P < 0.001): median concentrations (adjusted for specific gravity) for 1-hydroxypyrene, 2-hydroxyphenanthrene, 3-hydroxybenzo[a]pyrene, and 3-hydroxybenzo[a]anthracene were 0.56 µg/L, 0.78 µg/L, 4.75 ng/L, and 6.28 ng/L, respectively. Compared with controls, chimney sweeps had increased homocysteine, cholesterol, and HDL . In chimney sweeps, PAH metabolites correlated positively with the percentage of soot sweeping (P < 0.001). 2-hydroxyphenanthrene, 3-hydroxybenzo[a]pyrene, and 3-hydroxybenzo[a]anthracene were positively associated with diastolic BP . PAH exposure among chimney sweeps resulted in elevated levels of markers for CVD risk. These findings stress the need to reduce occupational exposure to PAH.Occupational exposure to soot, rich in polycyclic aromatic hydrocarbons (PAH), has been associated with increased risk of cardiovascular disease (CVD). However, our knowledge about PAH exposure and early markers of CVD remains limited. In this cross-sectional study of 151 chimney sweeps and 152 controls, we investigated occupational exposure to PAH and early markers of CVD. Blood pressure (BP) (chimney sweeps only), urinary PAH metabolites and serum biomarkers were measured . Chimney sweeps had up to 7 times higher concentrations of PAH metabolites in urine than controls ( In addition to the well-documented carcinogenic effects of PAH2, exposure to PAH in the general environment and at work may contribute to development of cardiovascular disease (CVD), including coronary heart disease, peripheral arterial disease, stroke, and myocardial infarction7. For example, PAH metabolites in urine, i.e. 2-OH-PH, correlated with increased CVD events in the U.S. general population 5. Moreover, a previous study of asphalt pavers reported an increased risk for fatal ischemic heart disease in relation to occupational exposure to benzo(a)pyrene 3. The underlying mechanisms are not understood, but oxidative stress and systemic inflammation have been suggested to play a role in PAH-induced CVD9. In this context, investigating early markers of CVD in relationship to PAH exposure will add important knowledge to understanding the mechanisms behind PAH-related CVD.Polycyclic aromatic hydrocarbons (PAH) are composed of two or more fused benzene rings. These omnipresent pollutants form naturally or by incomplete combustion of organic matter10. Occupational exposure has been of interest because workers in several professions, such as chimney sweeping, asphalt paving, and production of aluminum and coke, could receive high PAH exposures by inhalation, dermal contact, or even contaminated food at their places of work10. A previous study estimated that between 1990 and 1993, about one million workers in the European Union, including 18,000 from Sweden, were exposed to PAH at concentrations higher than background levels11.Humans can be exposed to PAH from many sources, such as ambient air, water, food, medication, and tobacco smoking, as well as from work13, particularly during soot sweeping, the removal of soot from chimneys and boilers in private homes and industrial buildings that predominantly use wood, petroleum oil, and coal as fuel. Chimney sweeps can also be exposed to other hazardous substances from work such as arsenic, lead, nickel, asbestos, carbon particles, sulfur dioxide, carbon monoxide, detergents, and degreasing materials15.Chimney sweeps are primarily exposed to PAH from soot19, chimney sweeps have increased incidence of myocardial infarction20, and excess mortality from CVD21. Nevertheless, some factors have likely altered the chimney sweeps’ exposure to PAH over time, including changes in the type of fuel used for heat production, along with increasing use of protective equipment by chimney sweeps22. Therefore, characterization of the exposure to PAH and potential risk of developing CVD, by linking individual data on exposure to PAH to individual data on early markers of CVD, will provide important insight into the effects of PAH on risk of CVD among chimney sweeps working today.Epidemiological studies have shown that, in addition to different types of cancerOur study aimed to: 1) evaluate occupational exposure to PAH among chimney sweeps by measuring PAH metabolites in urine and identifying the work tasks that might be associated with increased exposure, and 2) estimate the risk of CVD by measuring blood pressure and early serum biomarkers for risk of CVD.P < 0.05). Sampling season differed between groups as more controls (24%) were recruited during the summer than chimney sweeps (11%).The general characteristics of the chimney sweeps and controls are presented in Tables P = 0.594). However, it is worth mentioning that among chimney sweeps there were 4 cases of myocardial infarction but none among controls.CVD-related morbidities  in chimney sweeps did not differ from controls (P < 0.001). The use of fuel before and after 2002 changed, as the use of petroleum oil significantly decreased and the use of pellets and wood (for fireplaces) increased . The median values for chimney sweeps were 7-fold higher for 1-OH-PYR, 4-fold higher for 2-OH-PH, and 3-fold higher for 3-OH-BaP and 3-OH-BaA, compared with controls (specific gravity-adjusted concentrations). Comparable figures were seen for creatinine-adjusted concentrations , but not in the control group ; however, the correlations were weak for soot sweeping in industrial buildings . Non-soot sweeping tasks showed negative associations with PAH metabolites. We further evaluated the potential association between PAH metabolites and the use of gloves and masks during soot sweeping in the past 12 months but no significant associations were seen (Supplementary Table PAH metabolite concentrations in urine for chimney sweeps and controls are shown in Table P ≤ 0.001), HDL (P ≤ 0.004) and cholesterol (P ≤ 0.010) compared with controls (Table P ≥ 0.061). Chimney sweeps had lower non-significant CRP levels compared with controls.In general linear models, chimney sweeps had higher levels of homocysteine ( P ≤ 0.00 and cholP ≤ 0.044). However, the association remained significant only for 2-OH-PH in the fully adjusted model  than age  that can potentially lead to CVDet al. , Göen et al.  and Pavanello et al. 32. Workers at aluminum plants, coke ovens, and foundries, as well as workers laying asphalt pavement, experience considerable exposure to PAH. The median concentrations for 1-OH-PYR in urine ranged from 0.3 µg/L in foundry workers to 7.0 µg/L in coke oven workers33.Exposure to PAH is considerable among chimney sweeps in other countries and in other professions, suggesting that many workers are at risk for CVD-related changes: the 1-OH-PYR concentrations in our study  were lower than those reported by Letzel 36. This is of concern as benzo[a]pyrene is an established carcinogen in humans (IARC 2010) and our findings suggest that current chimney sweeps may be at risk for cancer as well as CVD.Compared with the majority of other professions where studies have produced data on 3-OH-BaP levels in urine, the chimney sweeps in our study showed relatively high 3-OH-BaP : median concentrations of 3-OH-BaP in urine were 1.1 ng/g creatinine in fireproof material production, 0.5 ng/g creatinine in coke oven plants, 1.3 ng/g creatinine in graphite electrode plants, 1.2 ng/g creatinine in converter infeed, below LOQ in steel plants, and 14 ng/g creatinine in fireproof stone plants41. A likely explanation would be that chimney sweeps reported the sampling’s week figures for the percentage of time for each task as a projection for the past 12-month period.The strong positive correlations between PAH metabolites and soot sweeping in the past 12 months were unexpected as PAH metabolites generally have short half-lives (3.9–35 h) and therefore only reflect recent exposure to the parent compounds42. In line with our finding, urinary 2-OH-PH concentrations were associated with increased incidence of hypertension in a population-based study on 4765 adults43. Working years with soot sweeping was highly correlated with age and it was therefore difficult to evaluate the effect of working years on blood pressure. However, when comparing the effect of age and working years on blood pressure separately, we could not see a clear influence of working years of soot sweeping. There are no clear mechanisms linking PAH exposure with changes in blood pressure, but it has been proposed that PAH can affect the autonomic nervous system44.Among chimney sweeps, we found some dose-response relationships with markers of CVD. PAH metabolites  consistently associated with diastolic blood pressure when adjusted for age, BMI, and smoking (or excluding smokers). 