chest Pain [Text Word]) OR  OR  OR  OR  OR (Thoracodynia [Text Word]); #4: (EDACS‐ADP [Title/Abstract]) OR (Emergency Department Assessment of Chest Pain Score‐Accelerated Diagnostic Protocol [Title/Abstract]); #5: (EDACS [Title/Abstract]) OR (Emergency Department Assessment of Chest Pain Score [Title/Abstract]); #6: ([Title/Abstract]) OR [Title/Abstract]); #7: #1OR#2OR#3; #8: #5 AND #6; #9: #4 OR #8; #10: #7 AND #9.Chinese databases: (“EDACS”) OR (“Emergency Department Assessment of Chest Pain Score‐Accelerated Diagnostic Protocol”) OR (“EDACS‐ADP”).This meta‐analysis was registered in PROSPERO (registration number CRD: 42021269142) and will be reported according to the PRISMA guidelines.2.2The following studies were included: (1) diagnostic studies published from China or internationally on the value of EDACS‐ADP for acute chest pain; (2) studies with 30‐day follow‐up on the outcomes of patients with acute chest pain; (3) major adverse cardiovascular events (MACE) within 30‐days of patients' ED presentation as an outcome event; and (4) studies with complete raw data, such that the true positive (TP), false positive (FP), false negative (FN), and true negative (TN) values of the screening test could be obtained directly or indirectly. The following were the exclusion criteria: (1) no 30‐day follow‐up or outcome results and (2) incomplete data or information.2.3Two researchers performed literature screening and data extraction independently, and their results were cross‐checked with all discrepancies resolved by a third party. Literature screening first involved removing duplicate studies, followed by reading the article title and abstract to remove clearly unrelated studies. Finally, the researchers read the full article to decide whether to include the study. The data extracted included the study characteristics , patient characteristics (age and sex), study setting, study methods, sample size, study quality, outcome measures, and results.2.4Using the RevMan 5.3 software (The Cochrane Collaboration), two researchers independently assessed the risk of bias and quality of the studies based on the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS‐2) tool.2.5χ2 and I2 tests. If considerable heterogeneity was observed, a regression analysis was performed to determine the cause. The summary receiver operating characteristic (SROC) curve was plotted to obtain the area under the curve, the sensitivity analysis graph was used to evaluate result stability, the Deeks' funnel plot was used to assess publication bias, and the Fagan plot was used to assess the clinical value of EDACS‐ADP.Stata 16.0 (StatCorp LLC) was used to collate the TP, FP, TN, and FN values for each study and obtain the corresponding combined sensitivity, specificity, positive diagnostic likelihood ratio (DLR), and negative DLR. The heterogeneity among study results was determined using the 33.1A total of 2025 studies were identified from the Chinese and English databases, including 44 Chinese studies and 1981 English studies. Among them, 12 studies met the inclusion criteria providing 13 data sets. The literature search process is shown in Figure 3.2The results of the quality assessment based on the QUADAS‐2 criteria  experienced MACE within 30 days of patients' ED presentation. Based on EDACS‐ADP, 7537 (52.74%) of the pooled patient group were classified as low risk, while 67 (0.89%) patients in the low‐risk group suffered from MACE within 30‐days of patients' ED presentation. The diagnostic accuracy of EDACS‐ADP was 63.5%. Figure p = .80). Supporting Information: Figure Figure Q statistics of sensitivity, specificity, positive predictive value, negative predictive value, positive DLR, and negative DLR all reached statistical significance (p < .001), with all I2 > 75%. These findings indicate a high level of heterogeneity among the studies. A regression analysis was thus conducted, which showed that the source of heterogeneity could have been the publication year, sex, and sample size. Subgroup analyses were performed based on the publication year, study type, country/region, sample size, age, sex, and outcomes. Details of subgroup analyses are shown in Supporting Information: Figure The 4Risk stratification tools for acute chest pain can reduce the rate of misdiagnosis and wrong treatment among high‐risk patients with chest pain in the ED, while enabling the early discharge of low‐risk patients with chest pain. Thus, their prospects for future applications are promising. EDACS‐ADP is a simple and flexible decision aid, which allows clinicians to perform risk stratification of patients with chest pain by combining the patient's troponin results at 0 and 2 hour to determine whether the patient should be admitted or discharged. However, the unstable performance of EDACS‐ADP in external cohorts has consistently been a key factor preventing its clinical application. To the best of our knowledge, Roche et al.As shown in Supporting Information: Table Owing to the enormous variations in the etiologies and disease severity of acute chest pain, as well as the potentially catastrophic consequences of high‐risk chest pain, risk stratification tools for chest pain emphasize sensitivity to avoid a missed diagnosis for patients with high‐risk chest pain to the greatest extent possible, which is a shared consensus among clinicians. A study has shown that a MACE incidence >1% among low‐risk patients was unacceptable to nearly half of the physicians, whereas an incidence >2% was unacceptable to most physicians.This study had some limitations. First, only the studies published in Chinese and English languages were included; thus, those in other