This study was designed to investigate the effect of neoadjuvant chemotherapy on the expression of hormone receptors and Ki67 in Chinese female breast cancer patients. The expression of estrogen receptor (ER), progesterone receptor (PR) and Ki67 among 525 neoadjuvant chemotherapy cases was studied by immunohistochemistry. Differences between specimens made through preoperative core needle biopsy and excised tissue biopsy were observed. The positive rates of ER, PR and Ki67 in core needle biopsy and excised tissue biopsy were 65.3% and 63.2%, 51.0% and 42.6%, 65.6% and 43.4%, respectively. The expression of ER, PR and Ki67 in core needle biopsy and excised tissue biopsy had no statistically significant difference. However, after neoadjuvant chemotherapy, the discordance rates of ER, PR and Ki67 were 15.2% (79/521), 26.9% (140/520) and 44.8% (225/502), respectively. The ER, PR and Ki67 status changed from positive to negative in 7.5% (39/521), 13.3% (69/520) and 21.1% (106/502) of the patients, whereas ER, PR and Ki67 status changed from negative to positive in 7.7% (40/521), 13.6% (71/520) and 23.7% (119/502) of the patients, respectively. These results showed that the status of some biomarkers changes after neoadjuvant chemotherapy and biomarker status needs to be reexamined to optimize adjuvant systemic therapy and better prognosis assessment. Neoadjuvant chemotherapy, the primary systemic treatment, has become a standard treatment to shrink the tumor and improve the chance of breast conserving surgery for operable breast cancer patients, because it can increase disease-free survival and overall survival rates compared with postsurgical adjuvant chemotherapy[2–. Neoadjuvant chemotherapy is completed before surgery, except for patients who have disease progression during treatment, because the progression may put them at higher risk of surgery. Meanwhile, hormone receptor (HR) assays have become a standard practice for clinicians in endocrine treatment. The hormone-dependent nature of breast cancer is the basis for endocrine therapy that benefits patients with positive estrogen receptor (ER) and progesterone receptor (PR)[6–. Previous studies have reported that neoadjuvant chemotherapy affects biomarker status in breast cancer, so a question has been raised about how neoadjuvant chemotherapy modulates these markers. However, the results of several studies on the effect of neoadjuvant chemotherapy on biomarker status in breast cancer are conflicting[8–.Breast cancer is the most common and deadly cancer among femalesIn this study, we retrospectively analyzed HR expression and Ki67 in specimens of core needle biopsy (CB) and excised tissue biopsy (EB) from 525 breast cancer patients who received neoadjuvant chemotherapy and evaluated the impact of neoadjuvant chemotherapy on the expression of these two biomarkers. We further determined whether these tumor specimens should be reexamined after neoadjuvant chemotherapy.Fig. 1). As for core needle biopsy, considering the heterogeneous nature of the tumor, four to six needles were punctured in the tumor as evenly as possible in each specimen. After neoadjuvant chemotherapy, the patients underwent tumor resection. The needle and excised biopsy specimens of these cases were preserved following the standards, and immediately transferred for pathology and immunohistochemistry (IHC) detection at the Clinical Pathology Diagnostic Center of Chongqing, Chongqing Medical University.We retrospectively collected data of patients with invasive breast cancer who had undergone preoperative biopsy and subsequent surgical resection after neoadjuvant chemotherapy at the First Affiliated Hospital of Chongqing Medical University . We reviewed pathology reports of breast cancer patients from April 2012 to December 2015 through the hospital medical record system, and extracted pertinent data. Eligible participants were breast cancer patients who had received neoadjuvant chemotherapy and had both core needle biopsy and excised tissue biopsy. After screening the cases with reliable history and unified pathology reports, we selected 525 neoadjuvant chemotherapy cases . All these were performed following the standard procedure at the Clinical Pathology Diagnosis Center of Chongqing Medical University. . The calculation was done in two steps. First, a proportional score, representing the proportion of positive-staining tumor cells , was determined. Second, a stage score, representing the average intensity stage of positive tumor cells , was determined. The Allred score is obtained by adding the proportional score to the stage score. An Allred score>2 was used to define tumor HR positivity. Cancer cell proliferation was assessed by identification of Ki67-positive cells within the tissue section. The proliferation rate is shown as the number of Ki67-positive cells in 100 carcinoma cells per section. In accordance with the study by Fasching et al., a proportion of ≥13% positively stained cells, the Ki67 index to distinguish luminal B from luminal A tumors, was used as the cut-off point for Ki67 status[11–.The Allred score is an indicator of HR status by immunohistochemistry to evaluate the potential magnitude of HR change. For statistical procedures, Microsoft Excel 2010 and SPSS 22.0 software were used. All statistical tests were two-sided and P<0.05 was considered statistically significant.The association of biomarkers between the CB specimens and the EB specimens were analyzed using the McNemar testTable 1. The mean age was 49±9 years (34.9% were 45 years or younger), and approximately half (50.9%) of the patients were premenopausal. Pathological tumor stage, nodal stage, clinical stage, histologic types and histologic grade were predominantly pT1 (61.7%), pN0 (36.4%), stage II (76.2%), ductal type (94.3%), and grade 2 (74.7%), respectively. All patients used anthracyclines or taxanes based regimens for neoadjuvant chemotherapy. Mastectomy was mostly performed after neoadjuvant chemotherapy.The demographic and clinicopathologic characteristics of the participants are summarized in Table 2 and 3. The positive rates of ER, PR and Ki67 in CB and EB were 65.3% and 63.2%, 51.0% and 42.6%, 65.6% and 43.4%, respectively. The expression of ER, PR and Ki67 between CB and EB was found to have no statistically significant difference (P>0.05).The expression of hormone receptors and Ki67 in breast cancer tissues are summarized in Table 3 presents changes in the status of ER, PR and Ki67 in CB and EB cases in detail. Comparing the concordance rates of the CB and EB groups, ER was 84.8% (442/521), PR was 73.1% (380/520) and Ki67 was 55.2% (277/502). The discordance rates of ER, PR and Ki67 were 15.2% (79/521), 26.9% (140/520) and 44.8% (225/502). Meanwhile, 7.7% (40/521) cases and 13.6% (71/520) cases had ER and PR positive conversion, respectively. In 21.1% (106/502) cases, Ki67 status changed from positive to negative after neoadjuvant chemotherapy, while in 23.7% (119/502) cases, Ki67 status changed from negative to positive.. Discordance of the hormone receptor status ranged from 8% to 33% in breast cancer patients who received neoadjuvant chemotherapy. About half of the studies reported a discordance rate of 2.5%–17.0% in ER receptor status and a discordance rate of 5.9%–51.7% in PR, respectively. Several studies have been done to evaluate the effect of neoadjuvant chemotherapy on biomarker status in breast cancer with conflicting results[9,. Because these studies were conducted using a limited number of specimens, there might not be sufficient evidence to detect significant differences. For instance, Arens et al. analyzed biomarkers of CB and EB in 25 patients receiving neoadjuvant chemotherapy and 30 patients without neoadjuvant chemotherapy, and concluded that neoadjuvant chemotherapy had no effect on the expression of biomarkers. By contrast, our study, with 525 neoadjuvant chemotherapy cases, is more likely to detect small but statistically significant differences.A number of studies have revealed concordance between HR and Ki67 after neoadjuvant chemotherapyAppropriate systemic treatment of breast cancer requires the knowledge of HR status, especially after neoadjuvant chemotherapy. In our study, the differences of ER, PR and Ki67 expression between CB and EB were not statistically significant. However, there is a certain discordance rate of ER, PR and Ki67 expression between CB and EB, which is in accordance with the results of some previous studies. These changes verify the presumption that the shift in biomarkers is elicited by neoadjuvant chemotherapy and random changes induced by heterogeneity, laboratory procedures and observer variability. Due to improvement in sampling and detecting techniques, tumor heterogeneity would be a negligible factor of biomarker shift. However, the effect of neoadjuvant chemotherapy on biomarker expression changes could hardly be ignored.; Another explanation would be selection of tumor cells clone during treatment, with selective disappearance of either HR-positive or HR-negative tumor cells. It is generally known that HR-negative tumors are more sensitive to chemotherapy than HR-positive ones. Chemotherapy could up-regulate some proteins favoring the expression or re-expression of HR in the tumor nuclei. The theory that HR-negative tumors are more sensitive to chemotherapy than HR-positive tumors is explained by up-regulation of HR. Although ER expression shift did not show significant difference, 7.5% positive cases became negative after neoadjuvant chemotherapy. A novel theory called neoendocrino chemotherapy can better explain these results. This novel theory focuses on enhancing tumor cell sensitivity to chemotherapy by providing endocrine hormones to patients before and/or during chemotherapy, and it may offer a new therapy for breast cancer. That is to say, some HR positive tumor cells may be more sensitive to chemotherapy. This novel theory is also concordant with the results from Kitagawa et al., indicating that negative conversion of HR positive cases were more frequently observed in patients under 50 years , since their hormone level is higher than those over 50 years.After neoadjuvant chemotherapy, 13.6% (71/520) initially PR negative cases became positive. As for ER expression, 7.7% (40/521) negative cases transformed to positive ones after neoadjuvant treatment. As previously reported, possible mechanisms for HR expression shift in breast cancer cells caused by chemotherapy were as follows: First, chemotherapy induced the regression to a positive hormone receptor status, since all cells originally derived from well-differentiated hormone receptor positive breast cancer cellset al. found patients with HR negative tumors which switched to a positive status after neoadjuvant chemotherapy had better overall survival and disease-free survival than patients whose tumors remained HR negative. Hirata