Here we developed SICT, a MATLAB program that automatically identifies rapid Ca2+ rises in time-lapse movies with low signal-to-noise ratios, using fluorescent indicators. A graphical user interface allows visual inspection of automatically detected events, reducing manual labour to less than 10% while maintaining quality control. The detection performance was tested using synthetic data with various signal-to-noise ratios. The event inspection phase was evaluated by four human observers. Reliability of the method was demonstrated in a direct comparison between manual and SICT-aided analysis. As a test case in cultured neurons, SICT detected an increase in frequency and duration of spontaneous Ca2+ transients in the presence of caffeine. This new method speeds up the analysis of elementary Ca2+ transients.Recent advances in live Ca In excitable cells, such as muscle cells and neurons, Ca2+ triggers contraction and neurotransmission, respectively5. Fluorescence microscopy allows the spatial and temporal measurement of intracellular Ca2+ dynamics in living cells7. Research questions linked to Ca2+ pathways are widespread in science and require different tissues or cell types, Ca2+ reporters and imaging hardware.Ca2+ transients, there is growing interest in various other types of Ca2+ signals and their putative role in synaptic plasticity and spontaneous neurotransmission11. In contrast to AP-evoked transients, smaller amplitude signals - referred to as Ca2+ sparks, Ca2+ puffs, Ca2+ syntillas or spontaneous Ca2+ transients (SCTs) - occur locally in restricted subcellular compartments and are relatively difficult to detect. Ca2+ sparks/sparklets are local Ca2+ transients that are commonly observed in skeletal and cardiac muscle cells. By definition, they originate from single Ca2+ release units in the sarco- or endoplasmic reticulum where each unit represents a cluster of Ca2+ release channels such as ryanodine receptors (RyRs), inositol 1,4,5-trisphosphate receptors (IP3Rs), or a combination of both13. These receptors are also expressed in nervous tissues and sparks have been observed in hippocampal and dorsal root neurons15. So-called Ca2+ puffs are thought to represent cytosolic Ca2+ rises released from intracellular Ca2+ stores, again with a contribution of IP3Rs16. Ca2+ syntillas are defined as Ca2+ sparks in presynaptic terminals, for instance in hypothalamic neurons, where they occur spontaneously in the absence of APs and extracellular Ca2+ by a RyR-dependent mechanism17. Finally, spontaneous Ca2+ transients (SCTs) occur in the absence of AP and involve RyRs. They were observed in cerebellar Purkinje cells18 and hippocampal neurons11. The recurrent role of intracellular Ca2+ channels in Ca2+ sparks, puffs, syntillas and SCTs suggests a shared mechanism for these types of events. Alternatively, spontaneous Ca2+ transients can also originate from the spontaneous opening of voltage-dependent Ca2+ channels at resting membrane potential, as demonstrated in cultured hippocampal neurons10. For simplicity, we will here collectively refer to all types of events as SCTs. The local and short-lived nature of SCTs (duration in the range of 40–260 ms depending on the reporter used), together with their low amplitude  and frequency (0.005–0.7 Hz) make the detection of these events challenging18.While classical neurotransmission is long known to be governed by action potential (AP)-evoked Ca2+ concentrations can be monitored with both chemical and genetically-encoded Ca2+ indicators (GECI), each with their own advantages and limitations22. In the latter class, the GCaMP family23 has been improved by altering its Ca2+ affinity, brightness, and subcellular localization26 to yield variants with high Ca2+ affinity and fast kinetics, including GCaMP6f27. Another key factor for the detection of intracellular Ca2+ fluctuations is related to imaging hardware. For optimal temporal resolution and light sensitivity, high-quality oil-immersion objectives offer high numerical apertures. Vacuum-cooled electron multiplying CCD (EM-CCD) cameras detect photons with quantum efficiencies above 90% while maintaining reasonable noise levels. Inevitably, there is a trade-off between the image capture time  and time resolution 19.Cytosolic Ca2+ imaging yields valuable datasets, but the large file sizes and the short duration of transient signals in noisy image data pose a challenge in data processing and storage. Considering the need to compare