Given the advanced-stage CKD with nephrotic proteinuria, we supplemented low-protein diet with high doses of a tailored essential amino acid mixture (AAs: 44 g per day) to improve weight reduction in the patient. After 20 months of conservative therapy, the patient lost 43 kg; despite two episodes of infection, albumin plasma levels increased up to 3.7 g per day. After a further 20 months of dialysis, the patient maintained a diet of 1800 kcal supplemented with 32 g of AAs and lost 47 kg, whereas both albumin (3.89±0.12 g dl−1) and C reactive protein returned to normal. During the follow-up period, anemia improved, erythropoietin was thus discontinued and insulin requirement decreased to 105 IU. This therapeutic option may be beneficial in advanced CKD patients with obesity and diabetes resulting from malnutrition.A 38-year-old woman, obese (219 kg), diabetic, hypertensive, chronic kidney disease (CKD) stage 4, with low plasma albumin level (2.9 g dl Moreover, we highlight the peculiarity of the measures adopted in clinical, diagnostic and therapeutic management.The patient had been affected by abdominal obesity since childhood, reaching a weight of 110 kg during puberty. At the age of 16 years, she went on a high-protein diet with amphetamine supplements and lost 50 kg. Over the following years, she attempted a series of diets and underwent implantation of a gastric balloon. However to no avail, as she progressively gained weight.−1, serum creatinine (sCr) 3.9 mg dl−1, blood urea nitrogen (BUN) 53 mg dl−1, abdominal obesity , BMI 81 kg m−2, nephrotic proteinuria (22 g per day), hypoalbuminemia (2.9 g dl−1), dyslipidemia and secondary hyperparathyroidism (SHPT)  per 20 kg of actual body weight. The singular composition of this AAs mixture was determined in a particular stoichiometric ratio, and contained all essential amino acids and two non-essential amino acids (tyrosine and cysteine).The patient, affected by arterial hypertension, insulin-dependent type 2 diabetes, diabetic retinopathy and Hashimoto's thyroiditis, came to our observation in October 2011, at the age of 38 years, for CKD stage 4 with glomerular filtration rate (GFR) 21 ml minm (SHPT) . sCr levm (SHPT) , glycemiAt this point, although the patient was able to manage her personal needs, she was only able to walk a few meters unaided.7 The patient began taking a dietary supplement of 44 g AAs divided into four daily doses in November 2011 (baseline). Within 3 months, the patient lost 10 kg, although her kidney function was stable . Following these results, the patient was strongly motivated to continue losing weight and in February 2012 (4th month) because of progressive respiratory failure with hypoxia and need to evaluate cardiovascular risk, the patient was admitted to hospital and started on low-calorie enteral nutrition (EN) via a nasal feeding tube to increase the speed of weight loss: 2000 ml of liquid per day, 56 g of AAs per day, electrolyte and vitamin supplements, and no food intake. Within 10 days, she lost 21 kg while her kidney function remained stable: sCr 3.3 mg dl−1, BUN 36 mg dl−1, GFR 24 ml min−1, proteinuria/24 h 16 g, plasmatic bicarbonate level 22 mEq l−1 and albumin level 3.0 g dl−1. Because of a condition of respiratory insufficiency, continuous O2 therapy was also prescribed. When EN was stopped, the patient restarted a supplemented LPD. In the 5th and the 15th months, the patient presented with a complication in the form of an infected ulcer in the first toe of her left foot, which was treated with protracted antibiotic therapy, but was resulted in persistently high CPR levels. In the 15th month O2 therapy was discontinued due to the resolution of respiratory insufficiency. After 20 months of conservative therapy, the patient had lost a total of 43 kg , and increase the utilization of lipid and glucose substrates.15 AAs moreover increase the production of adiponectin, leading in turn to a rise in insulin sensitivity.17 According to the literature, the use of AAs in our patient caused a sharp reduction in the need for exogenous insulin, whereas increasing the efficiency of endogenous insulin, as had already been observed in patients in highly critical conditions.18 In the particular AAs mixture formulation administered, the branched chain amino acids (BCAA) make up about 50% of total AAs. The resulting BCAA blood increase activates beta-oxidation, which enables the utilization of lipids for energy.19 Indeed, AAs regulate carbohydrate and fat metabolisms, thus contributing towards maintaining the basal metabolism by reducing fatty acid synthesis in the cytoplasm and providing for an augmented access into the mitochondria.20 Leucine, which the patient received at high doses (13.75±g per day), is essential for the promotion of protein anabolism and inhibition of their catabolism,23 and reduces appetite at the level of the hypothalamus.24 Our patient indeed confirmed that she had experienced a reduction in the sensation of hunger. In our opinion, the above-mentioned characteristics of AAs account for the remarkable weight reduction observed. In the patient, high doses of AAs met nitrogen requirements by minimizing urea synthesis due to the absence of arginine into the amino acids formulation, provided for an adequate caloric intake and produced a good nitrogen balance throughout the observation period, without eliciting protein catabolism. During the follow-up period, BUN remained within an acceptable range . This may have been due to a series of factors such as severe arterial hypertension, diabetes, obesity, persistent inflammatory status, high proteinuria and/or a potentially undiagnosed additional renal disease.26Nevertheless, the progression of CKD was unfavorable; even though a good metabolic balance was achieved, residual kidney function gradually but progressively decreased , whereas albumin levels stabilized (3.89±0.12 g d l−1). Administration of antihypertensive drugs was discontinued a few months after commencing HD in view of improved interdialytic weight loss (average 3.0% of body weight), progressively extended duration of dialysis session, and better modulation of dialysate sodium, assessed on the basis of blood pressure levels ranging between 140–142 mmol l−1. During the conservative phase, the patient received 300 mg die−1 of allopurinol, although she failed to achieve target values of uric acid .


Hepatocyte growth factor (HGF) counteracts peritoneal fibrosis in animal models and in-vitro studies, but no study explored effluent HGF in peritoneal dialysis (PD) patients with ultrafiltration failure (UFF). Our aim was to assess the relationship between effluent HGF with UF profile, free water transport (FWT) and small-solute transport.creatinine, FWT, small-pore ultrafiltration, and effluent HGF were quantified.We performed 4-hour, 3.86% PET with additional UF measurement at 60 minutes in 68 PD patients. MTACEffluent HGF negatively correlated with UF  and FWT . Patients with UFF had higher dialysate HGF  and, although not statistically significant, those with FWT compromise had also higher dialysate HGF compared with subgroup of UFF without FWT compromise . FWT ≤ 45% without clinical UFF was documented in some patients who also had increased effluent HGF.Dialysate HGF concentration is significantly higher among patients with UFF, specially, if FWT is impaired, being a sign of peritoneal membrane deterioration. Ultrafiltration failure (UFF) is still a challenging complication of peritoneal dialysis and its prevalence increases with time on PD–3. In loex-vivo study. This pPETs with an ultrafiltered volume ≤ 400 mL/4 h were considered to represent ultrafiltration failure (UFF).All patients provided written informed consent for participation, and the study was approved by the Ethics Committee of St. António Hospital – Oporto Hospital Center.Creatinine and sodium were measured both in plasma and dialysate. For creatinine, the Jaffé compensated method was used. The dialysate creatinine concentration was corrected for interference by glucose according to our laboratory standards. Sodium was measured using indirect ion-selective electrodes. Effluent samples taken at 4 hour were immediately stored at −70°C, until they were used to measure HGF, VEGF and CA125. Effluent CA125 was determined with an electrochemiluminescence method on an automated analyzer . Effluent HGF and VEGF levels were determined by ELISA technique according to the manufacturer’s instructions . The intra and inter-assay variations were 8,8%% and 10,0%, respectively for HGF and 5,9%, and 9,4% for VEGF. The sensitivity was 11 pg/mL for HGF and 1 pg/mL for VEGF. Both the assays are considered highly specific for the cytokines, and no significant cross-reactivity was observed.creatinine was calculated by the simplified Garred model[Patients were characterized by peritoneal transport status as described by Twardowski et al.. The MTAed model.FWT and UF through the small pores (SPUF) at 60 minutes were calculated as we previously described. Using aExcept for time on PD, HGF, HGF/CA125 and VEGF/CA125, all variables had normal distribution. Results are expressed as mean ± SD or as median and interquartile range.Pearson correlation analysis was used in order to explore possible relations between HGF (with logarithmic transformation) and ultrafiltration profile, FWT and small-solute transport.For comparison of small solute transport, water transport pathways and effluent markers between patients with and without ultrafiltration failure, Mann–Whitney U test was used.Creatinine ≥ 0.81 and patients without FWT compromise and non-fast transport category, using Mann–Whitney U test.In order to study our patients ultrafiltration failure profile we made a comparison of small solute transport, water transport pathways and effluent markers between patients with FWT ≤ 45% and D/PUnpaired Student t-Test or Mann Whitney U-test to compare patients with FWT ≤ 45% and FWT > 45%, as appropriate, according to the variables involved.Creatinine ≤ 0,49), 10 (14.7%) as slow-average , 41 (60.3%) as fast-average , and 16 (23.5%) as fast transporters . Concerning water transport pathways, FWT accounted for 37.15% of the UF at 60 minutes, and once corrected , its contribution increased to a mean value of 45.46%.Table creatinine . These correlations were even stronger when we focused the analysis in patients with ultrafiltration failure . No correlation was found between dialysate HGF concentration and small pore water transport .HGF measured in the effluent significantly correlated with total ultrafiltration at a 4 h, 3.86% glucose PET , with FWT  and MTACcreatinine and MTACcreatinine had UFF . Although not statistically significant, patients with UFF had been on PD for a longer time, had higher D/P% had UFFcreatinine ≥ 0,81). Those patients had significant lower ultrafiltration volume at a 4 h PET , lower FWT quantification  and higher MTACcreatinine   and increased . And still 3 incident patients (3 – 6 months on PD), were average transporters with preserved FWT, in whom higher lymphatic absorption was by exclusion presumed.From the 59 studied patients without UFF, 33 had a FWT ≤45%. Those patients had a mean UF volume at 4 h PET equivalent to patients with FWT > 45%  on top of increased effective capillary surface, also with higher values of effluent HGF. Three patients presented an increased effective capillary surface but FWT was not impaired. Another 3 incident patients were average transporters with normal FWT. A high effective lymphatic absorption rate could be the reason for UFF in those incident patients since high lymphatic absorption is recognized to be mostly a cause of inherent UFF% on top  that reccreatinine was higher but not statistically different between patients with and without UFF. This is due to the presence of many incident fast transporters (mean time on PD 6 ± 4 months) in the non UFF group.In contrast with previous studies, 6, 20, Report and interpretation of causes of UFF as well as absolute and fractional FWT may vary according to the PD vintage. Our results are in accordance with Parikova et al., that docMoreover, our study adds further clinical evidence to the recent report from Nakamura S. et al.. Those aThe effluent HGF concentration on patients under PD was addressed, until now, by one single clinical study. HoweverAs others we also We are aware that the mass of mesothelial cells could affect the levels of intraperitoneal growth factors in PD patients. Although there are some controversies about the use of CA125 as an index of mesothelial cells mass or their functional properties–26, we aSince there are no clinical studies that had examined plasma and effluent HGF, and we found a correlation with small-solute transport, we might question whether effluent HGF concentration could depend on plasma HGF levels. We think that this is not plausible for various reasons. First, the peritoneal permeability is expected to be poor, since HGF is a heterodimeric molecule composed of a 69 KDa alpha subunit and a 34 KDa beta subunit. Second and mostly important, human peritoneal cells constitutively synthesized HGF. For theThe use of effluent biomarkers as an early sign of peritoneal membrane alterations is currently under debate–29 speciIn a very recent report, MCP-1, corrected fraction of 45%, which is in line with our previous studies[In this study we reported a mean FWT studies, and wit studies. A FWT ≤ studies–35.Our study is limited by its cross sectional design and small number of patients with UFF. A longitudinal study is being conducted in order to document the dynamic profile of HGF production and its relationship with peritoneal membrane water transport changes. More studies are needed to increase the knowledge on HGF clinical value, in patients under PD, as it may possibly point to new diagnostic opportunities and therapeutic avenues in the ultrafiltration failure field.ex-vivo study[Since HGF ameliorated peritoneal fibrosis in an vo study, a clini


