Minimal interference of 80% aqueous methanol extract allows PLTX quantitation in mussels at concentrations lower than the maximum limit suggested by EFSA, with an LOQ of 9.1 µg PLTX equivalent/kg mussel meat. Given its high sensitivity and specificity, the cell-based ELISA should be considered a suitable method for PLTX quantitation.The marine algal toxin palytoxin (PLTX) and its analogues are some of the most toxic marine compounds. Their accumulation in edible marine organisms and entrance into the food chain represent their main concerns for human health. Indeed, several fatal human poisonings attributed to these compounds have been recorded in tropical and subtropical areas. Due to the increasing occurrence of PLTX in temperate areas such as the Mediterranean Sea, the European Food Safety Authority (EFSA) has suggested a maximum limit of 30 µg PLTX/kg in shellfish meat, and has recommended the development of rapid, specific, and sensitive methods for detection and quantitation of PLTX in seafood. Thus, a novel, sensitive cell-based ELISA was developed and characterized for PLTX quantitation in mussels. The estimated limits of detection (LOD) and quantitation (LOQ) were 1.2 × 10 Palythoa toxica. Ten years later, the chemical structure of PLTX isolated from this coral was reported [Palythoa [Zoanthus [Ostreopsis [Trichodesmium [S-OH-50S-PLTX), isolated from P. toxica [S-OH-50R-PLTX), isolated from P. tuberculosa [Ostreopsis siamensis [Ostreopsis cf. ovata in the Mediterranean Sea [Palytoxin (PLTX), a complex marine poly-ol toxin, is one of the most toxic natural compounds. The discovery of PLTX dates back to the 1960s, when, in a tide pool of Hana Bay , Prof. Paul Helfrich collected samples of a toxic soft coral, subsequently identified as Palythoa ,4,5,6,7 Zoanthus , in benttreopsis ,13,14,15odesmium . Only a . toxica ,17, and iamensis ,18,19,20nean Sea ,22,23. Ostreopsis blooms [Ostreopsis has been recorded since the early 1970s [The main public health concern associated with these toxins is their presence in marine organisms and potential entrance into the human food chain. Indeed, PLTXs have been detected in porifera and polychaete worms as well as in other edible species, including crustaceans, mollusks , and echinoderms  ,24,25. Ms blooms ,31. In ps blooms ,30,32,33ly 1970s , and in +/K+ ATPase [Despite their high toxicity, PLTXs are not regulated as seafood or environmental contaminants. However, the European Food Safety Authority (EFSA) has suggested a maximum limit of 30 µg PLTX/kg of shellfish meat . Moreove+ ATPase ,45. Howe+ ATPase ,47,48,49+ ATPase , while Z+ ATPase . Recentl+ ATPase . Another+ ATPase .+/K+ ATPase expressed on the cell surface. Given the high-affinity binding of PLTX to cells and the ability of a monoclonal anti-PLTX antibody to efficiently and simply quantify bound PLTX, a novel cell-based immunoenzymatic assay (cell-based ELISA) for PLTX quantitation was set up and characterized for its sensitivity, accuracy, reproducibility, and specificity. This novel method was further characterized for its suitability to quantify the toxin in mussels.Recently, we demonstrated the ability of specific anti-PLTX antibodies to measure and characterize the binding of PLTX to cultured cells . Using oThe cell-based ELISA was developed starting from the protocol for the characterization of PLTX binding to cultured cells reported by Pelin et al. . The ass−10 M (interquartile range = 2.2 × 10−10 to 2.4 × 10−9 M) and a median maximal binding of 0.015 (interquartile range = 0.0095 to 0.02738) were calculated. Binding parameters varied between the different cell lines, and HaCaT cells were the most sensitive cell line, as confirmed by the Kd values and maximal binding . On the contrary, the less sensitive cell models for PLTX binding were HepG2 cells  and MCF-7 cells . For these reasons, the HaCaT cell line was chosen as the most sensitive model to set up the cell-based ELISA.PLTX binding on cultured cells was evaluated using a panel of different cell lines. Cells were exposed to PLTX for 10 min at 37 °C and the toxin binding was subsequently evaluated as described in the Materials and Methods section. The obtained results for each cell model were normalized on the protein content of each sample. To improve sensitivity, the assay was carried out exposing HaCaT cells to PLTX, varying the fixing agents and incubation temperature (37–60 °C) with the primary and secondary antibodies. An increased signal  was observed with increased incubation temperature up to 50 °C, which subsequently decreased at higher temperatures . This tr−9 M PLTX as compared to the data recorded using 4% PFA, p < 0.05) and 4% PFA + 1% glutaraldehyde   D. Thus, The possibility of changing the sequence of cell fixation and exposure to PLTX was also evaluated: the cell-based ELISA was carried out fixing HaCaT cells with 4% PFA for 30 min before exposure to PLTX or exposing the cells to the toxin before fixation, as described in the Materials and Methods section. −10 M PLTX using a Tris-buffer saline (TBS) solution containing 0.2% Tween 20 and 1% or 2% dried milk powder.The influence of different blocking agents on the assay signal was also evaluated. To further increase the signal/background ratio, the influence of different dilutions of the primary antibody on the assay signal was evaluated. As shown in 4 cells/well) to PLTX (5.1 × 10−13–1.0 × 10−8 M) for 10 min at 37 °C, followed by two washes with PBS; (ii) fixation with 4% PFA (50 µL/well) for 30 min, followed by washing with PBS; (iii) blocking with TBB buffer containing 10% HS (200 µL/well) for 30 min, followed by two washes with PBS; (iv) cell incubation with 0.5 µg/mL mouse anti-PLTX monoclonal antibody (mAb)  for 1 h at 50 °C under gentle agitation; (v) after three washes with PBS containing 0.1% Tween 20 followed by three washes with PBS, cell incubation with 1:6000 HRP-conjugated anti-mouse IgG  for 1 h at 50 °C under gentle agitation; (vi) after three washes with PBS containing 0.1% Tween 20 and three washes with PBS, cell incubation with tetramethylbenzidine  for 20 min at room temperature; (vii) stopping the colorimetric reaction by 1 M H2SO4 (30 µL/well); and (viii) measuring the optical density at 450 nm.The optimized protocol of the cell-based ELISA consisted of: (i) exposing HaCaT cells  and 2.8 × 10−11 M (75.0 pg/mL), respectively. The working range was analyzed by linear regression, plotting the theoretical PLTX concentrations used in the assay against the measured toxin concentrations, and the obtained results revealed a good correlation coefficient   and quantitation (LOQ) of PLTX, as well as the accuracy, repeatability, and specificity of the assay. The calibration curve of PLTX is shown in  n = 10) B. A mean n = 10) .r2 = 0.9770 for intraassay and r2 = 0.9985 for interassay . As can be seen in Ouabain (OUA) is a known inhibitor of PLTX in vitro effects due to its binding to the common molecular target, Na+ ATPase ,55,56. Tr2 = 0.9734) and an excellent mean bias value  of the PLTX-free mussel extract in PBS were spiked with known PLTX amounts. Each extract was then analyzed by the cell-based ELISA and the OD values were compared to those obtained by analyzing the same PLTX concentrations without matrix. The minimum extract dilution that did not interfere with the assay was 1:10 . At the −11 to 1.1 × 10−9 M) and subsequently extracted as described above to obtain extracts containing 0.1 g meat equivalent/mL. Extracts were diluted 1:10 and analyzed by the cell-based ELISA. Recovery of PLTX from mussels ranged between 94% and 110% , pancreatic cancer (PANC-1), hepatic cancer (HepG2), and breast cancer  were considered. In addition, nontumor cell lines were used, such as the HaCaT skin keratinocyte cell line and the immortalized human hepatic (IHH) cell line. A wide distribution of binding parameters  was observed, suggesting a wide sensitivity range among the different cell lines. Consequently, the HaCaT cell line turned out to be the most sensitive to PLTX binding, corroborating literature data showing that HaCaT keratinocytes are among the cell models most sensitive to the toxin . For theTo optimize the method, the influence of the incubation temperature of the antibodies on the assay signal as well as the effects of different cell fixing agents were evaluated. The highest signal was obtained using 4% PFA as a fixative solution and incubating the primary and secondary antibodies at 50 °C. The decreased signal recorded after increasing the incubation temperature of the mAb is probably due to a thermal denaturation of the antibodies . For the+/K+ ATPase of HaCaT cells and its detection by monoclonal antibodies, the sensitivity of the cell-based ELISA was higher than that of other PLTX immunoassays [−11 M, 32.2 pg/mL) was more than 400 times higher than that of LC-HRMS [Once the final protocol of the assay was defined, an intralaboratory validation demonstrated good sensitivity, accuracy, and repeatability. As expected, with the combination of two sensitive features, PLTX binding to Nanoassays ,48. In anoassays . With re LC-HRMS . Similar LC-HRMS  and abou LC-HRMS . On the  LC-HRMS , who emp−11 M), even lower than that of the estimated LOQ, strengthening the ability of the cell-based ELISA to accurately quantify the toxin in mussels at very low concentrations. The specificity of the cell-based ELISA for PLTX was demonstrated by the lack of cross-reactivity toward other marine algal toxins contaminating seafood  and poteOn the contrary, despite its higher sensitivity, the immunoassay of Garet and colleagues  suffers +/K+ ATPase with PLTX detection by specific antibodies allowed the development of a novel and sensitive cell-based immunoenzymatic assay for PLTX detection. This combination resulted in higher sensitivity as compared to other very sensitive methods. Hence, the cell-based ELISA described here is sensitive, repeatable, and accurate, and is able to quantify PLTX in mussels at concentrations lower than the maximum limit suggested by EFSA (30 µg PLTX/kg shellfish meat). Thus, the high sensitivity, specificity, and rapidity make the cell-based ELISA a suitable method for PLTX screening in mussels during monitoring programs. Further studies are in progress to characterize the ability of the cell-based ELISA to detect and quantify PLTX in other frequently contaminated matrices, such as other shellfish, fish, crabs, and other seafood.In conclusion, the combination of PLTX binding to NaPalythoa tuberculosa (purity > 90%), was purchased from Wako Pure Chemicals Industries Ltd. . Yessotoxin and maitotoxin were kindly provided by Prof. T. Yasumoto . Brevetoxin-3, saxitoxin, and azaspiracid-1 were supplied by Dr. M. Poli , Dr. F. Van Dolah , and Dr. J. Kilcoyne , respectively. Okadaic and domoic acids were purchased from Sigma-Aldrich . The mouse monoclonal anti-palytoxin antibody 73D3 (mAb-PLTX) was produced and purified from a hybridoma cell culture at the U.S. Army Medical Research Institute of Infectious Diseases . The horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) was purchased from Jackson ImmunoResearch . HaCaT cell line was purchased from Cell Line Service , and all cell culture reagents were purchased from EuroClone . All other reagents were of analytical grade and purchased from Sigma-Aldrich .Palytoxin, isolated from −2 M), penicillin (1.0 × 10−4 g/mL), and streptomycin (1.0 × 10−4 g/mL). HepG2 and Caco2 cells were maintained in Eagle’s Minimal Essential Medium (EMEM) with the addition of 10% fetal bovine serum (FBS), L-glutamine (1.0 × 10−2 M), penicillin (1.0 × 10−4 g/mL) and streptomycin (1.0 × 10−4 g/mL), and 1% sodium pyruvate 100 mM. LoVo, MCF-7, and MDA-MB-231 cells were maintained in RPMI 1640 containing 10% FBS, L-glutamine (1.0 × 10−2 M), penicillin (1.0 × 10−4 g/mL), and streptomycin (1.0 × 10−4 g/mL). IHH cells were maintained in DMEM medium-high glucose with the addition of 10% FBS, 1.25% L-glutamine 200 mM, penicillin (1.0 × 10−4 g/mL) and streptomycin (1.0 × 10−4 g/mL), 1% HEPES buffer 1 M, 0.01% human insulin 10−4 M, and 0.04% dexamethasone 1 mg/mL.HaCaT, PANC-1, and HCT-116 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose, supplemented with 10% fetal bovine serum, L-glutamine  and cultured for 3 days. The assay procedure consisted of the following steps: (i) cell exposure to PLTX (5.1 × 10−13 to 1.0 × 10−8 M) for 10 min at 37 °C, followed by washing away unbound toxin with PBS; (ii) cell fixation with 4% PFA for 30 min; (iii) blocking with TBB buffer , and 0.2% Tween 20, pH 7.5) containing 10% HS for 30 min, followed by washing with PBS; (iv) incubation with mouse monoclonal anti-PLTX antibody  for 1 h at room temperature (RT) followed by washing with PBS containing 0.1% Tween 20 and PBS; (v) incubation with 1:6000 HRP-conjugated anti-mouse IgG (secondary antibody) for 1 h at 37 °C, followed by washing with PBS containing 0.1% Tween 20 and PBS; (vi) incubation with 60 µL/well of tetramethylbenzidine (TMB) chromogen and hydrogen peroxide substrate for 20 min; (vii) stopping the colorimetric reaction by 1 M H2SO4 (30 µL/well); and (viii) measuring the optical density at 450 nm .n et al. . Cells wAssay optimization. The assay was optimized by varying the following parameters: (i) type of cell line, (ii) fixative agents and temperature of incubation with primary and secondary antibodies, (iii) sequence of cell fixation and cell exposure to PLTX, (iv) blocking agents, and (v) dilution of the primary antibody.Assay characterization. The optimized assay was characterized following the Eurachem Guide [em Guide  as descrMytilus galloprovincialis collected in the Gulf of Trieste . Shucked mussel meat (200 g) was homogenized using an Ultra-Turrax  at 14,000 rpm and room temperature, until a homogeneous pulp was obtained (about 5 min). Mussel homogenate (1 g) was extracted 3 times with 3 mL 80% aqueous MeOH by Ultra-Turrax homogenization  followed by centrifugation at 5500 rpm for 30 min. The supernatants were then pooled and the volume adjusted to 10 mL with 80% aqueous methanol to obtain 0.1 g mussel meat equivalent/mL. The extract was analyzed by LC-MS/MS  to confirm the absence of PLTX before the matrix effect evaluation. Then, dilutions  of the PLTX-free mussel extract were spiked with known PLTX concentrations to prepare a series of matrix matched samples at PLTX concentrations ranging from 1.4 × 10−11 M to 1.1 × 10−9 M. These samples were then analyzed by the cell-based ELISA and compared to the same PLTX concentrations in solutions free of mussel matrix.The suitability of the cell-based ELISA for PLTX quantitation in mussels was assessed using different extracts from edible parts of −11 to 1.1 × 10−9 M). Each extract was then analyzed by the cell-based ELISA, as previously described.To evaluate the recovery of PLTX from mussels, samples of the PLTX-free mussel homogenate were spiked with known amounts of PLTX and then extracted as described above to obtain extracts containing different theoretic concentrations of PLTX (1.4 × 10r2) of the calibration curve was estimated by linear regression analysis, also using GraphPad Prism version 6.0. Data were compared by 2-way ANOVA followed by Bonferroni post test, and significant differences were considered at p values < 0.05.PLTX concentrations are reported as mean ± SE of at least 3 independent experiments performed in triplicate, unless otherwise specified. For binding experiments, dissociation constant (Kd) was calculated by a 1-site binding hyperbola nonlinear regression analysis using GraphPad Prism software version 6.0 . Maximal binding was evaluated as the maximal optical density (OD) normalized for the µg of proteins of each sample. Linearity (n = 6) and by different operators within a 6-month period  were evaluated. Accuracy was measured as % bias (n = 10), calculated as % difference between PLTX concentration measured by the assay and the theoretical concentration in the sample divided for theoretical PLTX concentrations.The optimized assay was characterized according to the international principles defined by the Eurachem Guide : limits


Deep ultraviolet AlGaN-based nanorod (NR) arrays were fabricated by nanoimprint lithography and top-down dry etching techniques from a fully structural LED wafer. Highly ordered periodic structural properties and morphology were confirmed by scanning electron microscopy and transmission electron microscopy. Compared with planar samples, cathodoluminescence measurement revealed that NR samples showed 1.92-fold light extraction efficiency (LEE) enhancement and a 12.2-fold internal quantum efficiency (IQE) enhancement for the emission from multi-quantum wells at approximately 277 nm. The LEE enhancement can be attributed to the well-fabricated nanostructured interface between the air and the epilayers. Moreover, the reduced quantum-confined stark effect accounted for the great enhancement in IQE. In the past decade, AlGaN-based UV LEDs have attracted wide attention because of their promising applications such as water purification, sterilization, and biochemical detection. –3. CompaFabrication of AlGaN-based deep UV nanostructured LEDs can be an alternative way to overcome QCSE and low LEE issues at the same time. Generally, nanostructured LEDs were fabricated by nanometers scale masks and top-down dry etching techniques. The masks were prepared via annealed metal nanoparticles such as nickel (Ni) or gold , 18, nanIn this work, we successfully prepared AlGaN nanorod (NR) arrays with MQWs embedded from planar AlGaN LED wafers. Compared with the planar (PLA) samples, 1.92-fold LEE enhancement and 12.2-fold relative IQE enhancement have been demonstrated. Cathodoluminescence (CL), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) measurements suggested that the enhanced LEE can be attributed to the improved interfacial quality between the air and the epilayers. The Raman measurements demonstrated that the strain in the MQWs is reduced from 0.42% to 0.13%, which is beneficial for IQE enhancement.c plane sapphire substrate, which is defined as the PLA sample. The epitaxy comprised a 900-nm undoped AlN buffer, a 400-nm graded Al composition AlGaN layer, a 1.4-μm-thick Si-doped n-Al0.5Ga0.5N, and 5 periods of Al0.35Ga0.65N/Al0.45Ga0.55N MQWs with well and barrier thickness of 3 and 10 nm, respectively, followed by a 100-nm Mg-doped p-GaN contact layer.The AlGaN LED wafer was grown by metal-organic chemical vapor deposition (MOCVD) on a 2 in. 2) by using plasma-enhanced chemical vapor deposition (PECVD) method  plasma was utilized to etch SU8 photoresist via reactive-ion etching (RIE) procedure  etching process. Finally, these SiO2 nanorod array masks were removed by HF solution, and AlGaN NR arrays were acquired as depicted in Fig. A soft UV-curing NIL and a post-growth etching approach have been employed to obtain the AlGaN NR arrays –28. As shod Fig. b. Then, hod Fig. c, with phod Fig. d. To remure Fig. e. After ask Fig. f. The press Fig. g. Subseq−1.The morphology of fabricated AlGaN NR arrays was characterized in a ZEISS SIGMA high-resolution field emission SEM system. TEM images were collected by FEI Titan 80-300 TEM system with electron beam operating at 200 kV. The CL spectra were collected by an electron beam-fiber probe system with electron beam operating at 10 kV and 922 pA. The spectra of Raman scattering was obtained in a Confocal Raman spectroscopy imaging system  with backscattering configuration, by using a 514-nm laser as excitation source. The Raman measurement was calibrated by a standard single crystal silicon sample with optical phonon mode at 520.7 cmFigure I1(NR300K)/I1(PLA300K)] and 2.78 [I1(NR10K)/I1(PLA10K)] times higher than that for the PLA sample at RT and LT, respectively. However, at RT, the integrated intensity of the emission from the MQW for NR sample is approximately 5.81 [I2(NR300K)/I2(PLA300K)] times higher than that of PLA sample, while the ratio is only 0.48 [I2(NR10K)/I2(PLA10K)] at LT.Figure I1(NR)/I1(PLA)]. Moreover, according to the geometric structure obtained from Fig. I2(NR10K)/I2(PLA10K)]. Clearly, the LEE enhancement of the n-type layer emission is higher than that of the MQW emission.Compared with PLA sample, the sidewalls of the NR sample are exposed to the air as shown in Fig. 0 that escape from the top surface, and the source power of dipole is defined as PS, so the LEE is η = P0/PS. And the extraction enhancement can be calculated by En = ηr/ηp, where ηp, ηr is the LEE of PLA and NR samples, respectively. Figure Finite-difference time-domain (FDTD) simulations were performed to clarify the LEE enhancement of AlGaN NR arrays. The diameter, period, and length of the NR arrays are set as 350 nm, 730 nm, and 1300 nm, respectively, to accord with the fabricated NR arrays as depicted in Fig. I1(PLA300K)/I1(PLA10K)] and 2.03% [I1(NR300K)/I1(NR10K)] for the PLA and NR samples, respectively. They are very close to each other because the QCSE does not exist in the n-type layer. However, the IQEs of the MQW emission at 300 K are approximately 1.32% [I2(PLA300K)/I2(PLA10K)] and 16.1% [I2(NR300K)/I2(NR10K)] for the PLA and NR samples, respectively. Thus the enhancement ratio of IQE is 12.2 for the MQW emission of the NR sample compared with the PLA sample. This great enhancement of the relative IQE should be attributed to the reduced QCSE of the NR sample. According to some similar works in blue/green LEDs [On the other hand, the reduced QCSE can enhance the IQE for the NR sample for the MQW emission. The IQEs of the n-type layer emission at 300 K can be estimated as approximately 1.96% [een LEDs , 31, a lE2(high) phonon mode is usually utilized to characterize the stress state in the epitaxial layers. Notably, three E2(high) phonon modes are shown in the Raman spectra for both PLA and NR samples, corresponding to the GaN contact layer, n-type layer, and AlN buffer layer. Clearly, the peak shifts of PLA and NR samples are different compared with stress-free E2(high) phonon modes, indicating that the stress state has changed after the PLA sample was fabricated into the NR sample. Usually, the in-plane stress of the epitaxial layers is expressed by the following equation [−1/GPa, − 3.1 cm−1/GPa, and − 3.25 