languages were not included, which may have affected our search results. Second, the gold standards used in the included studies were not completely consistent, which may have affected the strength of our results. Third, the statistical analysis revealed significant heterogeneity among the included studies; hence, more clinical studies are required to verify the reliability of our results. Fourth, the external cohort results for EDACS‐ADP were unstable, and the regression analysis identified the study type and country/region as possible sources of heterogeneity. This suggests that heterogeneity is related to the basic information of the enrolled patients. Thus, it is necessary to design larger‐scale prospective studies and reinforce the control of relevant factors. Further investigations should be conducted on the applicative value of EDACS‐ADP in the risk stratification of ED patients with chest pain to obtain more robust data that will enable the wider adoption of this tool among clinicians.In conclusion, despite the significant heterogeneity detected in the included studies, EDACS‐ADP was able to identify a considerable number of low‐risk patients for early discharge, which can help alleviate ED crowding and reduce the waste of medical resources. Moreover, MACE incidence within 30‐days of patients' ED presentation among low‐risk patients was 0.89%, which was not significantly higher than that for other ADPs. Thus, EDACS‐ADP is efficient for the risk stratification of patients presenting to EDs with chest pain and has an acceptable safety profile.Minghu Wang and Zhiwei Hu contributed to the study conception and design, searched the articles, independently reviewed all identified articles for eligibility, performed the data analyses, and wrote the original draft. Lihui Miao and Manman Shi assisted in data acquisition and review literature. Critically revised the work were performed by Qiang Gao. All authors read and approved the final manuscript.The authors declare no conflict of interest.Supplementary Fig. 1 Assessment of methodological quality of each study using the Quality Assessment of Diagnostic Accuracy Studies 2.Click here for additional data file.Supplementary Fig. 2 Forest plots for the combined positive and negative diagnostic likelihood ratios. CI, confidence interval; df, degree of freedom; DLR, diagnostic likelihood ratio; Q, Cochran's heterogeneity statistic.Click here for additional data file.Supplementary Fig. 3 Forest plots for the combined diagnostic odds ratio and diagnostic score. CI, confidence interval; df, degree of freedom; Q, Cochran's heterogeneity statistic.Click here for additional data file.Supplementary Fig. 4 Deeks’ funnel plot of Emergency Department Assessment of Chest Pain Score‐Accelerated Diagnostic Protocol chest pain risk stratification. ESS, effective sample size.Click here for additional data file.Supplementary Fig. 5 Forest plots for univariate meta‐regression and subgroup analyses. CI, confidence interval; I2, I2 tests; I2lo, I2 tests ; I2hi, I2 tests ; LRTChi2, χ2 tests.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


To evaluate the clinical utility of p16/Ki67 dual‐stain (DS) compared with cytology for detecting cervical intraepithelial lesion grade two or worse (CIN2+) in women with a transformation zone type 3 (TZ3).Cross‐sectional study.Colposcopy clinics in Central Denmark Region.Women aged 45 years or older referred for colposcopy because of an abnormal screening test.All women had a cervical sample collected for cytology and DS testing and underwent large‐loop excision of the transformation zone (LLETZ).Sensitivity, specificity and negative (NPV) and positive (PPV) predictive values of DS for CIN2+ detection were compared to those of cytology.p = 0.021) and NPV  compared with cytology for CIN2+ detection. In contrast, the specificity (63.5% versus 90.5% p < 0.001) and PPV  were lower for DS compared with cytology.Of 166 women eligible, 93 (56.0%) were included in the final analysis. Median age was 68 years (interquartile range [IQR] 63.4–70.5 years). Most women were postmenopausal (95.7%) and referred based on a positive human papillomavirus screening test (86.0%). Fifty‐two women (55.9%) were DS‐positive, 29 (55.8%) of whom had CIN2+ detected. Twenty‐seven (29.0%) women had atypical squamous cells of undetermined significance or worse (ASC‐US+), and CIN2+ was detected in 21 women (77.8%). DS had a higher sensitivity (96.7% versus 70.0% Dual stain may be a valuable risk marker to guide clinical management of women with a TZ3. The superior NPV of DS suggests that a diagnostic excision may safely be avoided in DS‐negative women. DS‐negative postmenopausal women with a TZ3 have a low risk of CIN2+ and may undergo follow up instead of LLETZ. Women aged 23–59 years are screened with liquid‐based cytology (hereafter ‘cytology’), whereas women aged 60–64 years undergo HPV‐based screening. In addition, from April 2019 women aged 65–69 were invited for one additional HPV‐based screening test as part of an intervention study in Central Denmark Region.2.2Samples used in the present study were collected as part of another clinical study.Women answered questions on basic characteristics . Further, information on previous screening history was retrieved from the National Danish Pathology Databank2.3Cytology slides were reviewed by experienced cyto‐technicians using computer‐assisted microscopy  and categorised using the Bethesda 2014 grading system.Dual‐stain slides were reviewed independently by two cyto‐technicians who were blinded to all study data  except for the age of the woman. Both cyto‐technicians received training in interpretation of DS slides