Amongst women without GDM, first-trimester mono-(2-ethylhexyl) phthalate and mono(carboxyisooctyl) phthalate levels were positively associated with 120-min plasma glucose  in mid-pregnancy. No other monotonic associations were detected between phthalate or phenol levels and fasting or stimulated plasma glucose, β-cell function, insulin resistance, or 60-min disposition index. Our results support a glycaemia-raising effect of phthalates during pregnancy, consistent with findings in non-pregnant populations and suggest a possible protective effect of exposure to TCS against GDM.Certain phthalates and bisphenol A (BPA) have been associated with insulin resistance and type 2 diabetes in non-pregnant adults, but studies of gestational diabetes mellitus (GDM) have reported conflicting results for phthalates and no associations with BPA. Our aim was to investigate the relationship between maternal serum levels of phthalate metabolites and phenols at 10–17 weeks of gestation and glucose homeostasis at 28 weeks of gestation. 232 women aged ≥16 years without type 1 or 2 diabetes with singleton male pregnancies were recruited from a single UK maternity centre between 2001 and 2009 as part of a prospective observational study (Cambridge Baby Growth Study). Serum levels of 16 phthalate metabolites and 9 phenols (including BPA) were measured using liquid chromatography/tandem mass spectrometry. Oral glucose tolerance tests were performed at 28 weeks. 47/232 (20.3%) women had GDM. First-trimester triclosan (TCS) was inversely associated with incident GDM (adjusted odds ratio per log increase in concentration 0.54, 95% confidence interval 0.34–0.86,  Late pregnancy is characterised by peripheral insulin resistance with a compensatory increase in insulin secretion and β-cell mass. Inadequacy of this response leads to gestational diabetes mellitus (GDM), which is associated with both perinatal and long-term adverse outcomes . The incIn vitro, phthalates activate peroxisome proliferator-activated receptors, which regulate cellular lipid and glucose metabolism (n = 350), second-trimester urinary monoethyl phthalate (MEP) was positively associated with impaired glucose tolerance (IGT), whereas di-(2-ethylhexyl) phthalate (DEHP) metabolites were associated with lower odds of IGT  observed no significant associations between first-trimester urinary phthalate metabolites and incident IGT/GDM  reported a positive association between second-trimester (but not first-trimester) urinary BPA levels and stimulated blood glucose levels (but not incident GDM) at 24–28 weeks of gestation , first-trimester urinary BPA was not significantly associated with incident IGT/GDM (n = 94), second-trimester urinary BPA was not associated with GDM or fasting blood glucose levels amongst controls , and triclosan (TCS)] have not been associated with T2DM or fasting serum glucose in humans .Our objective was to investigate the prospective relationships between maternal serum levels of phthalate metabolites, BPA, and other phenols at 10–17 weeks of gestation and glucose homeostasis at 28 weeks of gestation in a UK setting.n = 2,229) were recruited to the Cambridge Baby Growth Study (CBGS), a large prospective cohort study, from routine antenatal clinics at 10–17 weeks of gestation at the Rosie Maternity Unit, Cambridge, between April 2001 and March 2009. Characteristics of CBGS participants have previously been described (Pregnant women aged ≥16 years (escribed . Writtenp > 0.2).A subcohort of 330 mothers of 334 male infants was selected for a nested case-control study of EDCs and cryptorchidism . Of these, women were retrospectively included in the present study if they had a singleton pregnancy, no pre-existing diagnosis of type 1 or 2 diabetes mellitus or GDM, and undertook an oral glucose tolerance test (OGTT) at 28 weeks of gestation. This subset of 232 women was representative of the whole cohort with regard to age, ethnicity, pre-pregnancy body mass index (BMI), and deprivation index  as these women did not differ from others with regard to age, pre-pregnancy BMI, ethnicity, smoking status, parity, or prevalence of GDM .A questionnaire administered at ~36 weeks of gestation collected demographic information, including history of diabetes mellitus, ethnicity, smoking, and parity. The Index of Multiple Deprivation 2007 (IMD), which measures various economic, social, and housing parameters, was derived from residential postcodes . The samp > 0.1). Insulin and C-peptide levels were measured at 0 and 60 min in 158/232 (68.1%) women. Women with missing insulin and C-peptide levels did not differ from others with regard to age, BMI, IMD, ethnicity, smoking status, parity, or prevalence of GDM . Glycated haemoglobin levels were not measured.At 28 weeks of gestation, a 75-g OGTT was performed following an overnight fast. Venous whole blood glucose levels were measured at 0 and 60 min, and capillary blood glucose levels were measured at 0, 60, and 120 min. From May 2007 onwards, venous whole blood glucose levels were also measured at 120 min . Women completing OGTTs before May 2007 did not differ from those completing OGTTs during/after May 2007 with regard to age, BMI, IMD, ethnicity, smoking status, parity, or prevalence of GDM . Fasting and stimulated insulin and C-peptide levels were measured by enzyme-linked immunosorbent assay using commercial kits .n = 232), with the first quartile used as the referent category.Analyses of associations with parameters of blood glucose homeostasis included only those chemicals measured above the LOD in >60% of samples. Concentrations below the LOD were assigned a value equal to LOD/2 if the data were highly skewed or LOD/√2 if not . These sR2 = 0.790, F = 2,889.3, p = 2 × 10−262]. A linear regression model assessing the ability of fasting capillary blood glucose to predict fasting venous plasma glucose gave a different result: venous plasma glucose = 0.826 × capillary blood glucose + 0.996 ; however, we justified using the first equation to estimate the missing 120-min venous plasma glucose levels as across the entire CBGS cohort the mean 60-min venous plasma glucose (7.4 mmol/l) was closer to the mean 120-min venous plasma glucose (6.9 mmol/l) than the mean fasting venous plasma glucose (4.7 mmol/l) The updated homeostasis model assessment (HOMA2) was used to estimate steady-state β-cell function  and insulin resistance  . To estimate insulin secretion independent of insulin sensitivity, we calculated the disposition index as the 60-min insulinogenic index [(insulin 60 − insulin 0)/(venous glucose 60 − venous glucose 0)] divided by HOMA2-IR  ; softwarHOMA2-IR . HOMA2-BFor primary analyses, women were diagnosed with GDM if they met one or more of the following criteria: fasting plasma glucose ≥5.1 mmol/l, 60-min plasma glucose ≥10.0 mmol/l, or 120-min plasma glucose ≥8.5 mmol/l , 32. ForNewborns were examined by research nurses, as far as possible in the first 2 weeks of life, either in hospital or at home visits. They were then re-examined during clinic visits at 3, 12, and 24 months of age. Birth weight as measured at delivery by midwives was taken from health records. Cryptorchidism was defined as one or more testes not present in the inferior half of the scrotum for at least a few moments after manipulation.The study was approved by Cambridge Local Research Ethics Committee and adhered to the Declaration of Helsinki.t-test and Mann–Whitney U-test for continuous variables, and chi-square test for categorical variables.Distribution percentiles of phthalate and phenol concentrations were calculated for women with and without GDM, and differences in patient characteristics were assessed using the per se may cause insulin resistance and β-cell dysfunction (phet) and linear trend (ptrend) in outcome value across serum phthalate/phenol concentration quartiles were performed. Because numerous chemicals were significantly correlated and several have been previously associated with dysglycaemia  and a possible association between GDM and congenital cryptorchidism , we addip-values were two-sided and p < 0.05 was considered statistically significant. Data were analysed using SPSS, version 22.0 .All 232 women participated in the study, of whom 47 (20.3%) met one or more criteria for GDM , and several of the other chemicals were weakly correlated .Six phthalate metabolites  and three phenols  were detectable in >60% of samples (Table p = 0.010] (Table p = 0.028), third , and fourth  quartiles for triclosan, compared to the first quartile . Inspection of the data did not reveal evidence for a threshold or non-linear relationship. Conversely, women with MiBP levels in the second  and fourth  quartiles, but not in the third quartile, had higher odds of GDM .In continuous models, of these nine chemicals, only triclosan was associated with incident GDM [adjusted OR (aOR) per log increase in concentration 0.54, 95% CI 0.34–0.86, p = 0.005) and the categorical analysis . However, there was no significant association between MiBP levels and incident GDM (data not shown).To explore the robustness of our results, and in view of the high incidence of GDM in our sample compared to previous studies , we repen = 185), MEHP was positively associated with 120-min plasma glucose , explaining 6.9% of the variation in 120-min glucose . MCiOP was also positively associated with 120-min glucose , explaining 3.4% of the variation in 120-min glucose. Other phthalate metabolites (including MiBP) and all phenols were not significantly associated with fasting/stimulated glucose levels, HOMA2-B, HOMA2-IR, or disposition index .Among women without GDM at 28 weeks of gestation (p = 0.00008), explaining 7.6% of the variation in 120-min glucose, but not any other indices of glucose homeostasis.Experimental studies suggest that MEHP, the primary metabolite of DEHP, may have greater endocrine disrupting activity that is secondary metabolites, such as MECPP . We, thep = 0.12) nor MCiOP  was associated with incident GDM; and MCiOP was not associated with GDM in the categorical analysis . However, women with MEHP levels in the second  and fourth  quartiles, but not in the third quartile , had significantly increased odds of GDM .We hypothesised that the lack of association of MEHP and MCiOP with GDM, despite their positive associations with 120-min plasma glucose, may reflect the relatively small proportion of cases in our sample with abnormal 120-min glucose levels, owing to the use of a higher diagnostic threshold (≥8.5 mmol/l) , 32 thanp = 0.58) or fasting/stimulated glucose levels, HOMA2-B, HOMA2-IR, or disposition index (data not shown). When we remodelled the of odds of GDM in relation to serum phthalate/phenol concentrations in the 216 mothers whose sons did not have cryptorchidism at birth, our results were not substantially different from the primary analyses: triclosan was associated with incident GDM in both the continuous analysis  and categorical analysis , and women with MiBP levels in the second  and fourth  quartiles, but not in the third quartile, had higher odds of GDM  (other data not shown).Of the 232 male infants born to the women in our study, 16 (6.9%) had cryptorchidism at birth  phthalate and MCiOP were positively associated with 120-min plasma glucose. These findings are consistent with the thalates  and rodethalates . Two epithalates , 9—althothalates , 8, and thalates . The lacOur phthalate results do not corroborate with three other recent prospective studies of GDM, which variously reported inverse associations of MiBP and MBzP with 60-min glucose , a positThe prevalence of GDM in our subcohort 20.3%) was several-fold higher than the reported prevalence for England and Wales (~3.5%) . This ma0.3% was We did not have data for all possible confounders: for example, education, income, activity levels, diet, subfertility, family history of diabetes, and other EDCs , 18. ThiSerum phthalate metabolite levels in our study were of a similar order of magnitude to those reported in other cohorts of pregnant or postpartum women , 49. SerStrengths of our study include its prospective design; use of laboratory criteria to diagnose GDM; sample collection and storage in glass tubes ; use of a highly sensitive technique for measuring the chemicals; and our ability to account for a number of potential confounding variables. This is also the first study to examine the association between phthalate metabolites and phenols and markers of insulin resistance and β-cell function in pregnant women.p values were ≤ 0.01, making a type-1 error unlikely.Our study has several limitations. First, the sample size was relatively small, which will have limited precision and statistical power, although it was larger than two of the five previously published studies of phthalates/BPA and GDM , 19. SecIn conclusion, our study provides a novel and interesting demonstration of inverse association between maternal serum triclosan levels at 10–17 weeks of gestation and incident GDM, in addition to biologically plausible associations between MEHP, MCiOP, and the MEHP/MECPP ratio and stimulated blood glucose levels. However, these findings need to be confirmed by further carefully designed prospective studies in other settings, that use a variety of specimen types (including urine) and assess exposure levels at multiple time points. More evidence is required before specific recommendations can be made with regards to clinical practice in diabetes or public health policy.This study was carried out in accordance with the recommendations of Cambridge Local Research Ethics Committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by Cambridge Local Research Ethics Committee.BF, AT, and CA contributed to the conception and design of the study; BF, HF, A-MA, AJ, and AT performed the data analysis; KO provided statistical expertise; BF wrote the first draft of the manuscript; AT, IH, DD, KO, and CA contributed to the design of the CBGS; and HF, A-MA, AJ, IH, DD, KO, and CA contributed to data collection. All authors contributed to the interpretation of the results and revision of the manuscript for important intellectual content, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Optimization of outcomes of extra-abdominal STS is not clearly understood. We sought to determine whether hospital surgical volume and adherence to NCCN guidelines, or both, are associated with outcomes in the treatment of extra-abdominal soft tissue sarcoma (STS).  