cm−1/GPa for GaN, AlN, and Al0.5Ga0.5N, respectively) [ω0 are the Raman shifts for the E2(high) mode of the corresponding epitaxial layers in our study and the stress-free materials, respectively. The ω0 values for GaN and Al0.5Ga0.5N are reported to be 567.0 and 586.0 cm−1 at RT, respectively [σxx is the in-plane stress; εxx is the in-plane strain, and Cij is the elastic constants of GaN and AlN given in previous report [0.5Ga0.5N.Raman measurement was performed to confirm the strain relaxation in the NR samples. Figure equation :1\documectively) . \documeectively . The strectively :2\documes report , i.e., aUsing Eqs. , 2, the Ew in the quantum wells can be expressed aslw, lb, Pw, Pb, and εb, εw are the widths, total polarizations and dielectric constants of the wells and the barriers, respectively. Thus, not only the piezoelectric polarization but also the spontaneous polarization should be taken into account. The piezoelectric polarization is calculated by e31, e33, C31, and C33 are obtained by the linear interpolation from the related parameters of GaN and AlN [εxx is calculated by the Raman spectra using linear interpolation method. The spontaneous polarization is obtained by the linear interpolation from the spontaneous polarization of GaN and AlN [εGaN = 8.9 and AlN εAlN = 8.5 [According to previous investigation , the pol13C33εxx , where e and AlN , 38, the and AlN , 39. ThulN = 8.5 , the polIn summary, highly uniform AlGaN NR arrays with MQWs embedded have been successfully fabricated by NIL and top-down etching techniques. Two peaks corresponding to the emission from the n-type layer (at higher energy) and MQWs (at lower energy) are observed by the CL measurement for both NR and PLA samples at 300 K and 10 K. For the n-type layer emission, an over 2-fold LEE enhancement has been observed while the IQE is hardly enhanced via the NR fabrication. For MQW emission, the LEE enhancement ratio can be estimated around 1.9 and a 12.2-fold IQE enhancement is achieved. Raman spectra demonstrated that the strain is reduced from 0.42% to 0.13% by the NR fabrication, showing a strong evidence of reduced QCSE. Our results indicated that for the samples without great crystal quality, the spatial separation between the electrons and the holes caused by the QCSE would be an important factor for the IQE reduction.


This study aimed to investigate whether different levels of fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) in prediabetes are associated with hyperfiltration.A prospective cohort of 2,022 individuals aged 30–74 years took part in the PREDAPS Study. One cohort of 1,184 participants with prediabetes and another cohort of 838 participants with normal FPG and normal HbA1c were followed for 5 years. Hyperfiltration was defined as an estimated glomerular filtration rate (eGFR) above the age- and gender-specific 95th percentile for healthy control participants, while hypofiltration was defined as an eGFR below the 5th percentile. The prevalence of hyperfiltration was compared for different levels of prediabetes: level 1 of prediabetes: FPG <100 mg/dL plus HbA1c 5.7–6.0% or FPG 100–109 mg/dL plus HbA1c < 5.7%; level 2 of prediabetes: FPG <100 mg/dL plus HbA1c 6.1–6.4% or FPG 100–109 mg/dL plus HbA1c 5.7–6.0% or FPG 110–125 mg/dL plus HbA1c <5.7% and level 3 of prediabetes: FPG 100–109 mg/dL plus HbA1c 6.1–6.4% or FPG 110–125 mg/dL plus HbA1c 5.7–6.4%.The participants with hyperfiltration were significantly younger, had a higher percentage of active smokers, and lower levels of hemoglobin and less use of ACEIs or ARBs.Only level 3 prediabetes based on FPG 100–109 mg/dL plus HbA1c 6.1–6.4% or FPG 110–125 mg/dL plus HbA1c 5.7–6.4% had a significantly higher odds ratio (OR) of hyperfiltration (OR 1.69 (1.05–2.74); P < 0.001) compared with no prediabetes (FPG < 100 mg/dL and HbA1c < 5.7%) after adjustment for different factors. The odds ratios for different levels of HbA1c alone in prediabetes increased progressively, but not significantly.Level 3 of prediabetes based on FPG 100–109 mg/dL plus HbA1c 6.1–6.4% or FPG 110–125 mg/dL plus HbA1c 5.7–6.4% had a significantly higher OR of hyperfiltration compared with participants without prediabetes. Glomerular hyperfiltration, also called hyperfiltration, is a well-recognized early and reversible stage of kidney damage in subjects with diabetes and hypertension and is a marker for subsequent chronic kidney disease (CKD) progression –3.Previous studies addressing the association between levels of FPG and HbA1c in prediabetes and hyperfiltration have had inconsistent results –11. The Obesity and the metabolic syndrome (MetS) are also independent risk factors for CKD. The potential mechanisms involve inflammation, lipotoxicity, and hemodynamic effects; and perhaps other, unknown, mechanisms . MoreoveSince hyperfiltration is considered to represent an early and potentially reversible stage of kidney damage, identifying subjects with prediabetes and hyperfiltration might be helpful for implementing preventive and therapeutic strategies in this population.This study aimed to investigate whether different levels of FPG and HbA1c in prediabetes are associated with hyperfiltration.This sub study form part of the PREDAPS study  which is a prospective cohort study in which one cohort of 1,184 participants with prediabetes and another cohort of 838 participants with normal fasting glucose (NFG) were followed during 2012 and 2017. A total of 1,453 participants (71.9% of the baseline sample) attended the last follow-up visit: 622 of the cohort with no alterations in glucose metabolism and 831 of the cohort with prediabetes. Baseline data were collected in 2012, in the context of routine clinical practice, by general practitioners distributed across Spain. Participants aged between 30 and 74 years old who consecutively sought medical attention for any reason were invited to participate in the study. In both cohorts, participants were excluded if they had diabetes, terminal disease, pregnancy, surgery, or hospital admission in the previous 3 months at study entry, or any hematologic disease, which could alter HbA1c values. Information about the design and methods of the PREDAPS study have been previously published [Prediabetes was defined as anyone who fulfilled the following criteria: FPG levels between 100 and 125 mg/dL , and/or a glycated hemoglobin range from 5.7% to 6.4% (39 to 47 mmol/mol) . Patient2 and with central obesity, if waist perimeter was ≥ 102 cm in men and ≥ 88 cm in women. Hypertension was defined if the patient was on antihypertensive drugs or personal history of high blood pressure or had systolic blood pressure (SBP) ≥ 140 mmHg and diastolic blood pressure (DBP) ≥90 mmHg. Laboratory data collected included: FPG and HbA1c; serum hemoglobin; total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, non-high-density lipoprotein (non-HDL) cholesterol and triglycerides (TG); serum creatinine, estimated glomerular filtration rate and urinary albumin-to-creatinine ratio (UACR). Hypercholesterolemia was defined as total serum cholesterol ≥ 250 mg/ dL , low HDL as <40 mg/dL  in men and <50 mg/dL  in women, high LDL ≥ 100 mg/dL , and hypertriglyceridemia as serum TG ≥ 150 mg/dL . Metabolic syndrome was defined if the participants met three or more of the following criteria: waist circumference >102 cm in men or >88 cm in women; TG ≥ 150 mg/dL ; blood pressure ≥130/85 mmHg; HDL <40 mg/dL  in men or <50 mg/dL  in women; and FPG between 110 and 126 mg/dL  [For this sub study we used data previously collected during the baseline visit from PREDAPS Study . This da mmol/L) .The adherence to Mediterranean diet was measure according to the study ATTICA and its score published by Panagiotakos . For eacSmoking consumption was classified as: smokers, ex-smokers and never smokers. Alcohol consumption was divided in three categories: occasional drinkers, regular drinkers and no-drinkers which includes ex-drinkers and teetotalers. Physical activity was classified according to World Health Organization (WHO) recommendations. Subjects followed the recommendations if they practiced more than 150 minutes per week of moderate aerobic physical activity, more than 75 minutes each week of vigorous aerobic physical activity or an equivalent combination .The eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula . First, Participants, according to their FPG and HbA1c levels, were classified into three groups: level 1 of prediabetes: FPG <100 mg/dL  plus HbA1c 5.7–6.0% (39–42 mmol/mol) or FPG 100–109 mg/dL  plus HbA1c < 5.7% (39 mmol/mol); level 2 of prediabetes: FPG <100 mg/dL  plus HbA1c 6.1–6.4% (43–46 mmol/mol) or FPG 100–109 mg/dL  plus HbA1c 5.7–6.0% (39–42 mmol/mol) or FPG 110–125 mg/dL  plus HbA1c <5.7% (39 mmol/mol) and level 3 of prediabetes: FPG 100–109 mg/dL  plus HbA1c 6.1–6.4% (43–46 mmol/mol) or FPG 110–125 mg/dL  plus HbA1c 5.7–6.4% (39–46 mmol/mol).2 test. Patients with severe chronic kidney disease have not been excluded. Considering values of glomerular filtrate of the basal stage (CDKEPI), we did not have any case with values below 30 (severe kidney failures–G4 and G5-), and only 8 cases with values <45 (moderate-severe kidney failure–G3B).”A descriptive analysis was performed describing the baseline characteristics  in participants in each prediabetes group and in participants without prediabetes. Next, we estimated the distribution of these characteristics in the three categories of glomerular filtration . Data for the continuous variables were presented as means ± standard deviations (SDs) for categorical variables as percentages. Different types of analysis were used for comparison between the groups such as unpaired t-test, variance (ANOVA), or χWe then created a dichotomous variable of glomerular filtration rate—hyperfiltration versus non hyperfiltration- grouping hypofiltration and normofiltration into a single category. The association between levels of FPG and hyperfiltration and between levels of HbA1c and hyperfiltration was measured using logistic regression. We used two consecutive models: the first one (model 1) was adjusted by sex and age; the second one (model 2) was adjusted for the model 1 factors plus body mass index, waist circumference, metabolic syndrome, systolic blood pressure, diastolic blood pressure, alcohol consumption, smoking status, and physical exercise. The measure of association with its 95% confidence interval was the odds ratio, taking as reference the category of participants without prediabetes. All analyses were conducted using the IBM SPSS statistical package.The study cohort consisted of 2,022 participants (48.6% men). Of these, 1,184 had prediabetes, and 838 had normal FPG and normal HbA1c. Detailed information on the demographic and clinical characteristics of the participants is provided in in The prevalence of hyperfiltration was higher in subjects with prediabetes (8.8%) than in subjects with normoglycemia (6.9%), although the difference did not reach statistical significance (p = 0.073).Multiple logistic regression analyses for hyperfiltration according to different