according to the manufacturer's recommendations.Cytology, HPV testing and DS testing were performed at the Department of Pathology, Randers Regional Hospital, Denmark, which is responsible for analysing all cervical cytology samples in Central Denmark Region .Clinical management was not based on DS results.Histopathological examination of cervical biopsies and LLETZ specimens was performed in routine laboratories at the Departments of Pathology, Randers and Viborg Regional Hospital and graded according to the CIN classificationNo patients or patient organisations were involved in the development, design or implementation of this study.2.4For analysis, only women with valid results on cytology, HPV and DS were included. CIN2+ in the LLETZ specimen was used as the primary outcome instead of CIN grade 3 or worse because CIN2 is the threshold for excisional treatment in older women in most countries, including Denmark and the UK.For cytology and DS, we calculated specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) for CIN2+ detection with corresponding 95% confidence intervals (CI). Specificity and sensitivity for cytology and DS, respectively, were compared using the McNemar's chi‐square test. To test the robustness of our findings we conducted a sensitivity analysis excluding women with unclassifiable CIN. Further, we have restricted the analyses of DS positivity by cytology and histology to women who have undergone HPV‐based screening.Finally, we calculated sensitivity, specificity, PPV and NPV using different thresholds of DS‐positive cells  for CIN2+ detection and calculated the Youden's index (YI = sensitivity + specificity − 1). Cases with one DS‐positive cell served as reference for the comparison between different thresholds.p less than 0.05 were considered statistically significant.Data were entered and stored in REDCap.33.1One‐hundred and sixty‐six women were assessed for eligibility, 99 (59.6%) of whom were enrolled Figure . Six womThe median age of the included women was 68.0 years (IQR 63.4–70.5 years) with 89 (95.7%) women being postmenopausal Table . Most wo3.2p < 0.001), respectively  had normal cytology, 8 (8.6%) had low‐grade cytology (ASC‐US and LSIL) and 19 (20.4%) had high‐grade cytology (ASC‐H and HSIL) Table . DS posily Table .Restricting the analyses to women who have undergone HPV‐based screening showed similar results to those stated above Table .3.3p = 0.021), but less specific  (Table p = 0.018), whereas the PPV of DS was significantly lower compared with the PPV for cytology  (Table n = 80), we found no major differences in sensitivity, specificity, PPV and NPV compared with the overall results  were DS positive, of whom 29 (55.8%) had CIN2+ detected (Table p = 0.004) and cytology , respectively. The sensitivity, specificity and NPV for DS and cytology did not change (data not shown).Excluding unclassifiable CIN from analysis resulted in a significant decrease in PPV for DS  as well as increased specificity  and PPV . Sensitivity and NPV of two or more DS‐positive cells for CIN2+ detection were almost identical compared with one‐cell cutoff .In the present study, our results suggest that DS is of great value in clinical management because of a high sensitivity and because a negative DS test provides greater reassurance against CIN2+ than a negative cytology. Hence, DS may enable a risk stratification of women into those in need of immediate excisional treatment while allowing those with a negative DS test to undergo follow up. However, future studies are needed to determine the appropriate follow‐up interval and to replicate our findings focusing on women with a TZ3.5The high NPV of DS suggests that postmenopausal women with a negative DS can safely undergo follow up with repeated cervical sampling instead of diagnostic LLETZ. Therefore, DS may be a valuable risk marker to guide clinical management of postmenopausal women with a TZ3 at colposcopy.6The idea and the overall design of this study were conceived by LWG, MT, BA, PB, LKP, NW, MC and AH. Recruitment, data collection, data management and analysis of samples were by RB, PNC, PB, MT and LWG. Data analyses were conducted by MT, NW, MC, AH and LWG. LWG prepared the first draft with guidance/supervision from MT, NW, MC, PB, BA, LKP, PB and AH. Critical revision of the manuscript for important intellectual content was provided by NW, MC, MT, LWG, BA, RB, PNC, PB, LKP and AH. All authors approved the final manuscript before publication.8LWG, MT and LKP have received speaker's fees from Astra Zeneca, outside the submitted work. LWG, MT and BA are participating in other studies with HPV test kits and CINtec plus kits sponsored by Roche, outside the submitted work. MT has received honoraria from Roche Diagnostics for lectures on CINtec plus, outside the submitted work. AH has received reagents from Roche, Denmark for free, outside the submitted work. MC: Enduring Guidelines for Cervical Cancer Screening and Management . International Neoplasia Society's Task Force for the development of anal cancer screening guidelines (Working Group Co‐Chair). LKP: Speakers fee from Merck on subjects outside this study. PNC, RB, NW and PB report no conflict of interest. Completed disclosure of interest forms are available to view online as supporting information.910The project was listed at the record of processing activities for research projects in the Central Denmark Region (j.no. 1‐16‐02‐528‐18) and was deemed exempt from the need for ethics approval (j.no:1‐10‐72‐4‐17).Table S1Click here for additional data file.Table S2Click here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.ICMJEClick here for additional data file.