Piaractus mesopotamicus; piavussu, Leporinus macrocephalus, and curimbatá, Prochilodus lineatus) were exposed to an acute dose of 5 ppm methyl parathion organophosphate pesticide. Three to five individuals per species were exposed, one at a time, to 40 liters tap water spiked with Folidol 600. Pesticide concentrations and cholinesterase (ChE) activities were evaluated in serum, liver, brain, heart, and muscle. The bioconcentration of methyl parathion was similar for all studied fishes. Brain tissue showed the highest pesticide concentration, reaching 80 ppm after exposure for 30 min to methyl parathion. Three to 5 hours of 5 ppm methyl parathion exposure provoked the death of all P. lineatus at 92% brain AChE inhibition, whereas fish from the other two species survived for up to 78 hours with less than 80% brain AChE inhibition. Our results indicate that acute toxic effects of methyl parathion to fish are correlated with brain AChE sensitivity to methyl paraoxon.Three species of freshwater Brazilian fishes (pacu,  Pesticides are a group of toxic compounds with a deep effect on aquatic life and water quality. Organophosphates (OP) are a group of pesticides widely used in Latin America. For instance, in Brazil, methyl parathion  has extensively been applied in agriculture, food storage shelters, pest control programs, and aquaculture ponds to control aquatic insect larvae, which are predators of fingerling , 2. The  Girardinichthys multiradiatus captured in the Ignacio Ramirez Dam (Mexico) accumulated methyl parathion more than 13,000 times in relation to water levels of this compound [An important mechanism of acute toxicity by methyl parathion is the inhibition of acetylcholinesterase (AChE) activity. Methyl parathion is a weak acetylcholinesterase inhibitor, but it can be activated into a more potent metabolite, its oxon derivative, methyl paraoxon , by a desulfuration reaction catalyzed by cytochrome P-450 [compound , 7. Methcompound , whereascompound .Intoxication mechanisms of aquatic organisms by OP are not completely understood. Dembéle and coworkers have attributed fish death to asphyxia resulting from gill irritation, not dependent on brain AChE inhibition . Patil a p-nitrophenol (PNP), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and methyl paraoxon  were obtained from Sigma . Triton X-100 was acquired from Riedel of Haën AG (Hannover). Isooctane HPLC grade was acquired from Merck. Folidol 600  was purchased at the local market. All other reagents were of analytical grade.Acetylthiocholine iodide (ASCho), butyrylthiocholine iodide (BSCho), propionylthiocholine iodide (PrSCho), Piaractus mesopotamicus , commonly named pacu, and Leporinus macrocephalus Garavello & Britski, 1988 (piavussu), were supplied by the Morro Grande Fish Farm, RJ, Brazil. Specimens of curimbatá, Prochilodus lineatus , were supplied by the Sol Nascente Fish Farm, RJ, Brazil. Experimental procedures were carried out according to the ethical principles of animal experimentation elaborated by the Brazilian College for Animal Experimentation (COBEA), which is in agreement with the uniform requirements for manuscript submissions to biomedical journals.Common and scientific names, geographical origin, and size of fishes examined in this work are shown in 2 L−1 (measured with an oximeter). Fish were always on a 14 : 10-h light/dark cycle and fed with a commercial pellet once a day at 9 o'clock AM. Water pH was 6.4 ± 0.2.Prior to the assays, the fish were separated by species and kept into 500 L tanks with dechlorinated water at 25 ± 2°C for 15 days. Water was constantly aerated through a biological filtering system pushed by common pumps to produce 5 mg OIn order to be exposed fish were removed from the larger tanks and individually kept in 40 L aquaria filled with water obtained from the 500 L tanks. Methyl parathion (as Folidol) was added once to a final 5 ppm concentration. All fish were not fed 24 h prior to and during the experiments in order to limit organic matter in the water. Moreover, the water during fish exposure was not filtered to avoid removal of the pesticide. Control fish were submitted to the same conditions, without Folidol.The concentration of 5 ppm methyl parathion was chosen to elicit a quick response and to correlate the inhibition of cholinesterase with the bioconcentration of pesticide in several tissues and organs of the fish.The intervals of 30 min, 24 hours, and 78 hours were chosen to carry out laboratory analyses. Curimbatás showed more sensitivity to methyl parathion. This was noticed when they stopped moving their opercula. Once this happened, they were immediately removed and laboratory analyses were immediately carried out.−1 potassium phosphate buffer, pH 7.0. They were minced with scissors and homogenized with 20 strokes in a Potter-Elvehjem apparatus while being maintained in an ice bath at 5-6°C. ChE assay in brain and liver tissue were carried out using this crude homogenate as sample. Samples for assaying heart and muscle ChE were produced by mixing their homogenates with three volumes of 10 mmol L−1 Tris-HCl buffer, pH 7.0, containing 3% Triton X-100 and 1 mol L−1 sodium chloride. After centrifuging these mixtures at 3,000 ×g for 10 min at 5°C enzyme assays were carried out in the resulting heart and muscle Triton X-100 soluble supernatant fractions.Blood was collected by puncture of the dorsal aorta. Then, the fish were euthanized by quickly sectioning their spinal cord. Liver, brain, and heart were removed using scissors and tongs. Also, a portion of epaxial muscle (1 g wet tissue) was excised. All tissues were separately washed quickly with 50 mL of ice-cold saline (0.9% NaCl) and placed into cryogenic vials that were dropped into liquid nitrogen for storage. They were thawed separately by suspension in four volumes of ice-cold 0.1 mol LμL) were extracted by using a mixture of 6 mL of isooctane, 200 μL methanol, and 200 μL saturated sodium chloride solution. The extracts were centrifuged at 3,000 ×g for 10 min at 5°C. Three milliliters from each supernatant fraction was collected and evaporated under a gentle nitrogen stream. The residue was reconstituted in 200 μL of acetonitrile. From this reconstituted sample, 50 μL were injected onto a 200 mm × 4.6 mm ODS Hypersil RP-18, 5 μm particle size HPLC column, using (50 : 50 v/v) acetonitrile/ultrapure water as mobile phase with a flow-rate of 1 mL min−1, and examined under UV light with the detector set at 270 nm. Under these conditions, methyl parathion recovery was estimated at approximately 92%, following quantification based on a standard prepared with a 98% methyl parathion pure sample previously obtained by thin-layer chromatography.Serum and homogenate samples , with 0.32 mmol L−1 DTNB. Enzyme activity was continuously recorded up to 90 s, at 412 nm, using a Shimadzu spectrophotometer, model UV-160 A, according to the Ellman method [μL final volume with a 0.1 mmol L−1 sodium phosphate buffer, pH 7.5, at 25°C, and the thionitrobenzoate ion concentration was estimated using an extinction coefficient of 14,150 M−1 cm−1. One unit (1 U) of enzyme activity was defined as the amount that hydrolyzes 1.0 μmol of substrate per minute.Serum and tissue homogenate samples were placed in a medium containing 1.8 mmol Ln method . The reaThe studied fish species (control groups) exhibited remarkable differences in tissue-specific cholinesterase activity . The ChEMuscle, heart, brain, liver, and serum from pacu, piavussu, and curimbatá exhibited similar methyl parathion bioconcentration patterns . Brain tHeart, liver, and serum cholinesterase activities for pacu, piavussu, and curimbatá were greatly inhibited after 30 minutes exposure to Folidol. By contrast, skeletal muscle ChE activity for the three species and brain AChE activity for pacu showed no inhibition within this time period . ExposurAll curimbatás stopped moving their opercula between 3 and 5 hours of exposure to 5 ppm Folidol, showing more than 90% inhibition of brain AChE activity and no inhibition of skeletal muscle ChE activity . IncreasBrain tissue showed the highest pesticide bioconcentration, reaching 80 ppm after 30 minutes of exposure to methyl parathion . The thr−1 ethyl parathion Abbas and coworkers [The results demonstrate that the fishes exposed to Folidol rapidly absorbed methyl parathion, since concentrations of methyl parathion in the tissues and serum peaked within 30 min of exposure to Folidol. This concentration continued to increase only in liver, in which the amount of methyl parathion doubled after 24 hours . Such fioworkers  observedoworkers . This suThe results reported herein demonstrated that ChE inhibition in fish exposed to Folidol does not seem to depend on the methyl parathion bioconcentration in their tissues. No ChE inhibition in pacu brain was observed up to 30 min of exposure, despite the fact that this organ presented 80 ppm of methyl parathion at that time. Therefore, this data is important for further investigations. Tissues in which ChE inhibition occurred more quickly, such as pacu liver  showing Brycon cephalus) exposed to water with 2 ppm Folidol 600 for 96 hours. The present study indicates that piavussu and pacu might be more tolerant to Folidol than matrinxã, since piavussu presented 66% and pacu 74% brain AChE inhibition when exposed to water with 5 ppm methyl parathion for 78 hours. On the other hand, curimbatá showed a 92% brain AChE inhibition with only 5 hours of exposure to 5 ppm methyl parathion, probably due to the higher sensitivity of this species AChE to inhibition by methyl paraoxon [In their work, de Aguiar and coworkers  describeparaoxon .Mammals poisoned by OP usually die by asphyxia. However, fishes have been reported to be more resistant to poisoning by high levels of organophosphates compounds than rats . The actGambusia affinis) [+ K+ ATPase inhibition [This study suggests that a major contributing factor to acute fish toxicity by Folidol is brain AChE sensitivity to methyl paraoxon, the oxon derivative of methyl parathion. The same explanation, that is, the sensitivity of brain AChE to oxon derivatives, has been suggested for chlorpyrifos, parathion, and methyl parathion toxicity in mosquito fish (affinis) . Althoughibition  and tissProchilodus lineatus (curimbatá), Piaractus mesopotamicus (pacu), and Leporinus macrocephalus (piavussu) studied here showed similar capacities to bioconcentrate methyl parathion in their tissues after exposure to 5 ppm in water. However, only curimbatá, with the highest brain AChE sensitivity to methyl paraoxon, died after 5 hours of exposure to Folidol. Pacu and piavussu are more resistant to methyl paraoxon and were alive up to 78 hours of exposure to 5 ppm of methyl parathion. Brain AChE sensitivity to methyl paraoxon might be a decisive factor for determining the sensitivity of these species to poisoning by high concentrations of organophosphate compounds. The present study indicates that fishes whose brain acetylcholinesterase activity is more sensitive to oxon derivatives will suffer more severe impacts from environmental contamination by organophosphate pesticides.Measures reducing the use of organophosphate pesticides in fish culture should be adopted in order to minimize the discharge and consequent impact of these chemicals to natural communities inhabiting rivers and lakes, of which the sensitivity to intoxication by pesticides is largely unknown.