2-OH-PH showed the strongest association and the highest effect estimates. For every 1 µg/L increase in urinary 2-OH-PH, diastolic blood pressure increased by 1.7 mm Hg. That dramatic increase implies higher risk for developing future CVD among chimney sweeps, as observational studies with more than 1 million total participants have shown that every 10 mm Hg increment in diastolic blood pressure was associated with a 2-fold increase in mortality from CVD52. Increased homocysteine levels can also be driven by several factors, e.g. vitamin B deficiency, atherosclerotic events, medication, and diabetes48. We did not have data on vitamin B deficiency, but we evaluated the differences in homocysteine concentrations after excluding participants with diabetes and those using regular medication. Still, chimney sweeps had higher homocysteine levels in serum than controls. The observation that the chimney sweep group had higher levels of HDL compared with controls was intriguing, but when we restricted the analysis to chimney sweeps, we only found negative associations between HDL and PAH metabolites (model 1). This suggests that the higher HDL among chimney sweeps compared with controls is not linked to soot exposure. Studies in the general population have shown associations between PAH metabolites and increasing levels of GGT and CRP55 and a study on 489 coke oven workers showed a positive association between PAH metabolites and plasma CRP56. However, in our study the levels of other serum biomarkers i.e., GGT, CRP, LDL and TG were not different between chimney sweeps and controls.Also, PAH metabolites were positively associated with homocysteine, although significance was only reached in the non-smokers for one of the metabolites, suggesting that PAH impairs one-carbon metabolism. No dose-response effect was found for cholesterol with PAH metabolites despite the fact that the chimney sweeps had elevated serum concentrations of homocysteine and cholesterol compared with controls. One possibility for this finding is that other exposures, e.g. particles or degreasing chemicals, could have caused the elevation of cholesterol. Nevertheless, higher homocysteine and cholesterol is of major concern since there is strong evidence linking these biomarkers with adverse cardiovascular eventsIn conclusion, currently working chimney sweeps showed clear occupational exposure to PAH. Chimney sweeps had a higher risk for developing CVD, indicated by elevated homocysteine and cholesterol levels in serum. Our study shows that PAH metabolites were associated with increased diastolic blood pressure, suggesting exposure to PAH as a driving factor for developing CVD. These findings warrant further investigation of the cardiovascular profile of workers exposed to PAH. They also suggest the need for incentives to increase the proper use of protective measures during work and to develop different work routines.We enrolled 151 male chimney sweeps and 152 male controls from southern Sweden. Chimney sweeps were recruited from 29 different companies. Every chimney sweep performs multiple tasks: soot sweeping, white sweeping , and office work. Their work usually starts in the morning when chimney sweeps drive to their client’s premises, carried out the sweeping task, and then returned to their company to shower and do task-related office work. The controls were employees at ten companies  and four municipalities, and were not occupationally exposed to PAH.Between 2013 and 2015, we contacted, in coordination with the chimney sweeps’ trade union, managers of 39 chimney-sweeping companies to ask for participation in the study; 34 of them agreed, resulting in 87% company response rate. Five of the 34 companies were in the end not included in the study: one company had only female employees  and four companies were located far away and for logistic reasons not included in the study, leaving 29 companies. We instructed the manager at each company to invite all employees that currently were working.Thereafter we booked a visit at the company for a general medical examination and sampling of blood and urine of chimney sweeps that had agreed to participate. We sent informed consent forms and questionnaires to each participant in advance. The visits at the companies took place after the chimney sweeps had finished their shift on Wednesdays or Thursdays. On the day of visit, a trained nurse checked that the questionnaires were completed, sampled blood (non-fasting sample) and urine, and measured blood pressure, height, and weight of the participants.For the nonsmoking control group, we directly contacted the companies’ managers without involving the trade union. As a result, 7 companies were positive for participation out of 12, ending up with 58% company response rate for the nonsmoking controls. We instructed the manager at each company to invite all employees that currently were working. The controls were recruited during two periods: 127 nonsmoking controls were recruited in 2010–11 and 25 smokers in 2015. The smoking controls  were introduced into the study to match the smoking status of the chimney sweeps. The controls also filled in a questionnaire, donated blood and urine, and had their blood pressure, height, and weight measured.All participants gave informed consent before participation in the study. The study was approved by the Regional Ethics Committee at Lund University, Lund, Sweden and we confirm that all experiments were performed in accordance with relevant guidelines and regulations.The questionnaire for chimney sweeps and controls contained questions about age, weight, height, education , personal history of disease and symptoms (yes/no) , family history of CVD and cancer (yes/no), prescribed medicines (yes/no), over-the-counter medicines (yes/no), dietary habits regarding consumption of vegetables, fruits, and fish , physical activity , tobacco smoking , passive smoking (yes/no), use of snus (yes/no), current residency , employment history, and exposure from hobby .In addition, the questionnaire given to the chimney sweeps enquired about the details of chimney sweeping tasks . The questions about the aforementioned tasks concerned 6 periods . For instance, “what was the extent of soot sweeping in private homes on a scale from 0 to 100% in the past 12 months, considering that the total percentage for all tasks must be 100%”. Chimney sweeps were also asked, on a 0–100% scale, to what extent that their clients used different types of fuels  in private homes or industrial buildings during 3 different periods . Over the same 3 periods , the sweeps were asked to estimate how often they used gloves, masks, long-sleeved shirts, long pants, and protective overall suits, on a scale from 0–100% for each exposure-related task . However, questions about the use of vacuum machines concerned only soot sweeping in private homes and industrial buildings. Furthermore, information about the frequency of changing gloves  and type of mask  was collected.n = 151) after a rest of at least 15 min. Post-shift spot urine samples and venous blood samples were collected from chimney sweeps and controls (non-fasting samples). Buffy coat, EDTA, and heparin plasma, and serum were separated after 10 minutes and kept on dry ice until storage at −80 °C at the Division of Occupational and Environmental Medicine at Lund University, whereas urine and EDTA blood samples were kept at room temperature until storage at −20 °C. Nine participants abstained from giving blood samples (5 chimney sweeps and 4 controls) and 4 participants could not urinate during the visit (3 chimney sweeps and 1 control).Blood pressure was measured using a mercury sphygmomanometer in the upright (sitting) position for chimney sweeps  and 3-hydroxybenzo[a]anthracene (3-OH-BaA)36, respectively.Total exposure to PAH was evaluated by analyzing 4 different PAH metabolites in urine. These metabolites have been suggested as biomarkers for PAH exposure. 