conditions in many cells per groups, manual processing of Ca2+ imaging data is laborious and sensitive to human bias by gradual changes in decision-making. Many good programs have been developed to detect changes in Ca2+ imaging data. Most are open source and available for widely used platforms as ImageJ29, MATLAB39, Python40 and R29 or can be run as a desktop application41. Many excellent tools, such as SeNeCA30,NeuroCa31 and FluoroSNNAP32, were designed for the optical analysis of neuronal circuits or high-throughput analysis of cell cultures, typically reporting Ca2+-based activity patterns of single or multiple neuronal cell bodies detected in vivo, ex vivo or in vitro41. Other tools like PeakCaller35 and SamuROI40 analyse Ca2+ signals in predefined regions (ROIs), offering accurate detection of biological signals in noisy data on the macro- to micro-scale, as small as dendritic spines. Automated detection of micro-scale Ca2+ signals in spatiotemporal  datasets has been achieved with xySpark in cardiomyocytes loaded with Fluo-4-AM 28 and with CaSCaDe in astroglia expressing GCaMP3 33. For our purpose of studying spontaneous synaptic Ca2+ signals however, we were prompted to develop an algorithm combining all of the following features: (a) the ability to detect low-amplitude signals in noisy data with a small type II error ; (b) analysing full images without user-defined ROIs; (c) the ability to process large movies ; (d) analysing events in micro-scale subcellular compartments rather than somatic oscillations or patterns in neuronal ensembles; (e) the ability to visualize spatiotemporal signals and categorize the resulting events; and (f) tools to report descriptive parameters of the detected events including amplitude, frequency and kinetics.High quality real-time Ca36, non-negative matrix factorization (NMF)37 and constrained non-negative matrix factorization and deconvolution38 exist but were not evaluated in our study.In general, automated detection methods employ four steps. First, data are filtered to reduce noise. The standard deviation of a Gaussian filter is typically implemented as a user controlled setting and can be applied to the spatial or temporal dimension of a signal, or both. Second, a threshold is set based on data characteristics, such as the mean and the standard deviation of the spatial and/or temporal dimension of the data. Data are then divided into super threshold and sub threshold pixels, where super threshold pixels are considered to contain relevant biological signal whereas sub-threshold pixels are excluded from further analysis. Third, super-threshold pixels that are directly adjacent in the spatial or temporal dimension are grouped into individual segments, which can be further analysed as distinct units of the data (‘regions of interest’). A widely used method for grouping is connected-component analysis. Other strategies such as spatio-temporal independent component analysis (ICA)2+ event and classify events for further analysis. The validity and sensitivity of the methods were confirmed using both simulated and experimental data.Here we report SICT, an open source program that automatically detects low amplitude SCTs in noisy time-lapse imaging data. SICT allows to visually inspect each Ca2+ indicator GCaMP6f were studied by time-lapse imaging at ≈29 Hz. Exploiting repeated measurements in the time dimension, a 3D Gaussian filter effectively reduced noise as illustrated in Fig. 2+, occurring within less than 5 frames (0.17 s in our experiments), ΔF/F0 values were calculated using a moving average for F0 (see methods for details). Note that this definition of F0 is unsuitable to detect slow fluctuations. A threshold was set at the median plus 3 times the interquartile range for each frame  represented the most abundant class of events (59.69%). Other events appeared as a combination of fast and slow Ca2+ rises , included multiple fast Ca2+ transients  or were classified as slow isolated Ca2+ transients . Other event types had minor contributions . Common properties are shown in Supplementary Fig. 2+ transients based on experience and a list of guidelines: the Ca2+ event should be isolated (and therefore not surrounded by other Ca2+ events), spatially localized in the neuronal structure and short-lived (in the order of ms). The same four recordings were processed by each observer. Before inspection, ROIs were sorted from high to low amplitude. Observers were instructed