The National Cancer Database (NCDB) was queried for patients undergoing surgery for extra-abdominal STS diagnosed from 2003 to 2007. Mean annual hospital volume for STS surgery was divided into volume terciles . Adherence to NCCN guidelines was determined. Primary outcome was overall survival. p ≤ 0.001) than 1T hospitals. On multivariable analysis, adherence to NCCN guidelines was associated with improved survival , but hospital volume was not . Five-year overall survival was comparable for compliant groups at 1T, 2T, and 3T hospitals . 3T hospitals were not associated with a lower risk of 30-day mortality  compared to 1T hospitals but did have a higher R0 resection rate .  Our study population consisted of 13,684 patients with a median age of 56 years. 3T hospitals were more likely to adhere to NCCN guidelines for stage III patients (63% versus 47%;  Adherence to NCCN guidelines, irrespective of hospital volume, is associated with improved overall survival for patients with extra-abdominal STS. High-volume hospitals more often adhere to guidelines, but low-volume hospitals that follow national guidelines may achieve comparable outcomes. One of the challenges in managing complex cancers is identifying health care processes and structural measures that optimize outcomes. Hospital volume is a well-known measure that correlates with improved clinical outcomes for several cancer types . AlthougSoft tissue sarcoma (STS) is a group of over 50 rare mesenchymal malignancies that accounts for less than 1% of all new cancers. Optimal treatment of STS is based on an institution's experience and resources to provide a multidisciplinary effort to treat such a rare and complex malignancy. Although a volume-outcome relationship has been demonstrated for STS, optimizing cancer outcomes likely also depends on the appropriate use and expertise of a multimodality team that includes pathologists, radiation oncologists, medical oncologists, and surgeons , 11. HowConsequently, the purpose of this study was to analyze the impact of hospital surgical volume and adherence to NCCN guidelines on outcomes for extra-abdominal STS patients. We hypothesize that increased adherence to NCCN guidelines is associated with higher overall survival (long-term outcomes) and that high hospital surgical volume is associated with improved 30-day mortality and R0 resection rates (short-term outcomes).Data from the National Cancer Database (NCDB) was used to conduct this study. The NCDB, a joint program of the Commission on Cancer (CoC) of the American College of Surgeons (ACoS) and the American Cancer Society (ACS), is a nationwide oncology outcomes database for more than 1,500 commission-accredited cancer programs in the United States and Puerto Rico. Approximately 70% of all newly diagnosed cases of cancer in the United States are captured at the institutional level and reported to the NCDB. Variables in the database cover demographics, socioeconomic status, tumor stage, treatment received, and hospital characteristics. NCDB data contain no protected health information; hence, this study was exempt from formal IRB review.http://www.nccn.org). For this study, we utilized NCCN guidelines on the use of radiation therapy for these tumors with regard to margin status and tumor stage to divide patients into compliant and noncompliant groups . This time period was chosen to ensure up to date coding in the NCDB for the variables of interest for this study and to provide at least 5 years of follow-up for survival analyses. Histologies included were liposarcoma, fibrosarcoma, leiomyosarcoma, malignant fibrous histiocytoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, and not otherwise specified (NOS). Patients with stage I, II, and III were included, and patients with stage IV were excluded. Only patients who underwent curative intent surgery were included in the study cohort. NCDB surgical codes distinguish between curative intent surgery and procedures such as open biopsies. Patients undergoing palliative surgery were excluded. In order to provide valid volume-outcome comparisons, only patients who had all treatment at the reporting hospital were included in the study cohort.The exposure variables were hospital volume status and adherence to NCCN guidelines as outlined in a priori and before any analysis of the data was performed. Using this technique, the cutoff identified for high volume  was ≥11 cases per year.We adapted the methods from Birkmeyer et al. to compute the hospital volume . The nump value of <0.05 was set as our threshold for statistical significance. The analysis was performed using SAS 9.4 and R 3.1.3.Bivariate analyses were initially performed to identify demographic, tumor, and treatment differences between different terciles of volume using the chi-square test or analysis of variance. Logistic regression analyses were used to model the margin negative resection and 30-day mortality following surgery, and adjusted odds ratios (OR) and 95% confidence intervals (CIs) were reported. We excluded patients who underwent amputation from the margin status analysis. Overall survival was estimated by the Kaplan–Meier method, and the comparison in the survival curves between different surgical volumes and compliance to NCCN guidelines was assessed by the log rank test. Cox regression analyses were used to model overall survival, and adjusted hazard ratios (HRs) and 95% CIs were reported. For the multivariable logistic regressions and Cox regression, we included demographic and clinical characteristics that were considered as variables influencing 30-day mortality and margin status. A Our study population consisted of 13,684 patients who underwent surgical resection for extra-abdominal STS . The medN=7373). 3T centers were also more likely to see tumors that were >10 cm in size and hence had a higher proportion of patients with stage III tumors than 1T facilities . The use of chemotherapy and radiation therapy was also higher in 3T facilities. Statistically significant differences in socioeconomic characteristics were seen between the volume terciles. Finally, patients treated at 3T centers were more likely to receive care in accordance to NCCN guidelines in the use of radiation therapy than at 1T centers . Though this finding is likely not clinically meaningful, these differences were most pronounced for stage III patients between 3T and 1T centers . There was no difference in guideline compliance between 1T, 2T, and 3T centers in the management of stage I patients , or stage II patients .Following the division of the study cohort by volume terciles, some noticeable differences between groups emerged . Overallp=0.003) was seen from 1T to 3T centers. p < 0.001), respectively. The 5-year overall survival rates for NCCN guideline compliant and noncompliant patients were 72.4% and 67.2% (p < 0.001), respectively.A monotonic decrease in unadjusted overall mortality from 35.8% to 32.4% (p < 0.001). In contrast, hospital surgical volume was no longer associated with improved overall survival . As expected, larger tumor size, higher grade, and positive margins were all associated with a higher risk of overall mortality.p ≤ 0.001). A similar association was seen for 2T hospitals, with improved 5-year overall survival for the compliant group compared to the noncompliant group . There was no difference in 5-year overall survival for patients treated at 3T hospitals who received compliant care compared to those who received noncompliant care . It is important to note that 5-year overall survival for patients treated at 1T, 2T, and 3T hospitals was similar when treated in compliance with guidelines .p < 0.001)  . However< 0.001) , the 30-p < 0.001). After adjustment of variables, The overall rate of margin negative resection was higher at 3T versus 1T centers , yet the baseline overall rate remains low (0.9%). This is consistent with other published reports of low postoperative mortality following extra-abdominal STS surgery . ConsequOne advantage of the NCDB over other registry data is the inclusion of margin status. It can be reported as either (a) margin negative or margin positive or (b) margin negative (R0), microscopic positive (R1), and macroscopic positive (R2). For the sake of simplicity, we used the R classification system and found that the margin negative rate (R0) of a 1T center was lower than that of a 3T center (83% versus 90%). This association persisted following adjustment for other variables related to margin status, such as tumor location and size, with 3T centers 77% more likely to achieve negative margins as 1T centers. Related to this, we also noted that 3T centers were more likely to treat more advanced cancers and perform amputation. It is possible that the higher margin positive rate noted for 1T centers may be secondary to their reluctance to perform an amputation. Whether such cases would have been better treated with amputation is not known. Also, it is possible that hospitals planned for positive margins for those cases where the tumor is in close proximity to vital neurovascular structures, a strategy that one would expect to be more common at high-volume centers in the context of a multidisciplinary approach. Although we are unable to quantify the rate of planned positive margins using the NCDB, we expect that inclusion of these cases would artificially inflate the margin positive rate at 3T centers and bias our results towards no difference between 1T and 3T centers. In reality, the true negative margin rate may be even higher at 3T centers than reported in this analysis.Our study has limitations typical of large database studies. Incomplete clinical data as well as lack of granularity can bias our results. The NCDB does not report the cause of death; hence, our endpoint for survival analysis was overall survival and not the oncologically more relevant disease-specific survival. We are also unable to study local recurrence rates and correlate these with margin status or overall survival as recurrence information is not available in the database. Moreover, other measures highly relevant to extra-abdominal STS care such as limb function and treatment-related morbidity are not captured by the NCDB. It is quite possible that 3T centers would have improved local control, limb salvage, limb function, and treatment-related morbidity outcomes compared to 1T centers. The distribution of socioeconomic variables was significantly different between volume terciles. Although these variables were not significant predictors of overall survival, the fact that they were not balanced between groups may have introduced unknown bias. Also, we rely on data abstractors to determine whether surgery was performed for curative intent, and therefore it is not known if R2 resected tumors were for curative or palliative intent. Histologic subtype has been shown to have a significant change in 16% of cases referred to a sarcoma center, suggesting that the histologic subtypes reported by 1T centers may be unreliable in sarcoma cases . To addrSo what do we take from this data? First, the inclusion of guideline compliance is important to disentangle the true significance of hospital volume. A prior sarcoma study showed an association between hospital surgical volume and long-term outcomes . When guHigh-volume hospitals more often adhere to guidelines. Low-volume hospitals that follow national guidelines appear to achieve comparable survival, but data regarding the association between hospital volume and important outcomes such as local control, limb salvage, limb function, and treatment of related morbidities are lacking. This may help inform the debate on regionalization of sarcoma care. Rather than a focus on volume, centers should encourage expert multidisciplinary consultation and guideline-based treatment for extra-abdominal STS. Currently, adherence to guidelines for all volume terciles remains moderate at best, suggesting a potential avenue to improve outcomes across the board.