1-hydroxypyrene (1-OH-PYR) is a metabolite of pyrene and has been extensively used as a proxy for total exposure to PAH. For exposure to benzo[a]pyrene, we measured the metabolite 3-hydroxybenzo[a]pyrene (3-OH-BaP)57. For analysis of 3-OH-BaA and 3-OH-BaP, sample aliquots of 20 µL were injected onto a two-dimensional LC system with two analytical columns. Concentrations were determined by peak area ratios of the analytes versus the internal standards. All samples were prepared in duplicate and the average concentration of the duplicate samples was used. For more details, see Supplementary Methods and Supplementary Table Liquid chromatography coupled to tandem mass spectrometry  was used for measurement of PAH metabolites in urine. The urine samples were prepared in 96-well plates and hydrolysed using glucuronidase. Internal standards for all PAH metabolites were added. For analysis of 1-OH-PYR and 2-OH-PH, sample aliquots of 5 µL were injected onto a C18 columnadjusted = C(measured) × (1.020 − 1)/(ρ − 1), where C(measured) was the determined concentration in a urine sample, ρ was the measured SG for the same sample, and 1.020 was the average SG of all urine samples in this study. Urinary creatinine was measured by an enzymatic colorimetric method58. We adjusted PAH metabolites in urine to the creatinine concentrations for comparison with other studies (Supplementary Table A hand refractometer was used to measure the specific gravity (SG) of urine. PAH metabolite concentrations in urine (C) were adjusted for SG according to C60. CRP was quantified by using monoclonal CRP antibodies and by measuring the absorbance with immunoturbidimetry (limit of detection (LOD) <0.6 mg/L). Homocysteine was measured by an enzymatic reaction followed by quantification of the absorbance of NAD+ at 340 nm (LOD <3 µmol/L). GGT was determined by a method based on formation of 5-amino-2-nitrobenzoate, which was measured by kinetic photometry (LOD <0.05 µkat/L). Biomarkers of blood lipid status (low-density lipoprotein (LDL), high-density lipoprotein (HDL), cholesterol, and triglycerides (TG)) were also considered for analysis of dyslipidemia, which is a traditional risk factor for CVD61. LDL was measured by selective micellar solubilization (LOD <0.1 mmol/L); HDL by an enzymatic colorimetric method (LOD <0.08 mmol/L); cholesterol by a method based on formation a quinone dye measured photometrically (LOD <0.1 mmol/L); and TG by a method based on lipase and peroxidase (LOD <0.1 mmol/L). The levels of CRP, homocysteine, and LDL in serum had previously been determined in the non-smoking controls, but here we performed new measurements of these markers using fresh (not previously thawed) aliquots of the same serum samples. The correlations between old and new measurements were high , which strengthens our analysis.Aliquots from serum samples that had not been previously thawed were analyzed for seven different early biomarkers of CVD at the Department of Clinical Chemistry, Lund University Hospital. C-reactive protein (CRP), homocysteine, and gamma-glutamyltransferase (GGT) have been reported to be elevated in multiple CVD events2. For cigarette smoking, participants were categorized into 4 groups: smoker (daily smoker), party smoker (party smoker or ex-smoker who quit smoking less than a year before the day of recruitment), ex-smoker (quit smoking more than a year before the day of recruitment) and non-smoker (never smoked). For PAH metabolite concentrations below the LOD, measured concentrations were used. For CVD marker concentrations below the LOD, the values were replaced by LOD/√2. Median, minimum, and maximum values were calculated for continuous variables and frequencies for categorical ones.Age was calculated based on the birth and recruitment dates. BMI was obtained using the formula BMI = weight in kilograms/(height in meters)The Mann-Whitney U test was used to preliminarily compare differences in continuous variables between chimney sweeps and controls, i.e., BMI, age, and serum markers of CVD. Fisher’s Exact test was used to compare differences in distribution of categorical variables between groups. Spearman’s correlations were used to assess the correlations between PAH metabolites and between PAH metabolites and work tasks, use of gloves and masks during the past 12 months. Changes over time in work tasks, use of fuel by clients, and use of protective equipment were explored by Wilcoxon signed-rank tests having the year 2002 as an arbitrary cutoff.A general linear model was used to evaluate differences in CVD serum markers and PAH metabolites between chimney sweeps and controls, adjusting for potential covariates and confounders such as age, BMI, and smoking, which are well-known risk factors for CVD, as well as other lifestyle factors that could cause additional exposure to PAH. We performed the analyses in three different models: 1) adjusted for age (continuous), BMI (continuous), and smoking status ; 2) adjusted for age and BMI (excluding current and party smokers); and 3) adjusted for age, BMI, smoking status, use of snus (yes/no), physical activity , passive smoking (yes/no), current residency , education , family history of CVD (yes/no) and exposure to PAH from hobby (yes/no). A general linear model  was also used to evaluate whether working with soot sweeping correlated with PAH metabolites in urine. The sweeps were divided into two groups based on the percentage of time spent doing soot sweeping in private homes and industrial buildings in the past 12 months .In the chimney sweep group only, the same 3 models were adopted to assess associations between PAH metabolites and markers of CVD. We calculated the dose of exposure to soot over time (1963–2013), expressed as working years with soot sweeping, as follows: percentage of work with soot sweeping during the past 12 months divided by 100, plus percentage of work with soot sweeping in every 10-year work period divided by 10. Calculations were performed for the sum of soot sweeping in private homes and industrial buildings. For instance, one chimney sweep spent 20% of his work time doing soot sweeping in private homes and industrial buildings during the past 12 months, 40% during 2003–2012 and 80% during 1993–2002. Working years with soot sweeping for this participant would then be (0.2*1 + 0.4*10 + 0.8*10), which equals 12.2 years. We investigated the effect of working years with soot sweeping on blood pressure and serum markers using general linear model analysis as follows: first, we evaluated age versus blood pressure/serum markers (excluding working years from the models), and second, we evaluated working years versus blood pressure/serum markers (excluding age from the models).P-values < 0.05 were considered statistically significant (two-tailed).Statistical analysis was performed using SPSS 22.0 . All data generated or analyzed during this study are included in this article and its supplementary information files.Supplemental material


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.


The effects of statins in patients with diabetic nephropathy are controversial. With increasing interest in the potential therapeutic role of statins in diabetic nephropathy, it is essential to evaluate its real effects.PubMed, EMBASE, Web of Science databases, Cochrane Central Register of Controlled Trials and China National Knowledge Infrastructure were systematically searched for randomized controlled trials (RCTs) of statins in patients with diabetic nephropathy.P < 0.0001] and 1.68 , respectively. The reduction of albuminuria was greater in patients of type 2 diabetes mellitus with diabetic nephropathy  and the decrease was significant during the 1 to 3 years period of statin therapy . Subgroup analysis demonstrated the effects of statins were much stronger in subjects with pathologic albuminuria: change of −0.71  for those with urinary protein excretion 30 to 300 mg/day, −0.37  for those with excretion more than 300 mg/day and −0.29  for those with excretion less than 30 mg/day. In contrast, statins did not significantly reduce estimated glomerular filtration rate, serum creatinine and blood urea nitrogen levels.Fourteen trials with 2866 participants were included in our meta-analysis. Compared with placebo, albuminuria and urinary albumin excretion rates in the statin group were reduced by 0.46 [95 % confidence interval (CI),−0.68 to −0.25, Statins decrease the albuminuria and urinary albumin excretion rates significantly. The efficacy of statins on renal function is time dependent and better in type 2 diabetic patients with nephropathy. According to the International Diabetes Federation , it is pA number of potential mechanisms for kidney damage in DN have been identified. Hyperlipidemia may play an important role in