Hypoxic ischemic encephalopathy (HIE) after a perinatal insult is a dynamic process that evolves over time. Therapeutic hypothermia (TH) is standard treatment for severe to moderate HIE. There is a lack of evidence on the temporal change and interrelation of the underlying mechanisms that constitute HIE under normal and hypothermic conditions. We aimed to describe early changes in intracerebral metabolism after a hypoxic-ischemic insult in piglets treated with and without TH and in controls.Three devices were installed into the left hemisphere of 24 piglets: a probe measuring intracranial pressure, a probe measuring blood flow and oxygen tension, and a microdialysis catheter measuring lactate, glucose, glycerol, and pyruvate. After a standardized hypoxic ischemic insult, the piglets were randomized to either TH or normothermia.Glycerol, a marker of cell lysis, increased immediately after the insult in both groups. There was a secondary increase in glycerol in normothermic piglets but not in piglets treated with TH. Intracerebral pressure, blood flow, oxygen tension, and extracellular lactate remained stable during the secondary increase in glycerol.This exploratory study depicted the development of the pathophysiological mechanisms in the hours following a perinatal hypoxic-ischemic insult with and without TH and controls. Brain dll lysis . After tll lysis . Despitell lysis –8. Cell ll lysis , 10. In ll lysis , 12. Thull lysis . TH has ll lysis . Preclinll lysis –17. TH mll lysis , 19. Thell lysis . Basic kll lysis . We therWe used a piglet model of HIE, first described in 1996 and later modified by our group , 22. The2 of 4.5–5.5 kPa. Continuous infusion of propofol (4–12 mg/kg/h) and fentanyl (5–10 µg/kg/h) were used as sedation throughout the experiment. Using aseptic techniques, umbilical venous and arterial catheters were placed. Mean arterial blood pressure (MABP), SatO2%, FiO2, EtCO2, temperature, and electrocardiogram (ECG) were monitored and logged . Core temperature was measured continuously approximately 5 cm into the rectum. To prevent infection, all animals were treated with subcutaneous benzylpenicillin 15,000 IE/kg every 12 h or intravenous gentamicin 5 mg/kg once every 24 h and ampicillin 30 mg/kg every 12 h . A continuous 10% glucose infusion was administered at 5–10 ml/kg/h and continuously adjusted according to blood glucose measurements. Urine production was monitored through regular ultrasound-guided bladder punctures. Blood electrolytes, glucose, and gas composition were regularly monitored. We aimed to maintain a MABP > 40 mmHg. Hypotension was treated initially by reducing infusion of anesthetics. If insufficient, noradrenaline infusion (0.25–1.5 µg/kg/min) and/or dopamine infusion (5–15 µg/kg/min) was titrated to reach MABP > 40 mmHg , fentanyl (30 µg/kg), and rocuronium (1 mg/kg) was administered. The piglet was intubated and mechanically ventilated with a target end-tidal CO2). A single-channel amplitude-integrated electroencephalogram (aEEG) was acquired with an interelectrode distance of 3 cm and one electrode behind each eye . The aEEG was scrutinized for seizure activity, i.e., rhythmic, evolving activity of a minimum 2 µV and with a duration of >10 s. Due to the supine position of the animal, probes and microdialysis were placed without the use of a stereotactic frame. Standardized device placement was decided based on earlier post-mortem measurements. We have previously investigated the consequence of inserting devices into cerebral tissue and the effect on early measurements in the same animal model (Using cannulas (2.1 mm diameter for the first and 1.5 mm diameter for the second and third hole), three holes were made through the scalp 10 mm into the brain from the skin surface. The holes were made 10 mm to the left of the midline. The most rostral hole was made 5 mm posterior to a perpendicular line between the eyes, and the two consecutive holes were made in a line 3 mm posteriorly to the first hole and 3 mm apart. In the most rostral hole, a probe for measuring intracranial pressure (ICP) was installed . In the second hole, a probe for measuring cerebral blood flow (CBF) through laser Doppler, temperature, and oxygen tension was installed . In the last hole, a CMA 20 Elite 4 mm membrane microdialysis catheter connected to a CMA 402 syringe pump  was installed. Microdialysis was performed with central nervous system sterile isotonic perfusion fluid with a flow rate of 0.5 µl/min. The microdialysate was analyzed for lactate, pyruvate, glucose, and glycerol on a CMA 600 microdialysis analyzer . A cerebral near-infrared spectroscopy (NIRS) oximeter  was placed on the parietal region on the right side of the scalp, measuring regional cerebral oxygenation  during the 45-min period combined with hypotension (MABP < 70% of baseline) for at least 10 min. To ensure survival, FiO2 was shortly increased if MABP reached <50% of baseline, HR < 80 min−1, or aEEG < 3 µV. After 45 min, the piglet was resuscitated with room air. If needed, FiO2 was increased to keep SatO2 > 90%.A global HI insult was induced for 45 min . FiO2 waA total of 24 piglets were used. Four piglets were intended as controls without hypoxia or TH. The remaining 20 piglets were subjected to an HI insult and then randomized to normothermia or whole-body TH with a target temperature of 33.5–34.0°C. TH was achieved through passive whole-body cooling by turning of heating blankets and lamps to allow for the cold ambient air in the operation room to cool the piglet to target temperature. TH was continued throughout the experiment and kept stable by the use of alternating the heating blankets and lamps and the cold ambient air. Intracerebral measures were acquired at baseline, during hypoxia, and once