Further results indicate that, to some degree, such an accelerated warming trend might be attributable to cloud–radiation feedback. The increased nocturnal cloud over the northern Tibetan Plateau would warm the nighttime temperature via enhanced atmospheric back-radiation, while the decreased daytime cloud over the southern Tibetan Plateau would induce the daytime sunshine duration to increase, resulting in surface air temperature warming. Meanwhile, the in situ surface wind speed has recovered gradually since 1998, and thus the energy concentration cannot explain the accelerated warming trend over the Tibetan Plateau after the 1990s. It is suggested that cloud–radiation feedback may play an important role in modulating the recent accelerated warming trend over the Tibetan Plateau.The surface air temperature change over the Tibetan Plateau is determined based on historical observations from 1980 to 2013. In contrast to the cooling trend in the rest of China, and the global warming hiatus post-1990s, an accelerated warming trend has appeared over the Tibetan Plateau during 1998–2013 (0.25 °C decade Since 1998, however, both the global and Northern Hemisphere mean temperatures show only a small warming trend: 0.05 °C decade−1 during 1998–2013 globally and 0.10 °C decade−1 for the Northern Hemisphere. Such a result is consistent with that reported by the IPCC−1 for the 73-station average over the Tibetan Plateau and 0.35 °C decade−1 for the 563-station average in other parts of China . At the second stage (1998–2013), however, the warming rates in other parts of China have an opposite cooling trend (−0.20 °C decade−1), while the surface air temperature over the Tibetan Plateau exhibits an accelerated warming trend (0.25 °C decade−1), undocumented in previous studies.Under the background of global warming, the surface air temperature over both the Tibetan Plateau and China increased quickly during 1980–2013, with an even larger amplitude compared to the mean case for the Northern Hemisphere: 0.44 °C decadeof China . These r−1). Meanwhile, similar warming rates are found for four seasons. While the surface air temperature trends of China and the Tibetan Plateau are comparable in spring  and autumn , and the Tibetan Plateau has a larger warming rate in summer  and winter  than other parts of China, making it experiencing a larger warming rate than other parts of China in terms of annual mean temperature. At the first stage  after the abnormal minimal value removed in 2009. Meanwhile, the sunshine duration over the Tibetan Plateau decreases at the first stage, but it increases at the second stage  and the internal variability only explains 17% , including air temperature at 2 m above the surface, wind speed at 10 m above the surface, sunshine duration, low-level cloud amount, and 6-hourly total cloud for 636 stations (http://cdiac.ornl.gov/) during 1971–2009.We obtained the HadCRUT4 combined land air temperature and SST datasetstations , in whicyi=ati+b, where y is the climatic variable with a sample size n, parameter t is the corresponding time, a and b are the linear regression coefficient (i.e. Linear Variation Rate) and regression constant. They can be estimated by using the least squares method:In this paper, a simple linear regression equation is employed here to calculate the trend, i.e. wherehttp://dx.doi.org/10.5065/D6WD3XH5).It is worth to note that we obtain similar results from Theil-Sen trend estimation compared with the linear trend used in this paper. And all figures in this paper are created by an open access software NCAR Command Language  [Software]. (2014). Boulder, Colorado: UCAR/NCAR/CISL/VETS. How to cite this article: Duan, A. and Xiao, Z. Does the climate warming hiatus exist over the Tibetan Plateau? Sci. Rep. 5, 13711; doi: 10.1038/srep13711 (2015).


Whole brain irradiation (WBRT) either with or without resection has historically been the treatment for brain metastases from non-small cell lung cancer (NSCLC). The effect of gamma knife (GK) radiosurgery, chemotherapy, or the combination remains incompletely defined. In this study, we assessed the outcome of brain metastases from non-small cell lung cancer treated by WBRT followed by GK, gefitinib, or the combination of GK and gefitinib.We retrieved the records of NSCLC patients with brain metastases from the National Health Insurance Research Database (NHIRD) of Taiwan from 2004 to 2010. WBRT either with or without resection was the first line treatment for nearly all patients. The decision to add GK and/or gefitinib treatment was at the discretion of the treating physician and based upon a patient’s medical records and imaging data. These patients were classified into four groups including WBRT, WBRT + gefitinib, WBRT + GK, WBRT + gefitinib + GK. These data was evaluated for difference in survival and factors that portended an extended survival from the time of brain metastasis diagnosis.p < 0.0001). The hazard ratio (95 % CI) for survival was 1, 0.56, 0.43, and 0.40, respectively (p < 0.001). The adjusted hazard ratio (95 % CI) by age, sex and Charlson comorbidity index (CCI) was 1, 0.73, 0.49, and 0.42, respectively (p < 0.001).Of the 60194 patients with newly diagnosed NSCLC, 23874 (39.6 %) developed brain metastases. The distribution of patients for the groups was WBRT for 20241, WBRT + gefitinib for 3379, WBRT + GK for 155, and WBRT+ gefitinib + GK for 99 patients. The median survival for the time of brain metastasis diagnosis for WBRT, WBRT+ gefitinib, WBRT+ GK, WBRT+ gefitinib + GK groups was 0.53, 1.01, 1.46, and 2.25 years, respectively (Patients with brain metastases from NSCLC receiving GK or gefitinib demonstrated extended survival. The improved survival seen with GK and gefitinib suggests a survival benefit in selected patients receiving the combined treatment. Further Phase II study should be conducted to assessment these influence. Lung cancer harbored the highest incidence of brain metastasis in relation to all malignancies. Approximately 40 % of all patients with non-small cell lung cancer (NSCLC) will develop brain metastasis during the course of their disease . Even wiGK can be used to treat multiple metastases during the same procedure and permits treatment of deep seated lesion considered surgical inaccessible –6. SubseIn two randomized phase II trials, the efficacy of gefitinib showed encouraging activity, in term of the objective response rate and clinical benefit with symptomatic improvement in patients with advanced NSCLC after failure of one or two previous chemotherapy regimens , 12. SevThe combination of EGFR TKI and radiation has enhanced effects for inhibition of proliferative and antiapopotic signaling pathways downstream of EGFR in cancer cell lines , 24. A cSince 1995, Taiwan established its state-run National Health Insurance (NHI) program, which covers more than 99 % of the island’s population and has contracted with 97 % of the hospitals. Data analyzed in this study were retrieved from the Taiwan National Health Insurance Research Database (NHIRD), which is managed by the Taiwan National Health Research Institute (NHRI). Details of this population-based database have been described previously. Diagnoses were coded with the International Codes of Disease 9th Edition Clinical Modification (ICD-9-CM).The study subjects were retrieved the newly defined NSCLC with brain metastases from the NHIRD between January 1, 2004, and December 31, 2010. The diagnostic accuracy of NSCLC was confirmed by inclusion in the Registry for Catastrophic Illness Patient Database (RCIPD), a subpart of the NHIRD. Histological confirmation of NSCLC is required for patients to be registered in the RCIPD.n = 20241), WBRT + gefitinib (n = 3379), WBRT+ GK (n = 155), and WBRT+ gefitinib + GK (n = 99).There were a total of 60149 patients diagnosed as NSCLC and 23874 (39.6 %) with brain metastasis in the study cohort were divided into the aforementioned four cohorts. The WBRT was comprised of a radiation dosage of 24 to 30 Gy in 8 to 10 fractions. As first line treatment in Taiwan, WBRT either with or without craniotomy was delivered. In general, GK was utilized if the following criteria were met: number of lesions <3, individual size of tumor <20 cc, and KPS > 70. Gefitinib was used in patients if there was evidence of an EGFR mutation. Gamma knife or gefitinib could also be utilized at the discretion of the treating neurosurgeon and medical oncologist, and in such circumstances, the decision was based on the patient’s medical records and imaging data contained within the central bureau of Taiwan National Health Insurance Institute. Thus, patients were allocated into four treatment groups: WBRT  after nested polymerase chain reaction as previously described [Mutation analysis was conducted in an institutional core facility regulated by the Clinical Laboratory Improvement Amendments as below. The analysis of EGFR gene mutations were conducted in paraffin-embedded tissue sections from the primary lung cancer. Tumor tissue was scraped from the glass side under direct visualization or under a dissecting microscope. DNA was extracted with a QIAmp DNA Mini Kit . EGFR mutations were performed by DNA sequencing as follows. escribed .n = 1), C (n = 1), 4C (n = 3) and Perfexion (n = 2) (Elekta AB). All GK was delivered via a multidisciplinary approaching and the team consisted of a neurosurgeon, neuroradiologist, radiation oncology and medical physicist. The GK technique followed a treatment guideline developed by the Taiwan neurosurgical association. The dosage prescription and number of lesions treated were individually determined by expertise teams.All patient were treated with the Leksell gamma knife; Gamma Knife units