et al. verified patients whose HR status shifted from negative to positive after neoadjuvant chemotherapy, if administered endocrine treatment, had a better prognosis than patients who were HR-negative both before and after neoadjuvant chemotherapy; but for those who did not receive endocrine treatment, the prognosis was worse. These results indicate that it is necessary to assess lesion HR status both before and after neoadjuvant chemotherapy, and endocrine treatment is also essential for patients with positive HR status conversion. The poor prognosis for patients with positive HR conversion and without adjuvant endocrine treatment indicates the necessity to evaluate the biopsy specimens both before and after neoadjuvant chemotherapy; the pre- and post-neoadjuvant chemotherapy HR status would help determine the application of adjuvant endocrine treatment in patients. Endocrine treatment can be applied in patients with HR positive tumor at least once, that is, either before or after neoadjuvant chemotherapy.Tacca . In the neoadjuvant chemotherapy cases, there are 34.4% negative cases and 65.6% positive cases in CB specimens, while 56.6% negative cases and 43.4% positive cases in EB specimens after neoadjuvant chemotherapy. In addition, 21.1% (106/502) cases have negative conversion, which could be attributed to the disturbance of signal transduction pathways caused by chemotherapeutic agents. Meanwhile, 23.7% (119/502) cases have positive conversion, possibly due to the fact that some tumor cells were resistant to the chemotherapeutic agent. A recent research indicates that post-neoadjuvant chemotherapy Ki67 levels provided more prognostic information than pre-neoadjuvant chemotherapy ones. Patients with a high level of post-treatment Ki67 had higher risks for relapse and death compared with those who had a low or intermediate level of Ki67. The Ki67 index is a predictive marker for pathologic complete response. Patients with a low level of Ki67 showed a comparable outcome with patients with a pathologic complete response[19–. The 2011 and 2013 St. Gallen Consensus Conference added Ki67 as a proliferation marker for breast cancer subtypes like luminal A, luminal B, as well as triple negative basal-like and HER2 overexpressing types[21–. These breast cancer molecular subtypes have been proposed as risk factors and prognosis indicators. For the above reasons, it is necessary to reexamine Ki67 in patients with residual disease after neoadjuvant chemotherapy for better subsequent therapy planning and prognosis assessment.In most of the previously reported studies, a decrease in Ki67-associated proliferation was observed, which is in accordance with our studyThe limitations of our study are as follows: (1) Our study is retrospective, so biomarker information was not acquired from patients who received neoadjuvant chemotherapy. (2) We did not stratify the neoadjuvant chemotherapy patients based on their chemotherapy regimens. (3) Patients’ prognostic information was not available, so the impact of biomarker conversion on prognosis was not evaluated. Further research should be conducted prospectively on change of biomarker expression, and the prognosis of breast cancer patients after neoadjuvant chemotherapy should be assessed. Regardless of the limitations, our study has shown that neoadjuvant chemotherapy could modulate tumor biomarker status, indicating the significance of evaluating biomarker status both before and after neoadjuvant chemotherapy.In summary, in our study, the discordance rates of ER, PR and Ki67 were 15.2%, 26.9%, 44.8% before and after neoadjuvant chemotherapy, respectively. Meanwhile 7.7% and 13.6% cases showed positive conversion of ER and PR expression, respectively, after neoadjuvant chemotherapy. Regardless of the reasons for ER, PR and Ki67 status switch, changes of ER, PR and Ki67 expression should not be ignored by oncologists. Re-examination of the biomarkers should be conducted in certain situations to optimize the adjuvant systemic therapy and assess patient’s prognosis.


Physical activity is associated with health in women. Published MET-values  may assess physical activity and energy expenditure but tend to be too low for subjects with a high total body fat (TBF) content and therefore inappropriate for many contemporary women. The MET-value for an activity is the energy expenditure of a subject performing this activity divided by his/her resting energy expenditure, often assumed to be 4.2 kJ/kg/h. Relationships between TBF and MET have been little studied although overweight and obesity is common in women. Available data indicate that MET-values decrease during pregnancy but more studies in pregnant contemporary women are needed.Using indirect calorimetry we measured energy expenditure and assessed MET-values in women, 22 non-pregnant (BMI: 18–34) and 22 in gestational week 32 (non-pregnant BMI: 18–32) when resting, sitting, cycling (30 and 60 watts), walking (3.2 and 5.6 km/h) and running (8 km/h). Relationships between TBF and MET-values were investigated and used to predict modified MET-values. The potential of such values to improve calculations of total energy expenditure of women was investigated.p < 0.001) and 7–15% lower MET-values (p < 0.05) than non-pregnant women. MET-values of all activities were correlated with TBF (p < 0.05) in non-pregnant women and modified MET-values improved estimates of total energy expenditure in such women. In pregnant women, correlations (p ≤ 0.03) between TBF and MET were found for running (8 km/h) and for walking at 5.6 km/h.The resting energy expenditure was below 4.2 kJ/kg/h in both groups of women. Women in gestational week 32 had a higher resting energy metabolism  during pregnancy are warranted. Studies of how pregnancy and TBF affect MET-values of high intensity activities are also needed. Corrections based on TBF may have a potential to improve the MET-system in non-pregnant women. The physical activity of men and women is extensively studied since it is associated with chronic disease . Of specThe MET-value of an activity is often defined as the energy expenditure of a subject when performing this activity divided by his/her resting energy expenditure . PublishThe effect of variations in the body fat content on MET-values is of special concern in women. The prevalence of overweight and obesity in female populations has increased during recent years  and the Studies in pregnant women have sometimes been conducted using MET-values developed in non-pregnant subjects , 20. HowWe recruited two groups of women, one pregnant and one non-pregnant to a study with the following two objectives: 1) To assess the effect of gestational week 32 on MET-values for activities with varying intensity; 2) To identify relationships between TBF and MET-values for such activities. Using previously published data , we had Forty-four healthy, non-smoking women (22 non-pregnant and 22 pregnant) were recruited during 2007 and 2008 by means of advertisements in the local press. Results obtained in some of these women have been published , 24. All2) and carbon dioxide production (VCO2) were measured during 20 min in the supine position after an overnight fast and 45 min of rest using a ventilated hood system . To assess energy expenditure when performing standardised activities a spirometer  was used to measure VO2 and VCO2 during 15 s-periods for six minutes. The woman was connected to the spirometer through a mouthpiece and wore a nose clip. The session started with the sitting activity followed by standing. The woman was then transferred to a treadmill board, adjusted to make her walk at 3.2 km/h followed by walking at 5.6 km/h and then running at 8 km/h. Subsequently she was allowed to rest for 20 min after which she mounted a test bicycle where she cycled at 30 watts and then at 60 watts. VO2 and VCO2 were recorded throughout the six minutes when each standardised activity was performed and steady state was always reached after two to three minutes. The validity of the response, both of the Deltatrac and the Spiropharma, was controlled according to instructions provided by the manufacturer. This involved calibration using gases with defined concentrations of O2 and CO2 before each test. For each activity average of values recorded during the fifth of the six minutes was used to calculate energy expenditure and MET-values. The recorded VO2 and VCO2 values confirmed that all subjects were in a steady state when values used to calculate energy expenditure were recorded. Energy expenditure was calculated using the Weir equation [To assess BMR, oxygen consumption (VOequation  and MET-2H20 and 0.23 g H2180 per kg body weight) per os. Isotope enrichment of dose and urine samples was analyzed as previously described [2H-space (ND) and 18O-space (NO) were calculated from zero-time enrichments obtained from the exponential isotope disappearance curves providing estimates of the fractional turnover rates for the two isotopes [2H and 0.03 for 18O. Total body water was calculated as (ND/1.04 + NO/1.01)/2 and fat-free mass as total body water divided by 0.732 (non-pregnant) [D / NO was 1.032 ± 0.005 (n = 44). Analyzing dose and urine samples from one subject nine times gave the following CV: 0.3% , 0.3% or less  all well within the recommended criteria [Each woman consumed an accurately weighed dose of stable isotopes   or 0.747regnant) . TBF wascriteria . Assessmcriteria  and in gcriteria .When comparing MET-values of pregnant and non-pregnant women, 20 subjects in each group gave a power above 0.8 to detect a difference of 10% between groups while assuming the CV of MET-values to be 10%. A correlation coefficient of at least 0.45 for linear relationships between MET-values and TBF would be significant with 20 observations.2 and TBF 32.7 ± 7.8%. Their TEE was assessed during 14 days using the doubly labeled water (DLW) method (TEEDLW) [DLW was assessed [pubMET) or modified MET-values, predicted by means of the relationships between TBF (%) and MET-values (TEEmodMET) identified in the present study. Then TEEpubMET and TEEmodMET are both compared to TEEDLW.We have previously investigated and published the physical activity pattern and TEE of 23 women before pregnancy and in gestational week 32 . Before (TEEDLW) . A few d(TEEDLW) . The queassessed . In the assessed  during tt-test for paired or unpaired observations after confirming that values were normally distributed. If a normal distribution could not be confirmed Wilcoxon’s test was used. Linear regression analysis was also used. Significance (two-sided) was accepted when p < 0.05. Statistical analyses were conducted using Statistica, version 8.0 .Values are given as means ± standard deviations (SD). Significant differences between mean values were identified using n = 22) gained 12 ± 4 kg body weight during the complete pregnancy and all of them delivered one