P < 0.05; 95% CI, 2.3‐2.9 and 1.4‐1.8). The higher PPT1 enzymatic levels in FEP schizophrenia patients were positively associated with larger PANSS scaling scores . Higher enzymatic PPT1 in FEP schizophrenia patients is significantly associated with increased PANSS scaling values, indicating more serious rates of developing psychosis. Enzymatic activity of PPT1 may provide an important new view for schizophrenia disorders.Genome‐wide association studies have confirmed that schizophrenia is an inheritable multiple‐gene mental disorder. Longitudinal studies about depression, first episode psychosis (FEP) and acute psychotic relapse have mostly searched for brain imaging biomarkers and inflammatory markers from the blood. However, to the best of our knowledge, the association between enzymatic activities with diagnosis or prediction of treatment response in people with schizophrenia has barely been validated. Under the Longitudinal Study of National Mental Health Work Plan (2015‐2020), we have studied a subsample of approximately 36 individuals from the cohort with data on palmitoyl‐protein thioesterase‐1 enzymatic activity from FEP and performed a bivariate correlation analysis with psychiatric assessment scores. After adjusting for sex, age, body mass index (BMI) and total serum protein, our data demonstrated that PPT1 enzymatic activity is significantly associated with schizophrenia and its Positive and Negative Syndrome Scale (PANSS) scores. This longitudinal study compared the PPT1 enzymatic activity in FEP schizophrenia patients and healthy volunteers, and the former exhibited a significant 1.5‐fold increase in PPT1 enzymatic levels (1.79 mmol/L/h/mL, and 1.18 mmol/L/h/mL;  After centrifuging at 1000 rpm for 10 minutes, the plasma was stored at −80°C for later use.Before electrophoresis or ELISA assays, each serum sample was treated with a Qproteome Albumin/IgG Depletion Kit . For western blotting, primary antibodies were as followed: rabbit anti‐PPT1 ; rabbit anti‐transferrin . The secondary antibody was goat anti‐rabbit IgG . The PPT1 enzymatic assay  was performed after 8 months with no freeze‐thaw cycles during storage. All intra‐assay coefficiency of variations was less than 5%. Please also see in supplemental materials.2.3Details of the clinical evaluation in the schizophrenia patients have been published elsewhere.2.4t test was used to compare two groups to continuous variables. Chi‐square test was used to compare two groups in binary variables. PANSS scales  and S‐scales were regressed on age, sex, BMI, serum total protein levels. The resulting residuals were ranked and inverse normalized. The multiple linear regressions in SAS 9.14 were used for analyses of the transformed PANSS scales. The logistic regression model was used for the binary variable of FEP status with covariates of sex, age, BMI and serum total protein level. The Pearson correlation coefficient scores were calculated among PPT1, sex, age, BMI and serum total protein, regressed, ranked and inverse normalized PNASS scales. Receiver operator characteristic (ROC) curves analysis in R (pROC) was performed to evaluate if PPT1 activity was a predictor of SCZ. The data set was randomly divided into train (60%) and test (40%) sets. Parameters were estimated in a train set with covariant of sex, age, BMI and serum total protein, and the parameters were evaluated in test set. ROC and AUC (area under a curve) scores were calculated based on the predicted probabilities within a test set. P< 0.05 was considered statistically significant.The PANSS 3P < 0.01.Demographic and clinical details of the samples are presented in Table 3.1Palmitoyl‐protein thioesterase 1 was weakly correlated with functional magnetic resonance imaging in specific brain regions,3.2From the current subsample of the longitudinal study, the distributions of age and sex were not different between the SCZ patients and matched healthy volunteer subjects as a study designed. Basic study information is presented in Table P < 0.01 .Body mass index, total serum protein and PPT1 enzymatic value in the SCZ patients were significantly higher than those in the matched controls  and 9.51  for with and without adjustment of total serum protein levels respectively  scores for PPT1 enzymatic activity only , negative scaling scores , general scaling scores  and PNASS‐S scaling scores .Our findings demonstrated, in a longitudinal cohort study, that PPT1 enzymatic activity levels associated with FEP schizophrenia clinical assessment scores. This association persisted after adjusting for several potential confounders, including sex, age, BMI and total serum protein levels. Higher PPT1 enzymatic activity levels were extremely positively correlated with FEP schizophrenia with higher PANSS‐positive scaling scores . These confronted results of serum factors in oxidative and immune profiles will limit their value to determine whether oxidative and inflammatory markers as good candidates for diagnosing or indicating treatment responses. However, the enzymatic activity of PPT1 in the current study provides a potentially novel path for seeking diagnostic serum biomarkers of schizophrenia.To the best of our knowledge, this is the first longitudinal study of enzymatic activity levels on blood‐based assay in first episode patients of schizophrenia. We applied restricted categories of psychotic measurements to assess each FEP patient and the degree of severity according to the latest version of the PANSS scaling system.Schizophrenia alters basic brain processes of perception, emotion and judgement to cause hallucinations, delusions, thought disorder and cognitive deficits.Of all these related genes, PPT1 is one of the responsive genes that encode critical proteins for neural development, synaptic transmission and plasticity that are widely improperly expressed in the SCZ brain.The limitations of our present study are that these FEP patients are still enrolled in longitudinal treatment studies and that the 90% dropout rate makes the follow‐up evaluation of serum levels of PPT1 enzymatic activity unavailable in the current study. However, associations between PPT1 enzymatic activity and psychotic assessment scores are consistent with the clinical and genetic studies from Ibrahim's group that 33 genes, including PPT1, as the potential blood‐based markers that are related to clinical fMRI changes in left hippocampal ventricle integrity. This view of the association of PPT1 enzymatic activity with severity PANSS scores provides a novel scope to the diagnosis or prediction of prognosis outcomes of schizophrenia in clinics.5The strong association between the systemic enzymatic levels of palmitoyl‐protein thioesterase‐1 in FEP schizophrenia patients and the psychotic assessment PANSS values could be used for diagnosing or predicting the severity of schizophrenia. Further study of PPT1 enzymatic activity in schizophrenia patients may provide crucial new intervention and prevention targets for schizophrenia disorders.Uses of all human samples and blood tissues in this study complied with safety guidelines and regulations of the National Institute of Health (NIH). All experiments on human samples were approved by Xinxiang Medical University (XXMU) Ethics Committee.Authors of this paper claimed no potential financial and nonfinancial competing interest. Click here for additional data file.