utilized included a model B  were calculated. The proportional hazards assumption was tested graphically and by including the interaction of time with each covariate. A two-tailed p value of <0.05 was considered statistically significant. All statistical analyses were performed with SAS .The characteristics of patients stratified by age, sex, number of CCI, and status of decease was shown in Table The median and minimum follow up time in WBRT were 0.53 and 0.03 years, respectively. The median and minimum follow up time in WBRT + gefitinib were 1.01 and 0.05 years, respectively. The median and minimum follow up time in WBRT + GK were 1.46 and 0.074 years, respectively. The median and minimum follow up time in WBRT+ gefitinib + GK were 2.25 and 0.151 years, respectively.At last follow up, 61.9 % of patients were deceased in the entire study population. Those deceased comprised 62.9 % of the WBRT alone group, 56.8 % of the WBRT+ gefitinib group, 44.5 % of the WBRT + GK group, and 52.5 % of the WBRT+ gefitinib + GK group. The characteristic of patients was subcategorized by gender, and the data showed very similar demographics compared to the whole series.p < 0.0001). The crude hazard ratio (95 % CI) of WBRT+ gefitinib, WBRT+ GK, WBRT+ gefitinib + GK related to WBRT were 0.56 (0.62-0.68) (p < 0.0001), 0.43 (0.34-0.54) (p < 0.0001), and 0.40(0.30-0.52) (p < 0.0001). The adjust hazard ratio by age, sex, and CCI were 0.73 (0.70-0.78) (p < 0.0001), 0.49(0.36-0.66) (p < 0.0001), and 0.42(0.30-0.59) (p < 0.0001). In addition, if the hazard ratio was only adjusted by sex, the statistical data did not reveal a significant difference between male and female groups.The hazard ratio of death associated with different treatment modality is detailed in Table The rate of patients who underwent brain surgery were 2.1 % of WBRT group, 3.3 % of WBRT + gefitinib group, 12.9 % of WBRT + GK group, and 13.1 % of WBRT+ gefitinib + GK group, respectively. For examining whether “brain surgery” was a confounder of present study outcome, we conducted a multivariate model which adds “brain surgery” as a variable for adjustment (Table p < 0.0001). There was also significantly increased survival between WBRT + GK and WBRT (p < 0.0001), WBRT+ gefitinib and WBRT (p < 0.001). These data also demonstrate that WBRT followed by a combination of gefitinib and GK exerted a significantly increased survival as compared to gefitinib or GK alone . This finding indicates that therapeutic benefit of the GK and gefitinib combination on patient survival. In addition, the survival curves showed no significant difference stratified by sex (Fig. The number of deaths and cumulative death rate by follow-up time are shown in Table Brain metastases occur in as many as 47 % of patients with recurrence adenocarcinoma of lung . Even wiIn a trail of 1692 patients randomly assigned to receive either gefitinib or placebo without any selection according to molecular characteristic, the primary end-point of this study showed no significant difference between groups, neither in overall survival nor among the 812 patients with adenocarcinoma . AnotherCharlson comorbidity index was first developed to predict the risk of mortality using the medical record . The CCIIn recent years, management of brain metastasis from NSCLC has been refined and now includes surgical resection for single brain lesions . The surGefitinib has a low molecular weight and excellent cell penetration; animal studies have demonstrated a low concentration of 14C-labeled gefitinib in normal rat brain and spinal cord  and signControversy exists regarding the optimal treatment of brain metastases. Randomized trial comparing SRS alone to WBRT and SRS combined have shown conflicting results for patients with 1–4 brain metastases . AdditioIn 2013, RTOG published a randomized trial of 126 patients treated with WBRT and stereotactic radiosurgery alone versus WBRT and SRS with temozolmide or erlotinib for NSCLC. The results showed no improved survival and possibly deleterious effect with erlotinib . The resThe distributions of patients in four groups don’t seem appropriately balanced. The distribution of patients for the groups was WBRT for 20241, WBRT + IRESSA for 3379, WBRT + GK for 155, and WBRT + IRESSA + GK for 99 patients. There should present a debate that why more physicians/neurosurgery decided to treat those patients with WBRT + IRRESA instead of WBRT + GK. The way of the decision was based on our insurance policy that in those patients with EGFR mutation and brain metastases, the physician jumped to the decision of WBRT combined with IRRESSA. In the patients without ERGR mutation, the WBRT either combined with or without GK was then conducted. Hence, there were more patients treated with WBRT + IRRESA than WBRT + GK.There were some limitations in our study. First of all, in general, WBRT was the standard treatment in our patients. Gefitinib is persevered for patient with EGFR mutation and SRS for brain lesions less than 3. The better outcome in our combined treatment compared to WBRT alone cohort had the possibility of selection biases. Second, in our favorable outcome group as compared to WBRT alone, the age distribution seemed younger than those in WBRT. There will be a confounding factor in predicting the survival. Further Phase II study should be conducted to assessment these influence.Gamma knife radiosurgery or gefitinib when combined with WBRT increased the survival of NSCLC patients with brain metastases. The combination of GK and gefitinib following WBRT afforded the greatest survival benefit. Further study of therapies combined targeted therapies such as EGFR inhibitors and radiosurgery should be undertaken to identify brain metastasis patients most likely to benefit from aggressive, multimodality treatment. Further Phase II study should be conducted to assessment these influence.


Epidemiological and animal-based studies have suggested that prenatal and postnatal fluoride exposure has adverse effects on neurodevelopment. The aim of this study was to examine the relationship between exposure to fluoridated water and Attention-Deficit Hyperactivity Disorder (ADHD) prevalence among children and adolescents in the United States.Data on ADHD prevalence among 4-17 year olds collected in 2003, 2007 and 2011 as part of the National Survey of Children’s Health, and state water fluoridation prevalence from the Centers for Disease Control and Prevention (CDC) collected between 1992 and 2008 were utilized.State prevalence of artificial water fluoridation in 1992 significantly positively predicted state prevalence of ADHD in 2003, 2007 and 2011, even after controlling for socioeconomic status. A multivariate regression analysis showed that after socioeconomic status was controlled each 1% increase in artificial fluoridation prevalence in 1992 was associated with approximately 67,000 to 131,000 additional ADHD diagnoses from 2003 to 2011. Overall state water fluoridation prevalence (not distinguishing between fluoridation types) was also significantly positively correlated with state prevalence of ADHD for all but one year examined.Parents reported higher rates of medically-diagnosed ADHD in their children in states in which a greater proportion of people receive fluoridated water from public water supplies. The relationship between fluoride exposure and ADHD warrants future study.The online version of this article (doi:10.1186/s12940-015-0003-1) contains supplementary material, which is available to authorized users. Attention-Deficit Hyperactivity Disorder (ADHD) is the most common neurodevelopmental disorder of childhood . It is cADHD is considered to develop from an interaction between genetic and environmental factors -6, with The U.S. is one of the most widely fluoridated countries in the world, with approximately 74.6% of the population on public water systems and 67.1% of the total population receiving fluoridated water from public water systems for the prevention of dental caries . The vasRats exposed to fluoridation chemicals have been shown to exhibit ADHD-like symptoms. Male rats whose mothers were injected with 0.13 mg/L of sodium fluoride two to three times per day during gestation days 14-18 or 17-19 had symptoms of hyperactivity at nine weeks of age. Juvenile and adult rats who drank water fluoridated at 100 mg/L for six weeks and 125 mg/L for 11 weeks, respectively, exhibited hypoactivity and impaired attention . Althougα4 and α7 nAChR subunit genes  had attention deficit disorder or attention-deficit/hyperactivity disorder, that is, ADD or ADHD”. In the 2007 and 2011 NSCH, if the parent answered yes, he or she was asked whether the child was currently diagnosed with ADHD and, if so, how severe it is. In 2011, the responding parent was also asked the age of diagnosis . LifetimExtracted from the original sample of children aged 0-17, three subsamples of children aged 4-17 living in the U.S. were used to assess ADHD prevalence per state in 2003 , 2007 , and 2011 . The lifetime prevalence of ADHD increased over time and was 7.8% in 2003, 9.5% in 2007, and 11% in 2011. ADHD prevalence was also higher for males, children of lower socioeconomic status (SES), older children, and for children whose parents had a high school education as compared to those whose parents either did not graduate high school or attained postsecondary education [Data on the number of people receiving fluoridated water from public water supplies in each of the 50 states and the District of Columbia in 1992 , 2000 , 2002 , 2004 , 2006 , and 2008  was also obtained from the CDC website [To calculate the percentage of each state receiving optimally fluoridated (i.e. according to the DHHS