the progression of DN and it may impair the messangial cells through its lipotoxicity or by promoting intrarenal atherosclerosis –5. StatiTo assess whether statins have beneficial effects on renal outcomes in diabetic nephropathy, we performed this meta-analysis to investigate the potential therapy of statins in patients with diabetic nephropathy.http://www.clinicaltrials.gov). Clinical trials were included if the following criterias were met: (1) Primary study of statins versus control ; (2) Diabetic nephropathy patients with type 1 and type 2 diabetes mellitus at least 18 years old without pregnancy; (3) Patients with diabetic nephropathy in experimental group were defined as those who used statins, regardless of dosages, mode of administration or treatment duration; (4) RCT design; (5) Report of baseline and the end of follow-up data on renal function [estimated glomerular filtration rate (eGFR), urinary albumin excretion rates (UAER), serum creatinine (Scr), blood urea nitrogen (BUN) or albuminuria). Exclusion criteria included: (1) Kidney damage due to diseases other than type 1 or type 2 diabetes. (2) The final stage of diabetic nephropathy or end-stage-renal disease (ESRD), defined as onset of renal replacement therapy or death attributed to diabetic nephropathy.We conducted a search of PubMed, EMBASE, Web of Science databases, Cochrane Central Register of Controlled Trials and China National Knowledge Infrastructure (CNKI). All relevant articles were published in English and Chinese. The following Medical Subject Headings (MeSH) and text words were used: Hydroxymethylglutaryl-CoA reductase inhibitors, atorvastatin, simvastatin, rosuvastatin, pravastatin, lovastatin, fluvastatin, cerivastatin, mevastatin, pitavastatin, statin, kidney, renal, diabetic nephropathy, randomized controlled trial (RCT), controlled clinical trial and random allocation. We also searched the additional trials at the trial register centres  characteristics of trial subjects  and the trial’s inclusion/exclusion criteria; (2) type of intervention ; and (3) type of outcome and measurement.I2 was calculated as an index of heterogeneity between studies. If I2 was higher than 50 %, the sensitive analysis should be performed to find out the source of heterogeneity and to assess whether the results could be significantly influenced.The primary outcome was the change of albuminuria from the baseline. Other outcomes include: change from baseline in eGFR, UAER, Scr, BUN. The meta-analysis with fixed-model or random-model was performed by weighted mean difference (WMD), standardized mean difference (SMD) and 95 % confidence interval (CI) for outcome of continuous variables. Subgroup analysis by characteristics of patients  and study design  were performed. http://www.cochrane.org).Study quality and bias risk were assessed via predefined categories: randomization, allocation concealment, quality of blinding , withdrawal and loss and reporting bias. Two reviewers independently determined these items. Sensitive analysis was performed in studies with low quality. The analyses were performed using Review Manager 5.2 , and the standardized mean difference in change from baseline was −0.71  for those with excretion of 30 to 299 mg/d; and −0.37  for those with excretion of 300 or greater .Pooled data from 10 studies , 21, 22 P = 0.26, Fig. P < 0.00001, I2 = 14 %), while no significant improvement in diabetic nephropathy of T1DM subgroup  compared with those <1 year . Furthermore, different types of diabetic nephropathy, ethnicity, baseline of eGFR, treatment duration and dose of statins also did not influence eGFR in patients with statin and control therapy , we conducted a subgroup analysis and sensitivity analysis. In subgroup analysis of ethnicity, stage of diabetic nephropathy, treatment duration and dose of statins, the heterogeneity still existed . The fixed-effect model was used to merge SMD values and the pooled SMD was −1.16 , which indicated that statins reduced UAER significantly in patients with DN.The effect of statins on UAER was favorable in 5 studies , 20, 22.2 = 64.91, P <0.00001, I2 = 95 %), we removed 1 study at a time to identify the source of heterogeneity. When the Study by Tsukasa et al. (2001) [P = 0.50; Table We identified 4 randomized controlled trials –22 (253 . (2001)  was remoP = 0.20) and the heterogeneity among trials was not significant  indicated that baseline of UAER may be a source of heterogeneity , with the greater improvement of albuminuria among studies with greater baseline albuminuria. Notably, our results are consistent with the meta-analysis performed by Kevin Douglas et al. [As we all know, the degree of albuminuria is a risk factor for renal failure . Some sts et al. . The bens et al. . Keane es et al.  have shos et al. . Researcs et al. , 34.A recent review  reported2, p = 0.04), with the greatest GFR improvement after between 1 and 3 years of statin therapy . However, our meta-analysis found that statins did not improve eGFR significantly. Just as Satirapoj said [Some authors found statins may slow the decline in eGFR , 38. Nikpoj said , as a poFindings from our meta-analysis revealed that statins could reduce both albuminuria and the rate of progression of diabetic nephropathy. The benefits appear to supplement those derived from treatment with renin-angiotensin system (RAS) inhibitors . The patAs with any study, our meta-analysis had some limitations. Frist, the number of randomized controlled trials was small and only published data included. Second, the detection technique of albuminuria was different. Third, the results are heavily based on the findings of the CARDS trial, which represents more than 90 % of the population of the meta-analysis. So, more clinical researches with larger sample, higher quality and strictly RCT study should be taken in the future.Statins reduce albuminuria and UAER significantly . The beneficial effect of statins on renal function is time dependent and better in type 2 diabeties patients with nephropathy. Our findings, though exciting, still require larger and high-quality studies to confirm the kidney benefit of statins in patients with diabetes.


FT4 is negatively correlated with TG  while positively correlated with HDL cholesterol levels . TSH is positively associated with TG  and total cholesterol levels . Importantly, BMI modulates the effect of TH on lipid profile: the interaction of FT4 and BMI and the interaction of FT3 and BMI reach statistical significance in predicting TG and HDL cholesterol levels, respectively. Stratified according to BMI levels, most associations between TH and lipid profile are significant only in normal-weight group. In conclusion, in euthyroid adults, high normal FT3, TSH levels, and low normal FT4 levels are associated with unfavorable lipid profile. BMI mediates the effect of thyroid function on lipid profile in euthyroid adults.The impacts of thyroid hormones (TH) on lipid profile in euthyroid adults have gained much attention. It is currently unknown whether BMI influences such interaction. In the present study, we investigate the role of BMI in modulating the association between TH and lipid parameters in 1372 euthyroid healthy adults. Our results show that thyroid parameters are differentially associated with lipid profile. FT3 is positively correlated with total cholesterol ( Dyslipidemia constitutes a major risk factor for premature atherosclerosis and cardiovascular disease (CVD), which is a major cause of morbidity and mortality in both developed and developing countries . DyslipiThyroid hormones (TH) regulate multiple metabolic processes . It is wThe objective of the current study is to examine the association between thyroid hormones and lipid parameters in euthyroid adults. Furthermore, the role of BMI in modulating the association between thyroid hormones and lipid profile is determined.We conducted this retrospective study with subjects who participated in a routine health screening examination in Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine between 2007 and 2010. Individuals with previously diagnosed chronic cardiovascular diseases, diabetes mellitus, and liver and kidney diseases were excluded from our study. Women with diagnosed pregnancy were also excluded. Furthermore, individuals receiving thyroid hormone substitutions or antithyroid drugs, which may potentially interfere with thyroid function or having TPO antibodies, antithyroglobulin antibodies above