levels of FPG and HbA1c were analysed. As shown in Due to physiologic connection between the renin angiotensin system and hyperfiltration, we performed a sensitivity analysis including only 1367 subjects who did not use ACE-I / ARB. The trend of the association at different levels has been maintained, although the magnitude is greater. In prediabetes level 3 the ORs (95% CI) were 1.80 (1.19–2.98) and 2.04 (1.15–3.64) in model 1 and 2 respectively.In the current study, we examined the relationship between prediabetes and hyperfiltration. After multivariate adjustment, only the odds ratio of hyperfiltration was significantly increased in participants with FPG 100–109 mg/dL  plus HbA1c 6.1–6.4% (43–46 mmol/mol) or FPG 110–125 mg/dL  plus HbA1c 5.7–6.4% (39–46 mmol/mol). Based on these results, more attention should be paid to participants with FPG and HbA1c in order to maximize the detection and prevention of hyperfiltration in these participants.It has been reported that hyperfiltration is more prevalent among patients with prediabetes in various ethnic populations. In a Norwegian study  of a totIn addition to IFG, several parameters of MetS are significant risk factors for hyperfiltration. Different mechanisms are activated in the presence of MetS, leading to hyperfiltration ,24–26. IComparing our data on hyperfiltration with other studies is difficult for several reasons. First, none of these studies has used the gold standard of insulin clearance to measure eGFR. Second, there is no generally accepted cutoff of EGFR to define hyperfiltration, and studies have used different methods to measure eGFR ,30–31. EOur study has several strengths, such as large sample size from a geographically diverse Spanish population with a wide age range. Also, this study adopts the CKD-EPI creatinine-based equation, which is more accurate and has lower bias than the commonly used Modification of Diet in Renal Disease (MDRD) equation, especially at higher levels of GFR ,34, and However, several limitations warrant mention in this study. First, GFR was estimated rather than using the gold standard of insulin clearance for this measure. Although measured insulin clearance is more accurate than eGFR, it is quite cumbersome, costly and invasive, and is not used in clinical practice in Primary Care. Second, this study design limits inferences on causality. Third, the results cannot be generalized to other ethnic groups since the participants in this study are from the Spanish population, mainly Caucasians. Fourth, in some variables, such as urinary albumin, could not be obtained in a large percentage of subjects (41%) and for this reason this variable was excluded from the analyses. Finally, some participants with normal filtration may have already undergone the hyperfiltration stage.Level 3 of prediabetes based on FPG 100–109 mg/dL  plus HbA1c 6.1–6.4% (43–46 mmol/mol) or FPG 110–125 mg/dL  plus HbA1c 5.7–6.4% (39–46 mmol/mol) had a significantly higher odds ratio of hyperfiltration compared with participants without prediabetes. Our study is a cross-sectional study. Longitudinal studies are needed to confirm whether prediabetes is an independent risk of hyperfiltration and for development of CKD. Furthermore, hyperfiltration should be defined using age- and sex-specific reference values of eGFR and values that reflect the risk of CKD should be established in future investigations. These studies will help to determine whether prediabetes might be a target to prevent kidney damage at an early and reversible stage, and thus prevent the increasing burden of CKD.S1 Table(DOCX)Click here for additional data file.


In spite of the very high sintering temperature (1300 °C), no significant Ag losses were observed following this process. Moreover, Ag addition enhanced cation mobility during sintering due to the formation of a liquid phase. Microstructurally, it was found that Ag decreases porosity; this was confirmed by density measurements. Ag was also found to promote the formation of a Ca2Mn2O5 secondary phase. Despite the presence of this secondary phase, samples with Ag displayed lower electrical resistivity than Ag-free ones, without a drastic decrease in the absolute Seebeck coefficient. The highest thermoelectric performances, which were determined by power factor, were obtained in 1 wt.% Ag samples. These maximum values are slightly higher than the best of those reported in the literature for sintered materials with similar compositions, with the additional advantage of their being obtained using a much shorter sintering procedure.In this study, Ca These TE devices are built with p- and n-type TE materials connected electrically in series and thermally in parallel. At present, commercial devices are built with metal-based materials [2/ρκ ), where T, S, ρ, and κ are absolute temperature, Seebeck coefficient, electrical resistivity, and thermal conductivity, respectively [Thermoelectric (TE) devices can transform heat to electrical energy without using moving components. Consequently, they can increase the efficiency of energy-transforming systems  and decraterials , with Biaterials . On the ectively .3+ [These TE oxide materials belong to the family of transition metal oxide (TMO), due to the presence of transition metals within their composition. In these TMOs, p- and n-type conducting materials can be found, with the former consisting of either Bi–Ca–Co–O or Ca–Co–O ,6,7, and3+ ,20,21,223 sintering temperatures, no Ag losses are expected, as demonstrated in previous research [Another way to enhance electrical properties in ceramic systems is to use metallic additions when both materials do not react under sintering conditions. One of the most useful metals for this purpose is Ag, which, besides enhancing electrical properties ,24, is vresearch .0.9Yb0.1MnO3/Ag composites via a short sintering process. This process can be performed by taking advantage of the beneficial effect of using a liquid phase, which allows faster cation diffusion at sintering temperatures. The modifications in structure and microstructure, induced by Ag addition, were studied and related to electrical properties, using Ag-free samples as a reference.The objective of this work was to improve the performance of Ca0.9Yb0.1MnO3 + x wt.% Ag composites  were prepared using CaCO3 , Mn2O3 , Yb2O3 , and Ag  commercial powders. They were mixed in stoichiometric proportions and ball milled in water media in an agate ball mill for 30 min. The resulting slurries were dried using infrared radiation, followed by calcination at 900 °C for 12 h to decompose CaCO3. The powders were subsequently cold-uniaxially pressed at 275 MPa into pellets which were sintered at 1300 °C for 90 min. Following this, a final furnace cooling took place.Ca3 for Ca0.9Yb0.1MnO3 [3 for Ag [Identification of phases was made via powder X-ray diffraction (XRD), without any correction and with 2θ, from 10–70° using a Rigaku D/max-B X-ray powder diffractometer . Archimedes’ method was used for several samples of each composition to determine density values, using theoretical densities of 4.99 g/cmb0.1MnO3 , and 10.3 for Ag . MicrostElectrical resistivity and the Seebeck coefficient were determined in a LSR-3 system  between room temperature and 800 °C, under a He atmosphere, in steady state mode. These data were used to evaluate TE performance through PF values.Powder XRD patterns performed on sintered materials are displayed in 0.9Yb0.1MnO3 is the major phase (indicated by its diffraction planes) in all samples, independent of Ag addition, with a perovskite Pnma space group [2Mn2O5 [2Mn2O5 secondary phase when Ag is added to the samples. Furthermore, the amount of this secondary phase grew when Ag addition was increased. Consequently, it seems that the liquid phase  produces a less formation of the thermoelectric Ca0.9Yb0.1MnO3 phase. This lower level of the thermoelectric phase can be linked to a lower oxygenation of the bulk sample, as has been reported for different systems [2Mn2O5 phase. On the other hand, no shift in the XRD peaks was observed, indicating that Ag does not occupy any lattice site, and is maintained as metallic Ag.In ce group . Additio [2Mn2O5  and meta [2Mn2O5 , respect systems ,33, leadFESEM observations performed on transversal fractured sections of samples showed that no significant differences can be observed among them. The typical microstructure of these samples is illustrated in As can be observed, all samples possess well connected grains with relatively high porosity levels. To determine the density of the different samples, Archimedes’ method was used, and the results are presented in These data clearly indicate that Ag addition increases density up to 5 wt.% content, with a subsequent decrease for higher Ag additions. This trend can be explained by the formation of liquid Ag during sintering, which enhances cation mobility at high temperatures and helps to decrease porosity.These aforementioned features can be observed in representative SEM images obtained on polished longitudinal sections of samples. The images are displayed in 0.9Yb0.1MnO3 thermoelectric phase, while the dark grey and white sections  are associated with Yb-poor (Ca0.5Yb0.5MnO3) and Yb-rich (Ca0.95Yb0.05MnO3) compositions, respectively. When Ag was added to samples, modifications to these phases were produced. Yb-rich phase levels drastically decreased, and those of the Yb-poor phase disappeared, confirming the effect of the liquid phase on cation diffusion. In addition, a new Yb-free Ca2Mn2O5 secondary phase (#4) was formed in this process, probably due to a phase equilibrium modification induced by Ag. Moreover, very small metallic Ag particles (#5) can be found between the grains. These findings agree with XRD analysis, as previously discussed. Another characteristic which matches well with the density values displayed in In the Ag-free samples, three different phases were identified by their contrast levels via EDS see a. The maChanges in electrical resistivity with temperature and Ag content are presented in 2Mn2O5 secondary phase. Greater Ag content promotes a slight increase in resistivity, due to higher Ca2Mn2O5 secondary phase levels. It is also clear that Ag does not drastically change sample behavior, which was metallic-like for all samples (dρ/dT > 0) in agreement with previous research [3 (125 mΩ·cm) [In research ,20,21,225 mΩ·cm) , reflect5 mΩ·cm) ,18,19,205 mΩ·cm) , and are3. This is within the range of that reported for Yb-doped samples (90–100 µV/K) [Furthermore, absolute S values decreased with Ag addition, compared with Ag-free samples, which is in agreement with their lower electrical resistivity. Consequently, the highest absolute values have been determined as belonging to Ag-free samples across the whole measured temperature range. The highest absolute value at room temperature 90 µV/K) is much lower than that measured for undoped materials around 350 µV/K) [0 µV/K [30 µV/K is00 µV/K) ,19,20. A00 µV/K) ,20.Using these electrical data, sample performances were calculated using their PF values. a PF variation with temperature as a function of Ag content is illustrated in 2m) is higher than that reported for Yb-free CaMnO3 (0.1 mW/K2m) [2m) [2m) is still much higher than that reported for CaMnO3 (0.17 mW/K2m) [2m) [The highest PF values were found in 1 wt.