recommendations) water from public water systems  state population estimates were obtained from the United States Census website ,61. The Descriptive statistics were calculated for U.S. water fluoridation prevalence for all years examined. Statistical comparisons of ADHD prevalence and water fluoridation prevalence between geographic regions were determined using one-way ANOVA followed by Bonferroni post hoc test in all cases except for regional fluoridation prevalence comparisons in 2000 and 2002. In those cases Games-Howell’s test was used due to heterogeneous variances. Pearson correlations were used to examine relationships between state water fluoridation prevalence and state ADHD prevalence. These were not corrected for family wise error given the exploratory nature of this study. Hierarchical and multivariate regression analyses were conducted to examine the relationship between artificial water fluoridation prevalence and ADHD prevalence after controlling for natural water fluoridation prevalence and SES, and SES respectively. A one-tailed alpha level of 0.05 was the criterion for statistical significance for all analyses. A Bonferroni correction was applied to the univariate analysis of the multivariate regression however, making the criterion for significance for that analysis an alpha of 0.017.Median percentages and interquartile ranges of the U.S. population receiving optimally fluoridated water from public water systems in 1992, 2000, 2002, 2004, 2006 and 2008 are presented in Table ADHD and water fluoridation prevalence were organized in Tables Differences in water fluoridation prevalence between regions were also statistically significant in 1992  = 15.05, p = .000), 2000  = 12.21, p = .000), 2002  = 13.20, p = .000), 2004  =15.07, p = .000), 2006  = 13.28, p = .000), and 2008  = 8.88, p = .000). Similar to ADHD prevalence, water fluoridation prevalence in all years examined was lower in the West than in all other regions, but not significantly lower than the North East. In 2004, 2006 and 2008 water fluoridation prevalence was also higher in the South than in all regions, but not significantly higher than the Mid-West.Since artificial and natural water fluoridation prevalence per state was only distinguished in 1992, the relationship between each and ADHD prevalence was of primary focus and examined separately. States with higher artificial fluoridation prevalence had significantly higher ADHD prevalence in 2003 (r (49) = .46, p = .000), 2007 (r (49) = .42, p = .001), and 2011 (r (49) = .48, p = .000). Natural fluoridation prevalence in 1992 however, was not significantly related to ADHD prevalence in 2007 or 2011, r (49) = −.19, p = .09, and r (49) = −.22, p = .06 respectively, but was significantly negatively associated with ADHD prevalence in 2003, r (49) = −.29, p = 0.02.The relationship between overall state water fluoridation prevalence  and state ADHD prevalence in later years was also examined. Positive associations were found between the two for all years examined, except between water fluoridation prevalence in 2008 and ADHD prevalence in 2007 (p = .07). These correlations were numerically smaller however, than between artificial water fluoridation prevalence and ADHD prevalence  = 7.91, p = 0.000, and accounted for 33.5% of the variance in 2003 parent-reported health care provider-diagnosed ADHD. In the final model, artificial water fluoridation prevalence significantly and independently positively predicted 2003 ADHD prevalence, B = 0.017, t (47) = 2.16, p = 0.036, while natural water fluoridation prevalence and median household income in 1992 (measured in hundreds of dollars) significantly negatively predicted it, B = −0.071, t (47) = −2.21, p = 0.032 and B = − 0.010, t (47) = 2.62, p = 0.012 respectively. Therefore, while higher artificial fluoridation prevalence in 1992 was associated with higher parent-reported health care provider-diagnosed ADHD prevalence in 2003, higher natural fluoridation prevalence and median household income in 1992 were each associated with lower 2003 ADHD prevalence.A multivariate hierarchical regression analysis was also conducted to examine the unique relationships between artificial fluoridation prevalence and ADHD in all three years of interest after median household income in 1992 was controlled  = 8.71, p = 0.001), 2007  = 7.94, p = 0.001) and 2011  = 12.21, p = 0.000), accounting for 24%, 22% and 31% of the variance in ADHD prevalence respectively. In the final model, artificial fluoridation prevalence in 1992 significantly and independently predicted parent-reported health care provider-diagnosed ADHD in all three years examined, Wilks λ = .81, F  = 3.64, p = 0.02, while the predictive relationship between median household income in 1992 and ADHD prevalence in all three years was reduced to that of a trend, Wilks λ = .86, F  = 2.48, p = 0.07. After applying a Bonferroni correction, artificial fluoridation prevalence in 1992 significantly predicted ADHD prevalence in 2003,  = 3.05, p = 0.004), 2007  = 2.64, p = 0.011), and 2011  = 3.20, p = 0.002). Thus, after adjusting for socioeconomic status, a 1% increase in artificial water fluoridation prevalence in 1992 was associated with a 0.023% increase in ADHD prevalence in 2003 , a 0.031% increase in ADHD prevalence in 2007  and a 0.043% increase in ADHD prevalence in 2011 . Median household income in 1992 (measured in hundreds of dollars) did not meet the threshold for significance in predicting ADHD prevalence in 2003 (p = 0.061) or 2007 (p = 0.035), but did so in 2011    within sSince states of lower SES tended to have higher artificial water fluoridation prevalence and ADHD prevalence, another important area of investigation was whether artificial water fluoridation prevalence in 1992 still predicted ADHD prevalence after SES was considered. That is, did children and adolescents in states with higher artificial water fluoridation prevalence merely have higher rates of ADHD because they tended to be of lower socioeconomic status and therefore more likely to have additional ADHD risk factors? Results showed that this was not the case and prevalence of artificial water fluoridation in 1992 did indeed predict ADHD prevalence independent of SES. Moreover, artificial water fluoridation prevalence even appeared to be the more robust predictor.Although more research is needed to investigate the relationship between exposure to fluoridated water and increased ADHD prevalence, there are two main pathways by which exposure to fluoridated water could theoretically contribute to the disorder. First, silicofluoride-treated water has been shown to corrode lead-bearing plumbing, increasing the leaching of lead in the water . SilicofSecond, exposure to fluoridated water may contribute to ADHD via suppression of the thyroid gland. Fluoride reduces thyroid gland activity -75 and tEven though current findings indicate a relationship between ADHD prevalence and fluoride exposure that occurs through the optimal fluoridation of public water systems, there are several study design limitations that should be considered. First, this study is an ecological design that broadly categorized fluoride exposure as exposed versus non-exposed rather than collecting information related to concentration of fluoride and patterns and frequency of exposure or outcome at the individual level. Future research could explore the relationship between exposure to fluoridated water and the occurrence of ADHD at the individual level. Further clarification of a potential dose–response relationship between fluoride exposure and ADHD symptoms would also be important for determining causality. Second, given that fluoridation prevalence in neighboring years was highly correlated from 2000 onward and unavailable for the mid to late 90s, it could not be determined whether exposure to fluoridated water at a particular period of development was most associated with increased ADHD prevalence. Nevertheless, given that other research has demonstrated the developing brain’s particular sensitivity to the neurotoxic effects of fluoride, it is likely that prenatal and early postnatal development presents a window of vulnerability. Third, fluoridation prevalence data was analyzed with ADHD prevalence data from different years, and therefore, it cannot be confirmed that those surveyed in a given year were living in the same region as when the fluoridation data were derived. Fourth, we were unable to obtain reliable population-based data on blood lead levels among 4-17 year old children and adolescents, and therefore could not determine whether lead was mediating the relationship between exposure to fluoridated water and ADHD. Lastly, parent-reported health-care provider-diagnosed ADHD prevalence was used in this study which is not as precise a measure as others  or may be subject to potential parent biases regarding seeking or accepting an ADHD diagnosis for their child. Therefore, the survey method used in the current study may not completely capture ‘true’ ADHD prevalence. Despite these limitations, an association between exposure to fluoridated water and ADHD prevalence was still found, even after considering the increased tendency for children in low SES states to receive an ADHD diagnosis.In summary, this study has empirically demonstrated an association between more widespread exposure to fluoridated water and increased ADHD prevalence in U.S. children and adolescents, even after controlling for SES. The findings suggest that fluoridated water may be an environmental risk factor for ADHD. Population studies designed to examine possible mechanisms, patterns and levels of exposure, covariates and moderators of this relationship are warranted.