to continue searching for Ca2+ transients until no single transients were detected in the last examined 320 events.To evaluate the effect of differences between individual users during event inspection, four human observers were instructed to select all fast-isolated Ca2+ transients scored by different observers varies significantly. A similar difference was found for the amplitude, although with a small effect size. It is thus advisable that different experimental groups are analysed by the same person.Between the four human observers, of all 612 unique events scored by at least one observer  with ROIs generated by assisted processing as before; (2) with ROIs that were manually drawn as before; and (3) with ROIs identical to the manual method, but further processed by the assisted method. As expected, the lower amplitude of automatically processed ROIs  was restored to a similar amplitude when the ROIs were made identical . Differences in the reported peak duration were not statistically significant between the three groups  and even higher differences when the ROIs were made identical . The ROI area in presence of caffeine was 1.53-fold bigger than in its absence -automatic Ca41, such as FluoroSNNAP32, xySpark28 and NeuroCa31. All are able to detect SCTs. When adjusting parameters to strictly filter out true biological signals, we found that such filtering inevitably reduces the detection sensitivity, leading to false negatives. In addition, we did not want to make strict assumptions regarding the signal waveforms to be detected. Thus, we aimed to make the manual curation very fast by building a convenient graphical interface. Importantly, the automatically detected events are sorted for their likeliness to represent a biological signal. Various sorting methods can be chosen as explained in the section ‘ROI sorting methods’ of the SICT user manual . Events with the highest scores are shown first, typically representing convincing biological events, whereas later in the list, true events will be accompanied by more and more false positive hits. At some point, when biological signals are scarce and noise becomes predominant, the user can decide to stop the manual curation process. This decision can also be seen as a ‘filtering threshold’, but it is not a blind choice because the user is aware of the signal and noise patterns specific for the experiment.Events detected by the SICT algorithm still require manual curation. This was a deliberate decision and a key feature which distinguishes SICT from other excellent tools48 whereas our criterion for effective detection was set to 0.4 μm. Half-maximal performance with our program required SNRs of 1.91. Other published programs have claimed high performance (>98%) with SNRs as low as 0.2 albeit calculated differently49. However, the method in that study used predefined ROIs as input, and furthermore used manually selected example events to train a classifier. We avoided such conditions to be able to analyse large numbers of time-lapse movies. Furthermore, as illustrated by the example events in Fig. 2+ imaging experiments and should be reconsidered for each individual experiment. This dilemma underlines the value of visual event inspection to aid data interpretation.The assisted method was validated by testing its detection sensitivity using experimental data and simulated experiments with various SNRs. The program performance reached a maximum true positive rate (TPR) of 0.89, thus leaving 11% of true events undetected even at high SNRs. As a possible cause, local pixel noise may contribute to mislocation of peak signals in the x, y or time dimension. In another study, localization errors between 0 and 1 μm were reported2+ elevations with at least a similar sensitivity. SICT uses dynamic ROIs (i.e. changing over time) which are automatically generated instead of being user-defined. Higher Ca2+ signals yield larger ROIs. Therefore, the best measure of peak intensity is obtained by integrating the ΔF/F0 signal over ROI size. In contrast, ΔF/F0 amplitudes without integration should not be used as a direct measure of Ca2+ signal amplitude. For the same reason, the SICT method typically reports lower ΔF/F0 amplitudes (0.05–0.1) than manual methods (0.3–1)18. These lower ΔF/F0 amplitudes do not reflect lower Ca2+ peak concentrations but are a consequence of calculating the ΔF/F0 from a larger ROI, which helps to average out noise and improves the sensitivity of