Since the inception of psychology as an academic discipline, scholars have made extensive use of first-person (or introspective) enquiry: it is applied during theory-development and hypothesis-formulation; it permeates the data-collection stage; and it impacts on the process of data-interpretation and theory-development. In all these cases, the scientists' expertise, understanding, perspective, or expectations inform their scholarly practice. Sometimes this is the case in more explicit ways; sometimes, however, it can also occur in more implicit forms—and in turn it can color and even bias the research process.This universally applied impromptu or naïve form of introspection can thus be a burden to the research process and we hence considered it important to raise this issue more broadly. We were not only seeking to draw attention to the widely practiced, often unreflected and hence problematic form of first-person enquiry; rather, we were also hoping to advance methods that could systematically develop and advance this practice so that it can become a form of enquiry that may complement other, third-person forms of research. This was the methodological aim of our Research Topic.The other, theoretical aim was to explore more subtle facets of psychological phenomena that are difficult to access from a third-person point of view. Many such phenomena have first-person characteristics or are even of a primary first-person nature to begin with  and it appears inadequate to try and explore these topics while bypassing what a first-person exploration can uncover. First- and third-person research should not be seen as competing approaches but as providing complementary access routes in a pluralistic research culture.To bring together the available expertise and to explore current approaches on what is admittedly still a niche-theme—often even marred with notorious skepticism and apodictic rejection—we compiled the current Research Topic. From a competitive selection of contributions, six were successful in the review process and in what follows we provide a brief overview to orient the reader through these articles.Is there a problem in the laboratory?Wendt voices reservations against the common practice of reducing problem-solving research to laboratory settings, as this does not give justice to the experiential facets of many real-life problem-solving situations. Wendt proposes a phenomenologically oriented understanding and definition of what a problem is and proposes a set of empirical strategies that a revised approach should consider. These include the use of less intrusive methods of enquiry; and the inclusion of “atmospheric” characteristics of the experimental setup, for instance via the consideration of artwork.In his paper The Phenomenology of Habits: Integrating First-Person and Neuropsychological Studies of Memory, Tewes explores the possibility to integrate first-person research on embodied memory capacities with third-person research methods as commonly used in neuropsychology. Starting with the concept of habits, he analyzes phenomenological and embodied work of habitual memory capacities in order to show that next to rigid and stereotypical habits there are also flexible and adaptive ones, which are open to attentional control and accessible from the first-person perspective. In the last part of the paper he outlines how the method of front-loaded phenomenology—developed by Shaun Gallagher—could contribute to exploring this type of habitual body memory also with neurophysiological methods.In his contribution The relevance of explanatory first-person approaches for understanding psychopathological phenomena. The Role of Phenomenology, Schmidt draws on the Husserlian concept of genetic phenomenology to illuminate different psychopathological phenomena. He points to the importance of complementing biological/reductionist accounts of psychiatric conditions with phenomenological ones, as many experiential facets of psychiatric disorders have correlate symptoms that cannot be immediately traced back to the primary physiological deficiency. He points out how phenomenological research needs to be used to systematize psychopathological experiences rather than understand them via the detour of physiology.In his article Husserlian phenomenology as a kind of introspection, Gutland introduces phenomenology as a form of introspection and gives an overview over Husserl's phenomenological approach. He reviews the challenges of introspective enquiry and points to safeguards that Husserl introduced to shield phenomenological introspection against these challenges. One is the systematic scrutiny and reduction of accounts to those aspects that are fully compatible with what one experiences. The other is the exploration of different (imaginary) instantiations of a phenomenon and the attempt to identify patterns of lawfulness that permeate these—an approach Husserl termed eidetic variations. Gutland discusses the implications of these aspects and also places Husserl's account in the wider philosophical context.In his article The confluence of perceiving and thinking in consciousness phenomenology, Wagemann focuses on the phenomenon that similar dynamic patterns of separation and integration appear in the history of human consciousness as well as in elementary mental processes. Against this background—and substantiated with representative examples—he complements the standard approach to perception and thought as externally measurable processes with systematic first-person observations inspired by Steiner's and Witzenmann's Structure Phenomenology. In the context of voluntary perceptual reversals, this allows the author to trace back perceiving and thinking action to a diachronic structure of separating and integrating mental micro-gestures and to relate this to the historical development of psychology.In his article Pristine inner experience and descriptive experience sampling: Implications for psychology, Lesley-Carr and Heavey define pristine inner experience as experiences that is spontaneous and not yet colored by interpretation. Such experiences are theoretically important as they allow for an unobtrusive assessment of a momentary psychological state. The authors introduce descriptive experience sampling as a method to enquire into these spontaneously available states and illuminate how this method can help uncover facets of psychological functioning that are difficult to access with other methods.In their article Taken together, the Research Topic has implications for basic research as well as applied  practice and will equip the reader with an overview over the challenges and opportunities of introspection—and how these can be addressed and/or made use of.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The health and gender of older adults can elicit differing attitudes and emotions within young and middle-aged adults ; one’s own gender may also influence these differences . In this study, 287 participants , aged 19-55 years (M=32.8), were randomly assigned to read one description of an older adult that varied cognitive health status  and gender . Factorial MANOVAs examined differences by gender, health, and participant gender for participants’ (a) emotions about the older adult  and (b) negative perceptions about aging (ageist attitudes and aging anxiety). The first MANOVA found a significant main effect for health status; participants expressed more compassion (p=.013) and less emotional distance (p<.001) for the older adult with Alzheimer’s than for the healthy older adult. Also, the Target Gender X Participant Gender interaction was significant for emotional distance (p=.032), but not for compassion (p=.616); men reported more emotional distance than women for the female older adult, regardless of target health status, but men and women’s emotional distance were very similar for the male older adult. The second MANOVA showed only a significant health status main effect; ageist attitudes (p=.021), but not aging anxiety (p=.062), differed by health status of the older adult, with more ageist attitudes expressed for the healthy older adult than the older adult with Alzheimer’s. Overall, these results show that individual factors can influence young and middle-aged adults’ negative attitudes and emotions towards older adults. Implications will be discussed.


SMAD4 gene significantly contributed to the increased risk and might participated the pathological progression of TAAD. This investigation aims to test (1) the associations between rs12455792 and MØ recruitment, inflammatory response in aggressiveness of TAAD, and (2) the molecular mechanism accounting for their effects. In TGF-β signaling molecular detection, rs12455792 C>T variant activated the canonical and non-canonical TGF-β mediators. It also increased the secretion of chemotactic factors of HASMCs. To confirm the impact of this change, we detected MØ recruitment and infiltration in HASMCs and aortic tissues of TAAD patients. We found that MØ recruitment in cells and tissues with rs12455792 variant genotypes was increased than that in wild type groups. Moreover, rs12455792 variant increased M1 type inflammatory response, which might contribute much to TAAD progression. To mimic the SMAD4 suppression effect of rs12455792 in vivo, we constructed the SMAD4 KD mouse. After induction with Ang II for 4w, the thoracic aorta dilatation and vascular remodeling were more serious than that of wild type group. In conclusion, rs12455792 increased MØ recruitment, M1 type inflammatory response via activated TGF-β signaling, and further promoted vascular remodeling and pathological progress of TAAD.Thoracic aortic aneurysm and dissection (TAAD) is the most fatal macro vascular disease. The mortality of 48h after diagnosis of dissection is up to approximately 50-68%. However, the genetic factors and potential mechanism underlying sporadic TAAD remain largely unknown. Our previous study suggested rs12455792 variant of  Aortic aneurysm and dissection is the most fatal macro vascular disease, accounting for over 152,000 new deaths in the United States per annum . IncreasSMAD4, also known as DPC4 , is located at 18q21.1 [SMAD4 mutations were proven causing thoracic aortopathy and vascular malformation [SMAD4 haploinsufficiency resulted in aortic aneurysm exacerbation in a MFS mouse model [SMAD4 deficiency elevated MØ infiltration and initiated thoracic aortic aneurysm and dissection formation in mouse model [SMAD4 in the pathogenesis of human thoracic aortic disorders is largely unknown. 18q21.1 . It enco 18q21.1 . TGF-β/S 18q21.1 . Generou 18q21.1 . SMAD4 mormation -13 sinceormation . Furtherse model . Zhang ese model . HoweverSMAD4) C>T variant significantly increased TAAD risk and correlated with increased aortic diameter. We further detected the impact of other 4 SMAD4 SNPs on TAAD risk, and the potential impact of rs12455792 on SMAD4 expression and cell function [At first, we screened 20 SNPs  using recent Genome-wide associated studies (GWAS) based TAAD reports -18 and Ofunction . But theSMAD4 low expression generally, we constructed the SMAD4 KD mouse and detected the associated pathological progress of thoracic aortic aneurysm and dissection. Our study elucidated the potential mechanism for differences in susceptibility and prognosis of TAAD between patients with CC, CT or TT genotypes. These novel findings may shed light on the role of rs12455792 and SMAD4 in pathogenesis of TAAD and provide a predictive marker for optimizing clinical trial design and individualizing therapeutic plans.Here we identified 5 significant SNPs on the basis of GWAS-based TAAD reports. Among them, rs12455792 was correlated with aortic diameter of patients. We further explored whether rs12455792 contributed to the MØ recruitment, vascular remodeling and TAAD progression. To mimic the impact of rs12455792 - The demographic characteristics and clinical features of the study participants in screening and validation cohort were listed in P>0.05). Of the 20 SNPs, rs12913975, rs10757278, rs10770612, rs10733710 and rs12455792 were significantly associated with increased TAAD susceptibility, with ORs of 1.489 (P=0.012), 2.006 (P=0.005), 1.767 (P=0.003), 1.412 (P=0.005), 1.585 (P=0.011) adjusting for age and gender, respectively. Among them, the functional SNP rs12455792 C>T variant was correlated with increased aortic diameter of TAAD patients , yielding the most significant results  flag and piggyBac (PB) transposon in exon 8 of SMAD4   . Moreovew to 4w) . As the modeling . Notablyltration .SMAD6), rs10757278 (CDKN2B-AS1), rs10770612 (LINC02398), rs10733710 (TGFBR1) and rs12455792 (SMAD4). Among them, rs12455792 was most associated with thoracic aorta dilatation and was chosen for further analysis. Functional experiments demonstrated that rs12455792 C>T polymorphism activated TGF-β signaling, enhanced MØ recruitment and M1 type inflammatory response, thus facilitated vascular remodeling and pathological progress of TAAD. To the best of our knowledge, this is the first study to demonstrate potential impact of rs12455792 on TGF-β signalling, MØ infiltration and pathogenesis of TAAD.In the present study, we investigated the effects of 20 TAAD risk- related polymorphisms screened by GWAS and OMIM database. There was 5 significant SNP were identified using MALDI-TOF MS in 202 patients and 400 controls, rs12913975  of rs12455792 in  of TAAD . In bioipression . Numerourtopathy -13,15. SSMAD4 low-expression or rs12455792 variant groups. Activation of TGF-β signaling always contribute to uncontrolled cell growth and inflammatory response, leading to vascular disorders such as aneurysm [In detection for TGF-β signaling molecules, there was an enhanced canonical and non-canonical signaling activation in aneurysm . The reganeurysm ,22. TGF-aneurysm . Of noteSMAD4 low-expression , the first affiliated hospital of Soochow University (Jan. 2010 - Dec. 2016). All subjects were chosen from Han Chinese population of eastern China. 