healthy full-term infant, weight and length at birth being 3410 ± 390 g and 50 ± 2 cm, respectively.The pregnant and non-pregnant women recruited to the study are described in Table 2, VCO2 and energy expenditure at rest (BMR) and when performing the standardised activities. For pregnant women, BMR in kJ/kg/h was 94% of 4.2 kJ/kg/h and the corresponding value for non-pregnant women was 88%. When expressed in kJ/min, BMR was significantly (p = 0.000037) and 18% higher of pregnant than of non-pregnant women. For all activities except running energy expenditure (kJ/min) of the pregnant women was slightly higher than the corresponding values for the non-pregnant women but only the values for walking at 5.6 km/h were significantly different. In pregnant women, energy expenditure minus resting energy expenditure (the net energy expenditure) was on average 0.60 ± 0.58, 9.84 ± 0.89, 16.2 ± 1.0, 10.1 ± 1.0, 19.7 ± 2.7 (n = 21) and 36.4 ± 4.4 (n = 9) kJ/min when sitting, cycling at 30 and 60 watts, walking at 3.2 and 5.6 km/h and running at 8 km/h, respectively. In the non-pregnant group the corresponding values were: 1.04 ± 0.35, 10.1 ± 0.8, 16.2 ± 0.9, 10.1 ± 1.9, 18.2 ± 3.4 and 37.3 ± 5.9 (n = 21) kJ/min, respectively. The value for sitting was significantly lower for pregnant women (p = 0.0039). No significant differences between the groups were found for any other activity.Table Table p ≤ 0.003) for sitting and cycling activities and from 0.43 to 0.52 (p ≤ 0.044) for walking and running activities. For pregnant women significant correlations between MET and TBF (%) were found for the running activity (n = 9)  and for walking at 5.6 km/h (n = 21) . The correlation between MET and TBF (kg) was significant for the running activity (n = 9) . No other significant correlations between MET and TBF  were found in pregnant women.Table pubMET was 9710 ± 2110 kJ/24 h and significantly lower than TEEDLW. This value was 10,510 ± 1500 kJ/24 h [modMET was 10,060 ± 2660 kJ/24 h and not significantly different from TEEDLW.All investigated correlations between measured MET-values and TBF were significant for non-pregnant women while only a few such correlations were significant for pregnant women. Therefore, we investigated the possibility to improve calculations of TEE only in non-pregnant women. As shown in Table  kJ/24 h  while TEIn healthy women we found MET-values to be lower in gestational week 32 than in non-pregnant women, an observation mainly explained by the increased BMR during pregnancy. We identified significant relationships between TBF and measured MET in non-pregnant women while for pregnant women such relationships were found for activities with high intensity but not for activities of lower intensity. When relationships between measured MET and TBF were used to predict modified MET-values calculated TEE of our non-pregnant women was improved.When the inferences of our results are considered body fatness of our women becomes of interest. The average as well as the variability in their body fat content represents relevant information. In population studies body fatness is commonly assessed as BMI rather than by means of a suitable body composition method. This may be appropriate since relationships between BMI and TBF (%) have been identified  but is nWhen assessing activity energy expenditure of a woman to establish MET-values it is relevant to consider her feeding status. In the fed state the recorded energy expenditure will include dietary induced thermogenesis i.e. the increase in energy expenditure following food intake which represents about 10% of the energy content of the food consumed and lasts for at least six hours . PublishThe four-component model represents the best available method to assess body composition in vivo of human subjects while two- and three-component models are less accurate because they rely on assumptions . AssessmThis study is focused on the relationship between TBF and MET-values since published research shows that body fatness is a relevant factor when MET-values are assessed and applied. Note, however, that our study does not provide a complete description of how body composition and MET-values interact.For all investigated non-resting activities differences in energy expenditure between pregnant and non-pregnant women were small and the net increase in energy expenditure tended to be quite similar for the two groups. However, all measured MET-values were significantly lower in gestational week 32. Obviously, this is due to the increased BMR at this stage of pregnancy.An effect of pregnancy is to increase body weight and it has been suggested that pregnancy influences MET-values of weight bearing and non-weight bearing activities differently. For a non-weight bearing activity such as cycling, we found MET-values to be on average 0.42 (30 watts) and 0.62 (60 watts) units lower for pregnant than for non-pregnant women. This effect is similar to the corresponding average effect in gestational week 32 obtained in the studies compiled by Prentice et al. . For weiAll relationships between MET-values and TBF were significant in non-pregnant women, while only relationships for activities with higher intensity were significant for pregnant women. This may reflect that the influence on energy metabolism by factors associated with pregnancy, such as fetal metabolism, overrides the influence of TBF unless the activity is of sufficient intensity.In our study, sitting and cycling can be considered to represent non-weight bearing activities while walking and running represent weight bearing activities. For non-pregnant women it is of interest to note that relationships between MET and TBF (%) were negative for sitting activities (including cycling) and positive for walking and running. This suggests that TBF may affect MET-values for the two kinds of activities differently but studies with a larger selection of activities are required for firm conclusions on this issue.This study confirmed that the increase in BMR is an important factor behind the effect of pregnancy on MET-values. Thus it becomes important to investigate how pregnancy influences the one MET-value. Using published data  we foundOur observations suggest that the effect of pregnancy on MET-values may be influenced by the intensity of the activity and also that body composition may influence MET values of high intensity activities. Consequently, there is a need for systematic studies regarding the effect of body fatness and activity intensity on MET-values during pregnancy if appropriate modifications of the MET-system for pregnant women are to be developed.Women as well as overweight and obese subjects tend to have BMR below 4.2 kJ/kg/h  and highThis study showed that modified MET-values, predicted using the relationships in Table Strengths are that we measured MET-values of non-pregnant and pregnant women, both with valid estimates of TBF, and that both non-weight bearing and weight bearing activities were included. In non-pregnant women we established relationships between TBF (%) and measured MET-values for standardised activities and demonstrated that such relationships may improve calculated TEE. A limitation of our study is the small number of subjects.A valid MET-system is important when physical activity levels and energy intake of human subjects are assessed. This kind of information is needed by health workers and researchers within several different areas, for example nutrition and public health. This study deals with issues related to application of the MET-system and provides suggestions for improving this system. Correcting MET-values based on TBF of subjects may represent a possibility to improve this system for non-pregnant women. Our results indicate that for pregnant women more work is needed to develop a valid MET-system. Studies describing how body composition influences the one MET-value throughout gestation and how pregnancy and body fat affect MET-values for high intensity activities may be useful in this context.


Global action plan on antimicrobial resistance to ensure sustainable access to effective and safe antimicrobial medicines.The rapid spread of antibiotic resistance is a major public health challenge, because it endangers our ability to treat potentially life-threatening infections. In response to this challenge, in 2015 the World Health Assembly adopted the This action plan recognizes surveillance of antibiotic consumption and antibiotic resistance in humans and animals as a key strategy in addressing antibiotic resistance. Surveillance of antibiotic consumption can help countries identify problems relating to antibiotic use; direct their interventions to tackle these problems; and evaluate these interventions. Over time, surveillance allows comparisons between and within countries that help set common targets and identify changing trends in antibiotic use patterns.,Success stories where surveillance data have contributed to the improvement of antibiotic use are well documented. For example, surveillance data helped the governments of Belgium and France to realize that antibiotic consumption in their respective countries was among the highest in Europe, prompting them to launch national awareness campaigns that proved effective in reducing antibiotic prescribing.WHO Global report on antibiotic consumption, released in November 2018, during the World Antibiotic Awareness Week.While many high-income countries have ongoing surveillance of national antibiotic consumption, data from most low- and middle-income countries is absent. The lack of data on the quantity of antibiotics consumed at the national level is concerning: inappropriate prescribing and self-medication with antibiotics are prevalent in many of these countries,The report presents 2015 data on the consumption rates of antibiotics in 65 countries and areas, including 15 low- and middle-income countries that collected such data in a systematic manner for the first time with support from WHO.The recent implementation of surveillance on antibiotic consumption in the 15 low- and middle-income countries has also had a positive effect on strengthening pharmaceutical systems. For example, Côte d’Ivoire introduced a system to assign codes to authorized medical products to improve medicines tracking. Bangladesh is planning to improve quality assurance of medicines by prioritizing controls of the most sold antibiotic medicines identified in its surveillance data.While such progress is encouraging, there is a need for continued global commitment to support countries in developing national surveillance of antibiotic consumption across all sectors, including livestock and agriculture. Countries that already have existing surveillance systems should continue to share their data with WHO and the World Organisation for Animal Health (OIE), which oversees surveillance of antibiotic consumption by animals.