detection.Compared to manual processing, the SICT-assisted method readily detected CaThe SICT code is open source, allowing versatile downstream data processing in MATLAB. Both raw and analysed data are stored in a structure which allows easy data retrieval and event inspection. Overall, using the assisted processing and visual event inspection we could analyse 10 times more recordings compared to the manual method in the same amount of time. As may be expected, visual inspection by different users was a significant source of variation in the analysis, mostly affecting the event frequency. To avoid systematic differences, event inspection should be performed by the same user for all experimental groups.2+ syntillas in chromaffin cells previously showed a 1.73-fold frequency increase, without effect on the amplitude50. Nerve terminals stimulated with 20 mM caffeine exhibited a 2-fold frequency increase while the amplitude increased 1.18 fold17. Other studies on hippocampal neurons, which focused on SCTs, generally altered the SCTs using 30 μM ryanodine, which reduced the frequency of SCTs by blocking RyRs11. Finally, Llano and colleagues assessed SCTs in Purkinje cells after application of an agonistic concentration of ryanodine (5–10 μM), which induced a 3.8-fold frequency increase18. Our results thus agree with previous reports and demonstrate that SICT is well applicable to detect changes in SCT frequencies.As a test case, our program readily detected the expected increase in the frequency and duration of SCTs in paired recordings from hippocampal neurons after caffeine application. At the same caffeine concentration, Ca2+ transients or other fast local fluorescence increases in live cell imaging data, providing a new tool to accelerate the discovery of fast local signalling pathways in living systems.In conclusion, SICT-assisted image processing can be widely applied to investigate Ca2. After 8 days, half of the medium was replaced. Neurons were infected with lentiviral particles encoding GCaMP6f27 at 7 days in vitro (DIV) and analysed between DIV14-18.Animals were housed, bred and experimentally used according to institutional guidelines and Dutch and U.S. governmental laws. Prior approval was obtained from the animal ethical committee of the VU University and VU Medical Centre, named ‘Dier ethische commissie (DEC)’, approval license FGA 11-03. Hippocampal neurons were obtained from C57BL/6 mouse brain at embryonic day E18. Unless specified otherwise, all chemicals were from Invitrogen (The Netherlands). After removal of the meninges, the hippocampi were dissected in Hank’s balanced salt solution , which was buffered with 10 mM HEPES. Cells were dissociated with 0.25% trypsin for 20 min at 37 °C. After washing 5 times in HBSS, the cells were suspended in Neurobasal medium, supplemented with 2% B-27, 1.8% HEPES, 0.25% glutamax and 0.1% Pen-Strep. Cells were triturated by three passes through fire-polished Pasteur pipettes and counted using a Fuchs-Rosenthal chamber. High density continental cultures were created by seeding 25k neurons on a layer of 25k rat glia per well on 18 mm coverslips in 12-well plates or 50k neurons on 50k glia on 25 mm coverslips in 6-well plates. Glass coverslips were pretreated by washing in ethanol and sprayed with 0.1 mg/ml Poly-D-lysine, 0.2 mg/ml rat tail collagen (BD Biosciences) in 10.2 mM acetic acid. Cultures were kept in a humidified incubator at 37 °C and 5% CO2+ imaging was performed using a custom-built epifluorescence setup called FAINT . It uses an inverted microscope  illuminated by a polychromeV monochromator (TILL Photonics) with a 150 W Xenon high stability lamp in combination with a suitable GFP filter set . Coverslips were placed in the imaging chamber with artificial cerebrospinal fluid (ACSF) as extracellular solution containing (in mM): 140 NaCl, 2.4 KCl, 10 HEPES, 10 glucose, 4 CaCl2, 4 MgCl2 . 