% Ag samples, mainly due to a significant decrease in electrical resistivity. The maximum value at room temperature (0.17 mW/K mW/K2m) . However2m) [2m) ,18,19,20 mW/K2m) , and sli3 materials can be achieved via additions of Ag in small amounts. Metallic Ag promotes the formation of a liquid phase during sintering. This phase enhances cation mobility and density of samples within a very short processing time. However, it will still be necessary to optimize thermal treatment in order to decrease the amount of secondary phases before these materials can be used practically in thermoelectric devices. Even if Ag prices seem to be a drawback for their production, though, it should be mentioned that their costs are about 10 times lower than for those associated with the Yb-oxide.These data provide evidence that high thermoelectric performance in Yb-doped CaMnO3 thermoelectric ceramics with different metallic Ag additions were successfully prepared via the classical ceramic method. Microstructure observations showed that Ag addition improves Yb cation diffusion and decreases porosity, which was confirmed by density measurements. On the other hand, Ag addition promotes the formation of a Yb-free Ca2Mn2O5 secondary phase. Despite the presence of this secondary phase, electrical resistivity decreased significantly, without a drastic decrease in the absolute Seebeck coefficient. The greatest improvement in thermoelectric performance was observed in 1 wt.% Ag-added samples. These maximum values are slightly higher than the best of those reported for Yb-doped sintered materials, with the advantage of having required a much shorter sintering procedure . It can be concluded that this procedure is very advantageous to acquiring the best properties reported in the literature for CaMnO3 materials, while decreasing, at the same time, the energy consumption used during the sintering process.In this study, Yb-doped CaMnO


Recent studies have established that the phenotype of epithelial stem cells residing in the corneal periphery (the limbus) depends on this niche’s distinct biomechanical properties. However, the signaling pathways underlying this dependency are still poorly understood. To address this issue, we investigated the effect of substrate stiffness on the migration, proliferation, and molecular phenotype of human limbal epithelial stem cells (LESCs). Specifically, we demonstrated that cells grown on collagen-based substrates with limbus-like compliance showed higher proliferation and stratification and lower migration capabilities, as well as higher levels of pro-proliferative markers Ki67 and β-Catenin, and LESC markers ΔNp63, ABCG2, and CK15. In contrast, cells on stiffer substrates lost these stem/progenitor cell markers, but instead expressed the key mechanotransduction factor YAP, as well as elevated levels of BMP4, a promotor of cell differentiation known to be negatively regulated by Wnt/β-Catenin signaling. This data allowed us to propose a new model that integrates the various molecular pathways involved in LESC response to substrate stiffness. This model will potentially be a useful guide to future research on the mechanisms underlying LESC loss following fibrosis-causing injuries. The cornea is the outermost tissue of the eye, and its transparency is determinant for maintaining vision quality. On its anterior side, the cornea is covered by a nonkeratinized, stratified squamous epithelium that plays an essential role in the tissue’s function . In partA considerable amount of evidence has established that, in the human cornea, the limbus is populated by residing epithelial stem/progenitor cells ,3. MoreoThese findings highlighted the importance of corneal tissue biomechanics for maintaining epithelial homeostasis . This is2, with culture medium change every two days, to allow LESC attachment and expansion  were isolated from explants of cadaverous human corneal rings remaining from donor tissue  following removal of the central 7 mm for keratectomy, in accordance with the Newcastle University and Newcastle-upon-Tyne Hospital Trust Research Ethics Committees’ guidelines . Brieflyxpansion a. LESC mhenotype a.g at 4 °C for 5 min to remove air bubbles. This liquid gel mix was then distributed into 1 mL aliquots into 24-well tissue culture plates and incubated for 30 min at 37 °C to solidify. Subsequently, gels were plastic-compressed by applying RAFT absorbers  to their top surface for 10 min. The resulting ~150-µm-thick gels were then treated with a collagenase type I enzyme (Thermo Fisher Scientific) in order to soften them and obtain a limbus-like compliance (~15 kPa). Briefly, collagenase dissolved in phosphate buffer saline  at 25 mg L−1, and 1 mL of this solution was then used to treat the plastic-compressed gels for 1 h at 37 °C. Gels treated with PBS alone were used as stiffer (~65 kPa) substrate counterparts [−1 laminin solution (Thermo Fisher Scientific) for 1 h and at 37 °C to create a surface coating that promotes LESC adhesion.High-density collagen gels were prepared by mixing rat tail collagen type I , 10× MEM (Thermo Fisher Scientific), and 1M NaOH  solutions at a 8:1:1 volume ratio in a 50 mL falcon tube, followed by centrifugation at 1000× terparts . Followi5 LESCs in 1 mL of CnT-07 medium, and incubated for 24 h at 37 °C with the substrate held at an initial 45° tilt to ensure cells only attached to the lower half surface of the gel, thus forming a defined cell boundary with the upper half above the air–liquid interface. Subsequently, cultures were washed thrice with PBS to remove unattached cells, and incubated at 37 °C for 24 h fully submerged in CnT-07 medium. Cells were imaged every 10 min by time-lapse bright-field microscopy using a Lumascope 500 inverted microscope  to track their migration. Micrographs were binarized using the ImageJ v1.7 software to better determine the position of individual cells in each image frame. Cell speed (µm h−1) was evaluated by determining the position of 100 individual cells during the initial 6 h of migration, and tracing total distance covered by moving cells and their migration front using the standard parameters of the wrMTrck plugin for ImageJ v1.7. Data was expressed as the average ± standard deviation (s.d.) from three independent experiments (n = 3), each performed with cells from a different donor.Collagen gels treated with collagenase (softer) or PBS (stiffer) were seeded with 1 × 105 LESCs suspended in 1 mL of CnT-07 medium. Plastic coverslips  similarly coated with 1 µg L−1 laminin were used as infinite stiffness control substrates , each performed with cells from a different donor.Collagen gels treated with collagenase (softer) or PBS (stiffer) were transferred into Transwell culture inserts  and seeded with 1 × 10bstrates a. Cells Cells grown on either softer and stiffer collagen gels or plastic coverslips were washed in PBS, fixed in 4% paraformaldehyde for 20 min, washed with excess PBS, incubated with blocking solution comprising 5% bovine serum albumin (First Link) and 0.1% Triton X-100 (Merck) in PBS for 1 h, and then incubated overnight at 4 °C with the following primary antibodies diluted 1:500 in blocking buffer; mouse β-Catenin and CK15, rabbit BMP4 and Ki67, and rat ABCG2 ; rabbit ΔNp63, goat YAP, and mouse CK3 . Cells were then washed vigorously four times for 15 min in PBS containing 0.1% Triton X-100, and incubated for 2 h at room temperature and in the dark with corresponding secondary antibodies and bisbenzimide nuclear stain  diluted 1:1000 and 1:5000 in blocking buffer, respectively. After another four 15-min washes, both gels and plastic coverslips were immersed in antifade medium , mounted on glass slides, and imaged by confocal fluorescence microscopy using a Nikon A1, with 1 µm-thick optical sections and constant illumination and capture parameters. Data were analyzed using the NIS-Elements and the ImageJ v.1.7 software suite, with expression quantified by evaluating pixel intensity for each independent channel or by calculating the percentage of cells with nuclear ΔNp63, β-Catenin, Ki67, or YAP and normalizing it (in percentage) against the control (corresponding plastic substrates). Representative images were taken from each independent sample, for all three experiments (n = 3).post hoc test. Significance between groups was established for p < 0.05, 0.01, and 0.001, with a 95% confidence interval. For all assays, error bars represented the s.d. of the mean, analyzed a priori for homogeneity of variance.Data was analyzed using a one-way  or two-way analysis of variance (ANOVA) (marker expression) followed by Bonferroni’s multiple comparison −1, a significantly lower migration rate compared to the 26 ± 2 µm h−1 from cells on stiffer collagen substrates (p = 0.016). This latter condition supported a similar migration rate to that of cell on plastic (29 ± 2 µm h−1)  b.p = 0.036) (p = 0.044)  d. Specif= 0.044) d. The lo= 0.044) c.p = 0.001, 0.016, and 0.001, respectively), and 2- and 1.9-fold increases for Ki67 and β-Catenin, respectively    and significantly lower percentage of cells with nuclear YAP (p = 0.001)  b. Cells ctively) b. Furthe= 0.001) c. These = 0.001) a; insetsThese results indicated that, in the specific conditions of this in vitro setting, the phenotype of human corneal epithelial cells was highly dependent on the mechanical properties of their substrate, with tissue compliance promoting cell proliferation, stratification, and the expression of LESC-characteristic markers, and stiffer substrates promoting corneal epithelial cell migration and differentiation. Interestingly, no significant differences in marker expression profile were observed between cells on stiffer collagen gel and plastic substrates , indicatPrevious studies have established that the limbus represents as softer biomechanical niche compared with the central cornea ,11, suggFurthermore, our results suggested that the stiffness-induced behavior of LESCs may be stimulated by BMP4, an important pro-differentiation growth factor known to be tightly interconnected with mechanotransduction signaling during development , regenerThis data allowed us to propose a new mechanistic model to explain how substrate stiffness is able to regulate LESCs behavior and phenotype . In thisSimilarly, in cases where a stiffening of the limbus occurs, e.g., due to tissue fibrosis or chemical injury, the resulting YAP activation and nuclear translocation initiate a signaling cascade that alters LESC behavior (Overall, this integrated model establishes feasible links between YAP-dependent mechanotransduction, ΔNp63 and Wnt/β-Catenin signaling, and consequent BMP4-mediated cell differentiation. In this perspective, this study creates a new basis for future studies aiming at understanding in detail the molecular pathways controlling corneal epithelial cell in both health and disease states.