No validated molecular biomarkers exist to help guide prognosis of renal cell carcinoma (RCC) patients. We seek to evaluate the quality of published prognostic circulating RCC biomarker manuscripts using the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guidelines.The phrase “ AND circulating AND (biomarkers OR cell free DNA OR tumor DNA OR methylated cell free DNA OR methylated tumor DNA)” was searched in Embase, Medline and PubMed March 2018. Relevant manuscripts were scored using 48 REMARK sub-criteria for a maximal score of 20 points.The search identified 535 publications: 33 were manuscripts of primary research and were analyzed. The mean REMARK score was 10.6 (range 6.42–14.2). All manuscripts stated their biomarker, study objectives and method of case selection. The lowest scoring criteria: time lapse between storage of blood/serum and marker assay (n = 2) and lack of flow diagram (n = 2). REMARK scores were significantly higher in publications stating adherence to REMARK guidelines (p = 0.0307) and reporting statistically significant results (p = 0.0318).Most RCC prognostic biomarker manuscripts poorly adhere to the REMARK guidelines. Better designed studies and appropriate reporting are required to address this urgent unmet need. Renal cell cancers (RCC) are pathologically diverse with variable outcomes for patients with similar stage disease and treatment. Clinicopathological and radiological features comprise the primary RCC prognostic parameters. Despite these efforts, the natural history of RCC can still be unpredictable: small tumors < 4cm can harbor metastatic disease at time of diagnosis  and upwaAt least 16 RCC prognostic models exist that incorporate various clinical, laboratory and radiographic features, with each model examining discrete populations of patients  . HoweverOne strategy undergoing investigation to address these limitations is the development of minimally-invasive blood-based biomarkers. Yet despite a wide range of cellular and molecular features being studied in RCC for over 15 years, there still remains a paucity of validated circulating prognostic biomarkers. This is not unique to RCC: less than 1% of promising oncologic biomarkers become clinically useful . To helpOur study hypothesized the paucity of validated circulating RCC biomarkers is secondary to inadequate methodology and reporting, which could be demonstrated by failure of adherence to the REMARK guidelines. Hence, we performed a systematic review of the literature to determine the number of primary research manuscripts investigating RCC circulating prognostic biomarkers, and subsequently scored each valid manuscript using the REMARK criteria. The objective of our study was to review the quality of design and reporting in studies investigating prognostic circulating biomarkers in patients with RCC.a priori: inclusion criteria consisted of manuscripts analyzing RCC prognostic circulating blood-based biomarkers; exclusion criteria were duplicates, non-RCC papers, or RCC studies limited to case reports, review papers, or abstract publications. Search results were exported into CSV file format for review. All publications were independently reviewed and organized by two authors (MI and SP) into one of three possible categories: publications examining RCC circulating prognostic biomarkers, publications not examining RCC circulating prognostic biomarkers, and publications that were unclear about examining RCC circulating prognostic biomarkers. Any persistent disagreements in organization between authors MI and SP were adjudicated by author AH. Among the publications examining RCC circulating prognostic biomarkers, only primary research manuscripts were subjected to REMARK scoring; reviewed papers and abstract-only publications were not amendable to REMARK appraisal. Valid manuscripts were then sub-classified based upon their investigated biomarker; categories were created if ≥ 2 publications were analyzing the same biomarker, and papers analyzing a biomarker not examined in another publication were classified as “other”. Publications that were not suitable or of unclear significance were also sub-classified. Attempts to clarify publications of unclear significance were assessed by contacting the corresponding author or, in the case of unclear abstracts, subsequent publications were searched to determine if the abstract pertained to RCC prognostic circulating biomarkers.This literature search was guided by the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) statement . The S1 The REMARK criteria consists of a checklist of 20 items , and eacThe REMARK scores were summarized using descriptive statistics such as mean and range. Pearson’s correlation was used for continuous variable correlation with REMARK score: journal impact factor, year of publication and sample size studied were analyzed. Impact factors were determined on October 9, 2018 by searching either InCites Journal Citation Reports  or otherThe search identified 252 Embase results, 146 Medline results and 394 PubMed results. The PubMed results had 98 and 146 overlapping results with Embase and Medline, respectively. Embase had 13 repeated publications within its own Embase search: either the abstract was repeated twice (n = 5), or the abstract was repeated but under a different Unique Identifier . Note that one abstract had its Unique Identifier repeated twice and had the abstract repeated under a different Unique Identifier.In total, 535 unique publications were identified: 74 examined RCC prognostic circulating biomarkers, 438 did not examine RCC prognostic circulating biomarkers, and 23 were unclear if they examined RCC prognostic circulating biomarkers. Among the RCC prognostic circulating biomarkers publications, 33 were manuscripts of primary research and were organized into 10 different categories; manuscripts evaluating ≥ 2 biomarkers (n = 8) and those meeting our definition of “other”  (n = 7) comprised the majority. Although excluded from our final analysis, 26 review papers and 14 abstracts reported on RCC circulating prognostic biomarkers. One manuscript with a valid abstract was unable to be obtained despite attempts to contact the corresponding author. Publications not examining RCC prognostic circulating biomarkers were organized into 53 different categories see : publicaThirty three manuscripts were included for review. As displayed in the The majority of the studies were conducted in Europe , examined mixed RCC histologies  and had mixed RCC TNM staging . In total, 30 publications (91%) reported a statistically significant association between their circulating biomarker and a prognostic outcome, of which OS was the most common outcome reported . The mean impact factor was 5.85 with a large range (1.2–13.926). The mean year of publication was 2012 (range 2004–2018). There was a large variation in RCC patient sample size , and 18 manuscripts (55%) utilized RCC sample sizes < 100 patients. As shown in We report for the first time that translational studies analyzing RCC circulating prognostic biomarkers need to improve the quality of their methodology and reporting of results. Biomarker development is a challenging area of medical science that requires both step-wise methodological design , 26 and We explored several correlations with REMARK scores to explain possible causes underlying the paucity of good quality publications. We were only able to show that manuscripts acknowledging adherence to REMARK guidelines and those reporting statistically significant results had higher REMARK scores. However, this observation must be interpreted with caution given the few papers formally reporting adherence to REMARK guidelines (n = 3) or reporting non-significant results (n = 3). Although publications stating adherence to REMARK guidelines did score significantly better relative to publications not citing REMARK, these publications still only achieved a mean score of 13.03 (65%). Furthermore, the top scoring publication from our review did not formally state it was following REMARK guidelines. Our finding that publications stating adherence to REMARK guidelines are of improved quality is in contrast to the 2017 analysis of multiple tumor types and both circulating and non-circulating biomarkers . This diMedical journals with higher impact factors were not associated with better quality publications. This is consistent with a review that also determined poor REMARK scoring in higher impact journals , which ie(hazard ratio) [Encouragingly, our study identified a large proportion of publications reporting hazard ratios. This is in contrast to the neuroblastoma prognostic biomarker systematic review that showed approximately 10% of publications reported either a hazard ratio or logd ratio) . Howeverd ratio) , 41 thatd ratio) , 42–44. d ratio) . A complThis project has several limitations. Our subdivision of each REMARK point is based on the more elaborate REMARK publication , but to Although prognostic biomarkers are valuable tools to aid in clinical decision making, their development is inhibited by poor study design and incomplete reporting. Unfortunately our review shows RCC prognostic circulating biomarker investigations are fraught with similar limitations, possibly explaining their paucity of clinical validity. While publications stating adherence to REMARK guidelines and those reporting statistically significant results appear to be of better quality, they still fail to follow a large proportion of the recommendations. While good reporting will not compensate for poor study design or an ineffective biomarker, it will allow more rapid identification of problematic studies. We suggest that future efforts in RCC prognostic circulating biomarker design should consider the REMARK criteria when designing studies and reporting results to ensure their investigations are high quality, robust and reliable.S1 FileA list of all PRISMA items and location within the manuscript.(DOC)Click here for additional data file.S2 FileA summary of the terms used in the search, the rationale behind their choice, and associated MeSH terms.(DOCX)Click here for additional data file.S3 FileA list of all publications not examining RCC circulating prognostic biomarkers and their sub-classifications.(DOCX)Click here for additional data file.S1 TableA summary list of all abstracts, review papers and primary research publications analyzing circulating RCC prognostic biomarkers. The REMARK score for primary research publications is stated.(DOCX)Click here for additional data file.S2 TableA list of all valid manuscripts with their corresponding REMARK sub-criteria points obtained and correlative variables used in the statistical analysis.(XLSX)Click here for additional data file.