every hour for 24 h. After 24 h, the piglets were euthanized with intravenous pentobarbital injection (80 mg/kg). The investigators were not blinded to the group allocation.t-test for parametric data and Mann–Whitney rank sum test or Kruskal–Wallis test for non-parametric data. The use of inotropes was registered hourly and then averaged for the whole 24-h observation period. The number of animals that received inotropes was reported and for those the average infusion rate was calculated. The change in cerebral perfusion pressure (CPP) was calculated as the difference between MAPB and ICP . Intracerebral measurements are presented as mean with standard deviation (SD). The data set generated and analyzed during the current study is available from the corresponding author on request.A pilot study was performed including three piglets that were healthy and not subjected to HI or TH. Data from these pilot animals showed little variation in intracerebral measurements, and therefore we estimated that four animals were sufficient for the control group. As there was no data on animals with HI and TH, the resource equation method was used to determine the sample size for the two treatment groups . With an and ICP . The rel2 probe in the HI group and from one microdialysis catheter in the HI + TH group. aEEG recording during the observation period was impossible in one animal in the control group, one animal in the HI group, and one animal in the HI + TH group due to technical problems. Hypoxia resulted in EEG suppression, metabolic acidosis, and hypotension in both HI-groups ; 1.3 (0.4) vs. 4.0 (1.1) vs. 4.0 (1.5)] . There w2 remained stable in all three groups. Although not statistically significant, rSO2 tended to be higher in the HI-group during the observation period. During hypoxia, rSO2 was equally suppressed in the HI and HI + TH groups  Winter et al. and Tichauer et al. found that CMRO2 was decreased in piglets subjected to an HI insult compared with controls hours after the HI insult (2 occurred in the first hours after the HI insult, and during the same time period, CBF was equal to control animals (2 returned to baseline levels after the HI insult. A potential reason for this could be insult severity, i.e., the insult was sufficiently severe to provide a primary and secondary peak in glycerol, but not sufficient to elicit a change in cerebral oxygenation or CBF. Jinnai et al. measured the change in cerebral hemodynamics and oxygenation in piglets subjected to an HI insult similar to ours and found minor or no change in cerebral oxygenation during the first 24 h after the insult (In accordance with previous studies performed in a similar animal model, we found an instantaneous decrease in oxygen tension and rSOI insult –43. AfteI insult , 45. The animals . Howevere insult . They foe insult , 47, 48.e insult . In accoe insult , 50.2, CBF, or ICP in relation to seizures. In a child with HIE, seizures with a duration less than 7 min did not affect rSO2 (2 of approximately 2%–4% was found during seizures compared with pre-seizure measurements (A pathophysiological mechanism that constitutes the secondary deterioration after HI is the onset of seizures . In our ect rSO2 . In a courements . Thus, surements .A suggested neuroprotective mechanism of TH is reduction in cerebral metabolism as TH has been shown to reduce metabolism by approximately 5% for each 1-degree reduction in temperature . This ispost hoc power calculation based on microdialysis and NIRS data showed that the numbers of animals included in each group would need to be more than doubled for sufficient power. We believe that this would contribute with limited information to the manuscript—especially with regard to the 3R principle.In this study, we found some measurements with borderline significance when comparing the HI with the HI + TH group. A After a standardized HI insult, we found the presence of a progressive secondary deterioration with concomitant increase in markers of cell lysis, i.e., a secondary increase in glycerol levels in the normothermic piglets. We also found that intracerebral blood flow, pressure, and oxygenation were not influenced during this secondary increase in glycerol levels, and we found no secondary increase in extracellular lactate. TH treatment appeared to abolish the secondary increase in glycerol concentration. TH also tended to result in lower intracerebral pressure, glucose accumulation, and lower levels of extracellular lactate.


Urinary tract infections (UTI) are common types of bacterial infection in children. UTI treatment is aimed to prevent complications including hypertension, proteinuria, and progression to chronic kidney disease. Activated neutrophils release chromatin-based structures associated with antimicrobial proteins called neutrophil extracellular traps (NETs). We aimed to describe the role of NET-associated markers in children with UTI as well as the role of NETs formation in a mouse model of UTI.E.coli  and in healthy controls . Moreover, an acute experimental model of UTI was performed on PAD4 knock-out mice with diminished NETs formation (n = 18), and on wild-type mice (n = 15).Markers of NETs including extracellular DNA (ecDNA), myeloperoxidase (MPO) and cathelicidin were analyzed in children with febrile UTI caused by r = 0.53, p ≤ 0.001; r = 0.56, p ≤ 0.001, respectively) and the number of leukocytes in the urine . Moreover, urinary MPO was positively associated with cathelicidin . In the experimental model, bacterial load in the bladder (20-fold) and kidneys (300-fold) was significantly higher in PAD4 knock-out mice than in wild-type mice.Children with UTI had significantly higher urinary NETs markers including total ecDNA, nuclear DNA and mitochondrial DNA, altogether