202 cases of screening cohort, 227 cases of validation cohort and 400 healthy controls were included in this study. They have signed the informed consent. The inclusion criteria of sporadic TAAD patients was described in previous report . Controlhttp://www.omim.org/) and Han Chinese data from 1000 Genome Project resources (http://www.1000genomes.org). Among them, 10 SNPs were difficult to detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and excluded, by virtue of homologous interference, hairpin structure, short intervals and so on. All the candidate SNPs meet the following criteria: (1) the minor allele frequency (MAF)>0.05; (2) r2>0.80 for each paired SNPs. Functinal SNPs were bioinformatical analyzed using online tools- rsnp (http://rsnp.psych.ac.cn/) and snpinfo (http://snpinfo.niehs.nih.gov/cgi-bin/snpinfo/snpfunc.cgi).We selected 30 SNPs essential in pathogenesis of TAAD by analyzing GWAS-based TAAD reports -18, OMIMCandidate SNPs were genotyped using MALDI-TOF MS as previously described . BrieflySMAD4 knock-down (KD) mouse were designed and constructed by Biogle . All mice were maintained with C57BL/6J genetic background. Experiments regarding to animal model were performed according to institutional guidelines for laboratory animals. To induce aneurysm formation of TAA model, we chose 10-12w old mice and intraperitoneal injected with Angiotensin II (Ang II)  . All experiments were conducted using both male and female mice. Controls were sex-matched littermates. Four weeks later, mice were sacrificed, and thoracic aortas were histochemistry stained and evaluated.The Mice were anesthetized with 5% isoflurane by Small Animal Anesthesia Ventilator System . The thoracic aortic diameter was evaluated using a Vevo2100 cardiovascular ultrasound system with MS400 (30mHz) microprobe . Echocardiographic detection was performed in B mode. Each experiment was repeated at three times from 10 mice per group.2 pieces, then cultured in 10cm dishes with human SMC medium . Three days after primary culture, HASMCs were spread out from aortic tissues. With growth to 80% confluence, cells were digested by 0.25% trypsin-EDTA and passaged. In the experiments, HASMCs were used in passage 3-8 with at least 95% purity.Primary patients human aortic smooth muscle cells (HASMCs) were isolated from thoracic aorta of TAAD patients who received Bentall procedures. Normal HASMCs was obtained from aorta from patients during aortic valve replacement. In brief, the media of fresh thoracic aortas were tore off and cut into 1mmReal-time PCR was performed on an ABI StepOnePlus™ Real-Time PCR System . The primers used for amplification of chemotactic factors and TGF-β molecules were listed in 3) in 100 μl medium was added into each upper insert, and HASMCs in different group (5×104) in 500 μl medium were added into lower chamber. The plate were incubated at 37 °C, 5% CO2 for 24h. To examine MØ chemotaxic-migrating to lower chamber, cells was fixed by methanol for 10 minutes, washed, and stained with DAPI. Finally, the images of chmotaxic-migrated MØ were captured with an inversion fluorescence microscope . MØ was quantified by counting 10 independent visual fields per group via Image Pro Plus 6.0 software . Each assay was performed in triplicate.MØ was derived from THP-1 monocytes with 100ng/ml PMA induction for 48h. Cell chemotaxis were measured using 24-well Transwell® units . One hour before experiment, MØ was stained with Dil , a bioactive fluorescence probe. HASMCs were stained with Dio . Then, MØ .To evaluate the pathological performances of TAAD, murine aortas were separated and fixed in the 4% paraformaldehyde after 4w of treatment. Serial paraffin cross section (5 μm) of murine thoracic aortas were prepared for histological analysis. Aorta sections were stained with eosin and hematoxylin for morphological assessment. Immunostaining of CD86 and polarization markers   was performed to detect MØ infiltration in frozen sections of human thoracic aortas. The images of immunofluorescence were digitally captured on a Carl Zeiss LSM880 laser scanning confocal microscope . CD68+ Cell numbers or average integral optical density of CD206 and TNF-α in the aortic wall were quantitative analyzed from 10 samples per group using Image Pro Plus 6.0 software .t test (for continuous variables) or Chi-square test . The comparisons of cell numbers, mean intergral optical density, lesion areas and so on were using Student's t test (between 2 groups) or One way ANOVA (between more than 2 groups). Hardy–Weinberg equilibrium (HWE) was evaluated using online analytical tools. For analyzing main effect of candidate SNPs, univariate or multivariate logistic regression models were performed to generate ORs and corresponding 95%CIs with adjustment by possible confounders. All statistical tests were two tailed and conducted using Statistical Program for Social Sciences  or R software(http://www.r-project.org/). P value <0.05 was considered statistically significant.Differences of the demographic and clinical features, and frequencies of genotypes in case-control study were tested by Student's Supplementary FiguresSupplementary Tables


Thiodictyon syntrophicum” strain Cad16T, belonging to the Chromatiaceae, fixes around 26% of all bulk inorganic carbon in the chemocline, both during day and night. With this study, we elucidated for the first time the mode of carbon fixation of str. Cad16T under micro-oxic conditions with a combination of long-term monitoring of key physicochemical parameters with CTD, 14C-incorporation experiments and quantitative proteomics using in-situ dialysis bag incubations of str. Cad16T cultures. Regular vertical CTD profiling during the study period in summer 2017 revealed that the chemocline sank from 12 to 14 m which was accompanied by a bloom of cyanobacteria and the subsequent oxygenation of the deeper water column. Sampling was performed both day and night. CO2 assimilation rates were higher during the light period compared to those in the dark, both in the chemocline population and in the incubated cultures. The relative change in the proteome between day and night (663 quantified proteins) comprised only 1% of all proteins encoded in str. Cad16T. Oxidative respiration pathways were upregulated at light, whereas stress-related mechanisms prevailed during the night. These results indicate that low light availability and the co-occurring oxygenation of the chemocline induced mixotrophic growth in str. Cad16T. Our study thereby helps to further understand the consequences micro-oxic conditions for phototrophic sulfur oxidizing bacteria. The complete proteome data have been deposited to the ProteomeXchange database with identifier PXD010641.The microbial ecosystem of the meromictic Lake Cadagno  has been studied intensively in order to understand structure and functioning of the anoxygenic phototrophic sulfur bacteria community living in the chemocline. It has been found that the purple sulfur bacterium “ As an aThiocapsa roseopersicina istrain BBS  and Beggiatoaceae spp.  -hydroxyalkanoicacid synthase subunit PhaE and the phasin PhaP involved in the synthesis of PHB were found. In contrast, among the 17 proteins overexpressed under dark conditions, three enzymes of the dicarboxylate/4-hydroxybutyrate (DC/HB) cycle were detected. However, the absence of the complete set of DC/HB cycle genes in the genome of str. Cad16T indicates that these enzymes are rather involved in the reverse tricarboxylic acid cycle and the last step of the beta-oxidation of fatty acid and PHB granules  reaction center, the membrane-bound protein cascade of cyclic electron transport to generate ATP and reverse electron transport to produce NAD(P)H, and also contains a cbb3 type cytochrome c. The latter may enable microaerobic growth and Fe(III) oxidation of str. Cad16T  to study differences between day and night in the metabolism of PSB str. Cad16T incubated in situ at 12 m in the chemocline of Lake Cadagno and (ii) to monitor the environmental factors longitudinally that could influence the metabolic activity of the chemocline community. In order to quantify differences in metabolic activity, we used a combination of CO2 assimilation analysis using radioactive isotope 14C and label-free quantitation tandem mass spectrometry (LFQ-MS2) based proteomics. Relative light intensity and temperature at the depth of the chemocline were measured constantly and several physicochemical profiles were taken during the period of incubation. The unique micro-oxic in situ conditions measured in summer 2017 were reflected in the carbon assimilation rates and protein profiles obtained and revealed mixotrophy in str. Cad16T.With this study, we aimed at elucidating the microaerobic metabolic mechanisms of PSB in the presence and absence of light, using str. Cad16in situ incubations were performed in Lake Cadagno between 13 July 2017 to 12 September 2017 with dialysis bags attached to a mooring   equipped with temperature, salinity, oxygen and turbidity sensors was used to measure physicochemical profiles from a working platform at the anchor site of the mooring. Profiles from 13 July 2017 were taken as an estimate of chemocline depth (−2 s−1) were used for the surface and below the water, respectively as in Thimijan and Heins (The th 18 m) . From 13ne depth . HOBO UAne depth . Empiricnd Heins . The HOBWe additionally had access to CTD data from a parallel project from Oscar Sepúlveda Steiner and colleagues from EAWAG  where two CTD profiles were taken daily.−1.The analysis was performed with the BD Accuri C6 Plus software . Two lasers (488 and 680 nm), two scatter detectors, and two fluorescence detectors (FL3 = 670 nm and FL4 = 670) were used. To exclude abiotic particles a forward scatter threshold of FSC-H 10,000 was applied. A red fluorescent (FL3) threshold above 1,100 was applied to select for cells emitting autofluorescence due to Chl and BChl.Flow-cytometry (FCM) based cell counting was performed as in Danza et al. . In shorC. okenii and GSB C. clathratiforme to be 70 and 15% of the total daily CO2 photo-assimilation, respectively . Cells were grown up to a concentration of around 3 × 106 cells mL−1. Cell concentrations repeatedly were measured by flow cytometry.Str. Cad162CO3 (40 g L−1) and 0.01 M EDTA at 60°C while stirring. The bags were cleaned with ddH2O, cut into 0.6 m long pieces, closed by a knot on one end and autoclaved for 20 min at 121°C. On site, about 80 mL of bacteria culture (3 × 106 cells mL−1) were filled randomly in each bag, which was closed, attached to a rig and installed in the chemocline within 30 min  were rinsed for 1.5 h in Nan 30 min . In tota14C-radioisotope uptake experiment was performed as in Storelli et al.  were added and thereof, six technical replicates were transferred to 50 mL translucent Duran glass bottles . The bottles were then incubated at a depth of 14 m in a mesh basket on the rig for 4 h during day (1:00–4:00 p.m.) and night (9:00 p.m.