the clinical cutoff for positivity, or with TSH levels outside the normal reference range of our laboratory (TSH: 0.35–4.94 mIU/L), were also excluded from our analyses, finally leaving 1372 subjects for inclusion in this investigation.The study was approved by the Medical Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine and was conducted in accord with institutional guidelines. The study was in accordance with the principle of the Helsinki Declaration II. All study participants provided written informed consents.Data on gender and age were obtained from all the subjects. Standing height (cm) was measured using a wall-mounted Harpenden Stadiometer. Body weight (kg) was measured in light indoor clothing without shoes. Body mass index is defined as the body weight (kg) divided by the square of the body height (m).2, while overweight and obese are defined as BMI ≥ 25 kg/m2 according to the WHO criteria [Normal weight is defined as 18.5 ≤ BMI< 25 kg/mcriteria .3), free thyroxine (FT4), and thyrotropin (TSH) levels. Serum total cholesterol, low-density lipoprotein cholesterol (LDL cholesterol), high-density lipoprotein cholesterol (HDL cholesterol), and TG were measured using an autoanalyzer  in the Shanghai Institute of Endocrine and Metabolic Diseases.Blood samples were obtained from all the subjects after overnight fasting. Chemiluminescent methods  were used to quantitate thyroid function based on free triiodothyronine ) were performed using SAS 9.2 . Data were given as mean ± standard deviation for normally distributed continuous parameters, and for skewness distribution data, median and interquartile range was used. Descriptive statistics were used to describe the study population at the baseline.P for interaction resulted from linear regression analysis with FT3∗BMI  as independent variable and lipid profile as dependent variables, after multiple adjustment for age, gender, BMI, and FT3 (or FT4 and TSH). We further divided the population into subgroups according to gender, and P for interaction was calculated after adjustment for age, BMI, and FT3 (or FT4 and TSH).Linear regression analysis was used to identify the association between different thyroid parameters and different lipid parameters. These models were all adjusted for age, gender, and BMI. Each BMI subgroup was sequentially assigned to tertiles according to their plasma FT3, FT4, and TSH levels. Associations between thyroid function parameters and lipid profile among tertiles were identified using one-way ANOVA in each BMI subgroup. P values reported were two sided. Statistical significance was assumed for P values < 0.05.The 2) and 4.2% were obese (BMI ≥ 30 kg/m2). There were evident differences in lipid parameters and thyroid hormone levels between subgroups with BMI ≥ 25 kg/m2 (overweight/obese group: average BMI 27.28 ± 1.96 kg/m2) and with 18.5 ≤ BMI< 25 kg/m2 . Subjects in the overweight/obese group had worse lipid profile compared to the normal-weight controls: they had significantly higher TG levels (1.90 (1.32–2.86) versus 1.27 (0.87–1.89) mmol/L, P < 0.001), total cholesterol levels , and lower HDL cholesterol levels  . There was no significant difference in LDL cholesterol levels between the two groups .Characteristics of the study population, their anthropometric data, metabolic parameters, and descriptions for thyroid hormones were shown in < 0.001) . There wo groups .P < 0.001), but decreased FT4 levels . Moreover, TSH levels were slightly lower in the overweight/obese group but did not reach statistical significance .Thyroid hormone levels between subjects in different BMI categories also varied significantly. Subjects in the overweight/obese group showed increased FT3 levels  and LDL cholesterol concentrations . On the contrary, FT4 levels were negatively correlated with TG  but positively associated with HDL cholesterol levels . TSH levels were significantly and positively related to both TG  and total cholesterol levels .To understand the relationship between the levels of thyroid hormones and lipid parameters, we performed linear regression analyses, adjusted for age, gender, and BMI in euthyroid subjects. As shown in P < 0.05) and TG concentrations (P < 0.05), respectively, after multiple adjustment for age, gender, BMI, and FT3 (or FT4)  . We also(or FT4) .https://doi.org/10.1155/2017/8591986, the interaction of FT3 and BMI remained significant in predicting serum HDL cholesterol in male subjects (P < 0.05), whereas the interaction of FT4 and BMI was significant in modulating TG concentrations in females (P < 0.05). Interestingly, the interaction of TSH and BMI also appeared statistical significance in both male and female subjects: predicting total cholesterol and LDL cholesterol levels in males (both P < 0.05), while predicting TG levels in females (P < 0.01) (Table S1).It is known that thyroid function and lipid profile can be influenced by gender , 12, 14.Since BMI modulates the interaction between thyroid parameters and lipid profile, we further checked whether the association between thyroid parameters and lipid profile varied in subjects with different BMI. We performed linear regression analyses separately in the normal-weight and overweight/obese groups . In the β = 0.145 ± 0.071, P < 0.05;  and LDL cholesterol levels (P < 0.01) compared to the lowest tertile (T1) in both normal-weight and overweight/obese groups  as well as FT4 with HDL  were only detected in the normal-weight subjects (both P < 0.01), but not in the overweight/obese group (P < 0.01) were observed in the highest tertile of FT4 only in the normal-weight group (We then stratified the population according to their serum FT4 levels. A significant correlation between FT4 and TG (se group . Similarht group .β = 0.110 ± 0.039, P < 0.01; P < 0.01; A significant positive association between serum TSH levels and total cholesterol levels was only found in the normal-weight group (2. The observations strongly indicate that body weight might interfere with the effect of TH on lipid profile.Thyroid hormones influence key metabolic pathways which control energy balance by regulating energy storage and expenditure . Overt t2 in the current study. However, the influence of body weight on the interaction of TH and lipid profile has not been clearly clarified in the literature. Importantly, we showed that serum FT4 levels and BMI had interactions in predicting serum TG levels, whereas FT3 and BMI had interactions in predicting HDL cholesterol levels in euthyroid population. Such interactions remained significant and even more pronounced when we performed gender-based interaction analyses: TSH and BMI reached significance in predicting total cholesterol and LDL cholesterol levels in males, as well as TG levels in females.The thyroid and adipose tissue are organs producing thyroid hormones and adipokines, respectively . Both ofAs generally accepted, obesity is closely associated with dyslipidemia, manifested as elevated fasting and postprandial TG in combination with the preponderance of small dense LDL and low HDL cholesterol . ObesityThe novelty of the study is that, to the best of our knowledge, we are the first to identify the role of BMI in modulating the effect of thyroid hormones on lipid profile in euthyroid adults. However, our study has several limitations. First, due to the small sample size of obese population, we could not compare overweight and obese subjects separately in our analyses. Second, lacking of smoking status information impeded us to identify the effect of smoking habit on such relation. Third, it would be ideal to initially exclude the subjects on lipid-lowering therapy, although we estimate that this percentage is very low in our study population. Fourth, as a cross-sectional study, the causal relationship could not be inferred. Further studies are needed to clarify whether adjusting certain TH parameters in euthyroid normal-weight subjects do bring benefits in maintaining lipid homeostasis.In conclusion, in euthyroid adults, low normal FT4 levels, high normal FT3, and TSH levels are associated with less favorable lipid parameters. BMI modulates the effect of thyroid hormones on lipid metabolism. The findings that the associations between TH and lipid parameters are not identical in normal-weight and overweight/obese subjects provide clues for individualized lipid-lowering therapy in clinic.Table S1: The interaction between BMI and thyroid parameters in modulating lipids profile.