with MPO and cathelicidin. The concentrations of MPO and cathelicidin positively correlated with ecDNA (Higher urinary NETs makers—ecDNA, MPO and cathelicidin and their correlation with leukocyturia in children with UTI confirmed our hypothesis about the association between NETs and UTI in children. Higher bacterial load in mice with diminished NETs formation suggests that NETs are not only a simple consequence of UTI, but might play a direct role in the prevention of pyelonephritis and other UTI complications. Urinary tract infections (UTI) are the most common type of bacterial infection in the pediatric population , 2. DuriApart from antimicrobial defense of neutrophils , 7, extrTherefore, we have aimed to investigate the role of NETs formation and inflammation in UTI. Firstly, we investigated the presence of urinary markers of NETs in children with UTI in order to clarify the associations between UTI severity and NETs formation. Secondly, the role of NETs in the pathogenesis of UTI was further elucidated in an animal UTI model using protein-arginine deiminase type 4 (PAD4) deficient mice. PAD4 enzyme is crucial for chromatin depolymerization during NETs formation , 19. We E. coli were enrolled to this study.In this cross-sectional study, 179 consecutive pediatric patients with acute pyelonephritis were enrolled. Patients were admitted to the Department of Pediatrics of the National Institute of Childreńs Diseases in Bratislava, Slovakia between March 2019 and March 2022. Diagnosis of pyelonephritis was based on the medical history and physical examination, a urine microscopy and urine culture. Patients had to meet all of the following criteria: presence of nonspecific symptoms , C-reactive protein above 50 mg/L, pyuria and significant bacteriuria. Pyuria was defined as positive leukocyte esterase (LE > trace) on dipstick analysis and/or >10 white blood cells (WBC) per one power field in centrifuged urine. Significant bacteriuria was defined as >50,000 CFU/ml of a single uropathogen from a catheterized specimen (E. coli (40 patients), and the co-existence of other acute or chronic disorders (3 patients). Ninety-eight patients  with acute pyelonephritis caused by E. coli strain were included for further analysis , significant growth of other uropathogens than analysis . None ofA control group of 50 healthy children was recruited among children attending regular check-ups during visits at general pediatrician or from the children attending outpatient visits at the Department of Pediatrics due to disorders unrelated to UTI . None of the healthy controls had a history of UTI.3EDTA, lithium-heparin and SST™ ΙΙ Advance tubes . Urine samples were collected into sterile Falcon tubes  via urinary catheterization or via sterile bag specimen . Standard biochemical analysis and urine microscopy were performed immediately. If urine cannot be cultured within 4 h of collection, the sample was refrigerated. Aliquots of blood and urine were centrifuged at 1,600 g for 10 min. The supernatants were stored at −20°C until further analysis.All blood and urine samples were collected at the time of admission, i.e., before initiation of antibiotics treatment. Venous blood was collected from either dorsal superficial vein or cubital vein into KComplete blood count with differential leukocyte count was assessed using a Sysmex XN-1000™ Hematology Analyzer . Serum creatinine, BUN, uric acid, total proteins, albumin, bilirubin, C-reactive protein, natrium, potassium, calcium and phosphorus were analyzed using standard laboratory methods .Urinary creatinine was measured using Jaffé reaction as described previously . BrieflyConcentration of myeloperoxidase (MPO) in urine was determined using commercially available spectrophotometric assay according to manufactureŕs protocol . Absorbance was measured at 450 nm. Limit of detection was 62.5 pg/ml.Similarly, cathelicidin in urine was measured by commercial Elisa kit as recommended by the manufactureŕs protocol . Urine samples were 10-times diluted. Absorbance was measure at 450 nm. Limit of detection was 0.14 ng/ml.via fluorometric method using Qubit Fluorometer and Qubit dsDNA HS Assay Kit . Cellular origin of ecDNA was determined using quantitative polymerase chain reaction recorded in real time. Real-time PCR was performed using SsoAdvanced Universal SYBR Green Supermix  on MasterCycler RealPlex . To quantify ncDNA, primers encoding human beta-globin gene  with following program: initiation 3 min at 98°C, 40 × 15 s at 98°C for denaturation, 30 s at 51°C for annealing, 30 s at 60°C for polymerization, were used. To determine mtDNA, primers were designed to target mitochondrial D-loop amplification  using following program: initiation 3 min at 98°C, 40 × 15 s at 98°C for denaturation, 30 s at 47°C for annealing, 30 s at 60°C for polymerization. Concentrations of ncDNA and mtDNA in urine are expressed in genomic equivalent per milligram of creatinine in urine (GE/mg of creatinine).To remove apoptotic bodies, aliquots of plasma and urine used for the ecDNA isolation were centrifuged second time at 16,000 g for 10 min. ecDNA was isolated from 200 microliter of samples based on column method using commercial kit  as recommended by manufactureŕs protocol. Total ecDNA was assessed n = 15) and PAD4−/− KO (n = 18). Mice were housed in cages with a controlled temperature and humidity and 12/12 h light/dark cycle. All animals had free access to standard chow and water during the whole experiment.All procedures were performed on 7–8 weeks old mice obtained from Animalab  strains C57BL/6 (E. coli strain CFT073 isolated from patient with acute pyelonephritis (600nm = 1–8 × 108 colony-forming units (CFU)/ml.Uropathogenic ephritis . Bacteri7 CFU. After 24 h the animals were