Recent emphasis has been placed on elucidating the biologic mechanism linking socioeconomic status (SES) to cardiovascular disease (CVD). Positive associations of inflammatory biomarkers provide evidence suggestive of a biologic pathway by which SES may predispose to CVD. African Americans have disproportionately lower SES and have a higher prevalence of CVD risk factors compared to most ethnic/racial groups. Adiponectin (an anti-inflammatory marker) is also lower. The objective of this study was to assess the association of adiponectin with SES among African American men and women using the Jackson Heart Study.Study sample included 4340 participants. Linear regression was performed separately by SES and stratified by sex. Annual household income and level of education was used as proxies for SES. Crude, age, health behavior and health status adjusted models were analyzed. The main outcome was log-transformed adiponectin.p value = .16/.08, p = .0008. Men with < high school level of education had significantly higher adiponectin in the crude and age adjusted models than those with ≥ college degree . Women with some college or vocational training in the crude and age adjusted models had lower adiponectin compared to women with ≥ college degree .Men in the lowest income group had significantly higher adiponectin than those in the highest income group in the fully adjusted model (ß/standard error [se], Findings suggest a potential inverse biologic pathway between annual household income and adiponectin among African American men. There was no such finding among women. Findings suggest interventions should be targeted for higher SES African American men to improve adiponectin levels. Research has consistently demonstrated an inverse association with socioeconomic status (SES) and cardiovascular disease (CVD) related mortality, morbidity and risk factors –4. More Data from the Jackson Heart Study (JHS) was used which is a single-site, prospective cohort of risk factors and causes of heart disease in adult African Americans. A probability sample of 5301 African Americans, aged 21–95, residing in three contiguous counties surrounding Jackson, Mississippi was recruited and examined at baseline from 2000 to 2004 by certified technicians according to standardized protocols , 20. TheAdiponectin measurement was derived from venous blood samples drawn from each participant at baseline after more than 8 h of fasting. Vials of serum were stored at the JHS central repository in Minneapolis, MN, at −80 °C until assayed. Adiponectin concentration was measured in 2008–2012 as total plasma adiponectin by an ELISA system  and educational level was divided into four categories , some college or vocational school, ≥ college graduate).2). Cardiovascular disease health status variables include hypertension, type 2 diabetes, low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride, homoeostasis model assessment-insulin resistance (HOMA-IR), and C-reactive protein (CRP). Hypertension was based on a systolic blood pressure of ≥140 mmHg, diastolic blood pressure of ≥90 mmHg, or self-reported medication use for elevated blood pressure [All covariates were collected at baseline and were chosen because they are associated with adiposity and hypertension related adiponectin . Age waspressure . Fastingpressure . CRP waspressure . The intt-test for continuous variables and chi-square for categorical variables. Characteristics of men and women stratified by annual household income and level of education were performed with one-way ANOVA for continuous variables and chi-square for categorical variables. Differential age adjusted mean levels of adiponectin for men and women by the three categories of income and four categories of education were graphed for descriptive purposes via one-way ANOVA. Adiponectin was based on sample mean.All analyses were sex stratified because of the differential levels of adiponectin between men and women . Descripp ≤ .05. Analyses were conducted using SAS version 9.3 [Multiple linear regression analysis was utilized to test the association separately between two SES groups  and log-transformed adiponectin with crude, age, health behavior and health status adjusted variables; ≥ $50,000 and ≥ college graduate were entered as referents for annual household income and level of education, respectively. Four separate models per SES group were utilized to test the effects on log-transformed adiponectin in a stepwise fashion. Model 1 was crude and included annual household income and educational level, while model 2 added age entered as a continuous variable. Model 3 added health behavior variables . Body mass index ≥25, smoking status, and alcohol consumption were entered as binary variables; physical activity was continuous. The fully adjusted model added health status variables . Triglycerides, HDL, LDL, HOMA-IR and CRP were entered as continuous variables; hypertension and type 2 diabetes were entered as binary variables. Tests of linear trends were also conducted to examine the association of SES sub-groups with levels of log-transformed adiponectin which were exponentiated (i.e. geometric means) after model adjustments based on general linear model. This was done to assess change in the magnitude of difference in adiponectin level between the highest and lowest SES sub-groups and to assess threshold effects between levels after adjustments as a measure of SES disparity and adiponectin level. A two-tailed level of significance was established as p = .002). A higher proportion of men had a higher household income of ≥ $50,000 compared to women . A higher proportion men were current smokers (17.7 % versus 11 %), consumed alcohol (60 % versus 40 %) and had a higher mean physical activity score (8.7 versus 8.2) when compared to women . Women on the other hand, had significantly higher mean BMI . A higher proportion of women were also hypertensive (64 % versus 60 %), had type 2 diabetes (19 % versus 16 %) and had higher mean HDL cholesterol (55 mg/dL versus 46 mg/dL), HOMA-IR (3.7 versus 3.4), CRP (.60 mg/dL versus .37 mg/dL), and plasma adiponectin (6.0 ug/mL versus 4.1 ug/mL) when compared to men . Men had higher mean systolic and diastolic blood pressure , LDL cholesterol (128.7 mg/dL versus 125.5 mg/dL), and triglyceride (114.1 mg/dL versus 100.3 mg/dL) .The sex-stratified characteristics of the study population are presented in Table p = <.0002, .0012, .0006, .003, respectively). On the other hand, physical activity, alcohol consumption, BMI, diastolic blood pressure, and LDL cholesterol was significantly higher among men in the highest annual income category of ≥ $50,000 when compared to those in the other income groups . Type 2 diabetes, and triglyceride were higher among those with an income of $20,000–$49,000 . Relatively the same pattern was observed among women. The exception was that those in the lowest income group of ≤ $19,000 had significantly higher BMI, type 2 diabetes, and triglyceride when compared to women in the other income groups . Educational level with mean age and health behavior and health status patterned similarly among men and women as indicated in Table There was a significant differential pattern of mean age and health behavior and health status according to annual income among men and women as revealed in Table p < .0001). There was no difference among women. Mean adiponectin was also significantly different among men and women based on level of education with those with < high school level of education having higher adiponectin .Figure p < .0001) and persisted in the fully adjusted model (β (se) = .16 (.05); p = .0008). The reduction in model coefficients ranged from 19 to 38 %. Those with a medium annual income of $20,000–$49,000 had marginally higher plasma adiponectin than men with a higher annual income of ≥ $50,000. Men with < high school level of education had significantly higher adiponectin in the crude model (β (se).25 (.05); p < .0001) and remained significant when adjusted for age (β (se) .14 (.05); p = .005), but significance disappeared after adjustment for health behavior and health status; the coefficients between both models were reduced by 44 %. There was no association among men with high school or GED level of education or among those with some college or vocational training.Table p = .004). When adjusted for age, women with < high school level of education and those with some college or vocational training had significantly lower plasma adiponectin than women with ≥ college level (β (se)−.08 (.04), p = .03,−.06 (.03), p = .04, respectively). A 33 % reduction in coefficients between crude and age adjusted models was observed among those with some college or vocational training. There was no association in the models adjusted for health behavior or health status.There was no significant association between annual household income and plasma adiponectin observed among women in any of the models. In the crude model, women with some college or vocational training had significantly lower levels of adiponectin than women with ≥ college level of education (β (se) .09 (03), p < .0001) in the crude unadjusted model. This pattern persisted in each of the subsequently adjusted models. There were no differences observed among women by level of income in the crude, age, health behavior, and health status adjusted models. In the crude model, men who had an education level of < high school and those with high school or GED, had higher levels of mean adiponectin than men with ≥ college level of education . A similar pattern was observed in the age-adjusted model (p = .006). But differences by education level were attenuated in the models adjusted for health behavior and health status. Women with an education of < high school, high school or GED, and those with some college or vocational training had lower levels of adiponectin than women with ≥ college level of education  after adjustment for age. This significance, however, disappeared in the fully adjusted model.Table Consistent observations of SES gradients in health outcomes has led to the search for pathways through which social status may impact health-including biological mechanisms through which social characteristics and experiences may affect functioning and disease outcomes. One biological pathway that is gaining increasing interest is subclinical inflammatory processes that are thought to be involved in both the development and progression of a number of diseases, including cardiovascular, for which there are known SES gradients –10. AdipTo our knowledge, there are only three publications that investigated the relationship between SES and adiponectin. Khanolkar, Vagero and Koupil investigated the association of occupational class and educational level among a sample of Swedish men 50 to 70 years of age . They foThe strength of our investigation is that findings were from the largest community-based sample of African Americans, a cohort with strict protocol and high quality-control. It also addresses a risk factor (i.e. adiponectin) associated with CVD that disproportionately affects African Americans. In addition, it presents contrary new insight into historical findings concerning SES gradients and health-particularly among men. Finally, it is among the first to report on the association between SES and adiponectin based on a sample population in the United States. One limitation of the study is that findings cannot be generalizable to other ethnic groups. Secondly, this is a cross-sectional analysis; thus, we cannot establish a causal relationship between SES and adiponectin. Furthermore, residual confounders may have impacted the results. It is also important to mention that we use level of education and annual household income as proxy measures of SES and not a composite measure of SES. Although we adjusted for several known confounders, our study did not adjust for other factors such as dietary intake, sex hormones, and specific adiposity measures such as visceral fat. Finally, our study used total adiponectin rather than high molecular weight (HMW) adiponectin, which is considered the most biologically active form. This could potentially affect our findings since some studies suggest differences in biological activity between different isoforms of adiponectin and metabolic abnormalities . HoweverThe major finding of our study revealed that, after adjustments for age, health behavior and health status, adiponectin was significantly higher in African American men in the lowest category of income compared to those in the highest income category. Adiponectin was also higher among men with the lowest level of education after adjustment for age. Women with < high school and some college or vocational level of education had lower adiponectin than women with the highest level of education after age adjustment. Findings suggest a potential inverse biological pathway associated with annual household income as a measure of SES and adiponectin in men. Findings further suggest that perhaps intervention should be targeted to higher SES African American men to improve adiponectin level.BMI, body mass index; CRP, C-reactive protein; CVD, cardiovascular disease; GED, graduate equivalency diploma; HDL, high-density lipoprotein; HOMA-IR, homoeostasis model assessment – insulin resistance; JHS, Jackson Heart Study; LDL, low-density lipoprotein; SES, socioeconomic status