1 μM of tetrodotoxin  and 10 mM caffeine were added when specified. GCaMP6f was excited at a wavelength of 480 nm. Imaging was performed using a 40x-oil immersion objective  at RT (22–24 °C). Images were acquired by Live Acquisition software  at a frequency of 28.77 Hz for 3–5 minutes using an Andor Ixon Ultra 897 electron-multiplying CCD (EM-CCD) camera with the following settings: vacuum cooling to −70 °C, EM gain of 200, pre-amplification of 2, 40% lamp intensity, 30 ms exposure. Images had a spatial resolution of 512 × 512 pixels each calibrated to cover 0.16 μm2 of the specimen. Intensity values were recorded in 16-bit unsigned integer format, producing roughly 10 gigabytes per time-lapse movie. Data were exported in ‘.raw’ format using the ‘Offline Analysis’ data streaming option in Live Acquisition software, together with its associated ‘.mtd’ file which contains all metadata of the experiment. To investigate the effect of caffeine on SCTs, the same cell was first perfused in presence of 1 µM TTX for 3 min, followed by 3 min in 1 μM TTX and 10 mM caffeine.Ca51 unless specified otherwise. The automatic detection of ROIs is run by a graphical user interface . Data was imported as a 3D matrix  of unsigned 16-bit integer numbers indicating GCaMP6f fluorescence intensity. Instead of the often used 2D filter48 we used a 3D Gaussian filter (imgaussfilt3) to reduce high-frequency noise by weighted averaging each pixel with its neighbours, using a standard deviation (sigma) of 3 pixels for spatial, and 2 frames for temporal filtering. To calculate ΔF/F0 for each pixel, the baseline intensity (F0) was determined as a moving average of 15 to 5 time points prior to the current data point. The ΔF/F0 was then calculated as (F − F0)/F0. For an example of typical raw and calculated data values, see Supplementary Fig. 2+ transients was calculated for each frame by taking the median plus three times the interquartile range. The interquartile range is an effective measure of noise levels because it is not affected by the occurrence of Ca2+ fluctuations or baseline drift.Source code is accessible in the Supplemental Software. All computations were performed in MATLAB version 2016b2+ elevations. Information for each ROI was saved to an output file for further analysis, including raw intensity values, the peak x, y and t coordinates and the weighted centroid  represent putative Ca0 trace; the 10–90% rise time, 90-10% decay time and the full width at half maximum (FWHM). For kinetic parameters, time values were estimated beyond framerate resolution by linear interpolation. To report descriptive parameters of selected SCTs, the parameters were first calculated per event, averaged per cell and then averaged to get the overall mean.Kinetic parameters for single events are calculated as detailed in the Supplementary Table 0 was calculated using a F0 value calculated as the average from the last 25 data points of the trace. Peak amplitude was calculated as maximum ΔF/F0 during the peak minus a baseline value . The FWHM was calculated as the time of the closest data point between the half-maximal signal in the rising and decay phase of the peak. The frequency of events was calculated as the total number of events divided by the total imaging time in each experiment. The parameters were calculated per event, averaged per cell and then averaged to get the overall mean.For comparison, we also performed manual processing by opening the image stack in ImageJ (NIH). The first 500 frames (17s) were removed to allow removal of a bleaching component by subtraction of a mono-exponential curve fitted to the average frame intensity over time. ROIs were manually drawn, together fully covering visible neuronal structures in the image. Average pixel intensities of each ROI were calculated for each frame, graphed and visually inspected. The ΔF/F2+ events at 10 Hz (on average 0.35 per imaging frame) were generated by adding values of 1 at random locations within a cell mask in a matrix of all zeros. The mask corresponded to all pixels that had, on average in the time dimension, a higher-than-average intensity compared to the whole field of view (likely corresponding to a fluorescently labelled cell). The events were then spread out in the spatial dimension using a 2D Gaussian filter with a standard deviation of one pixel in both the x and y direction. Subsequently, the signal was convoluted in the time dimension with a normalized example event, for which we used the Ca2+ signal from a neuron firing a single action potential. This example event was multiplied by the standard deviation of the corresponding pixel and multiplied with a SNR factor. Finally, the matrix that contained background noise was summated with the matrix that contained simulated events and saved in the same format as experimental data files.To assess the detection performance, a range of signal-to-noise ratios (SNRs) was tested using simulated data. Background noise was simulated by taking raw data frames and calculating mean and standard