sacrificed by cervical dislocation and bladders and kidneys were harvested. Organs were immediately homogenized in sterile ice-cold phosphate buffered saline and serial dilutions of the homogenates were plated on agar plates. The plates were incubated overnight at 37°C, bacterial colonies were counted, and the number of CFU per organ were calculated.For induction of UTI in mice, surgical approach of bladder inoculation was used . BrieflyU test was used. Data with high variability were log transformed. Associations between variables and clinical parameters were determined using Spearman’s test (skewed data). p value less than 0.05 was considered as a limit of statistical significance. Data are presented as median with interquartile range.GraphPad Prism software 9.4.1 was used for statistical analysis . Normality of data was tested using D'Agostino-Pearson omnibus test. Outliers were identified using Grubbś test. Differences of non-normally distributed variables between two independent groups were evaluated by Mann-Whitney All procedures performed in this study involving animals were conducted in accordance with the ethical standards of the institutional and national research committee and was approved by the Ethics Committee of the Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia and the State Veterinary and Food Administration of the Slovak Republic. Clinical part of the study was performed according to guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of the National Institute of Children’s Diseases at the Faculty of Medicine, Comenius University, Bratislava, Slovakia. Consent to participate of all children was provided by a parent or legal guardian.U = 1,924, z score = −0.86, p > 0.05, U = 556, z score = −3.12, p < 0.01, U = 488, z score = −2.48, p < 0.05, U = 638, z score = −2.36, p < 0.05, U = 85.5, z score = −6.82, p < 0.001, U = 612, z score = −2.44, p < 0.01, Children with UTI had similar concentration of total ecDNA in plasma as healthy children . Similarly, total ecDNA in urine did not correlate with inflammatory parameters .Total plasma ecDNA in UTI patients did not correlate either with WBC, ANC, or with CRP  and uropathogenic E. coli (MOI 20) for 4 h. Colocalization of signal from DNA-binding dye SYTOX GreenTM (green channel) with strong signal from antibody against citrullinated histone H3 (red channel) was observed in PMN isolated from WT mice after incubation with PMA and E. coli, but not in PMN from PAD4−/− KO mice (Polymorphonuclear cell isolated from WT and PAD4 KO mice .−/− KO mice tended to be higher than in WT mice (Mann-Whitney U = 78, z score = −1.82, p = 0.06, −/− KO mice had significantly higher concentration of bacteria present in kidneys compared to WT mice (Mann-Whitney U = 77, z score = −2.09, p < 0.05, Twenty-four hours post-infection, bacterial load in bladder in PAD4The analysis of our data has shown that the concentration of total ecDNA in the urine rises with increasing inflammation. Plasma ecDNA concentrations did not change, which was in line with our hypothesis to focus on urine ecDNA as a suitable biomarker for the early detection and severity of acute pyelonephritis. Elevated levels of plasma ecDNA have been described in association with systemic infectious processes such as sepsis , 25. UriThe rise of urine ecDNA was further accompanied by increased concentrations of MPO and cathelicidin. MPO as a well-established marker of inflammation and oxidative stress plays a central role in the microbial killing. Multiple studies display the presence of MPO in a variety of renal diseases –29. Cirain vitro. We also showed that PAD4 KO mice have higher bacterial load of the kidneys suggesting that NETs formation is not only a consequence of UTI, but it is directly involved in the immune protection against pathogens and prevention of UTI complications in our animal model. NETs seem to be an important component involved in the bacteria trapping in UTI. Our results are in line with previous study dealing with the role of NETs in pathogen-induced acute lung injury where PAD4 deficiency was associated with the increased bacterial load and inflammation in lungs (In our study, we confirmed using fluorescent microscopy that PAD4 KO mice do not form NETs in lungs . Similarin lungs . On the in lungs . Correspin lungs . Likewisin lungs , 40. Thein lungs , 42.E. coli were excluded from analyses. Children with UTI, instead of adults, represent a uniform cohort with bacterial inflammation of only one organ without any comorbidities that could affect concentration of NETs markers. Thus, unique design of the study minimized external factors of variability. Using NETs markers for an early diagnostic of UTI without the need to wait for the results from the urine culture might be of practical importance. PAD4 deficiency was associated with increased bacterial burden in kidneys, which suggests a protective role of NETs during acute phase of UTI, where they would halt the dissemination of bacteria. On the other hand, excessive NETs formation has been linked to tissue damage and exacerbation of pathological inflammation and further studies that will monitor NETs formation in a longer time-period are needed. Persistent NETs formation that might have potentially detrimental effects could thus be targeted by NETs formation inhibitors, further highlighting the importance of establishment of reliable urinary NETs markers.The main limitation in the experimental study is that small volume of urine obtained from mice prevented evaluation of NETs, and thus only bacterial burden was reported. Limitation of the clinical part of the study is its cross-sectional design. Long-term observations would be valuable for gaining further insight. On the other hand, the clinical study was performed on a large cohort of pediatric patients that were strictly selected—all patients were aged under 2 years and were diagnosed with the first febrile UTI without known congenital abnormalities of kidneys or urinary tract. Moreover, patients with other uropathogen than