Meanwhile, phase composition, types of hydration products and microstructure characteristics of samples before and after the carbonation were analyzed by XRD and ESEM. Research results demonstrate that temperature, CO2 concentration and relative humidity influence the carbonation depth and compressive strength of concrete significantly. There is a linear relationship between temperature and carbonation depth, as well as the compressive strength of concrete. CO2 concentration and relative humidity present a power function and a polynomial function with carbonation depth of concrete, respectively. The concrete carbonation depth increases with the increase of relative humidity and reaches the maximum value when the relative humidity is 70%. Significant differences of phase composition, hydration products and microstructure are observed before and after the carbonation. Carbonization products of samples are different with changes of temperatures . The result of crystal structure analysis indicates that the carbonation products are mainly polyhedral spherical vaterite and aragonite.The influence of temperature, CO Concrete carbonation easily induces a decrease of the system pH, decomposition of hydration products, depassivation of deactivated film of reinforcing bar and cracking of the concrete cover. Therefore, concrete carbonation is a serious problem for the durability of concrete structures [Factors like environmental or exposure conditions are found to be governing the service life of reinforced concrete structures significantly. One of the major processes influencing the condition of steel bars is carbonation of concrete surrounding steel bars in reinforced concrete structures. Hence, considerable research efforts on the carbonation of concrete in the laboratory and field are carried out around the world . Concretructures .Many achievements on concrete carbonation have been reported in the world ,5,6. Lee2 concentration on the products, microstructure and depth of concrete carbonation. Ronaldo et al. [2 concentration. Andreas et al. [2 concentration and ambient relative humidity on accelerated and natural carbonation of 18 concrete mixtures produced with nine different cement types, and the results indicated that the water-to-cement ratio and cement-specific effect affected the relative carbonation resistance between the concrete mixtures.Although the analysis of the influences of environmental factors on concrete carbonation has been conducted ,19,20, eo et al.  proposedo et al.  investigs et al.  discusseThose above research shortages determine the poor accuracy of the theoretical model of concrete carbonation and the great gap between prediction results and measured results. This makes prediction of the endurance lifetime of actual engineering more apt for the concrete carbonation empirical model of concrete. It is urgent to study the influences of environmental factors on concrete carbonation performances for the sake of better evaluation on concrete endurance, disclosure of the carbonation mechanism and prediction of the service life of the concrete engineering structure.2 concentration and relative humidity on concrete carbonation was investigated. Carbonation depth and compressive strength of concrete with different strength grades under different environmental conditions were tested. Moreover, phase composition, as well as types and microstructure of hydration products before and after carbonation were analyzed by X-ray diffraction (XRD) and the environment scanning electron microscope (ESEM).In this study, the influence of temperature and CO+O 42.5 Portland cement was provided by China Building Materials Academy. The fly ash produced by Hunan Xiangtan Power Plant was low calcium fly ash . The physical properties of cement and fly ash are listed in The P2 concentration was adopted. Therefore, the corresponding concrete carbonation of this study belonged to the accelerated carbonation test method, and the results were applicable to characterize the change of accelerated carbonation test. Carbonation testing conditions of concrete were acquired by the environmental simulation test system. After the concrete carbonation test reached the preset carbonation age (28 d), the compressive strength and carbonation depth of specimens were tested subsequently. Carbonation depth of concrete was tested by 1% phenolphthalein reagent, which was prepared by 95% ethyl alcohol. Ten points on the cross-section of specimen were measured, and the corresponding average value was set as the carbonation depth with a precision of 0.1 mm. The cement paste with a water to cement ratio of 0.258 was prepared to investigate the hydrations before and after carbonation by XRD analysis. The carbonation conditions of cement paste were the same as the corresponding concrete specimens. Meanwhile, the concrete sample was prepared to carry out ESEM analysis. The mechanical properties of concrete were tested according to the GB/T50081-2002 Standards on the Test Method of Ordinary Concrete Mechanical Properties under the loading rate of 5 kN/s. The average value of three samples was set as the compressive strength of each group.Concrete specimens (100 mm × 100 mm × 300 mm and 100 mm × 100 mm × 100 mm) were cast according to GB/T 50082-2009 Standards on the Test Method of Ordinary Concrete Long-term Performance and Endurance. Specimens were demolded after 24 h of casting, and 120 specimens were cast for each concrete grade. Then, they were cured at a temperature of (20 ± 1) °C and a relative humidity of (95 ± 5)% for 28 d. Subsequently, the specimens were dried for 48 h at 60 °C. After drying, the specimens were placed in the environmental simulation test chamber for a week to ensure the same temperature and humidity between the concrete interior and external environment. In order to obtain the carbonation result of concrete in a short time, the high COTest instruments mainly included a Quanta-200 environment scanning electron microscope (ESEM) made by FEI Company , a WAW-DP Universal tester made by Shanghai Sansi Co. Ltd.  and the environmental simulation test system made by Wuhan Jinyatai Instrument Co. Ltd. . Moreover, BD-86 X-ray diffraction (XRD) produced by Rigaku Company  was used to investigate the hydration products. The corresponding scanning step was 0.02°, and the scanning range was from 5°–60° with a tube voltage and rated power of 60 kV and 4 kW, respectively.2 concentration. The variation curves of carbonation depth and compressive strength of concrete with temperature are shown in Generally, concrete carbonation is a complicated physical and chemical reaction. Temperature can influence the chemical reaction significantly. In this paper, the influences of temperature  on concrete carbonation were analyzed by the changes of carbonation depth and compressive strength of concrete, which had been carbonized for 28 d under 70% relative humidity and a 20% CO2 concentration and carbonation reaction rate in concrete were higher as a response to the high transmission coefficient of CO2 and the chemical reaction coefficient under high temperature. Although the carbonation reaction rate of concrete presented a nonlinear relationship with temperature, CO2 concentration in concrete took the dominant role, and the whole concrete carbonation was controlled by the transmission of CO2. Generally speaking, the transmission coefficient of CO2 was closely related to the temperature. Hence, there was a basically linear relationship between the carbonation depth of concrete and temperature. With the increase of concrete strength grade, the microstructure of concrete was manifested by higher density, lower porosity, higher complexity of pore structural characteristics and sinuosity, as well as smaller openness of pores. Combined with 2 in concrete decreased. If the concrete carbonation process was controlled by CO2 transmission in concrete, the carbonation depth of concrete was negatively correlated with concrete strength grade. At the same time, products from the reaction between hydration products and CO2 in the concrete surface filled in some pores, which further increased the surface density of concrete and decreased the CO2 transmission coefficient in concrete. Hence, the carbonation reaction of concrete declined.It can be seen from 2 with hydration products, such as calcium hydroxide , calcium silicate hydrate , ettringite  and calcium aluminate hydrate  in concretes, through which gelling hydration products were decomposed and carbonates and calcium silicates without gelling properties were produced. This induced deterioration of the concrete microstructure changes of the hydration products and destroys the chemical balance in the system. Hence, the compressive strength of concrete declined as the carbonation continued. The CO2 transmission coefficient and chemical reaction coefficient in concrete increased with the increase of carbonation temperature, which resulted in a higher concrete carbonation degree. Comparing with In The influencing law of relative humidity on carbonation depth and compressive strength of concrete was analyzed, as shown in 2 transmission coefficient in liquid water in concrete pores was very small, which decreased the CO2 transmission quantity and rate in concrete accordingly. Therefore, the concrete carbonation depth decreased under relatively high relative humidity (>70%). When the relative humidity was low, water vapor in concrete pores may cover up pore walls and form a liquid film. Different hydration products of cement (mainly CH) were dissolved in the film, and the corresponding oversaturated solution was formed. CO2 was transmitted into concrete pores and dissolved in the film to react with CH to generate calcium carbonate. The continuous carbonation consumed abundant ions. This induced decomposition of hydration products in the system and caused further changes of the chemical equilibrium state in the system. If the relative humidity was low, the film area and thickness on the pore wall in concrete were too small to support the carbonation of the system. Therefore, the corresponding concrete carbonation was slow. With the increase of relative humidity, the water content in concrete pores increased, which was conducive to concrete carbonation. Macroscopically, this was manifested by the increase of the carbonation depth of concrete with the increase of relative humidity. The carbonation depth of concrete began to decrease after the relative humidity exceeded a fixed value (about 70%). This deduction could be verified by the changes of the compressive strength of concrete with relative humidity . It could offer places for dissolution and reaction of hydration products , which increased carbonation depth and rate accordingly. As the carbonation continued, microstructure and hydration products of the system changed, which were manifested by a reduction of the compressive strength of concrete macroscopically.2 concentration on the carbonation depth and compressive strength of concrete were also investigated. The variation curves of carbonation depth and compressive strength of concrete at 28 d and the CO2 concentration are shown in The effects of CO2 concentration. A power function between them was observed. Under the same CO2 concentration, carbonation depth of concrete was negatively correlated with the strength grade of concrete. Given the higher CO2 concentration, the inner-outside concentration gradient was larger, and more CO2 could diffuse into the concrete and react with the hydration products in concrete. The growth rate of carbonation depth decreased as CO2 concentration increased. This was because there was a low CO2 concentration in the early carbonation of concrete and the CO2 quantity transmitted into concrete was consumed by a fast reaction. As the CO2 concentration increased, the CO2 quantity transmitted into the concrete was higher than the CO2 consumption by concrete carbonation. Concrete carbonation was controlled by chemical reaction rather than the CO2 transmission. As a result, the change rate of the concrete carbonation depth curve decreased gradually with the increase of CO2 concentration. A high strength grade of concrete implied a higher density, lower porosity and more closed pores in concrete, as well as the lower CO2 transmission coefficient in concrete. Hence, CO2 quantity transmitted into concrete decreased accordingly. Moreover, a higher strength grade of concrete demanded greater cement consumption and more hydration products in the hydration reactions, which helped the system to absorb more CO2. Therefore, the carbonation depth of concrete was negatively related to the strength grade of concrete. This could be verified by the relation curves between the compressive strength of concrete and CO2 concentration. Changes