Enterococcus faecalis biofilm formation. Sixty dentin blocks with 3-week E. faecalis biofilm were divided into six equal groups and flushed with irrigant for 2 min or left untreated. A blank control group was also established. Antibacterial activities of the irrigants were evaluated by counting colony forming units. To test residual antimicrobial activities, 280 dentin blocks were divided into seven equal groups and flushed with irrigant for 2 min or left untreated and then incubated with E. faecalis suspension for 48 h, or used as a blank. No bacteria were observed in the blank control group. The number of viable E. faecalis was significantly fewer in the irrigant-treated groups compared with the untreated control (P < 0.05). Among the five irrigants, QMix had the strongest antibacterial activity. Residual antimicrobial activities of CHX were significantly higher at 12 h, 24 h and 36 h compared to untreated control (P < 0.05). All five root canal irrigants were effective to some extent against E. faecalis, but QMix and CHX had the strongest, and CHX the longest (up to 36 h), antimicrobial activity.We compared the antibacterial and residual antimicrobial activities of five root canal irrigants  in a model of  Enterococcus faecalis is the predominant bacterial species in teeth with persistent periapical periodontitis23Enterococcus faecalis is detected in 24%–77% of the filled root canals with persistent periapical lesions and usually exists in the infected root canals in the form of a biofilm6E. faecalis and provides a matrix for deeper bacterial infection, thus influencing the effect of root canal irrigants on bacterial removalE. faecalis biofilm has strong viability and drug resistance.The root canal biofilm is the main reason for root canal treatment failureE. faecalis absolutelyE. faecalis at some extent10E. faecalis are weaker than those of MTAD and TetracleanE. faecalis rapidly, as can 0.2% CHX13Several root canal irrigants have antimicrobial activities when contacting microorganisms directly, but few can eliminate E. faecalis biofilm infection of dentin blocks.Currently, most studies focus on the antibacterial activities of different irrigating solutions. However, there are few studies on the residual antimicrobial activities of root canal irrigants. Therefore, this study aimed to compare the antibacterial and residual antimicrobial activities of five root canal irrigants  in a model of A total of 200 freshly extracted, intact maxillary and mandibular molars without preoperative treatment, root caries, and cracks were collected from the maxillofacial department of Tianjin Medical University Dental Hospital. The study was approved by the Medical Ethics Committee of Tianjin Medical University and the methods were carried out in accordance with the Declaration of Helsinki (2008). All patients have obtained the informed consent.The dental calculus and soft tissue remnants of all molars were mechanically cleaned with a periodontal curette, and then stored in a 0.1% thymol solution at 4 °C until required. Next, a total of 350 dentin blocks (2 mm × 2 mm × 1 mm [width × length × height]) were prepared by selecting the coronal one-third of the roots (from the cementoenamel junction); then, the pulpal side of the dentin blocks was polished with 600-grit silicon carbide abrasive paper . The dentin blocks were immersed into 3% NaOCl  for 1 min, and then transferred into 17% EDTA  for 2 min to remove the smear layer. Next, the dentin blocks were steam autoclaved for 30 min under 15 psi pressure at 121 °C to ensure that no bacteria remained. All samples were preserved in 37 °C brain heart infusion  for 24 h to test for the presence of bacteria. After confirming complete sterilization, all dentin blocks were put into sterile saline.E. faecalis preserved at −80 °C were thawed, they were inoculated into freshly prepared BHI and cultivated under anaerobic condition at 37 °C for 24 h. Then, a bacterial suspension with 1 × 107/ml E. faecalis was obtained by dilution with BHI broth.After the standard strains of Five root canal irrigants were used in the present study: 17% EDTA , 2% CHX (Sigma-Aldrich), 0.2% CTR (Sigma-Aldrich), MTAD , and QMix .E. faecalis suspension was added to each tube. After sealing with Parafilm®, the tubes were incubated under aerobic condition at 37 °C for 3 weeks. During this period, BHI broth was changed every other day to maintain normal growth of E. faecalis. Before each exchange, two samples of broth culture were collected randomly. A portion from each sample was inoculated into BHI, and further cultivated at 37 °C for 24 h. Finally, some of the bacterial colonies were dyed with Gram stain. Finally, the E. faecalis-infected models were established.A total of 60 dentin blocks were randomly selected from the initial 350 blocks and put into 200 μl tubes (one block per tube). Then, 180 μl The 60 infected dentin blocks were flushed with 180 μl sterile saline for 2 min, and put into 96-well plates (one block per well). The dentin blocks were randomly divided into six groups: EDTA, CHX, CTR, MTAD, QMix, and untreated control groups (n = 10 per group); ten sterile dentin blocks were used as the blank control group. The five different root canal irrigants or BHI broth were added into the corresponding wells (100 μl per well). After 2-min incubation, the dentin blocks were removed and dried with aseptic absorbent paper. Next, the dentin blocks were centrifuged in 200 μl BHI for 2 s and vortexed  for 1 min. The supernatants in the centrifuge tubes were removed by pipette and diluted ten times. The diluted supernatants were inoculated into BHI medium and cultivated under anaerobic conditions at 37 °C for 24 h. Finally, the colony-forming units (CFUs) were counted.Enterococcus faecalis suspension (180 μl) was added to the treated and untreated control groups, while an equal volume of BHI broth was added to the blank control group.The remaining 280 sterile dentin blocks were randomly divided into seven groups: EDTA, CHX, CTR, MTAD, QMix, untreated control, and blank control groups (n = 40 per group). The dentin blocks were put into 96-well plates (one block per well). The five different root canal irrigants or BHI broth were added into the corresponding wells (100 μl per well). All blocks were removed after 2 min and put into 200-μl tubes. All samples were incubated at 37 °C and cultivated under anaerobic conditions for 48 h. Every 12 h, ten samples were collected from each group to count the CFUs using the same method as described above.P-value of less than 0.05 was considered to be statistically significant.After the values of CFUs were converted into lgCFUs, data expressed as mean ± standard deviation were analyzed by SPSS version 17.0 statistical software . The differences in the antibacterial and residual antimicrobial activities were analyzed by one-way and multivariate ANOVA. A E. faecalis IgCFUs in dentin blocks treated with EDTA, CHX, CTR, MTAD, and QMix were significantly reduced (P < 0.05) (P < 0.05), except between CHX and QMix groups (P > 0.05).No bacteria were observed in the blank control group. Compared with the untreated control group,  < 0.05) , Fig. 1.P > 0.05). On the other hand, the lgCFUs of the CTR and QMix groups were significantly lower than those of the untreated control group  at 12 h. There was no significant difference in the residual antimicrobial activities among the EDTA, MTAD, QMix, and the untreated control groups at 24 h (P > 0.05). However, the lgCFUs of the CHX and CTR groups were significantly lower than those of the untreated control group  at 24 h. At 36 h, only the lgCFUs of the CHX group was significantly lower than those of the untreated control group . There was no significant difference in the residual antimicrobial activities among the treated and untreated control groups at 48 h (P > 0.05).Differences in the residual antimicrobial activities among the six groups at 12 h, 24 h, 36 h, and 48 h are shown in Enterococcus faecalis, which usually exists in the form of a biofilm, is considered one of the most intractable bacteria. Kishen et al. have determined the chemical composition of E. faecalis biofilms and observed apatite deposition on mature biofilmE. faecalis adhesion and drug resistance, making it more difficult to remove. This study was designed to identify the most effective root canal irrigant against E. faecalis, in the hopes of improving root canal treatment outcomes.E. faecalis biofilmWe discovered that 2% CHX had similar antibacterial activity to QMix. It has been reported that 2% CHX can sterilize 99.93% of the bacteria in E. faecalis in many studies10E. faecalis was weaker than that of the other four irrigants. As a chelating agent removing the inorganic components in the smear layer, EDTA could change the permeability of cell membrane; this is the mechanism for its antibacterial activityE. faecalis exhaustively.EDTA has not shown an obvious antibacterial effect against E. faecalis on the dentin13Previous studies have shown that 0.1% CTR and 2% CHX could sterilize similar amount of E. faecalis biofilm formation. At 12 h, the fewest viable bacteria were detected in the QMix group, followed by the CTR group. However, at 24 h, the number of viable bacteria in QMix group increased significantly, suggesting that residual antimicrobial activity of QMix was strong, yet of short duration. Although there were more viable bacteria at 24 h than at 12 h, the CTR group had the lowest CFUs at 24 h, and residual antimicrobial activity of CTR remained until at least 36 h. Therefore, residual antimicrobial activity of CTR was more stable and durable.Residual antimicrobial activities of the five irrigants were also examined to evaluate their effects on inhibiting E. faecalis at 12 h or CHX had a weak residual antimicrobial activity. Since the number E. faecalis increased gradually, but the residual antimicrobial activity of CHX did not change over time, the effect of CHX became gradually decreased. Therefore, compared with CTR and QMix, CHX had the weakest yet the most stable residual antimicrobial activity. Baca et al. have tested the residual antimicrobial activities of CHX and CTR with a fresh bacteria suspension and found that CHX and CTR can last for more than 40 daysE. faecalis gradually increases as nutrients are depleted and toxic waste products accumulate, and E. faecalis ultimately enter the decline phase. Residual antimicrobial activities of EDTA and MTAD may not have been detected because of this experimental limitation. In addition, a relatively large volume of bacterial suspension was added, and the small amount of irrigating solution remaining on the dentin blocks was diluted, which would decrease its residual antimicrobial activity.Residual antimicrobial activities of irrigants weaken gradually and finally disappearE. faecalis in the matured biofilm at different levels. Their antibacterial activity from strong to weak was CHX and QMix, MTAD, CTR, and EDTA. However, only CHX, CTR, and QMix had residual antimicrobial activities that lasted for at least 36 h, 24 h, and 12 h, respectively.In conclusion, EDTA, CHX, CTR, MTAD, and QMix can kill How to cite this article: Zhang, R. et al. Antibacterial and residual antimicrobial activities against Enterococcus faecalis biofilm: A comparison between EDTA, chlorhexidine, cetrimide, MTAD and QMix. Sci. Rep.5, 12944; doi: 10.1038/srep12944 (2015).