This study aimed to determine whether the immune prognostic index (ECIPI), based on hemoglobin (Hb) and neutrophil‐to‐lymphocyte ratio (NLR), could predict the prognosis in patients with advanced esophageal squamous cell carcinoma (ESCC) receiving programmed cell death‐1 (PD‐1) inhibitor treatment.p‐value was below 0.05 in the univariate analysis. All statistical comparisons were two‐way, and a p‐value below 0.05 was set as statistical significance.Advanced ESCC patients who had been treated with PD‐1 inhibitors from Jan 2016 to Oct 2021 were included. Kaplan–Meier method and Cox proportional hazards regression were used to analyze progression‐free survival (PFS) and overall survival (OS). The overall response rate (ORR) was the percentage of complete and partial responses. Univariate and multivariate analyses were used for estimating hazard ratio (HR) and 95% confidence interval (CI). Patients were grouped by ECIPI . Variables for the multivariate model were selected if the p < 0.01). Multivariate analysis showed that ECIPI was an independent influential factor for PFS (p = 0.004) and OS (p < 0.001). Kaplan–Meier curves demonstrated that patients with ECIPI good had the longest PFS  and OS  compared with those with ECIPI intermediate and ECIPI poor. Subgroup analysis indicated that ECIPI good was associated with improved PFS and OS in patients with ECOG 0–1, PD‐1 inhibitor plus chemotherapy, first‐line treatment, and smoke .Totally, of 123 ESCC patients with stage III or IV were included in the study. Efficacy evaluation showed that patients with pretreatment ECIPI good had the best ORR compared with those with ECIPI intermediate and ECIPI poor (53% vs. 22% vs. 8%, Pretreatment ECIPI was associated with the prognosis in advanced ESCC patients with anti‐PD‐1 therapy, suggesting that ECIPI may be a useful tool to identify patients likely sensitive to PD‐1 inhibitors. Pretreatment ECIPI predicts the prognosis in advanced ESCC patiens treated with PD‐1 inhibitors. Patients should be excluded according to the following criteria: (I) patients received one cycle of PD‐1 inhibitor treatment; (II) baseline Hb, ANC, and ALC were not measured before initial immunotherapy (within 5 days); (III) treatment efficacy was not evaluated after initial immunotherapy ; (IV) patients with TNM stage I or II.2.2Two independent investigators (J.Y. Lu and Z.B. Zhang) extracted data from medical records, including age, gender, clinical stage, smoking history, Eastern Cooperative Oncology Group performance status (ECOG PS), treatment strategy (PD‐1 inhibitor plus chemotherapy or radiotherapy), treatment line of immunotherapy , treatment response evaluation, blood results of Hb, ANC, and ALC at baseline. Disagreements were resolved through discussion with the other two investigators (X. Lei and L.H. Du).The ECIPI was developed based on Hb and NLR, and the cutoff values of Hb and NLR were estimated by X‐tile v3.6.1.2.3p‐value of below 0.05 in the univariate analysis. All statistical comparisons were two‐way, and a p‐value of below 0.05 was set as statistical significance.All statistical analyses were performed using SPSS v23.0 and graphs were drawn using GraphPad Prism 8.0. The cutoff values for Hb and NLR were determined with X‐tile v3.6.1. Comparations between categorical variables were performed using Pearson's chi‐square test or Fisher's exact test. The Kaplan–Meier method was used for survival analysis and the log‐rank test for comparison. Cox proportional hazards regression models were used for identifying independently influential factors and estimating their hazard ratios (HR) and 95% confidence intervals (CI). Variables included in the multivariate model were selected if the 33.1Initially, 158 ESCC patients treated with PD‐1 inhibitors were identified, of which, 35 patients were excluded including 11 patients with one cycle of immunotherapy, 5 patients without treatment assessment, 12 patients without blood results at baseline, 2 patients with stage I, and 5 patients with stage II. Ultimately, 123 patients were used for analysis in this study , and multivariate analysis revealed that ECOG 0–1 , first‐line treatment , and ECIPI good  were independently associated with prolonged PFS in comparison with ECOG ≥ 2, third‐line and above treatment, and ECIPI poor, respectively (Table p < 0.05), after multivariate analysis, the results showed that ECOG 0–1  and ECIPI good  or ECIPI intermediate  were independently correlated with improved OS in comparison with ECOG ≥ 2 and ECIPI poor . Kaplan–Meier survival curves demonstrated that patients with ECIPI good had the longest PFS  and OS  compared with those with ECIPI intermediate and ECIPI poor . The proportion of ECOG 0–1 , PD‐1 inhibitor plus chemotherapy , and first‐line treatment  were higher in patients with ECIPI good than that of ECOG ≥ 2, without chemotherapy, second‐line treatment, and third‐line and above treatment. Subgroup analysis showed that ECIPI good was associated with improved PFS and OS in patients with ECOG 0–1, PD‐1 inhibitor plus chemotherapy, first‐line treatment, and smoke with all p < 0.05 ; writing – review and editing . Lehui Du: Writing – review and editing . Xiao Lei: Writing – review and editing . Zhibo Zhang: Conceptualization (lead); formal analysis (lead).None.All authors declare no conflicts of interest in this study.This study was approved by the Ethics Committee of Chinese PLA General Hospital, and informed consent was not required because the study was retrospective.