deviation. These values were used to generate a matrix with the same spatial dimensions. To reduce processing time, a decreased time dimension was used. Per simulation, 100 CaThis procedure was repeated with SNRs varying from 0.01 to 7 in 0.01 increments. The files were automatically processed by SICT and the output automatically analysed to determine how many events had been detected, using the criterium that SICT detected a ROI with a spatial accuracy of 1 pixel (0.4 µm) and a temporal accuracy of 10 frames (±348 ms).Statistical analysis was performed using SPSS v.23.0 . Data are reported as mean ± SEM, except when specified otherwise. Data were checked for normality using the Shapiro-Wilk or Kolmogorov-Smirnov test. When required, other assumptions were tested: homogeneity of the variance was assessed with Levene’s test; the sphericity assumption was tested with Mauchly’s test. For normally distributed data, a parametric test was run; otherwise a non-parametric test was performed. If the sphericity assumption was not met, the Greenhouse-Geisser correction was used to adjust the degrees of freedom. For two groups, a paired samples t-test or Wilcoxon signed-rank test was performed. In case of more than 2 groups, one-way repeated measures ANOVA or Friedman test was used.Where necessary, the alpha significance threshold was adjusted for multiple testing. In case of more than 2 groups, a Bonferroni correction was used to adjust the p-value for pairwise comparison, keeping the alpha significance threshold at 0.05. The effect size was calculated for the paired samples t-test as 2+ transients”, https://hdl.handle.net/10411/UA6UZG, DataverseNL). An instruction is included in the Supplementary Information.MATLAB code can be retrieved without restrictions from the VU Institutional Research Data Management system (Groffen, AJA, 2018, “Replication data for: SICT: automated detection and supervised inspection of fast CaSupplementary Information


Protein tyrosine phosphatase 1B (PTP1B) negatively regulates leptin signaling by dephosphorylating JAK2, and the increased activity of PTP1B is implicated in the pathogenesis of obesity. Hence, inhibition of PTP1B may help prevent and reduce obesity. In this study, we revealed that phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate in certain cruciferous vegetables, potently inhibits recombinant PTP1B by binding to the reactive cysteinyl thiol. Moreover, we found that PEITC causes the ligand-independent phosphorylation of Ob-Rb, JAK2, and STAT3 by inhibiting cellular PTP1B in differentiated human SH-SY5Y neuronal cells. PEITC treatment also induced nuclear accumulation of phosphorylated STAT3, resulting in enhanced anorexigenic POMC expression and suppressed orexigenic NPY/AGRP expression. We demonstrated that oral administration of PEITC to mice significantly reduces food intake, and stimulates hypothalamic leptin signaling. Our results suggest that PEITC might help prevent and improve obesity.Obesity, a principal risk factor for the development of diabetes mellitus, heart disease, and hypertension, is a growing and serious health problem all over the world. Leptin is a weight-reducing hormone produced by adipose tissue, which decreases food intake  Phenn the US . After ohe brain . In the 3VO4) and corn oil were purchased from Wako Pure Chemical Industries (Osaka Japan). Benzyl isothiocyanate (BITC) was obtained from Thermo Fisher Scientific . All of the other reagents used in the study were of analytical grade and obtained from commercial sources.Fetal bovine serum (FBS) was obtained from Sigma-Aldrich . Dithiothreitol (DTT), the protease inhibitor cocktail, the phosphatase inhibitor cocktail, Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, Triton X-100, PEITC, allyl isothiocyanate (AITC), fluorescein isothiocyanate (FITC), phenyl isothiocyanate (PITC), and Nonidet P-40 (NP-40) were purchased from Nacalai Tesque . Sodium orthovanadate (Nap-nitrophenol phosphate (pNPP) as a substrate according to the published procedure [The recombinant PTP1B activity was assayed using rocedure .S-Carbamidomethylated PTP1B was prepared as previously described [Reactive cysteine labeling assay was performed according to the published procedure . Iodoaceescribed .After the reaction of recombinant PTP1B with FITC, the reaction mixtures were subjected to SDS-PAGE. Then, the fluorescence intensity was detected using a Typhoon FLA-7000 fluorescence imager (Fujifilm).2 atmosphere. The