TFPI-2−/− mice are more susceptible to pulmonary Pseudomonas aeruginosa bacterial infection. No anti-coagulant role of TFPI-2 was observed in these models in vivo. Furthermore, in vivo, the mouse TFPI-2-derived C-terminal peptide VKG24, a homolog to human EDC34 is protective against systemic Escherichia coli bacterial infection. Moreover, in sputum from cystic fibrosis patients TFPI-2 C-terminal fragments are generated and found associated with immunoglobulins. Together our data describe a previously unknown host defense mechanism and therapeutic importance of TFPI-2 against invading Gram-negative bacterial pathogens.Tissue factor pathway inhibitor-2 (TFPI-2) has previously been characterized as an endogenous anticoagulant. TFPI-2 is expressed in the vast majority of cells, mainly secreted into the extracellular matrix. Recently we reported that EDC34, a C-terminal peptide derived from TFPI-2, exerts a broad antimicrobial activity. In the present study, we describe a previously unknown antimicrobial mode of action for the human TFPI-2 C-terminal peptide EDC34, mediated via binding to immunoglobulins of the classes IgG, IgA, IgE, and IgM. In particular the interaction of EDC34 with the Fc part of IgG is of importance since this boosts interaction between the immunoglobulin and complement factor C1q. Moreover, we find that the binding increases the C1q engagement of the antigen-antibody interaction, leading to enhanced activation of the classical complement pathway during bacterial infection. In experimental murine models of infection and endotoxin challenge, we show that TFPI-2 is up-regulated in several organs, including the lung. Correspondingly,  The exploration of immuno-modulating proteins and peptides has attracted a significant interest within the past years , 2. To tPseudomonas aeruginosa .The peptides listed in Table Escherichia coli was grown to mid-logarithmic phase in 10 ml (3% w/v) of trypticase soy broth (TSB). Bacteria were washed and re-suspended in 10 mM Tris, pH 7.4 to obtain a dilution of 1% of bacterial suspension (1–2 × 109 colony forming units cfu/ml). Subsequently, 6.6 μl of bacteria were added to 15 ml of melted underlay agarose gel consisting of 0.03% (w/v) TSB, 1% (w/v) low electroendosmosis type (EEO) agarose and 0.02% (v/v) Tween 20 (Sigma) with or without 150 mM NaCl. The mixture was poured to a 144 mm sterile petri dish and left to solidify. After that, 4 mm diameter wells were punctured and 100 μM peptides were added to each well. Plates were incubated for 3 h at 37°C and then covered by adding 15 ml of over-lay gel consisting of 6% TSB and 1% Low-EEO agarose in Millipore water. After 16–24 h of incubation at 37°C, the peptides diffusion around each well formed the zone of inhibition, which corresponded to the peptide's antimicrobial activity.Escherichia coli bacteria were grown to mid-logarithmic phase in 10 ml of (3% w/v) Todd-Hewitt (TH), bacteria were washed and diluted in buffer 10 mM Tris, pH 7.4, containing 150 mM NaCl, either alone or with the presence of human 20% citrate plasma, lepirudin, heparin, EDTA plasma, or serum. 1–2 × 106 cfu/ml bacteria were incubated in a total volume of 50 μl with the indicated concentrations of C-terminal TFPI-2 derived peptides for 2 h at 37°C. Alternatively, 1–2 × 106 cfu/ml of E. coli bacteria in the presence or absence of EDC34 were incubated either in PBS alone or PBS supplemented with 25% of human citrated plasma, immunoglobulins depleted citrated plasma, or immunoglobulins depleted citrated plasma supplemented with 10 μg of IgG/μl, for 2 h at 37°C. Serial of dilutions of the reaction mixture were plated on TH agar plates, followed by the incubation at 37°C overnight. The number of colony forming units was determined as a percentage to control sample.Three μg of IgG or IgA or IgE or IgM were incubated in 20 μl of buffer , either alone or with 0.5 μg (6 μM) of EDC34 for 1 h at 37°C. Samples were diluted in SDS buffer and analyzed under non-reducing conditions by SDS-PAGE on Criterion TGX Any kD Gel (BioRad). Alternatively, EDC34 (6 μM) was incubated either alone or in PBS containing 20% serum, citrate, lepirudin, EDTA plasma at different time points . Samples were analyzed under non-reducing conditions.Six μM of EDC34 was either incubated alone or with IgGs in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer, under similar concentrations for 15, 30, 60, 120 min or 24 h at 37°C. In another experiment, 6 μM of EDC34 was pre-incubated with 50 μM of TCEP (tris(2-carboxyethyl)phosphine) for 30 min at room temperature before IgG (6 μM) was added. The mixture was further incubated for 24 h at 37°C. In another experiment, 6 μM of IgG or Fab-IgG or Fc-IgG were incubated with or without 6 μM of EDC34 in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer for 30 min at 37°C. Proteins and peptides were transferred to nitrocellulose membranes (Hybond-C), blocked by 5% (w/v) skimmed milk, washed and incubated with rabbit polyclonal antibodies to CAK27 or rabbit polyclonal antibodies to IgG  (Dako) or rabbit polyclonal antibodies to C1q  (Dako). Polyclonal swine anti-rabbit immunoglobulin secondary HRP antibody  was used for detection and an enhanced chemiluminescent Substrate (LumiGLO) developing system .E. coli bacteria (1–2 × 106 cfu/ml) were added, and then the mixture was incubated for additional 1 h. In a parallel experiment, the bacteria in the presence of absence of EDC34 were incubated for 30 min at 37°C before IgG was added for 1 h at 37°C. Samples were then centrifuged, bacterial pellets were washed twice with PBS and then re-suspended in 360 μl of 0.1 M glycine, pH 2.0 to elute the bound proteins. The pH of the eluted material was raised to 7.5 with 1 M Tris. Thereafter, 100 μl of 100% TCA were added to precipitate the eluted proteins followed by incubation for 10 min at −20° C followed by a centrifugation step for 20 min at 15,000 × g (4°C). The pellets were washed with acetone and left to air dry before they were dissolved in loading dye and analyzed under non-reducing conditions by SDS-PAGE, followed by Western blot analysis.In a total volume of 500 μl PBS buffer, 2 μM of EDC34, and 8 μM of IgG were incubated for 24 h at 37°C. Subsequently, TM Human Albumin/Immunoglobulin Depletion Kit (Millipore).The experiments were performed according to the manufacture's instructions. Briefly, total albumins and (>98%) and immunoglobulins  were depleted in human citrated plasma using PureProteomeIgG was immobilized (15 μg/mL) on a CM5 sensor chip via amine coupling in a 10 mM sodium acetate buffer (pH 4). A blank immobilization was performed in flow cell one. EDC34 was injected at different concentrations (25–400 nM) at a flow rate of 30 μl/min and a temperature of 25°C over the flow cells using a running buffer containing 10 mm Hepes, 150 mm NaCl, 0.005% surfactant P20, and 3.4 mM EDTA (pH 7.5). Regeneration of the sensor surface was conducted in 0.5 M sodium chloride. The analysis was performed using a Biacore X100 instrument  and the data was fitted using a 1:1 binding model.TFPI-2+/−, were mated with each other to yield complete knockout mice TFPI-2−/− (Data sheet 1). All experiments were performed facilitating female and male mice at the age between of 6 and 8 weeks according to protocols approved by the Animal Ethics Committee, Lund, Sweden. Animals were housed under standard conditions of light and temperature and had free access to standard laboratory chow and water.TFPI-2 conditional knockout mice were generated by Deltagen . Male chimeric mice were mated with C57BL/6J female mice, and F1 heterozygous mice Escherichia coli ATCC 25922 bacteria were grown to reach mid-exponential phase (OD620~0.4), washed twice in PBS and then diluted in PBS. Balb/c mice  were intraperitoneally (i. p) injected with 100 μl (8–8.5 × 108 cfu/ml) of E. coli. One hour after bacterial injection, VKG24 (500 μg per mouse) or PBS buffer alone were injected (i. p). Mice were sacrificed after 8 h post-infection and cfu were evaluated in blood, lung, spleen, kidney and liver.AIN-93M-Purified diet (ENVIGO) was provided for 1 week to mice, after 8 h post-infection mice were anesthetized and L-021 dye 25 mg per kg (Wako) was injected subcutaneously (s. c.), after 10–12 min of incubation. Reactive oxygen species (ROS) production was detected by measuring fluorescence in radiance (p/sec) for control and treated mice using IVIS® Spectrum . Imaging was performed in groups of 2 and 3 animals at a time.Escherichia coli ATCC 25922 bacteria were grown to mid-exponential phase (OD620~0.5), harvested, washed in PBS and diluted in the same buffer to 3–4 × 108 cfu/ml. Two hundred microliter of the bacterial suspension was injected intraperitoneally (i. p) into C57BL/6 mice. In another experiment, C57BL/6 mice were i. p. injected with 8 mg/kg of E. coli O111:B4 LPS (Sigma). In both cases, for evaluation of TFPI-2 expression, mice were sacrificed 0, 1, 2, 4, 6, and 12 h post-infection, and lung, brain, spleen, liver, kidney, small, and large intestine were harvested in trizol reagent. Before isolating RNA all organs were stored at −80°C.ad libitum. Pseudomonas aeruginosa Xen41 bacteria were grown to logarithmic phase (OD620~0.5), harvested, washed in PBS, pH 7.4 and diluted in PBS to either 3–4 × 108 cfu/ml, and kept on ice until injection. Fifty microliter of the bacterial suspension was administered intra nasally into mice. The survival data were obtained by following the animals daily up to 7 days monitoring. Mice reaching the pre-defined endpoint criteria were sacrificed and counted as non-survivors. For bacterial dissemination, mice were sacrificed after 12 h and cfu were determined in BALF and lung.Animals were housed under standard conditions of light and temperature, with access to chow and water P. aeruginosa using the Cytometric bead array (CBA) Mouse Inflammation Kit # 552364 (Becton Dickinson AB) according to the manufacturer's instructions on a FACSCalibur flow cytometer (Becton Dickinson AB). All plasma samples were stored at −80°C before the analysis.The cytokines IL-6, IL-10, MCP-1, IFN-γ, TNF-α were measured in plasma and BAL fluid from mice intra-nasally infected with *p < 0.05, **p < 0.01, *** < 0.001, ****p < 0.0001, and ns, not significant.Values are shown as mean with SEM. For statistical evaluation of two experimental groups, one-way with Tukey's multiple comparisons post-test was used and for comparison of survival curves the Mantel-Cox's