Mesenchymal stem cells (MSCs) are one of the most promising adult stem cells for clinical application in a cell therapy. The development of large-scale cryopreservation techniques, such as vitrification, for MSCs is a prerequisite for clinical therapies. Dimethyl sulfoxide (DMSO) and ethylene glycol (EG) are two types of cryoprotectants widely used for cell vitrification. However, the effects of DMSO and EG on the biological characteristics and transcriptome profiles of MSCs after cryopreservation remain unknown. In the present study, the viability, immunophenotype of cell surface markers, proliferation, differentiation potency, and global gene expression of rhesus macaque bone marrow-derived MSCs vitrified using DMSO and EG were studied. The results showed that vitrification did not affect the morphology, surface markers, and differentiation of the MSCs, and compared to DMSO, EG better protected cell viability and proliferation. Most importantly, vitrification resulted in changes in a large number of transcripts of MSCs either preserved using DMSO or EG. This report is the first to examine the effects of DMSO and EG on global gene expression in stem cells. These results will be beneficial to understanding the biological process involved in MSC vitrification and will contribute to improving cryopreservation protocols that maintain transcriptomic identity with high cryosurvival for preclinical research and clinical long-term storage. Mesenchymal stem cells can be differentiated into various cell types in vitro and in vivo under appropriate conditions, reflecting their multipotent capacity [6 MSCs per kilogram of body weight and 108 MSCs for one patient were infused in cell therapy. However, the long-term cultivation of MSCs can result in the loss of progenitor properties and generate malignant transformation due to changes in gene expression related to cell differentiation [Mesenchymal stem cells are spindle-shaped fibroblast-like adult stem cells that are easy to isolate, culture, and expand capacity . In addicapacity . Therefontiation , 4, altentiation . TherefoCryopreservation provides a practical and effective method for maintaining the potency of stem cells with low cost and less labor. Traditionally, MSCs are cryopreserved at a slow cooling rate using DMSO as a cryoprotectant. Cells in freezing medium containing 5–10% DMSO are packed into cryovials and frozen in a computer programmed freezer at a cooling rate of −1°C/min to −80°C prior to freezing in liquid nitrogen for storage , 8. VitrThe rhesus macaque is one of the most widely used laboratory animals in biomedical research because of its genetic, physiological, behavioral, and neurological similarities to humans, and the macaque provides excellent translational validity in preclinical studies . The preThree male rhesus macaques (2 years old) were used as bone marrow donors. The procedure for bone marrow retrieval was approved through the Institutional Animal Care and Use Committee of Kunming University of Science and Technology and performed in accordance with the Guide for the Care and Use of Laboratory Animals.v/v) fetal bovine serum (FBS) (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). The cell suspension was subsequently centrifuged at 500g for 5 minutes, and the supernatant was discarded. Next, the marrow cells were mechanically dispersed into a single-cell suspension and seeded onto 10 cm plastic dishes at a density of 1 × 106 cells/ml. The cells were cultured in DMEM medium supplemented with 10% FBS at 37°C in an incubator with a humidified atmosphere of 5% CO2. The nonadherent cells were removed, and the medium was refreshed every 48 hours. Ten days later, the primary cell culture (passage 0) was passaged at 80% confluency using 0.25% trypsin (Gibco). The cells were resuspended in culture medium at a dilution ratio of 1 : 3 and expanded on a new plastic petri dish to passage 1. The morphology, surface markers, and differentiation potency of MSCs were identified at passage 3. The MSCs were expanded up to passage 5 and subsequently subjected to vitrification and global gene expression examination as described below.The bone marrow-derived MSCs were isolated from the tibias of the young rhesus macaques. The muscular tissues on tibias were carefully removed. The ends of the bones were cut, and bone marrow was aseptically flushed ten times using a sterile syringe containing 10 mL of Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% . The washed cells were resuspended in 100 μL of PBSF for the analysis of surface markers of MSCs. Each cell sample was incubated with 5 μL (10 μg/μL) of antihuman PE-CD44, APC-CD73, FITC-CD90, PE-CD105, PE-CD105, PE-CD59, HLA-A,B,C, PE-CD45, PE-CD14, PE-CD34, PE-CD11b, PE-CD19, and PE-HLA-DR antibodies for 1 h on ice, and isotype control antibodies were used in parallel. Unbound antibodies were washed off with PBSF, and subsequently, the cells were resuspended in 500 μL of PBSF.The expression of surface markers of MSCs was examined using flow cytometry analysis . All antibodies were purchased from BD Biosciences. Approximately, 5 × 104 cells per well for 12 h. Subsequently, the cells were cultured in adipogenic differentiation medium  for 7 days [v/v) for 15 min and washed 3 times with PBS after fixation in 4% methanol. The adipogenic differentiation was confirmed as the appearance of cellular accumulation of neutral lipid vacuoles was stained red with Oil Red O .The bone marrow-derived MSCs were seeded onto 24-well plates and cultured at a density of 8 × 10r 7 days . The med4 cells per well for 12 h. Subsequently, the culture medium was replaced with osteogenic differentiation medium  and further cultured for 21 days. The medium was refreshed every three days. The cells were stained with fresh 0.5% alizarin red solution and washed 3 times with PBS, followed by fixation with 4% methanol. The osteogenic differentiation was confirmed as the appearance of alizarin red staining.The bone marrow-derived MSCs were seeded onto 24-well plates and cultured at the density of 4 × 105 per tube and subsequently cultured in chondrogenic differentiation medium . The medium was refreshed every three days. After 21 days of differentiation induction, the chondroid pellets were generated and washed with PBS and fixed in 4% paraformaldehyde, embedded with optimum cutting temperature (OCT) embedding material . The pellets were sectioned using a freezing microtome, and subsequently, sulfated proteoglycans were visualized by staining with 1% toluidine blue  for 10 min [The bone marrow-derived MSCs were collected in 15 mL centrifuge tubes containing approximately 2 × 10r 10 min . These s6 cells/mL. One of the aliquots without cryopreservation was subcultured in fresh medium for 24 h, and cell viability, immunophenotype surface markers, proliferation, and metabolic activity were subsequently examined as a nonvitrified control (VC). The other two aliquots were vitrified using either DMSO  (VD) or EG  (VE) as a penetrative cryoprotectant, respectively. The vitrification protocol contained a two-step exposure to equilibration and vitrification solutions, respectively [6 MSCs were suspended in 50 μL of equilibration solution for 5 min and subsequently mixed with 500 μL of vitrification solution for 40 s (step 1). The suspended MSCs in vitrification solution were immediately transferred to 1 mL cryovials  and directly frozen in liquid nitrogen (step 2). After storing in liquid nitrogen for 24 h, the cells were rapidly warmed by immersing the cryovial in a 37°C water bath for 5 min [The bone marrow-derived MSCs from the three donors were harvested at passage 5 for the vitrification assay when the cells reached 80% confluency. The cell suspension was divided into three equal aliquots at a density of 2 × 10ectively . The equor 5 min , 29. TheThe percentages of viable cells from vitrified  and control (VC) groups were assessed using a trypan blue dye exclusion assay at 1 : 1 dilution (0.4% trypan blue in PBS). The stained and total numbers of cells (approximately 100 cells) were counted using a hemocytometer under a microscope. This assessment was repeated three times.μL of cell suspension was seeded onto a 96-well plate at a density of 104 cells/well. Subsequently, 20 μL of CellTiter 96 AQueous One Solution Reagent  was added to each well and incubated for 2 h. The quantity of formazan product is directly proportional to the number of living cells in culture. The colored formazan was measured at 490 nm in a 96-well microplate reader at 0, 12, 24, 48, and 72 h [The proliferation ability and metabolic activity of MSCs from vitrified (VD and VE) and control (VC) groups were determined using an MTS -5-(3-carboxymethoxyphenyl)-(4-sulfophenyl)-2H-tetrazolium) assay. Briefly, 200 and 72 h . This asμg RNA per sample was used as an input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  according to the manufacturer's instructions, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was performed using divalent cations under an elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After the adenylation of 3′ ends of DNA fragments, NEBNext Adaptors with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferential 150~200 bp in length, the library fragments were purified using the AMPure XP system . Subsequently, 3 μL of USER enzyme  was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C prior to PCR. Subsequently, PCR was performed using Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer. Finally, the PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on the cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.The cells from the VC, VD, and VE groups were collected and resuspended in Trizol Reagent  and stored in a −80°C freezer, respectively. The total RNA was extracted from each sample. The RNA quality of each group was assessed using agarose gel electrophoresis, and the RNA purity was assessed using the NanoPhotometer® spectrophotometer . The concentration of RNA was measured using the Qubit® RNA Assay Kit in Qubit 2.0 Fluorometer . RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . Finally, RNA-seq procedures were performed at Novogene Co. . A total amount of 3 −△Ct procedures.Several selected differentially expressed genes  among thP value less than 0.05 was considered statistically significant. Differential gene expression analysis of the VC, VD, and VE groups was performed using the DESeq R package (1.18.0). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P value < 0.05 according to DESeq were assigned as differentially expressed. Gene ontology (GO) enrichment analysis of differentially expressed genes was implemented using the GOseq R package, in which gene length bias was corrected. GO terms with corrected P values less than 0.05 were considered significantly enriched with differentially expressed genes.The results from these experiments are presented as the means ± SD. The statistical significance of the cell viability, proliferation, and metabolic activities between the VC, VD, and VE groups were determined using SPSS 17.0 software with one-way analysis of variance (ANOVA) and Fisher's protected least significant difference test. A During primary culture, the MSCs derived from macaque bone marrow grew adhesively in plastic dishes in a scattered manner. The cells that formed colonies and appeared heterogeneously were referred to as passage 0. The MSCs started to appear homogenous fibroblast-like, elongated, and spindle-shaped with single nuclear features following subsequent culture . The MSCP < 0.05). The overall cell metabolic activities of VC, VD, and VE at different time points after a 24 h culture are shown in P < 0.01). However, no differences were observed between the cells of VC and VE at any time point (P > 0.05). The morphology of warmed cells after a 24 h subculture is shown in Vitrification significantly decreased the viability of MSCs using either DMSO  or EG  as penetrating cryoprotectants compared to that of the nonvitrified control group , and compared to EG, DMSO showed less cryoprotection of cells . The MSCs of all groups showed a positive immunophenotype of CD44, CD73, CD105, CD59, HLA-A,B,C, and CD90 and a negative immunophenotype of CD45, CD14, CD34, CD11b, CD19, and HLA-DR .https://doi.org/10.1155/2017/3893691). After osteogenic induction, the vitrified MSCs (VD and VE) and control cells (VC) presented an aggregation of micronodules or calcium deposits that was stained with alizarin red. The chondrogenic differentiation of warmed MSCs and control cells could be observed using alcian blue stain.Similar to the cells in the VC group, the vitrified MSCs from either the VD or VE group differentiated into adipocytes, osteocytes, and chondrocytes . After aThe number of significantly modulated genes among MSCs from the VC, VD, and VE groups is summarized in The differentially expressed genes were separated into several categories according to their functions, including immune pathway, cell signaling, epigenetic regulation, cell differentiation, cell adhesion and signal transduction, metabolic pathway, and cell apoptosis. The 10 selected genes were determined using qRT-PCR to confirm the results of the transcriptome profiles of MSCs according to original analysis results of RNA-Seq, including the up- or downregulated differentially expressed genes and corresponding multiples. The expression levels of the 10 genes among MSCs from the control (VC) and vitrified groups (VD and VE) are summarized in in vitro differentiation and global gene expression of MSCs. The MSCs derived from the bone marrow of macaques presented features of heterogeneous, fibroblast-like, and spindle-shaped morphology, consistent with the morphology of the bone marrow-derived MSCs as previously reported [In the present study, we used rhesus macaque as a model to establish bone marrow-derived mesenchymal stem cell lines and investigate the effects of vitrification using two common penetrating cryoprotectants on the self-renewing capacity and reported . The celreported . These rreported . The isoreported .v/v). The optimal concentration of cryoprotectant for primate MSC vitrification should be further explored and optimized.Cryopreservation plays an important role in maintaining cell function for tissue engineering, cell transplantation, pharmacological testing, and future therapeutic indications . TraditiIn the present study, the viability of MSCs vitrified using DMSO was significantly lower than that using EG, and the cells of VD groups showed sparse confluency after culture for 24 h and a low proliferation metabolic activity compared to MSCs of the VE and control groups. During vitrification, a high concentration of cryoprotectant was used, which may lead to cytotoxic effects. Thus, DMSO-free protocols for either traditional slow freezing or vitrification have been developed to optimize stem cell cryopreservation , 39. ComRecent studies have demonstrated that, although the basic characteristics of plasticity and multipotency of MSCs were not altered after cryopreservation with DMSO by traditional slow freezing, the expression of apoptosis-related genes, such as BAC, BCL-2, BAX, P53, and P21, was affected , 19. HowIn conclusion, vitrification using either DMSO or EG did not affect the morphology, surface markers, and differentiation of MSCs. However, MSCs vitrified using DMSO showed poor cell viability and proliferation ability compared to those vitrified using EG. The vitrification of MSCs using either DMSO or EG leads to changes in a large number of transcripts compared to those of control cells. This report is the first to show the different effects of DMSO and EG on the global gene expression and impact of EG on the epigenetic profile of stem cells. These results will be beneficial to understanding the biological process involved in the vitrification of MSCs and contribute to improved cryopreservation protocols that maintain transcriptomic identity with high cryosurvival for preclinical research and clinical long-term storage.Figure S1. The MSCs vitrified with DMSO (a) and EG (b) were cultured in DMEM medium supplemented with 10% FBS for 7 days without differentiation induction as negative controls. No adipogenic differentiation was observed evaluated with oil red staining.


Rct) over time indicating that the GPn acted as an efficient physical and chemical barrier preventing corrosive species from reaching the copper surface. The GPn-coated copper was composed of many PANI-coated graphene planes stacked parallel to the copper surface. PANI exhibits redox-based conductivity, which was facilitated by the high conductivity of graphene. Additionally, the GPn surface was found to be hydrophobic. These properties combined effectively to protect the copper metal against corrosion. We expect that the GPn can be further applied for developing smart anticorrosive coating layers capable of monitoring the status of metals.A smart and effective anticorrosive coating consisting of alternating graphene and polyaniline (PANI) layers was developed using top-down solution processing. Graphite was exfoliated using sonication assisted by polyaniline to produce a nanostructured, conductive graphene/polyaniline hybrid (GPn) in large quantities . The GPn was coated on copper and exhibited excellent anticorrosion protection efficiencies of 46.6% and 68.4% under electrochemical polarization in 1 M sulfuric acid and 3.5 wt% sodium chloride solutions, chosen as chemical and seawater models, respectively. Impedance measurements were performed in the two corrosive solutions, with the variation in charge transfer resistance ( In general, corrosion consists of the irreversible deterioration of metals resulting from chemical, or often electrochemical, reactions induced by the surrounding environment. Material properties such as appearance, chemical reactivity, mechanical strength, and electrical conductivity are significantly affected by corrosion. In particular, metallic corrosion in key conducting components, such as conductors and electrodes in devices, can cause unexpected and serious failure in device performance. It is also important to note that the corrosion of electrical/electronic components is insidious and cannot be easily detected in sophisticated devices. A rich variety of anti-corrosive coatings consisting of small molecules, polymers, carbon species, oxides, and alloys are available15. Anticorrosive mechanisms involve barrier, inhibitive, and galvanic effects, depending on the nature of the anti-corrosive coating material6. In most cases, these coatings render the metal surface chemically impermeable and/or electrically insulating to prohibit undesirable chemical/electrochemical reactions.Metal corrosion is a major problem in many industries, and it remains a popular research topic in materials science, with emerging applications16. While insulating coating agents inhibit the flow of corrosion current, conducting polymers allow current flow at the metal surface. The redox-reaction-based conductivity of conducting polymers involves introducing/releasing dopant ions17, which can provide a unique protective mechanism against corrosion. A plausible protective mechanism for conducting polymers includes anodic protection and/or a barrier effect, which is highly dependent on the dopant ions18. Polypyrrole and polyaniline (PANI) are the main conducting polymers that have been investigated for corrosion control20. The greatest benefit of using conducting-polymer coatings could be their electrical conductivity and electrochemical properties, such as capacitance and impedance, which makes them a unique and versatile class of anticorrosive coating