Cell proliferation was assayed over time, and migration was evaluated by Live Cell imaging microscopy. Fibroblasts grown in collagen-gels were treated with ORC or ONRC, and ECM contraction was measured utilizing a contraction assay. An absolute pH decline was observed with both ORC and ONRC after 1 hour. Mean daily cell proliferation, migration and matrix contraction were more strongly inhibited by ONRC when compared with ORC (p < 0.05). When control media was pH-lowered to match the lower pH values typically seen with ORC and ONRC, significant differences in cell proliferation and migration were still observed between ONRC and ORC (p < 0.05). However, in these pH conditions, inhibition of matrix contraction was only significant for ONRC (p < 0.05). We find that ORC and ONRC inhibit fibroblast proliferation, migration and matrix contraction, and stronger inhibition of these essential cellular processes of wound healing were observed for ONRC when compared with ORC. These results will require further validation in future in vivo experiments to clarify the clinical implications for hemostat use in post-surgical wound healing.In this study we investigated how hemostats such as oxidized regenerated cellulose  and oxidized non-regenerated cellulose  influence local cellular behavior and contraction of the extracellular matrix (ECM). Human stromal fibroblasts were inoculated  Vascular surgery patients are at elevated risk to develop superficial and deep incisional surgical site infections, with increased morbidity leading to prolonged hospital stays. These complications are common, with an overall risk of 3–44%, leading to enormous costs for health systems124Wound healing is hierarchically regulated based on an interplay between cellular, humoral and molecular mechanisms, and begins immediately after wounding88in vivo; however non-oxidized hydroxyl groups remain as a fibrous component which require phagocytosis by macrophages prior to hydrolysisin situ remaining hemostats on the postoperative wound healing process is poorly investigated so far.Chemical hemostats are often used by surgeons for local bleeding control after performing vascular anastomoses. Although, newer materials such as plasma-derived thrombin and fibrinogen have shown satisfying results, cellulose-based hemostats are still commonly used, as they are highly cost-effectiveThe objective of this study is to compare an ONRC-based hemostat  with an ORC-based hemostat  regarding their effects on fundamental processes of deep incisional wound healing (DIWH), including cell proliferation and migration, and contraction of the extracellular matrix (ECM).Postoperative wound healing is highly dependent on the interaction between different cell lines; however, fibroblasts seem to be of particular relevance for DIWH. For that reason, human fibroblasts were used for analyzing key cellular sub-processes. As degradation of ORC and ONRC initially consists of hydrolysis with release of acidic groups, we investigated the pH-value time course for both hemostats.A significant decline in absolute pH was observed for ONRC and ORC compared to controls. Over the first hour there was a significant pH drop with both hemostats in normal saline. After 1-hour absolute pH value remained constantly at approximately 2.5 for ONRC and ORC  p < 0.0.3 Δcells/day ± 180 Δcells/day) and ORC (1.6 × 103 Δcells/day ± 790 Δcells/day) as well as for the second week  when compared to controls (1.2 × 104 ± 1.3 × 103 Δcells/day: 3.8 × 103 ± 1.4 × 103 Δcells/day) (p < 0.05). Overall, ONRC displayed lower mean daily cell proliferation compared to ORC over both weeks (p < 0.05). There was near-total inhibition of cell proliferation within the second week for ONRC, whereas proliferation remained similar for ORC over the full duration . After 24 h, coverage of the inter-chamber gap was 98.1% ± 2.3% for controls, and 88.1% ± 5.7% and 80.5% ± 4.2% for ORC and ONRC respectively .2 ± 24.3 mm2; 48 h: 127 mm2 ± 24 mm2) matrices showed a greater surface area after 24 and 48 hours compared to ORC  and controls  (p < 0.05). Differences between ORC and controls were not significant at either time point  (p < .05). During the second week, this effect could only be seen for ORC  (p < 0.05)  or 48 hours . In contrast, there was significant inhibition of contraction for ONRC compared to low-pH controls at 24 hours  (p < 0.05), and near-significance at 48 hours  (p = 0.052)  . Cell mi < 0.05) . Matrix = 0.052) .et al. describe superior hemostasis for ONRC, with equivalent bactericidal effectiveness when compared to ORC13in vitro to clarify their potential impact in impaired DIWH.Cellulose-based hemostats are known for high clinical convenience, as they are storable at room temperature and can be quickly deployed or removed. The macroscopic appearance of RESORBA CELL is more frayed at the edges, and overall more flexible compared to TABOTAMP. These agents are primarily used for small amounts of bleeding1516in vivo in the presence of fibroblasts utilizing cell cultures. ONRC showed lower pH values in cell culture throughout the investigation period. The hemostats showed opposite effects on pH value, particularly in the time period between 1–4 hours. Within this time frame, ONRC continued to lower the pH value whereas ORC showed an increase in pH value, similar to non-hemostat controls. Two different hypotheses might explain this behavior. The observed effect could be based on the greater surface area of the frayed fibers found in ONRC. This might lead to an increased release of acidic-groups per time unit, exceeding the buffering capacity of the culture media and lowering the pH valueAs degradation of both hemostats results in a release of acidic groups, we investigated the effect of ORC and ONRC on the pH value over time. In the absence of cells and cell culture media, there was a sudden drop in pH value within the first hour for both hemostats. After this initial decline, no further changes in pH were noted; however, we later observed a marginally lower pH for ONRC. Next, we modeled the pH values observed in humans In vivo degradation of cellulose-based hemostats was studied by Pierce et al. and Dimitrijevich et al., who showed that beyond enzymatic degradation there is a fibrous component of hemostats which requires phagocytosis by macrophages for complete clearance14Another possible explanation of the divergence in pH value in cell culture media of fibroblast cultures between the hemostats might be that alterations occur in the cellular function of fibroblasts, meaning the fibroblasts themselves might acidify the environment. in vivo. For that reason, this approach might better mimic in vivo conditions. More importantly, depletion of buffer capacity might even be essential to the findings we observed. For example, fibrous degradation components might have variable influences at different pH values.In this study we also modulated the pH value to investigate whether hemostats themselves, rather than the associated changes in pH value could be responsible for the effects of both hemostats on cell proliferation, migration and matrix contraction. To investigate this, we elected to lower the pH value of the controls rather than adding buffer to the hemostat samples, as total depletion of buffer capacity due to massive acidic group release over a short time might also occur et al. described reduced fibroblast proliferation at lower pH values, as there is a greater tendency towards contact-inhibition and decreased sensitivity to growth stimulation once confluentet al. and Liu et al. investigated the effect of ORC on fibroblast proliferation in animal models. Both authors described either no significant effect or even enhanced remodeling of the extracellular matrix, which they conclude resulted from increased cell proliferation22et al. demonstrated for fibroblast sub-populations within the skinin vivo conditions, where wound healing depends on multicellular interactions. Nevertheless, our results regarding hemostats and fibroblast proliferation stand in clear contrast to some published findings.Fibroblast function is crucial for several aspects of healthy post-surgical wound healing. Overcoming the spatial defects through generation of cell-rich tissue is aided by adequate cell proliferation. We therefore studied the influence of ONRC and ORC on cell proliferation, and demonstrated that both hemostats inhibited fibroblast proliferation compared to controls. This effect was larger for ONRC compared to ORC. Froehlich et al. found that wounds in diabetic animals treated with ORC/collagen sponges demonstrated significantly accelerated wound closure when compared to control wounds in similar animals. Histological analysis of the wound tissues suggested that using ORC/collagen sponges enhanced the formation and maturation of granulation tissue as a result of increased cell migrationet al. showed that using ORC in oncoplastic breast surgery could promote dermal fibroblast proliferation and cell migration which resulted in beneficial effects for adjustment of the shapeet al. described ORC as detrimental to wound healing in vivo, and uncovered no advantageous effects for ORC on cell proliferation and migrationin vivo results have limited applicability to our study. Their authors found that the prevention of postoperative hematoma led to increased cell proliferation, a process we did not investigate. Rather we concentrated on the direct inter-dependency between hemostats and cellular function. Moreover, the above authors used diabetic mice to study wound healing with cellulose hemostats. In that model, the antibacterial effect of hemostats might have outweighed the potential inhibiting effects on wound healing. Our study found that cell migration was inhibited in vitro for ORC and ONRC compared to controls. This effect remains after buffering controls to similar pH values produced by hemostats. Therefore, we suggest that there exists a material-based negative effect on fibroblast cell migration, which is more evident for ONRC compared to ORC.Fibroblast migration is also crucial for DIWH, helping to develop bands of connective tissue and establish immature granulation tissue. The current literature provides variable results with regard to the influence of hemostats on cell migration. Hart A balanced level of wound contraction is essential for optimal healing. Fibroblasts promote wound contraction, through activation of signaling pathways promoting a shift from fibroblast to myofibroblastin vivo models before their clinical impact can be assessed.In conclusion, both ORC and ONRC inhibit fibroblast proliferation, and migration. These effects do not exclusively depend on acidosis, suggesting that material-specific compounds such as fibrous hydrolysis end-products are responsible. Further, ONRC inhibits matrix contraction, unlike ORC. ONRC may more strongly inhibit essential cellular sub-processes, which are fundamentally important for functional wound healing. However, these findings will require further validation using Employed hemostats included RESORBA CELL (oxidized non-regenerated cellulose -ONRC) and TABOTAMP (oxidized regenerated cellulose - ORC).2 5%, with medium changes every second to third day. Medium changes were suspended during subsequent experiments – detailed below. Cells were subcultured at 80–90% confluence, using 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) . Morphological cell assessment was performed using phase-contrast microscopy .Human stromal fibroblasts were purchased from PromoCell  and propagated using Dulbecco’s Modified Eagle Medium  supplemented with 20% fetal bovine calf serum , and 10 Units/ml Penicillin/0,1 mg/ml Streptomycin (PAN Biotech). Conditions included 37 °C, COin vivo conditions, fibroblasts were seeded into 12-well plates  and sections were placed in culture medium . The pH-value was measured at baseline and hourly for the first 1–6 hours, with further measurements being performed at 12 h, 48 h, 96 h, 144 h, 192 h, 240 h, 288 h and 336 h using a pH meter .ONRC (RESORBA CELL) and ORC (TABOTAMP) hemostats were sliced into 1 × 1 cm sections using a sterile scissor . Sections were placed in normal saline (NaCl 0.9%) to study whether there was a difference in the acidifying behavior of the hemostats. To better imitate 3) seeded in 12-well plates . After 7 and 14 days, total cell number was measured using the WST-1 proliferation assay  according to the manufacturer’s instructions. Briefly, all culture media was removed and replaced by 450 μl of fresh medium, supplemented with 50 μl of WST-1 reagent. After incubation for 2 h, the supernatant was mixed at 500 rpm for 3 min using an orbital shaker . An aliquot of 100 μl of the supernatant was measured at 450 nm by microplate reader . Mean daily proliferation was calculated using the difference in total cell number between measuring points divided by days of cultivation.As described above, hemostat sections were placed in the medium of fibroblasts . The concentration of collagen I in the matrix was set to 1.5 mg/ml. During gelation, the mixture was pipetted into 24-well plates  and supplemented with culture medium to a final volume of 300 μl. Mixtures were then placed in a cell culture incubator  for 30 minutes. 1 × 1 cm hemostat slices were placed in the supernatant and an additional 2 ml of culture medium were added.Fibroblasts at 10 × 10After 24 h, the adherent matrices were gently detached using a sterile spatula and photographed . Matrices were placed in the incubator for 48 h, with images obtained at 24 and 48 h. Matrix surface area was calculated using Axio Vision 40 × 64 V 4.9.1.0. .3) suspended in a volume of 70 μl were placed in both chambers of a μ- dish insert  and incubated  for 24 h. The insert was removed using a sterile tweezer  and 1 × 1 cm hemostat slices together with 3 ml of culture medium were added. The μ- dishes were placed under a live-cell-imaging microscope  for 24 h, taking 1 picture per hour and documented the gap between the two cell fractions. Picture analysis was performed using Wound Healing Image Analysis, WimScratch .Fibroblasts  or ORC (medium pH 7.0). We imitated pH values obtained over time, matching values for the experiments described above for each measuring point independently, utilizing a 5N HCL solution . Differences in pH value of less than 10% for each measuring point were considered acceptable.The data are shown as the mean ± SD. Statistical analysis was performed using GraphPad Prism 6.0 . Kolmogorov–Smirnov test was used to test for the normality of data. One way ANOVA and multiple t-test with Bonferroni correction was used to compare differences between ORC, ONRC and controls  in a time course for normally distributed data. Kruskal-Wallis test with Dunn’s post test and Mann–Whitney U test were applied for not normally distributed data. The level of significance was set to p < 0.05.How to cite this article: Wagenhäuser, M. U. et al. Oxidized (non)-regenerated cellulose affects fundamental cellular processes of wound healing. Sci. Rep.6, 32238; doi: 10.1038/srep32238 (2016).