test. Viable count and radial diffusion assay data are presented as mean with SD. All statistical evaluations were performed using the GraphPad Prism software 7.0 with Many factors including pH, salt concentrations, or interacting proteins, have been described to alter the antimicrobial activity of a number of AMPs . To testIgG is one of most abundant proteins in plasma and has a molecular weight in the range of 180 kDa. Thus, we hypothesized that EDC34 can interact with IgG or even with other immunoglobulins. In order to test this hypothesis, EDC34 was incubated with different classes of human purified immunoglobulins, i.e., IgA, IgG, IgE, and IgM and binding was tested by immunoblotting. Our results show that EDC34 avidly binds to all immunoglobulins tested Figure . When thE. coli bacteria. Figure E. coli bacteria and then later mixed with human IgG  was employed lacking the first seven amino acids, including two cysteine residues. Notably, AKA27 consists of several patches with clustered positively charged amino acids. Earlier studies have demonstrated these amino acids play an important role for the antimicrobial activity of many AMPs   12 h post-infection IL-6, IL-10, and MCP-1 was higher in TFPI-2−/− mice and no significant changes were observed in INF-γ, TNFα, and IL-12p70  Figure . In addi) Figure . Indeed 0 Figure , wherease Figure . No signe Figure , cytokins Figure , indicatTFPI-2−/− mice we for the first time show a protective role of TFPI-2 during Gram-negative infection. Studies using artificial membranes and live organisms have illustrated that the vast majority of AMPs exert their antimicrobial activity by permeabilizing the membrane of the pathogen. Thus a mechanism, leading to a burst of the bacterial cell wall, has been described for instance for defensins . Surprisingly, n TFPI-1 . PlateleE. coli under in vitro conditions (E. coli sepsis model. Our results show that bacterial growth was reduced in blood, liver, kidney, spleen, and lungs when infected mice were treated with VKG24. Along these lines we also noted that ROS production was notably lower in these mice, which indicates that VKG24 exerts an antimicrobial activity by lowering inflammation at the site of infection. Our current study describes increased cytokine levels detected in TFPI-2−/− bacterial challenged mice. This is in line with previously published study where we showed reduced cytokine levels in LPS challenged mice treated with VKG24 peptide (In vivo experiments further reveal the peptide motif exerts a systemic bacterial killing activity combined with a non-pathological inflammatory response, which in combination provides a promising approach for future therapeutic applications. Taken together, the evidence presented in this work demonstrates that TFPI-2 is mainly a host-defense protein, while the anti-coagulative functions likely play a minor role in normal hemostasis.As a proof of concept from a therapeutic perspective, we employed an animal model of infection using VKG24, the murine TFPI-2-derived homolog to EDC34 . Previounditions . Based o peptide , indicat peptide . In vivoPP and AS designed the research. MA, GK, ME, SA, SJ, and PP performed the experiments. AE, HH, AS, and PP contributed analytic tools,reagents,materials,analysis tools. PP analyzed the data and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer AT and handling Editor declared their shared affiliation.


Canarium tramdenum are commonly used as foods and cooking ingredients in Vietnam, Laos, and the southeast region of China, whilst the leaves are traditionally used for treating diarrhea and rheumatism. This study was conducted to investigate the potential use of this plant bark as antioxidants, and α-amylase and α-glucosidase inhibitors. Five different extracts of C. tramdenum bark (TDB) consisting of the extract (TDBS) and factional extracts hexane (TDBH), ethyl acetate (TDBE), butanol (TDBB), and water (TDBW) were evaluated. The TDBS extract contained the highest amount of total phenolic , while the TDBB extract had the most effective antioxidant capacity compared to other extracts. Its IC50 values were 12.33, 47.87, 33.25, and 103.74 µg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (ABTS), reducing power (RP), and nitric oxide (NO) assays, respectively. Meanwhile, the lipid peroxidation inhibition of the four above extracts was proximate to that of butylated hydroxytoluene (BHT) as a standard antioxidant. The result of porcine pancreatic α-amylase inhibition showed that TDB extracts have promising effects which are in line with the commercial diabetic inhibitor acarbose. Interestingly, the inhibitory ability on α-glucosidase of all the extracts was higher than that of acarbose. Among the extracts, the TDBB extract expressed the strongest activity on the enzymatic reaction (IC50 = 18.93 µg/mL) followed by the TDBW extract (IC50 = 25.27 µg/mL), TDBS (IC50 = 28.17 µg/mL), and TDBE extract (IC50 = 141.37 µg/mL). The phytochemical constituents of the TDB extract were identified by gas chromatography–mass spectrometry (GC-MS). The principal constituents included nine phenolics, eight terpenoids, two steroids, and five compounds belonging to other chemical classes, which were the first reported in this plant. Among them, the presence of α- and β-amyrins were identified by GC-MS and appeared as the most dominant constituents in TDB extracts (1.52 mg/g). The results of this study revealed that C. tramdenum bark possessed rich phenolics and terpenoids, which might confer on reducing risks from diabetes. A high quantity of α- and β-amyrins highlighted the potentials of anti-inflammatory, anti-ulcer, anti-hyperlipidemic, anti-tumor, and hepatoprotective properties of C. tramdenum bark.The fruits of  Diabetes or diabetes mellitus has become a burden for the global economy in recent decades. According to the World Health Organization’s report, this disease and its complications cause substantial economic loss through direct medical costs and loss of work and wages . Among dAs an endogenous toxin, oxidative stress is considered to be an important determinant of type 2 diabetes complications . The cauCanarium tramdenanum Dai & Yakovlev, a synonym of Canarium pimela Koenig, a woody tree belonging to Burseraceae family, is not an exception. This plant is widely distributed in subtropical and tropical regions of China and Indochina [C. tramdenum are commonly used as foods and cooking ingredients, whilst leaves are traditionally used for treating diarrhea and rheumatism [C. tramdenum or “Chinese black olive” is used in folk medicine as an anti-bacterial, anti-viral, anti-inflammatory, and detoxifying substance [C. tramdenum kernels were also documented [C. tramdenum bark extracts through in vitro assays of α-amylase and α-glucosidase suppression. Among natural sources, plants have been the most thoroughly scrutinized thanks to their vast diversity and wide distribution across the Earth. It is easy to derive antioxidant and nutrient components from every part of plants as fruits, leaves, stems, and roots which exhibit a wide range of biological effects such as anti-inflammatory, antibacterial, antiviral, anti-aging, and anticancer . Nonethendochina . In Vieteumatism ,11. In Cubstance . Recentlubstance ,12. The cumented ; howeverFrom 30 g dry TDB, five extracts with different yields were obtained by using an extraction method based on various polarity solvents. The yield of TDBS was 20% (6 g), followed by TDBW , TDBE , TDBB , and TDBH . The total phenolic contents (TPC) of TDB extracts were shown in The antioxidant capacities of TDB extracts by DPPH, ABTS, reducing power and β-carotene bleaching assays are displayed in 50=12.33 µg/mL) which was significantly higher than standard BHT (IC50=14.99 µg/mL) and other extracts, see DPPH is a stable reagent and widely used in most antioxidant tests. The principle of this sensitive assay is based on the reaction of samples with the organic radical DPPH . The ant•+ are the basis for this test. In the mixture, with the presence of antioxidants, the nitrogen atom of ABTS•+ quenches the hydrogen atom of antioxidants yielding the solution decolorization [•+ solution illustrates the antioxidant capacity of samples. As shown in 50 values of TDBB and TDBW were 1.7-times higher than that of BHT. Additionally, the TDBS extract was 1.3-times the antioxidant activity of BHT.In the case of the ABTS assay, antioxidants in samples reacting with an organic cation radical named ABTSrization . The dec3+) to potassium ferrocyanide (Fe2+). The final reaction with ferric trichloride results in a mixture of Fe3+ and Fe2+, a blue solution which can be spectrophotometrically determined at 700 nm [2+ in the final reaction) indicates a strong antioxidant activity. By comparing IC50 values, see 50 value of BHT was 38.34 µg/mL.Reducing power assay or potassium ferricyanide reducing power is based on the competence of antioxidants in converting potassium ferricyanide  of the linoleic acid peroxidation process can gradually decolorize the β-carotene color by time. The process can be delayed by the presence of antioxidants and the reaction can be recorded at 492 nm. It is apparent that, at the same concentration of 1000 µg/mL, all total extract and fractional extracts of TDB exhibit a similar inhibitory level of lipid peroxidation to the standard BHT, see 50 = 103.74 µg/mL), followed by TDBW (IC50 = 112.54 µg/mL), and TDBE (IC50 = 131.43 µg/mL). The activity of the total extract TDBS (IC50 = 116.80 µg/mL) was significantly higher than that of TDBE but lower than those of TDBB and TDBW. In the body, NO associates with many biological systems including neuronal messenger, vasodilatation, and antimicrobial and antitumor activities [Sodium nitroprusside in phosphate buffer saline (pH 7.2) generate nitric oxide (NO) which can be spontaneously converted into the more stable forms of nitrate and nitrite ions under the aerobic reaction with oxygen ,17. The tivities ,19. Henc50 = 257.20 µg/mL) while TDBW represented the lowest one (IC50 = 555.02 µg/mL). In this study, starch-iodine method was applied to examine the inhibitory effect of TDB extracts on porcine pancreatic α-amylase. The degradation of starch by enzyme activity was visually observed or spectrophotometrically measured at 565 nm based on changing dark-blue color to yellow color of reaction solution. Accordingly, in mixtures with inhibitors, the solution with higher absorption (darker