agents.Conducting polymers have been investigated for their ability to suppress corrosion reactions, taking advantage of their inherent reversible redox activity25, which can synergistically couple with a PANI barrier effect28. Lastly, the GPn coating allowed nondestructive, electrochemical monitoring of the metal-surface status.Herein, we examine the ability of a nanostructured, conductive graphene/polyaniline hybrid (GPn) coating to protect a metal against corrosion. Graphene sheets were physically exfoliated using soluble PANI in an organic solvent, resulting in a graphene dispersion stabilized by PANI. The GPn dispersion exhibited high colloidal stability and was readily coated as a thin film on copper metal, with the coating consisted of alternating graphene and PANI layers. The high conductivity of graphene contributed to an increase in the PANI conductivity, which strengthened the anticorrosive behavior of the GPn. Furthermore, the two-dimensional graphene layers provide a physical barrier against the migration of corrosive speciesN-methyl-2-pyrrolidone (NMP) solution) via a single production process using an efficient physical approach , indicating that the graphene sheets acted as a conductive filler and formed conducting networks in the hybrid. In addition, graphene can act as a two-dimensional structural barrier and PANI possesses chemical properties originating from its unique conjugated system. These characteristics of the GPn coating can elicit a synergistic effect between the graphene and PANI that limits metal corrosion. Copper corrosion is mediated by electron-exchange reactions that are accompanied by ion and small-molecule transport. Electrons released from the anodic oxidation of copper are consumed by the cathodic reaction; consequently, impeding one or both anodic/cathodic reactions will retard the overall corrosion process.A large quantity of GPn solution was first prepared  and PANI-coated copper (P-Cu) were employed as control samples for comparison. Representative Tafel plots of polarization curves recorded in a 1 M H2SO4 solution are presented in Fig. Icorr), corrosion potential (Ecorr), Tafel slopes (ba and bc), polarization resistance (Rp), and protection efficiency (PE), were calculated by extrapolating the linear sections of the Tafel plots, and are presented in Table Copper was chosen as a representative metal substrate because of its widespread use in various industries. The GPn coating showed strong attachment to the copper substrate due to the adhesive properties of PANI Fig. . PANIs pThe ability of GPn to protect the copper substrate in a sodium chloride solution was also explored using the same method. Figure 2SO4 and 3.5 wt% NaCl electrolyte solutions for 48 h. Corrosion of the copper in the samples is caused by time-dependent physical (e.g. mass transport and adsorption) and chemical (e.g. charge interactions and oxidation/reduction) processes, including i) water uptake, ii) penetration/diffusion of corrosion-related ionic and molecular species through the coating layer; and iii) formation and diffusion of corrosion products. Figure Rs), charge transfer resistance (Rct), capacitance (Ccl), and constant phase element (CPE) components, while two additional circuit components, double-layer capacitance (Cdl) and a Warburg element (W), were used for the sodium chloride solution. Ccl and Cdl are the capacitance of the coating layer and the double-layer capacitance on the copper surface, respectively, while the CPE describes non-ideal capacitance behavior. Four circuit components, Rs, Rct, Ccl and Cdl, were used in the GPn-Cu sulfuric acid system. However, in the sodium chloride solution, a CPE was used in place of Cdl to model the capacitance of the GPn layer. Values for all equivalent-circuit components were calculated through fitting  after 48 h, while the P-Cu Rct increased to an order of 101 Ω. This indicates that GPn-Cu plays a role in preserving the initial surface characteristics of copper in the sulfuric acid solution. The overall Rct trends for the systems were different in the sodium chloride solution. The corrosion behavior of copper in the sodium chloride solution is clearly different to that in the sulfuric acid solution. For example, chloride ions from sodium chloride can form copper chloride on the copper surface, which in turn reduces the redox potential of copper species from 0.52 V (Cu+/Cu vs. SHE) or 0.34 V (Cu2+/Cu) to 0.14 V (CuCl/Cu). The most striking difference between the impedance data for the two different corrosive electrolytes is that Rct is one to two orders of magnitude higher in sodium chloride than in sulfuric acid for all samples. More importantly, the variation in Rct as a function of time was completely different for GPn-Cu compared with B-Cu and P-Cu. B-Cu exhibited an initial steep increase in Rct, followed by a gradual decrease, with P-Cu exhibiting a similar trend. However, the GPn-Cu Rct constantly increased over time. This is a clear indication that the GPn coating slowed the corrosion of copper in the sodium chloride solution. We expect that the GPn acts as both a physical and chemical diffusion barrier, effectively preventing the chloride ions from interacting with the copper surface. The rate of change of Rct (dRct/dt) is plotted as a function of time for each system in Fig. Rct was negligible over the entire 50 h in comparison with the control systems.Electrochemical impedance spectroscopy (EIS) was used to investigate the effect of time on the copper-corrosion protection of GPn-Cu. The samples were immersed separately in 1 M Hg Tables –S6. The 1 to 105 Hz and the low-frequency region of less than 101 Hz. The high-frequency region appears to be more sensitive to corrosion, with the impedance increasing with exposure time. Calibration curves of log|Z| vs. exposure time at a constant frequency were linear for GPn-Cu, while they were non-linear for P-Cu  was used to directly assess the effect of the nanostructured GPn coating on copper corrosion. The three samples  were exposed to the same two corrosive solutions of sulfuric acid and sodium chloride for 72 h and the coating layer was then peeled off the copper surface prior to SEM observation Fig. . After ites Fig. .Figure 12SO4 and 3.5 wt% NaCl solutions, respectively. The GPn can be easily produced using an efficient top-down approach and is adaptable to solution processing34. Therefore, we believe that commercialization of our material will be possible in the near future. Furthermore, the conductivity of our material renders it suitable for use in developing smart protective layers capable of monitoring the corrosion status of metals.A GPn was successfully prepared and used to protect copper metal against corrosion in sulfuric acid and sodium chloride solutions. Owing to its conductivity, potentiodynamic polarization and impedance measurements were used to monitor the behavior of the GPn layer as a corrosion barrier. Contact angle and SEM measurements were used to characterize the hydrophobicity of the GPn layer and assess the extent of copper corrosion, respectively. The copper corrosion mechanism was different in the two corrosive solutions. Nevertheless, the GPn coating provided superior corrosion protection, compared with a pure PANI coating, in both solutions. Under induced electrochemical polarization, the protection efficiency of the GPn coating was 17.0% and 20.9% larger than that of pure PANI in 1 M H4OH, 25%), and sodium chloride (99.5%) were obtained from OCI Co. Ltd. Copper foil  was purchased from Alfa Aesar.Graphite flakes, aniline (≥99.5%), ammonium persulfate (APS) (≥98.0%), NMP (99.5%), (1S)-(+)-10-CSA, 4-DBSA, and D-sorbitol (≥98%) were purchased from Sigma-Aldrich. Sulfuric acid (95.0%), hydrochloric acid , ammonium hydroxide solution (NH31: APS (0.10 mol) was dissolved in 1 L of 1 M HCl solution, and aniline (0.44 mol) was dissolved in 1.5 L of 1 M HCl. The two solutions were then mixed and stirred for 90 min at 1.5 °C. The resulting solution was filtered and then washed with excess distilled water and ethanol, which yielded a dark-green filter cake. The dark-green powder was washed several times with 2.5 L of 0.1 M NH4OH solution and the resulting product was dried under vacuum at 25 °C. Next, 1 g of the obtained PANI powder was mixed with CSA (3.1 wt%) and DBSA (1.5 wt%) in NMP (92.5 wt%), and undesirable residues or precipitates were removed from the PANI/CSA/DBSA solution. D-sorbitol was dissolved in NMP in a 1:1 weight ratio (PANI/CSA/DBSA-to-sorbitol) at 90 °C, and this solution was mixed with the PANI/CSA/DBSA solution at room temperature.First, PANI was synthesized as follows30. First, 0.5 g of graphite flakes were added to 0.5 L NMP and sonicated for 30 min. The resulting graphene dispersion was added to the PANI solution in a 1:60 graphite:PANI-solution weight ratio, which is the optimized graphene-to-PANI amount for achieving balanced electrical and adhesive properties. The mixed solution was sonicated for 30 min to produce a homogeneous GPn solution. The GPn solution was transferred onto the copper substrate (0.5 mL cm−2), which was dried for 5 min under a loading of 4 metric tons at 25 °C. The average coating layer thickness was 15 μm.GPn was then fabricated by exfoliating graphene using the synthesized PANI solution2SO4 or 3.5 wt% NaCl solution as the electrolyte, with a Pt auxiliary electrode and a Ag/AgCl reference electrode. All electrochemical measurements were performed using a Metrohm Autolab B.V. PGSTAT101 potentiostat/galvanostat. The EIS equivalent circuits and component values were calculated using NOVA 2.1.2 software.EIS was performed using a three-electrode cell containing a 1 M HSEM was performed using a JEOL JSM-7500F microscope. Contact angle measurements were conducted using a SurfaceTech GSA-X goniometer.Supplementray Information