−12:00 a.m.), respectively. Chemocline background fixation rates were determined in 50 mL chemocline samples. Filtered chemocline lake water (0.45 μm) was used as negative control. Upon retrieval, the amount of β-activity (14C) assimilated by microbes during the incubation time was measured in the laboratory following standard method that included acidification and bubbling of the samples  running with the WinSpectral v.1.40 software. Raw data was statistically analyzed using t-tests in Excel .Scintillation was done on a 3Merck Spectroquant kit No. 1.01758.0001 and the Merck spectroquant Pharo 100 photospectrometer . Samples were taken at 14 m depth, filtered with 0.45 μm filters, pH was tested with indicator paper  to lie within 6.8–7.0 and triplicate samples were measured.The inorganic dissolved carbon concentration was determined with the CaCOv/v; Thermo Fisher Scientific, Reinach, Switzerland) and frozen at −20°C until further processing.Equal volumes of subsamples were transferred to 50 mL tubes upon retrieval and stored at 4°C in the dark. They were then brought to the lab within 30 min and centrifuged 10,000 g at 4°C for 10 min. The supernatant was discarded and the pellets were re-suspended in 1× PBS pH 7.0 and 1% EDTA-free Protease Inhibitor Cocktail  and sonicated for 15 min at 200 W at 10°C with a Bioruptor ultrasonicator . Samples were then shipped to the Functional Genomic Center Zurich (FGCZ) on dry ice for further processing. Protein concentration was estimated using the Qubit Protein Assay Kit . The samples were then prepared using a commercial iST Kit [PreOmics, Germany -analysis.The cells were thawed, lysed in 5% sodium-deoxycholate (w/w) in 100 mM ammonium-bicarbonate buffer containing 1% EDTA-free Protease Inhibitor Cocktail . Initial solvent composition was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% acetonitrile for channel B, respectively. For each sample 4 μL of peptides were loaded on a commercial Acclaim PepMap Trap Column  followed by a PepMap RSLC C18 Snail Column . The peptides were eluted at a flow rate of 300 nL min−1 by a gradient from 5 to 22% B in 79 min, 32% B in 11 min, and 95% B in 10 min. Samples were acquired in a randomized order. The mass spectrometer was operated in data-dependent mode (DDA), acquiring a full-scan MS spectra  at a resolution of 70,000 at 200 m/z after accumulation to a target value of 3,000,000, followed by HCD (higher-energy collision dissociation) fragmentation on the twelve most intense signals per cycle. HCD spectra were acquired at a resolution of 35,000, using a normalized collision energy of 25 a. u. and a maximum injection time of 120 ms. The automatic gain control (AGC) was set to 50,000 ions. Charge state screening was enabled and singly and unassigned charge states were rejected. Only precursors with intensity above 8,300 were selected for MS2 (2% underfill ratio). Precursor masses previously selected for MS2 measurement were excluded from further selection for 30 s, and the exclusion window was set at 10 ppm. The samples were acquired using internal lock mass calibration on m/z 371.101 and 445.120.MS2 data were processed by MaxQuant v.1.4.1.2 (RRID:SCR_014485), followed by protein identification using the integrated Andromeda search engine. Each file was kept separate in the experimental design to obtain individual quantitative values. Spectra were searched against a forward str. Cad16T database , concatenated to a reversed decoyed fasta database and common protein contaminants . Carbamidomethylation of cysteine was set as fixed modification, while methionine oxidation and N-terminal protein acetylation were set as variable. Enzyme specificity was set to trypsin/P allowing a minimal peptide length of 7 amino acids and a maximum of two missed-cleavages. Precursor and fragment tolerance was set to 10 ppm and 0.05 Da for the initial search, respectively. The maximum false discovery rate (FDR) was set to 0.01 for peptides and 0.05 for proteins. Label free quantification was enabled and for the quantification across multiple run-files the “match between runs” option with a retention time window of 2 min was enabled. The re-quantify option was selected. For protein abundance, the intensity (Intensity) as expressed in the protein groups file was used, corresponding to the sum of the precursor intensities of all identified peptides for the respective protein group. Only quantifiable proteins (defined as protein groups showing two or more razor peptides) were considered for subsequent analyses. Protein expression data were transformed (hyperbolic arcsine transformation) and missing values (zeros) were imputed using the missForest R-package v.1.4  was used to validate MS2 based peptide and protein identifications. Peptide identifications were accepted if they could be established at >42.0% probability to achieve an FDR <0.1% by the Peptide Prophet algorithm with Scaffold  t-test on transformed protein intensities (hyperbolic arcsine transformation). Proteins were called significantly differentially expressed if linear fold-change exceeded Two-fold and the q-value from the t-test was below 0.01.Scaffold v.4.8.4  and eggNOG v.4.5.1  were used to classify the proteins into functional categories. The complete KEGG-dataset for str. Cad16T can be found under KEGG PATHWAYThiodictyon syntrophicum).BlastKOALA v.2.1  was measured. At 12.4 m depth, a mean of 1.3 μmol quanta m−2 s−1 (0.04–7.8) was registered (a profiles in the daily CTD profiles indicated that the chemocline had been sinking from 12 to 13–14.5 m depth (−2 s−1 (0.2–4.0) was measured at 14 m depth from 24 August to 13 September 2017, and no light was measured at 14.4 m depth after 23 August 2017. Temperature was stable around 5°C (4.6–6.1) at both incubation depths with a positive trend over the months (Fluctuations in the relative light intensity were positively correlated with the measured surface radiation and negatively associated with cloud cover . Daily rgistered . Within  m depth . To ensue months .−1 (265.6 μM) throughout the mixolimnion. From 7 to 9.5 m depth, DO-concentrations steadily declined to 6 mg L−1, and then more rapidly to 2 mg L−1 at 10.5 m depth, at both time points measured. At 14 m depth, 0.4 mg L−1 (12.5 μM) and 0.3 mg L−1 (9.4 μM) dissolved oxygen were measured during day and night, respectively. Conductivity increased along the profile from 0.13 in the mixolimnion to 0.22 mS cm−1 hypolimnion, both at day and night. In contrast, a pronounced turbidity peak (28 FTU) was observed at a depth of 13 m at 1:30 p.m. whereas a broader distribution of the FTU values (6–16 FTU) from 13 to 14 m depth, with a maximal peak of 18 FTU at 13 m, was observed at 9:00 p.m. Water samples taken at 1:30 p.m. and 9:00 p.m. from 14 m depth showed a milky and pink coloration, characteristic for a concentrated PSB at high FTU values. The total inorganic dissolved carbon concentrations measured at 14 m depth were 1.26 mM at 1:30 p.m. and 1.46 mM 9:00 p.m.Physicochemical measurements and endpoint-sampling was done on the 12 and 13 September 2017. The weather was cloudless with weak wind. Two CTD profiles at 1:30 p.m. and 9:00 p.m. showed a comparable situation for temperature, dissolved oxygen and conductivity . Water tT cell concentrations were on average 3.1 × 106 cells mL−1 in July, as measured by flow cytometry. The rigged cultures were checked on 23 August 2017 and all dialysis bags were found intact and cells were judged healthy due to the turbid-pinkish appearance. When retrieved for sampling on 12 September 2017, all dialysis bags were still intact, the population was uniformly distributed within the dialysis bags and the cells grew to a mean concentration of 9.3 × 106 cells mL−1. In total, the number of str. Cad16T cells increased three-fold from July to September.Initial str. Cad165 cells mL−1 at 1:30 p.m., whereas it was 2.73 × 105 cells mL−1 at 9:00 p.m., corresponding to 33.4 and 23.5% of the total events count by FCM, respectively. Moreover, FCM revealed that the phototrophic microbial community at 1:30 p.m. consists mainly of C. okenii, Chlorobium sp., and cyanobacteria spp. with 1.31 × 105, 1.49 × 105, and 1.57 × 105 cells mL−1, representing 10.3, 11.8, and 12.4% of the total cell counts, respectively. At 9:00 p.m. the population at 14 m was found to consist of 5.37 × 104 cells mL−1C. okenii, 9.82 × 104 cells mL−1Chlorobium sp. and 1.03 × 105 cells mL−1 with 4.6, 8.5, and 8.9% of all phototrophic cells, respectively. Together C. okenii and Chlorobium sp. corresponded to the 66.1 and 55.7% of the total anoxygenic phototrophic sulfur bacteria cells, during the day and at night, respectively. No significant difference in total cell concentration and internal complexity was measured between the two sampling groups (P = 0.74).The average concentration of phototrophic cells in the lake sample taken at 14 m (12 September 2017) was 4.23 × 10−1, respectively  during the day, and 834 amol C cell−1 h−1  during the night  and Cad16T_dia_12 (“dark”) were identified as outliers in cluster analysis and were excluded from further data analysis. Therefore, the data analysis was made with four samples of the category “light” and five samples of the category “dark.” Overall a total of 1,333 proteins—21% of the total protein coding sequences—with at least two peptides were identified.A total of 11 samples, five samples for light period and six samples for the dark period, were processed for quantitative bottom-up proteomics and protein quantification was performed using the MaxQuant package. We used corrected Peptide identifications were accepted if they could be established at >42.0% probability to achieve a false discovery rate (FDR) <0.1% with Scaffold delta-mass correction, resulting in 12,576 spectra included. Protein identifications were accepted if they could be established at >54.0% probability to achieve a FDR <1.0% and contained at least two identified peptides. The number of quantified proteins per condition was similar, with an average of 374 for the day and 354 for night period, respectively. Between 102 and 663 proteins were quantified in each biological replicate . Consequently, 684 proteins were quantified over all samples. Thereof, 21 contaminants were excluded. The remaining 663 proteins were classified with blastKOALA and EggNOG, with 627 annotated proteins for (99%) COG and 460 annotated proteins (69.4%) for blastKOALA, respectively.0F1 ATPase subunits  and chaperons . Since cell growth depends on protein synthesis, we found 36 ribosomal subunits as well as elongation factor Tu (AUB80476.1) to be equally abundant. The cells always contained the established components of the dissimilatory sulfide oxidation pathway such as ATP-sulfurylase Sat (AUB82369.1), the adenylylsulfate reductase AprAB , and the dissimilatory sulfite reductase (Dsr) complex (AUB83448.1–AUB83455.1). A Sqr sulfide:quinone reductase and a glutathione amide reductase GarA homolog to A. vinosum putatively involved in intracellular sulfur shuttling  and PuhA (AUB85431.1) subunits forming the reaction center II and six different PufAB antenna proteins  were expressed.As expected, many of the proteins with unchanged abundance belonged to the functional categories energy conversion, genetic information processing, carbohydrate and amino metabolism and protein modification . Among tcbb3 cytochrome c oxidase subunits. PSB use the ATP and NAD(P)H derived from photosynthesis to fix CO2 through the CBB cycle. For str. Cad16T a complete CBB cycle with the key enzymes CbbM/RbcL RuBisCO form II (AUB81831.1) and phosphoribulokinase PrkB (AUB79979.1) were present. The fixed carbon enters the central carbon metabolism as 3-phospho-D-glycerate. In both growth conditions, str. Cad16T contains enzymes for glycolysis and gluconeogenesis, as well as pyruvate oxidation, the glyoxylate cycle and the citrate cycle (TCA) in unvaried abundance. Additionally, the presence of malic enzyme  may allow the entry of malate into the central carbon pathway via pyruvate, as shown for A. vinosum. In both conditions examined, the PHB