of the compressive strength of concrete after 28 d of carbonation under different CO2 concentrations are shown in In 2 concentration. Under the same test conditions, the influences of the carbonation effect on concrete strength intensified in response to the decreasing strength grade of concrete. Since concrete carbonation was the reaction between gelling hydration products  and CO2, hydration products were decomposed and decreased. The system microstructure was loosened and deteriorated accordingly, thus decreasing the compressive strength of concrete. With the increase of CO2 concentration, the CO2 concentration gradient inside and outside of the concrete surface, as well as the CO2 quantity transmitted into the concrete increased. Therefore, there was more CO2 participating in the reaction and decomposition of the abundant hydration products that caused the microstructure failure of concrete. Macroscopically, this was manifested by a reduction of the compressive strength of concrete. For C20 concrete, a low CO2 concentration may increase the concrete strength. This might be caused by CO2 reacting with the hydration products in the concrete surface. The reaction products were generated and filled in some part of the pores, which enhanced the density of the concrete microstructure. The positive effect of carbonation in a short period was caused by the dominant role of the concrete microstructure.It can be seen from The types of hydration products and the phase composition of specimens before and after the carbonation were analyzed to disclose the carbonation mechanism of concrete and its performance changes. The XRD spectra of the phase composition before and after the carbonation are shown in 2 concentration were different. These were mainly manifested by the attenuation  and disappearance  of the diffraction peaks of some cement gelling hydration products, as well as the appearance and strengthening of the diffraction peaks of carbonation products . When the temperature and relative humidity were fixed, the diffraction peak intensity of CaCO3 was positively related to CO2 concentration. The CaCO3 content in carbonized samples increased. This was because more and more CO2 may be transmitted into samples and react with hydration products in the system under the high CO2 concentration. 2 concentration and relative humidity, the phase composition and diffraction peaks of hydration products changed more significantly with the increase of temperature. This was caused by the faster decomposition of the carbonation products and hydration products of the system under the higher temperature.It can be seen from The microstructure and product morphology before and after the carbonation were studied to investigate the influences of temperature on concrete carbonation. The ESEM spectra of specimens before and after the carbonation under different temperature are shown in 3 in the pores. The carbonation products of specimens under different temperatures were also different to some extent. Specifically, polyhedral vaterites could be observed in the ESEM spectra of carbonized samples under 10 °C and 20 °C (2 and hydration products in the system took place under different temperatures, which produced CaCO3 crystals of different forms.In the ESEM spectra of non-carbonation samples, the hydration products were mainly hexagonal plate-like CH, rod-like AFt, flocculent CSH and CAH a. Nevertnd 20 °C b,c. Undend 20 °C d. Hexago12 concentration and relative humidity influence the carbonation depth and compressive strength of concrete significantly. Temperature has a linear relationship with the carbonation depth and compressive strength of concrete. CO2 concentration and relative humidity present power and polynomial functions with the carbonation depth of concrete, respectively.Temperature, CO22 concentration, but the compressive strength of concrete is negatively correlated with the strength grade of concrete. The carbonation depth of concrete increases with the increase of the relative humidity and reaches the peak when the relative humidity is 70%. This is because the CO2 transmission coefficient and chemical reaction coefficient may increase with temperature. Besides, the increase of the CO2 concentration may bring the increase of the concentration gradient and the CO2 concentration in concrete, as well as the intensified carbonation. Concrete density is positively related to the strength grade of concrete. The CO2 transmission coefficient in concrete is low, which may decrease the carbonation depth of concrete.The carbonation depth of concrete is positively correlated with temperature and CO33 takes the dominant role in hydration product composition after the carbonation. Temperature affects the crystal form of carbonation products. Polyhedral spherical vaterites are major carbonation products under 10 °C and 20 °C, whereas aragonites are the major carbonation products under 30 °C.The phase composition, hydration products and microstructure of concrete change significantly before and after the carbonation. Such changes are mainly manifested by the disappearance and attenuation of the diffraction peak of some hydration products. XRD and ESEM spectral analysis reveal that hexagonal plate-like CH, rod-like AFt, flocculent CSH and CAH are major hydration products before the carbonation, but particulate CaCO


To study different lateral restraints, different constituents of expansion agents, the influence of different steel ratios, and concrete creep properties, we carried out experiments with lateral restraint and without lateral restraint conditions separately on 12 specimens with the expansion agent content accounting for 4%, 8%, and 12% respectively. In addition, the creep tests were performed on specimens with different steel ratios of 0.0%, 3.8%, 6.6%, and 9.2%. The test results show that the lateral restraint improves the strength of the system (concrete-filled steel tubes) which resists further load after the concrete ultimate strength is surpassed and reduces the creep. The creep degree of the concrete-filled steel tube with lateral restraint is about 0.09–0.30 times smaller than that of the tube without lateral restraints. The creep degree of the concrete-filled steel tube increases as the steel ratio decreases. Creep tests with different amounts of expansion agent indicate that the creep degree of the concrete structure increases as expansion agent content decreases. To study the internal mechanism of the creep of concrete-filled steel tubes with different lateral restraints and different expansion agent concentrations, a microscopic pore structure test on the steel core concrete was conducted using the RapidAir457 pore structure instrument. Microscopic studies show that the air content and the length of the bubble chord of the laterally restrained core concrete are lower than those without lateral restraint core concrete. The amount of air content and the length of the bubble chord of core concrete specimens increase as the expansion agent content in the core concrete specimens decreases from 12% to 4%. Under the same external loading conditions, as steel ratio increases, the lateral restraint causes a further reduction of creep. The results of this study suggest that the creep of concrete can be reduced by selecting appropriate lateral restraint conditions and an optimal amount of expansion agent in the mix design of concrete for concrete-filled steel tubes. A concrete-filled steel tube (CFST) is an effective composite structure that is manufactured with advanced technologies to combine and optimize both the high strength and ductility of steel and the high-pressure resistance of concrete in the composite structure. According to the triaxial stress state of concrete specimens under loading, the lateral restraints of steel tubes can improve the compressive strength and the elasticity modulus and increase the plasticity of concrete, especially for high-strength and ultra-high-strength concrete. Such concrete has been widely used in the composite structures in steel tubes, which provide lateral restraints to increase the toughness and reduce the brittleness of the steel structures. The composite structures are manufactured entirely by filling the steel tubes with concrete to avoid local buckling in the steel. A CFST is the best combination of two materials that are concrete and steel tube  and has In the prestressed concrete structure, concrete creep causes a loss of prestress of the structure by redistributing internal forces that directly affect the long-term performance and shorten the service lifetime of the prestressed concrete. Long-term creep is a significant material response that needs to be accounted for in the concrete structural design . TherefoCFSTs are composite structures. Steel tubes provide lateral restraints inside concrete structures in multiple directions. Concrete is a typical heterogeneous porous medium composed of solid materials , liquid (water), and gas (air). Expansion agents are often added to concrete mixtures to provide micro expansion and to limit shrinkage of the concrete, making the steel tube tightly contact concrete and avoiding any debonding between the two materials. The effects of expansion agents and the lateral restraint effect by steel tubes on creep are different from those of ordinary concrete. Lateral restraint conditions, expansion agent amounts, and steel ratios are recognized as key factors that cause creep and shrinkage in CFST structures. However, the deformation mechanism caused by these factors has not yet been exploredThis paper mainly investigates the creep of CFSTs with two lateral restraint conditions , three expansion agent concentrations , and four steel ratios . Air void characteristics in concrete with or without lateral restraints, mixed with different concentrations of expansion agents, were also studied, and air-void parameters were determined with a RapidAir457 analyzer . The main goal was to explore the mechanism of creep under combination conditions of varied lateral restraint, different expansion agent concentrations, and different steel ratios. The outcomes of this study can contribute to a better understanding of the long-term mechanical performance of CFSTs in structural infrastructures.A 480-day experiment on the creep of concrete with lateral restraints was carried out.It was observed that the lateral restraint greatly reduces the creep degree in concrete.The influence of the mechanism of lateral restraint on creep was analyzed by means of the microstructure of concrete.Highlights:3 and the mud content was 0.5%. The coarse aggregate was demolished basalt with particle sizes within a range of 5–20 mm, apparent density was 2680 kg/m3, crushing index was 3.2%, and alkali activity was 0.07. The properties of cement, fly ash, mineral powder, expansion agent, air-entraining agent, and water reducing agent are summarized in Mechanical properties of raw materials were measured before every test. Fine aggregates were of natural sand with a fineness modulus of 3.2, where the apparent density was 2716 kg/mAll steel tubes used in this study are 140 mm in diameter and 350 mm in length, but the thickness of the tubes varied by 1.3, 2.2, and 3.0 mm, which resulted in differences in the steel ratio of the CFST of 3.8%, 6.6%, and 9.2%, respectively. In addition, the water cement ratio was 0.3, whereas the expansion agent content in the core concrete varied by 4%, 8%, and 12%. Twelve specimens were prepared with a combination of two lateral restraint conditions, three varied expansion agent concentrations, three steel ratios, and two diameter sizes. All the information about each specimen including mix design of the concrete is summarized in A dial indicator with an intelligent memory  and a string strain gauge were mounted on the specimen to measure deformation stress, and vertical deformation at the middle of the specimen  while maEffects under varied lateral loading were studied in two ways. First, the creep properties of the concrete specimens with and without lateral loading were compared. The steel tube was fixed on the concrete specimen to provide lateral restraint. Second, the lateral loading concrete with different steel ratios, which can result in different degrees of lateral restraint, was investigated. The results of all 12 creep tests are shown in the three subfigures of According to the test results and analysis shown in When all specimens had 4% expansion agent, the creep ratio of the specimen with a steel ratio of 6.6% was about 0.42–0.71 times that with a steel ratio of 3.8%, while the creep ratio of the specimen with a steel ratio of 9.2% was about 0.44–0.58 times that with a steel ratio of 3.8%.When all specimens had 8% expansion agent, the creep ratio (coefficients) of the specimen