color) signifies the higher inhibitory activity. As shown in p-nitrophenol, a yellow product that can be measured at wavelength 405 nm [50 values 18.93, 27.27, 28.17, 141.37, and 145.35 µg/mL, respectively.The α-glucosidase inhibitory activity of TDB extracts was assayed using a synthetic substrate pNPG. In physiological buffer (pH 7), α-glucosidase hydrolyzes pNPG to release h 405 nm . The lowp < 0.05). Especially, antioxidant assays including DPPH, ABTS, reducing power, and nitric oxide were strongly correlated with the α-glucosidase inhibitory assay at p < 0.05. Meanwhile, ABTS assay was not significantly associated with the α-amylase inhibitory assay.According to Phytochemical compositions of TDB extracts analyzed by GC-MS were listed in To the best of our knowledge, except for α-cubebene and (-)-spathulenol that were previously identified in the essential oil of TDB , the remAmong the identified compounds, phenolics accounted for the highest amount with nine compounds, followed by terpenoids (eight compounds), steroids (two compounds), carbohydrates (glycerol), monosaccharides (levoglucosan), phenylpropanoic acids (ibuprofen methyl ester), carboxylic acids (acetyltributyl citrate) and one unidentified compound. All phenolic compounds were detected in fractional extracts TDBB and TDBW while all terpenoids were identified in the TDBE extract. Although the phenolic components were scant in the total extract TDBS, they were accompanied with greater amounts in the fractional extracts of TDBB and TDBW. This is reasonable when lower polar components have been eliminated through hexane and ethyl acetate solvents. Whereas, 2-propylphenol and acetyltributyl citrate were only detected in fractional extracts TDBB and TDBE, respectively, but were not recognized in the total extract TDBS. Significantly, β-amyrin and α-amyrin were the most dominant compositions in the TDB extract, which showed the highest contents in TDBS  and TDBE  extracts.m/z 189, 203, 218, and 426, respectively. Meanwhile, β-amyrin data were 15, 52, 100, and 5, respectively at m/z values similar to the peaks described above, see The GC-MS analysis on the crystal mixture isolated from F15–F18 of TDBE extract confirmed the presence of α- and β-amyrins (with a 3:4 ratio) as the dominant components. MS data showed that the relative intensity corresponding to the main fragments of α-amyrin were 27, 25, 100, and 13 for peaks at Nelumbo nucifera (3 mg/g), respectively [C. tramdenum bark involves a greater amount of these terpenoids than C. subulatum (0.03 mg/g of β-amyrin), a species that belongs to the same Canarium genus  was collected at 21.07°N, 106.10°E, Bac Ninh province, Vietnam in 2016. The species was identified at a study field based on Vietnam Plant Data Center (http://www.botanyvn.com) and Plants Database Missouri Botanical Garden, United States (TROPICOS—http://www.tropicos.org). The specimen with voucher number TDB-J2016 was deposited at the Plant Physiology and Biochemistry Laboratory, IDEC, Hiroshima University, Japan.Stem bark of All extraction solvents including methanol, hexane, ethyl acetate, and butanol were purchased from Junsei Chemical Co., Ltd., Tokyo, Japan. The analytical reagents for antioxidant assays were acquired from Kanto Chemical Co., Inc., Tokyo, Japan while those of enzymatic assays were procured from Sigma-Aldrich, St. Louis, MO, USA.® R-300, Nihon Buchi K.K., Tokyo, Japan), a microplate reader , and GC-MS system  were the main instruments used in this study.A vacuum evaporator  and water. After blotting with tissues, bark samples were dried at 45 °C for 1 week by an oven, then ground to make the powder. The TDB sample (30 g) was extracted using 1 L of methanol in one week. Afterwards, dry methanol crude extract (TDBS) was blended with distilled water (200 mL), then successively fractionated by an equal volume of hexane, ethyl acetate, and butanol. Each fractionation step was conducted three times. After filtrations, they were separately dried under vacuum by an evaporator at 50 °C. Eventually, five dried extracts including TDBS , hexane (TDBH), ethyl acetate (TDBE), butanol (TDBB), and water (TDBW) extracts were obtained as fractional extracts. They were preserved in sterilized vials and kept in a refrigerator (−20 °C) for further tests on the physical properties, biological activities, and identification of phytochemical components.2CO3 (w/v), respectively. The mixture was then shaken and incubated at room temperature for 30 min. The absorbance was measured at 765 nm using a microplate reader. TPC was expressed as mg gallic acid equivalent per g dry weight of sample (mg GAE/g DW).The total phenolic content (TPC) was calculated according to the Folin-Ciocalteu method described previously . Specifi50 value was calculated as the concentration required to reach a 50% reduction of DPPH.DPPH assay was evaluated following Elzaawely et al.  with a s•+) decolorization was measured as described by Pellegrini et al. [•+ solution was prepared by mixing aqueous ABTS (7 mM) solution with 2.45 mM potassium persulfate (1:1 v/v) and incubating in darkness at room temperature for 16 h. The working solution was then obtained by diluting ABTS•+ solution in methanol to an absorbance of 0.70 ± 0.05 at 734 nm. In each well of a 96 well-plate, 25 µL of TDB sample was added to 200 µL of the working solution. After a slight shake, the plate was covered by an aluminum foil and kept at room temperature for 30 min. Subsequently, the absorbance was recorded by a MultiskanTM Microplate Spectrophotometer . The ABTS radical decolorizing activity was calculated by the following formulasample is the absorbance of reaction with momilactones or positive control (BHT) and Acontrol is the absorbance of reaction without momilactone or positive control. The IC50 value was determined as the concentration needed to bleach 50% of ABTS•+.2,2′-Azino-bis radical cation  were combined and incubated at 50 °C for 30 min. Subsequently, the reaction mixture was stopped by 500µL of trichloroacetic acid  and centrifuged for 10 min at 4000 rpm. Eventually, 100µL of the supernatant was double diluted with distilled water (100 µL) followed by adding 20 µL of ferric chloride solution . After shaking, the absorbance of the resulting mixture was read at 700 nm by a microplate reader. BHT was used as a standard reference. The IC50 value was calculated as mentioned above.The reducing capacity was carried out referring to the modified method of Minh et al.  with a 10 is the absorbance of reaction at the zero-minute time and A180 is the absorbance of reaction at the 180-min time. Methanol was used as negative control, whilst BHT was the positive control.The β-Carotene bleaching activity of TDB extracts was assayed using a β-carotene linoleate bleaching system  with tri50 value was obtained in the same way as described above.Nitric oxide scavenging was measured following the method of Govindarajan et al.  with som50 value were calculated as described previously [The α-amylase inhibitory activity was assayed based on the starch-iodine method . α-Amylaeviously . AcarbosSaccharomyces cerevisiae, Sigma-Aldrich, St. Louis, MO, USA). After 6 min of incubation at 25 °C, a 20 µL aliquot of 5 mM p-nitrophenyl-α-D-glucopyranoside (pNPG) substrate in the buffer was added and the mixture was incubated for another 8 min. Finally, the reaction was terminated by adding 100 µL of 0.1 M Na2CO3, and absorbance was recorded at 405 nm. Inhibition percentage was calculated by:sample is absorbance of the reaction with samples or positive controls (acarbose) and Acontrol is absorbance of reaction with 10% methanol. The enzymatic inhibitory activity of TDB extracts was expressed as IC50 value as well.The anti-α-glucosidase activity of TDB extracts was measured using a modified version of the method described by Johnson et al. . In brieThe phytochemical components of TDB extracts were identified by GC-MS analysis. The GC-MS system  with an autosampler coupled with a 30 m x 0.25 mm I.D. x 0.25 μm film thickness DB-5MS column . A concentration of 1000 µg/mL of each TDB extract was used for the initial injection. Helium was used as a carrier gas at split ratio 5:1. The GC oven conditions were as follows: The initial temperature was 50 °C without hold time, the boosted temperature was up to 300 °C at 10 °C /min, and held for 20 min. The injection port and detector temperature were set at 300 °C and 320 °C, respectively. The mass range scanned from 29 to 800 amu. The control of the GC-MS system and the confirmation of analytes were conducted using JEOL’s GC-MS Mass Center System Version 2.65a .v/v) which yielded 8 fractions (F1–F8) and 12 fractions (F9–F20), respectively. The elution step for every fraction was 100 mL. Fractions F15–F18 afforded a mixture of colorless crystal (45.6 mg) which was subsequently identified as α-amyrin and β-amyrin (75% purity) by GC-MS, see By preliminarily screening the GC-MS results, we found that two major compounds in TDB extracts were tentatively the pentacyclic triterpenes α-amyrin and β-amyrin. Therefore, we implemented an isolation of these two compounds by column chromatography. In brief, The TDBE extract (1.2 g) was premixed with 5 g silica gel . The mixture was then loaded onto a normal phase silica gel (40 g) column (2 × 50 cm). The mobile phase was 100% hexane and hexane:ethyl acetate  were calculated by using the same software.Data were elaborated on the Minitab 16.0 software . All assays were thrice implemented, and results were displayed as means ± standard errors (SE). Significant differences among tests were determined by one-way ANOVA using Tukey’s test at Canarium tramdenum bark possessed potent antioxidant and α-glucosidase inhibitory activity in in vitro trials. It was found that the fractions of ethyl acetate, butanol, and water extracts enriched with terpenoids and phenolics appeared as a promising source of natural antioxidants, α-amylase, and α-glucosidase inhibitors. In vivo tests and clinical trials should be elaborated to affirm the bioavailability of C. tramdenum bark for the development of food additives and supplements to reduce the risks from type 2 diabetes. The isolation of novel constituents, as well as investigations on potent pharmaceutical properties of C. tramdenum bark, should also be conducted. The contribution of α- and β-amyrins to biological activities of C. tramdenum bark should be further investigated.This study documented that