synthase subunits PhaC and PhaE are expressed (AUB80707.1 and AUB84676.1). Also enzymes necessary for amino acid biosynthesis and Co-factor and vitamin synthesis were expressed under both groups analyzed.Additionally we found enzymes for BChl synthesis, terpenoid backbone biosynthesis, and carotenoid synthesis. Noteworthy, elements of the reduction pathways driven by photosynthesis are shared with oxidative phosphorylation in PSB. We found in total 23 protein subunits involved in substrate respiration including the NADH dehydrogenase subunits NuoCDEFG and HoxFU, the cytochrome reductase CytB and Cyt1, seven F-type ATPase subunits and two t-test on transformed protein intensities (q.mod <0.01). The expression dataset was alternatively analyzed using correlation-adjusted t-scores (CAT scores) in order to additionally address the correlative structure of the dataset as only 4.5% of the proteins are differentially expressed. Sixty proteins were found differentially expressed  at a local false discovery rate of lfdr <0.05  corrected dr <0.05 , 3.T. Thereof, all protein-coding sequences were annotated with eggNOG . Additionally, chaperone-type proteins DnaJ and proteolytic ClpX were also over-expressed. During the night period 39 proteins were shown to be more abundant and thereof 35 entries (89%) were successfully annotated to COG categories with eggNOG (T further expressed proteins associated to cell division (FtsZ), cell wall formation CpxP, Lysine and branched amino acid synthesis and nucleotide metabolism . Elements of two secretion pathways were identified, a putative polar amino acid transport system and type II general secretion system. Additional three proteins detected to be more abundant in the dark are involved in stress response. The BolA-family transcriptional regulators, is a general stress-responsive regulator. Rubrerythrin may provide oxidative stress protection via catalytic reduction of intracellular hydrogen peroxide and a ATP-dependent serine protease mediates the degradation of proteins and transitory regulatory proteins, and ensures cell homeostasis.During the day period 21 proteins were found relatively more abundant for str. Cad16h eggNOG . In presh eggNOG . Analysih eggNOG . Str. CaT through a combination of longitudinal monitoring of physicochemical conditions, FCM counts of the microbial population, 14C-radioisotope uptake and quantitative proteomics of cultures incubated in situ in the Lake Cadagno chemocline.We compared light to dark carbon fixation metabolism under micro-oxic conditions of PSB str. Cad162 L−1 (19 μM) below a depth of 12 m. The chemocline also retained some of the produced oxygen through the night  sunk from around 12–14 m depth. Taken together, a dense microbial population inflicted increased self-shading below a depth of around 14 m. For this reason, we decided to lower the dialysis bags from 12 to 14 m at the 23 August 2017. Subsequently, the average light availability measured at the chemocline was 10× lower than previously recorded at 12 m  and at night  previously obtained for Lake Cadagno, respectively    and was as such 8× longer than observed in vitro at room temperature  seems to be the main carbon storage regulator where it was detected under both conditions. Glycolysis under mixotrophic conditions might thereby be regulated through mRNA transcription and stability as in A. vinosum  and CysP (AUB80377.1) and oxidation to sulfite via the intermediate S-sulfocysteine by cysteine synthase B CysM (AUB82938.1) and possibly monothiol glutaredoxin of the Grx4 family (AUB83488.1) as suggested by Dahl (Additionally, str. Cad16 by Dahl . HoweverT is metabolically flexible in situ and growths phototrophically as well as chemotrophically in the light as shown in this study. In dark conditions, low levels of oxygen may enable respiration of different small organic molecules. In summary, the 60 proteins found differentially expressed between night and day period represent only about 1% of all protein coding sequences and about 5% of the identified proteins. Therefore, their impact on metabolic pathways of str. Cad16T is unclear and has to be further examined. In wider perspective, the role of PSBs in large water bodies has to be studied in more detail as we demonstrated that that low levels of oxygen can be used for respiration. As for other stratified lakes it was found that after a bloom of PSB anoxia can prevail (Bush et al., T to mixotrophy.PSB str. Cad16SL, NS, FD, JP, and MT conceived the study. SL, NS, FD, and SR installed the mooring and performed field measurement and sampling. NS and FD prepared scintillation samples. FD and NS did flow cytometry cell enumeration. SL extracted total protein and performed scintillation measurements. SL, MW, JP, and MT analyzed physicochemical, proteomic, and scintillation data. SL, NS, FD, and MT prepared the manuscript. All authors contributed to writing and agreed on the manuscript before review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The objective of this study was to evaluate the effects of glycolic acid (GA) (with pH 1.2 and 5) and ethylenediaminetetraacetic acid (EDTA) on the chemical and mechanical properties of dentin to investigate the potential use of GA as final irrigant in the root canal therapy. Specifically, changes in microhardness, smear layer removal, erosion, mineral content distribution, apatite/collagen ratio and flexural strength of mineralized dentin treated with GA were assessed. Saline solution was used as a negative control. Knoop microhardness (KHN) was measured on the root canal lumen of root segments. Dentin beams were used for 3-point flexural strength (σ) test. Scanning electron microscopy (SEM) images of root sections were obtained for evaluation of smear layer removal and dentin erosion on root segments and energy dispersive X-ray spectroscopy (EDS) was used for mineral content distribution. The apatite/collagen ratio (A/C) in dentin powder were examined by Fourier transform infrared (FTIR) spectroscopy. KHN, σ and A/C results were statistically analyzed with ANOVA and Tukey tests (α = 0.05). Smear layer and dentin erosion scores were analyzed with Kruskal-Wallis and Dunn tests (α = 0.05). Root dentin treated with EDTA and GA presented similar KHN regardless of the pH (p > 0.05). However, KHN was significantly reduced in EDTA and GA groups when compared to control group (p<0.001). GA showed the same ability to remove the smear layer and to cause dentin erosion as EDTA. EDS results showed that the GA and EDTA solutions did not alter the dentin mineral content distribution. The apatite/collagen ratio reduced with all irrigant solution and was the lowest with GA pH 5 (p<0.001), while σ was not significantly affected by the experimental solutions (p = 0.559). It can be concluded that GA has similar ability to remove the smear layer than EDTA. GA does not affect negatively the chemical/mechanical properties and it does not increase dentin erosion. The use of GA with low pH seems to promote less change in collagen/apatite ratio, but further studies are needed to establish an ideal clinical protocol. Therefore, this study supports the potential use of GA as an alternative final irrigation solution for root canal preparation. However, this step results in the deposition of a smear layer on the root canal walls. The presence of the smear layer keeps bacteria and their byproducts in the root dentin, hinders the penetration of intracanal disinfectants and cements into the dentinal tubules, and decreases the sealing capacity of root canal sealers2. Therefore, smear layer removal during chemical and mechanical preparation of root canals has been proposed by the use of final irrigants. Different final irrigant solutions such as citric acid, MTAD , etidronate and ethylenediaminetetraacetic acid (EDTA) are used for removal of the smear layer, being the EDTA still the most widely well-known in endodontics3.The main purpose of the root canal preparation is cleaning and disinfection by using endodontic files and root canal irrigants3, it has unfavorable features such as denaturation of collagen fibrils4 and peritubular and intertubular dentin erosion when used for more than three minutes3. These mineral changes in root canal dentin could have an effect on the adhesive properties of the dentin surface and reduce root canal sealing5. Furthermore, extrusion of EDTA solution beyond the apical foramen should be avoided because of its cytotoxicity6. Another important factor is that EDTA is produced on an industrial scale from ethylenediamine, formaldehyde, and sodium cyanide leading to the formation of contaminants that are considered some of the important pollutants dispensed in water7. Thus, EDTA plays a part in aquatic toxicity and may cause chronic effects, including the disequilibrium of body calcium in other organisms8. The investigation of alternative final irrigants that are biocompatible, effective in removing the smear layer without causing damage to the root dentin structure and properties is needed to ensure success of the root canal therapy.However, an effective final irrigant solution must act only on superficial dentin removing the smear layer without causing damage to the internal portion of the root dentin. Although EDTA has good capacity of smear layer removal9. GA is found in concentrations of 2 to 10% in dermocosmetics and used in higher concentrations for chemical peeling (from 20 to 70%)10. In dentistry, recent studies showed GA to be suitable for enamel and dentin etching in restorative procedures and as efficient as EDTA in removing smear layer from root canals walls12. In addition, cytotoxic results indicated that GA has less toxic effects on fibroblasts when compared to EDTA13. Furthermore, GA showed pH stability for 90 days when stored at 4, 25 and 37 °C, which may facilitate its clinical use in endodontics, and did not promote alterations in dentin flexural strength14. Due to its positive characteristics, and considering the need for biologically compatible endodontic materials and substances, GA may be a suitable agent to remove the smear layer from the root canal walls with minimal negative biological effects.The glycolic acid (GA) is an alpha hydroxy acid (AHA) extracted from sugar cane and other sweet vegetables. It is uncolored, odorless, has only two carbons in its molecular structure, and can be easily dissolved in water. GA is commonly used in dermatology for applications that range from skin moisturizing to deep chemical peeling, a common esthetic procedure. As the smallest AHA, GA has great penetration potential and its absorption on skin and mineral surfaces is faster than other AHA14. However, its application in endodontics relies on the establishment of a clinical protocol that details the concentration, pH, and irrigation time. The objective of the present in vitro study was to evaluate the effects of irrigation with 17% GA used with different pH (1.2 or 5.0) compared to 17% EDTA  on their capacity of smear layer removal from the root canals, changes in erosion, microhardness, mineral content distribution, apatite/collagen ratio and flexural strength of mineralized dentin. The tested null hypotheses were: (1) the 17% EDTA and 17% GA have similar capacity to remove the smear layer in all root canal thirds, regardless of the pH values of GA (1.2 or 5.0); and (2) there is no significant change on chemical and mechanical properties of root dentin when using 17% EDTA or 17% GA, regardless of the pH values.Previously published studies from our research group suggest the use of GA as a final irrigant in the root canal therapy, since it demonstrated ability to remove the smear layer without negatively affecting the dentin properties when used at low pH (around 2)Tables The mean and standard deviation of the KHN values, flexural strength and apatite/collagen ratio of the experimental groups are presented in Table Figure Table The erosion scores for the experimental groups are summarized in Table Figure In order to evaluate whether the irrigation with GA at acidic or neutral pH is effective in removing the smear layer and its potential effects on dentin surface integrity and chemical/mechanical properties when compared to EDTA, the pH values of 1.2 and 5.0 for GA were tested in the present study. The ability to remove the smear layer of EDTA was similar