with steel ratio of 6.6% was about 0.48–0.74 times that with a steel ratio of 3.8%, while the creep ratio (coefficients) of the specimen with a steel ratio of 9.2% was about 0.44–0.48 times that with a steel ratio of 3.8%.When all specimens had 12% expansion agent, the creep ratio (coefficients) of the specimen with a steel ratio of 6.6% was about 0.51–0.76 times that with a steel ratio of 3.8%, while the creep ratio (coefficients) of the specimen with a steel ratio of 9.2% was about 0.45–0.55 times that with a steel ratio of 3.8%.Moreover, creep variation caused by different steel ratios were also compared in pairs:In this paper, based on the ferrule effect of the combined structure, the mechanical properties of the concrete under different lateral restraints are calculated based on the confinement effect of the combined structure. The influence of the steel tube’s lateral restraints on the creep characteristics of the concrete was studied via detailed calculation and analysis considering the load distribution between concrete and steel tube. The results of the analysis are summarized in All creep tests on CFST specimens with different steel ratios show that, except for the first 10 days, the creep ratio (coefficients) decreased as the steel ratio increased up to 9.2%. The creep ratio (coefficients) in the first 10 days did not decline when the steel ratio increased from 6.6 to 9.2% in the concrete with 4% and 12% expansion agent. The reason for this phenomenon is that lateral restraint enhances with the increase in steel ratio. Lateral restraint improves the strength of the core concrete. Furthermore, the steel tube improves the lateral restraint by increasing the steel ratio, which effectively shares the external load. Consequently, less stress in the core concrete leads to a creep ratio (coefficients) reduction under the same external loads. Triaxial stresses were applied to the specimen because of a combination of both lateral constraints and vertical loading, and these stresses affect the strength of the core concrete, which in turn affects creep behavior.The results are summarized in four groups in When the steel ratio was 3.8%, with expansion agent concentrations of 8% and 12%, the creep ratios were about 0.865–0.910 and 0.825–0.863 times those of the 4% expansion agent, respectively, except for the first 240 h.When the steel ratio was 6.6%, with expansion agent concentrations of 8% and 12%, the creep ratios were about 0.895–0.932 and 0.869–0.964 times those of the 4% expansion agent, respectively, except for the first 2400 h.When the steel ratio was 9.2%, with expansion agent concentrations of 8% and 12%, the creep ratios were about 0.932–0.976 and 0.873–0.915 times those of the 4% expansion agent, respectively, except for the first 240 h.When the concrete specimens contained 8% and 12% expansion agent but no lateral loading, the creep ratios were about 0.841–0.886 and 0.778–0.817 times those of the 4% expansion agent, respectively.Creep ratios of the two concrete specimens with different expansion agent concentrations are compared in pairs in In general, increasing expansion agent concentrations in the concrete effectively reduced the creep ratio as lateral loading was fixed. However, when the expansion agent concentration increased from 8% to 12%, the reduction of the creep ratio was much smaller than when the agent concentration increased from 4% to 8%. In other words, the decrease in creep ratio was not proportional to the increase in expansion agent concentration. Therefore, there is an optimum rate of expansion agent that brings about an optimal effect on creep ratio.2O3 was applied to the specimen after the ink dried. Afterwards, Al2O3 was removed, and the specimen was placed under an air-void analyzer (RapidAir457) for parameter measurement. The test equipment with specimens is shown in Eighteen cube specimens  with a side distance of 100 mm that have the same lateral restraint conditions and expansion agent concentrations as the creep test specimens were cured for 28 days and prepared for pore structure analyses. Each subfigure in As The self-stress effect of the core concrete with lateral restraint contributes to further hydration of the expansion agent in the concrete structure. Therefore, there will be more crystallized expansion agent to make the concrete structure denser with lateral restraints than without lateral restraints. This also explains why creep ratio is smaller for the concrete with lateral restraint than that without lateral restraint, as shown in Air content and the number of bubble chords with an expansion agent concentration of 8% (or 12%) are 0.63 and 0.78 times (or 0.84 and 0.75 times) higher than those with an expansion agent concentration of 4%, respectively, for concrete specimens with lateral restraint, as Analyses on 12 creep test results reveal that creep ratios of the concrete specimens with lateral restraint are about 0.09–0.30 times smaller than those without lateral restraint. Meanwhile, the creep ratio of CFST specimens decreases as the steel ratio increases.Creep ratio decreases as expansion agent concentration increases.According to analyses on the microscopic pore structures of concrete specimens, the percentage of air content and the number of bubble chords of the core concrete with lateral restraint are lower than those without lateral restraint. Air content and the number of bubble chords reach the highest values in the core concrete with a 4% expansion agent, followed by core concrete with 8% expansion agent and then with 12% expansion agent.The lateral restraint and the expansion agent concentrations have a combined effect on the creep of the core concrete, so selecting the appropriate lateral restraint and the best content for the expansion agent can effectively reduce the creep of concrete.The key contribution of this study is the finding that CFSTs require an optimum combination of proper lateral restraint and an optimum ratio of expansion agent in order to effectively reduce the creep and thus improve the long-term service of concrete structures.This paper investigates the creep behavior in CFSTs. Specimens with different expansion agent concentrations and different steel ratios were tested, and the results were compared in pairs. Meanwhile, air-void analysis in the concrete structure in a microscope is also carried out. The following conclusions are drawn from this study:


Immunoglobulin G4-related disease (IgG4-RD) is a fibroinflammatory disorder which has been first reported in 2001 by Hamano and colleagues in a patient with autoimmune sclerosing pancreatitis. Almost every organ in the human body can be affected by IgG4-RD from infiltration with IgG4-positive plasma cells. Involvement of lungs with IgG4 is reported previously, but still, there is no clear picture of the pathophysiology behind lung involvement. Here, we are presenting a patient who has IgG4-RD presenting as pseudotumor of the lungs. The IgG4-related disease is characterized by increased serum IgG4 concentration along with pathologic findings of lymphoplasmacytic infiltration of IgG4-positive plasma cells, storiform fibrosis, and obliterative phlebitis in different organs. Some of the manifestations of IgG4-related disease include but not limited to autoimmune pancreatitis, tubulointerstitial nephritis, Mikulicz's disease, pseudotumor, aortitis, pericarditis, meningitis, Riedel's thyroiditis, and retroperitoneal fibrosis . The IgGWe are reporting a patient who presented with lung nodule along with mediastinal lymphadenopathy. We confirmed the diagnosis with lung biopsy status post lower lobe resection.A 76-year-old African American male presented to the rheumatology clinic with severe fatigue, unintentional weight loss, memory loss, and ambulatory dysfunction secondary to generalized weakness. He lost 40 pounds in the last two years. Past medical history includes hypertension, hyperlipidemia, COPD, and diabetes mellitus. His medications were glipizide, metformin, bupropion, atorvastatin, aspirin, lisinopril, salbutamol, senna, and docusate. No pertinent family history. He stopped smoking 1½ years ago, with 120 pack-year smoking history, quit drinking three years ago, and smokes marijuana. He was a construction worker in the past. Physical examination was unremarkable except subjective weakness. Labs are significant for elevated creatinine level of 1.49 mg/dl (0.5–1 mg/dl), elevated IgE level of 234 (1–180 KU/L), proteinuria, and glucosuria.Prior to this presentation, he was found to have worsening renal function with a creatinine level of 1.4 as an outpatient. An ultrasound image of Kidneys showed echogenic foci in the right kidney, which was further evaluated with CT (computerized tomography) scan of the abdomen and pelvis with contrast. It showed a scar in the interpolar region of the right kidney and also showed incidental findings of bronchiectasis and centrilobular emphysematous changes at lung bases. Later, CT scan of the chest with contrast was done which showed a similar finding and a 10 mm low-density area in the right thyroid lobe. Further evaluation by thyroid ultrasound showed bilateral thyroid nodules, including a 3.5 cm hypoechoic nodule in the lower pole. Fine-needle aspiration cytology of the nodule showed benign follicular nodule.He had a repeat CT chest with contrast after 20 months concerning his progressively worsening weight loss and smoking history which showed 1.4 cm right lower lobe lung nodule, bilateral interlobular septal thickening with lower lobe predominance, multiple bilateral ground-glass opacities, cystic changes, and multiple enlarged mediastinal lymph nodes   along wiHe was started on 40 mg of prednisone, which resolved his fatigue and had significant weight gain, and his prednisone was tapered down and stopped in 4 months. He was not placed on any immunosuppression and was monitored for any further relapse. He is in remission for last one year off any medications without any relapses.IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory disease with protean manifestations involving virtually any organ in the body. Hallmark of IgG4-related disease is lymphoplasmacytic tissue infiltration, fibrosis (often in the storiform pattern), obliterative phlebitis, and elevated serum IgG4 concentration. The disease is most commonly seen in middle-aged and older people (mean age from 59 to 68 years) and is more common in men (70 to 80 percent) , 4. The Sarles et al. first described a form of IgG4-RD as sclerosing pancreatitis in 1961 . It was β (TGF-β) which results in increased eosinophils as well elevated IgG4 and IgE as concentrations. It also contributes to the activation of fibroblasts leading to fibrosis [The pathophysiology of IgG4-related disease is still not well understood as it is a newly recognized disease. Autoimmunity is one of the commonest triggers for IgG4-related disease with a key role of T helper-2 (Th2) cell involvement in the pathophysiology. Th2 cells overexpress interleukins 4, 5, 10, and 13 as well as transforming growth factor fibrosis .Most of the patients with lung involvement in IgG4-RD are asymptomatic or with nonspecific symptoms like cough, fever, chest pain, and dyspnea . There aIgG4 : IgG tissue plasma cell ratios of >40% and >50%, and >10 IgG4 plasma cells per HPF have been used for the diagnosis of IgG4-RD. Masaki et al. found ratios >40% in tissue plasma cells had a sensitivity and a specificity of 94.4% and 85.7%, respectively, in the diagnosis of IgG4-RD. It was also demonstrated that fibrotic tissue areas have decreased IgG4 plasma cells .Radiological features of IgG4-related lung disease are variable. The following findings have been reported on CT scan of the chest—solid nodular density, ground-glass opacities, bronchiectasis featuring like interstitial lung disease, honeycombing simulating pulmonary fibrosis, and thickening of bronchovascular bundles and interlobular septa. In a single patient, multiple radiological features can be found . PleomorSystemic corticosteroids are the first-line treatment in IgG4-RD. Although spontaneous remission can occur, adequate treatment with steroids is highly recommended to prevent fibrosis. In IgG4-related lung disease, prednisone 30 mg or higher is prescribed daily for 1-2 weeks following by tapered dose. No randomized clinical trials have been conducted, and there are no formal treatment guidelines. Anecdotal data and multiple case reports mention the use of other immunosuppressive agents like methotrexate, mycophenolate mofetil, cyclophosphamide, and tacrolimus . BiologiWe report an unusual case of IgG4-related disease presenting with symptoms, signs, and radiological findings mimicking lung malignancy. It is imperative to differentiate both diseases as management as well as prognosis is quite different.


Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor  is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT. Photodynamic therapy (PDT) is a type of cancer treatment in which tumor cells are killed by reactive oxygen species, such as singlet oxygen, formed by the local and light-induced activation of a photosensitizer (PS) . By locaCamelidae family  or VHHs (variable domain of the heavy chain from heavy chain-only antibodies)) . After ectodomain coupling, excess reactive esters were deactivated with 100 mM ethanolamine pH 8. The amount of immobilized protein ranged from 700 to 3000 response units (RU); 1 RU corresponds to approximately 1 pg of protein per mmVHHs were site-directionally conjugated to the photosensitizer IRDye700DX–maleimide (LI-COR Biosciences). First, the VHHs were incubated with an 2.75-fold molar excess of TCEP (tris(2-carboxyethyl) phosphine hydrochloride)  to reduce the C-terminal cysteine upon which the VHH were incubated with a four-fold molar excess of IRDye700DX-maleimide for 16 h at 4 °C. Free label was removed by size-exclusion chromatography using three consequent Zeba Desalting Columns (Thermo Fisher Scientific) according to the manufacturer’s protocols. Degree of conjugation was determined using the Multiskan Go spectrophotometer (Thermo Fisher Scientific), and the amount of free dye was determined after size separation by SDS-PAGE (Bio-Rad) on the Odyssey scanner (Li-COR Biosciences). Afterwards, the SDS-PAGE gel was stained with Page Blue (Thermo Fisher Scientific) to show total protein. For the internalization assay, G2c was conjugated to HiLyte Fluor 647 (HL647)–maleimide  according to the protocol described above.MKN45 cells were seeded in eight-well chamber slides  two days before the experiment. Cells were incubated with 1 µM G2-Alexa647 conjugate for 2 h at 37 °C. Unbound conjugate was removed by washing three times with PBS. Cells were fixed in 4% PFA for 10 min at room temperate (RT). PFA-induced autofluorescence was quenched using 100 mM glycine in PBS . Cells were washed with PBS and then incubated with DAPI  for 10 min at RT. The slides were mounted using SlowFade (Invitrogen) and pictures were taken with a LSM700 confocal microscope using a 63× oil immersion objective . The images were analyzed with ImageJ.2 fluence rate for a total light dose of 18 J/cm2, then were incubated overnight at 37 °C in the cell culture incubator. The next day, the cells were screened for viability. For this, cells were incubated with calcein (Thermo Fisher Scientific) and propidium iodide (Thermo Fisher Scientific) to stain live and dead cells, after which they were imaged on an EVOS fluorescence microscope. Cell viability was quantified using the alamarBlue reagent (Thermo Fisher Scientific), which was quantified using a FLUOstar Optima plate reader . Data were plotted and fitted using Prism 7 software .Photodynamic therapy was performed similarly to the process previously described ,8. CellsEscherichia coli production vector, and binding to the human Met ectodomain was assessed with ELISA (D) of 7 ± 8 nM and 4 ± 3 nM, respectively. The KD value of F5 could not be determined properly, but was estimated to be at least higher than 50 nM. To investigate which subdomain these VHHs bind to, ELISAs on chimeric human/llama Met ectodomains were performed. In these chimeric constructs , either the first 122 (LS1) or 473 (LS5) amino acids of human Met were replaced by those of llama Met . In conclusion, addition of a cysteine residue to the C-terminal region of G2 did not affect binding affinity to human Met nor did it affect domain specificity.We examined the propensity of G2c to interact with the four variants of the Met ectodomain  using surface plasmon resonance (SPR). In agreement with the ELISA data , the SPRSubsequently, the free thiol in G2c was conjugated to the water-soluble PS IRDye700DX using maleimide chemistry, resulting in a degree of conjugation of ~1, as determined spectrophotometrically. However, because ~40% of the signal in the solution was still free PS A, a degrPDT is a valuable method for inducing local cell death of malignant cells by the local activation of PS. Unfortunately, clinically employed PS are administered systemically, are relatively hydrophobic, and passively accumulate in the tumor as a result of their hydrophobicity in combination with the enhanced permeability and retention (EPR) effect ,37. ThisClinicalTrials.gov: NCT02422979). These results are eagerly awaited, as these are the first tests in patients and thus could significantly advance the field of targeted PDT. Nevertheless, we consider further improvements a necessity, and in the current study we have employed nanobodies or VHHs as targeting agents. Conventional antibodies of the IgG-type are large, dimeric molecules, typically designed for extended blood circulation. These characteristics may lead to slow tumor penetration/accumulation, poor tumor to normal tissue ratios, and long photosensitivity [Targeted PDT using antibodies as targeting agents  is currently being clinically evaluated for the treatment of recurrent head and neck cancer  was provided with a C-terminal tag containing an unpaired cysteine to allow conjugation to the PS using maleimide chemistry. The addition of this thiol-containing C-Direct tag did not affect the binding characteristics of the VHH, which is in line with previous studies of C-terminal-labeled VHHs via an unpaired cysteine ,15,16. MD values determined under equilibrium by SPR. The SPR analysis does, however, allow the determination of association and dissociation rate constants, which provided additional information on the binding kinetics of G2c on wild-type and chimeric Met proteins. Moreover, similar affinities for G2 were found for binding to Met-expressing cells [The binding affinity of G2c for Met was determined on purified ectodomains using two different technologies: ELISA and SPR. In these experiments, the apparent binding affinity of G2c in ELISA was comparable (low nanomolar range) to the Kng cells , indicatApplication of the anti-Met conjugate G2c-PS in in vitro PDT resulted in the efficient and specific killing of the Met-overexpressing MKN45 cells and the Met-expressing TOV + Met and HepG2 cells, while the Met negative cell line TOV112D remained undisturbed. The observation that the treated cells could be stained with propidium iodide suggests that cells could have died through necrosis. Further studies would be needed to determine the exact mechanism of cell death. The potency of G2c-PS in killing MKN45 cells was in the nanomolar range. In the two other cell lines, the Met-expressing HepG2 tumor cells and the TOV112D cells stably expressing Met (TOV + Met), the observed potencies were lower. These differences in potencies might be related to the relative Met expression levels of these cells. MKN45 is a highly Met-overexpressing cell line due to genomic amplification of the Met gene . It woulIn conclusion, this study has demonstrated that targeting Met using site-directionally conjugated VHH-PS has the ability to specifically kill Met-overexpressing tumor cells. Follow up studies should evaluate the potency of this approach in more relevant models (in vivo). Taken together, Met-targeted PDT might serve as an alternative or complementary approach for combating cancer.