to GA at both pH values in each root third evaluated, so the first null hypothesis was accepted. Considering the chemical and mechanical properties evaluated in the present study, EDTA and GA irrigants caused significant reduction on the dentin microhardness, showed similar potential to promote erosion of root dentin walls, and no clear differences were noted in mineral composition on dentin surface. However, when comparing the collagen/apatite ratio, the most significant change was observed for GA at pH 5. Therefore, the second null hypothesis was rejected.15. EDTA has the ability to combine with calcified components of dentin through a chelating mechanism resulting in demineralization and softening of tissue15. GA also showed significant reduction in microhardness than the control group, with similar changes regardless of the solution pH. GA has shown potential to demineralize dentin and remove smear layer in previous studies. When used as an etchant for smear layer removal prior to application of etch-and-rinse adhesive system, GA showed decrease of dentin microhardness comparable to phosphoric acid and ability to promote adequate bond strength11. In the same previously published study, GA was less aggressive in promoting enamel demineralization than PA11. Overall, irrigant solutions that present ability to remove the smear layer facilitate the access and action of endodontic instruments until the apical foramen, which is critical in narrow and calcified root canals15, but may cause reduction of dentin microhardness. Nevertheless, this reduction caused by the final irrigants does not seem to be detrimental to the fracture resistance of endodontically treated teeth16 when these solutions are used for a short period inside the root canal. For example, the resistance to fracture of endodontically treated roots significantly decreased when 17% EDTA persisted for 10 minutes or more inside the root canal17. Different irrigation time with GA should be explored in future studies and its clinical application should consider short irrigation time as proposed for EDTA.The decrease of dentin microhardness values promoted by EDTA compared to control  is in agreement with other studieset al.14. On the other hand, when comparing EDTA and GA with saline solution (control group), there were significant changes in the apatite/collagen ratio. These findings are agreement with others and support the hypothesis that the use of NaOCl prior EDTA and GA may promote collagen degradation and/or extraction and lower the apatite/collagen ratio18. Although is expected that this collagen degradation would also affect the mechanical property, the low concentration of NaOCl used in the present study (2.5%) might have resulted in less collagen breakdown, not enough to significantly reduce the flexural strength when compared to the control group, as previously suggested19. When comparing the FTIR spectra among the groups, the most significant changes are observed for the GA pH 5  and teeth collection were performed in accordance with relevant guidelines and regulations as per the ethical and research board committee instructions. All teeth were obtained from the teeth biorepository of the School of Dentistry of the University of Passo Fundo under informed consent of the patients. After the extraction, the teeth were stored at 4 °C in 0.9% NaCl solution and were used within 1-month period.For the treatment of dentin samples, the GA  was used at pH 1.2 and pH 5.0. To obtain GA solution at higher pH (5.0), a neutralizing agent, aminomethyl propanol, was used. As a positive control, 17% EDTA  was selected as the conventional final endodontic irrigant. Saline solution was used to treat samples in the negative control group.The dentin surface demineralization was indirectly assessed by the microhardness test. The smear layer removal capacity and dentin erosion were evaluated by analyzing images obtained with Scanning Electron Microscopy (SEM). The analysis of mineral dentin distribution was assessed using Energy-Dispersive X-ray Spectroscopy (EDS). The apatite/collagen ratio and flexural strength of mineralized dentin were evaluated by Fourier-transform infrared spectroscopy (FTIR) and flexural strength test, respectively.Twenty canine human teeth were sectioned transversely below the cementum-enamel junction producing 15-mm-long root segments. Thereafter, the roots were sectioned longitudinally in two, creating 40 specimens from the buccal and lingual segments. Each root specimen was embedded in self-curing acrylic resin, leaving the root canal dentin exposed on the surface. The dentin surfaces were then flattened using silicon carbide paper  under constant water irrigation, and polished using a suspension of 0.1-mm alumina on a rotating felt disc.In the negative control group (10 root segments), dentin surfaces were irrigated only with a saline solution. Dentin specimens in the other three groups (30 root segments) were irrigated with 2 ml of 2.5% sodium hypochlorite (NaOCl) for 1 min, followed by irrigation with 5 ml of saline solution and randomly divided according to the final irrigant (n = 10): 17% EDTA, 17% GA at pH 1.2, and 17% GA at pH 5. All final irrigations were performed with 2 ml of solution for 1 min. Finally, all samples were rinsed with 5 ml of saline solution.15. The values were averaged to generate one hardness value per specimen. Microhardness data showed normal distribution using the Shapiro-Wilk Normality and Equal Variance tests . Data were statistically analyzed using one-way analysis of variance (ANOVA) and Tukey’s post-hoc test for multiple comparison (α = 0.05).Dentin microhardness was measured using a Knoop indenter with 40× magnification  under a 25-g load and a dwell time of 15 s. Three indentations were performed on each specimen along lines parallel to the edge of the root canal in the apical direction. The first indentation was performed at 1.000 µm distance from the entrance of the root canal, and the other two indentations were performed at a distance of 200 µm from each other21.The chemical-mechanical preparation of 40 mandibular first premolar roots was performed using nickel-titanium rotatory instruments  following the manufacturer’s recommendations. The sequence of files used was X1, X2, and X3 at a speed of 300 rpm and torque of 2 N until the working length was achieved. At each change of instruments, the root canal was irrigated with 2 ml of 2.5% NaOCl. Thereafter, the 40 roots were randomly divided into four groups and the final irrigation was performed with 2 ml of the experimental solutions as previously mentioned. The solutions were applied inside the root canal as follows: 29-gauge needles  were inserted up to 3-mm short of the working length and the test solution was introduced until it fully filled the root canal. The test solution was inserted using up-down movement and remained within the root canal for one minute. Final irrigation was made with 5 ml of saline solution, and the canals were dried using absorbent paper cones21. Two blinded independent investigators analyzed the presence or absence of the smear layer and verified root dentin erosion on the respective areas. The Kappa coefficient test showed a high agreement between the investigators regarding the interpretation of erosion scores (k = 0.876).Thereafter, the 40 roots were divided into two halves resulting in 80 halves (20 in each group). The specimens were dehydrated using increasing concentrations of ethanol . Each half root was mounted on metal stumps, covered with palladium gold , and examined using SEM . Images were obtained to observe the morphology of the surface of the canal wall at 2000× magnification and 20 kV along the coronal (10–12 mm from the apex), middle (6–7 mm from the apex), and apical (1–2 mm apex) thirds of each specimen27: Score 1: no smear layer, dentinal tubules wide open; Score 2: small quantity of smear layer, some dentinal tubules open; Score 3: homogenous smear layer covering the root canal wall, only few dentinal tubules open; Score 4: entire root canal wall covered by a homogenous smear layer, no open dentinal tubules; and Score 5: heavy, non-homogenous smear layer covering completely the root canal wall. The images were classified according to the following specification for dentin erosion23: Score 0, smear layer covering almost all dentin surface, with few or no open tubules; Score 1, no erosion: all tubules without alteration in appearance and dimension; Score 2, moderate erosion: the peritubular dentin was eroded; and Score 3, strong erosion, the intertubular dentin was destroyed and the tubules were connected to each other.For the smear layer analysis the following criteria was usedSmear layer and dentin erosion data were determined to be non-parametric based on the Shapiro-Wilk Normality test (p<0.001). Therefore, data were analyzed with Kruskal-Wallis test and Dunn´s Method (α = 0.05), and results were presented using medians and quartiles (to represent data dispersion).The same specimens prepared in the previous test  were used for the evaluation of surface mineral content. The entire area of the dentin matrix was visualized using SEM at a standard magnification of 200× (650×420 µm), and analyzed using EDS to determine the atomic ratio (at. %) of calcium (Ca), phosphorus (P), sodium (Na), chlorine (Cl), and magnesium (Mg). Changes in the mineral levels were recorded and differences among the groups were qualitatively analyzed.13.Twenty non-carious extracted human mandibular third molar teeth were selected for this test. Enamel and cementum were removed from the teeth using a diamond bur #2215 in a high-speed handpiece under refrigeration. Dentin powder (90 µm) was obtained with a high-speed handpiece and diamond bur #3145 F without refrigeration. The powder went through a 90 µm sieve, so the dentin grains were equal to or smaller than this size. The dentin powder was divided in four groups of 9 mg each. One group remained untreated (NT), and the other three groups were rinsed with the GA and EDTA solutions. For irrigation, dentin powder was placed over a filter paper and fixed on a glass Becker. Irrigation was performed with 5 ml of the experimental solutions using a 25×4 mm needle for 1 min. After irrigation, the powder was washed three times with 5 ml of deionized water to remove residue of the experimental solutions and air-dried at 37 °C−1 resolution, using 48 scans . The IR spectrum is produced as a result of the absorption of electromagnetic radiation at frequencies that correlate to the vibration of chemical bonds within a molecule. Thereby, when IR radiation is absorbed by an organic molecule, it is converted into molecular vibration energy, and the spectrum portray the vibrational motion and usually appears in the form of bands28. The range for the characterization of organic compounds is mid-IR (4000 to 400 cm−1)29.FTIR spectra of the dentin powder were collected for each group (n = 3). Spectra were obtained between 650 and 4000 cmApatite/collagen ratios derived from FTIR spectroscopy showed normal distribution using the Shapiro-Wilk Normality test (p = 0.882) and Equal Variance test (p = 0.419). Data were statistically analyzed using one-way ANOVA and Tukey’s post-hoc test for multiple comparison (α = 0.05).13. Two beams were obtained from each tooth, totaling 40 beams, which were randomly divided into four groups (n = 10) following the same irrigation protocols previously mentioned.Twenty human mandibular third molars were used for the flexural strength test. Mid-coronal dentin disks were cut perpendicular to the longitudinal axis of each tooth with a slow-speed diamond saw under constant water-cooling. The disks were trimmed to a final rectangular-shaped beam 2, where P = load at fracture (N), L = length of support span (mm), b = beam width (mm)13.Flexural tests were conducted using a three-point flexure device with a 3-mm support span. The specimens were tested at a crosshead speed of 0.5 mm/min using a universal testing machine . Flexural strength (in MPa) was calculated using the following equation: 3PL/2bdFlexural strength data was normally distributed as observed in the Shapiro-Wilk Normality test (p = 0.05) and Equal Variance test (p = 0.405). Data were statistically analyzed using one-way ANOVA and Tukey’s post-hoc test for multiple comparison (α = 0.05).