cells were differentiated by treatment with 100 nM of all-trans retinoic acid (atRA)  between 1 and 6 days after plating on a collagen-coated culture plate [SH-SY5Y human neuroblastoma cells  were grown in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% heat-inactivated FBS, 100 units/mL streptomycin, and 100 μg/mL penicillin at 37°C in a humidified 5% COre plate . The difSDS-PAGE and immunoblot analysis were performed as previously reported . The antCellular PTP1B activity was measured by malachite green assay for detection of free phosphate from PTP1B phosphopeptide substrate (Santa Cruz Biotechnology) as previously described . Biomol The cytoplasmic and nuclear extracts for immunoblotting were prepared as previously described . ProteinPOMC , NPY , and AGRP  were purchased from Thermo Fisher Scientific. Primers for human ribosomal protein S18 (RPS18) (Primer Set ID: HA067807) were purchased from Takara Bio, Shiga, Japan. qRT-PCR was performed on the Thermal Cycler Dice Real Time System Single (Takara Bio) using the THUNDERBIRD SYBR qPCR Mix (Toyobo). The Ct value was normalized with the housekeeping gene RPS18 and the relative fold change was computed by the ΔΔCt method.The total RNA was isolated from differentiated SH-SY5Y cells using the SuperPrep Cell Lysis Kit for qPCR  according to the manufacturer’s protocol. The total RNA was converted to cDNA using the SuperPrep RT Kit for qPCR (Toyobo). Primers for the human The immunocytochemical study was performed according to previously described methods . Antibodad libitum access to water and a standard powdered diet . Animal experiments were carried out according to a protocol approved by the Animal Care Committee of Osaka Prefecture University (permission number: 28–184).Six-week-old male C57BL/6J mice were purchased from Kiwa Laboratory Animals . All mice were individually housed at 24 ± 2°C under 12:12-h light-dark cycle with lights on 7:00 AM-7:00 PM, and had via oral gavage (10 mL/kg body weight), followed by further 1-h fasting. At 7:00 PM, the mice were allowed ad libitum access to a standard powdered diet. After feeding at 0 h (at 7:00 PM), 1 h (at 8:00 PM), 4 h (at 11:00 PM), 14 h (at 9:00 AM), and 24 h (at 7:00 PM), food intake and body weight were measured. In addition, after oral dosing at 2 h, the blood sample was obtained from tail veins, and plasma leptin concentrations were measured using a Murine Leptin Standard ABTS ELISA Development Kit .After 1 week of environmental acclimation, the mice were fasted for 6 h (food removed at 12:00 AM). At 6:00 PM, the mice were divided into 2 groups and given either corn oil (vehicle) or 2.5 mg/mL PEITC-corn oil (25 mg/kg PEITC) g for 15 min at 4°C, and the supernatants were analyzed by immunoblotting as described above.The mice were orally infused with either corn oil (vehicle) or 2.5 mg/mL PEITC-corn oil (25 mg/kg PEITC) as described above. After further 3-h fasting, the mice were sacrificed by cervical dislocation. Then, the hypothalamus was quickly dissected out, frozen in liquid nitrogen, and kept at –80°C. The frozen hypothalamus was homogenized in radio-immunoprecipitation assay (RIPA) buffer containing 1% Triton X-100, the phosphatase inhibitor cocktail, and protease inhibitor cocktail using a bead-beater type homogenizer. The homogenates were centrifuged at 13,000 × Fig 1B, the incubation of PEITC with recombinant PTP1B at 37°C for 30 min caused a concentration-dependent reduction in the enzymatic activity with a half-maximal inhibitory concentration (IC50) value of 26.2 ϋM. Recent studies have revealed that PTP1B activity is subject to regulation by post-translational modification such as oxidation and alkylation of a reactive deprotonated cysteine residue at the catalytic center [Fig 1C, we speculated that this inactivation could be due to the reaction of ITC with the cysteinyl thiols to form an unstable thiocarbamoyl adduct. To examine whether or not the active site of PTP1B is modified by PEITC, we determined the reactive cysteinyl thiol in PTP1B using the cysteine-reactive probe iodoacetyl-LC-Biotin [Fig 1D and S1A Fig). Moreover, we also observed that BITC, PITC, and AITC inactivate PTP1B in a concentration-dependent manner, and cause a significant decrease in the reactive cysteinyl thiol . To further verify whether or not ITC directly inactivates PTP1B by the covalent modification, we fluorometrically assessed the modification of PTP1B by a fluorescent FITC . We confirmed that FITC inhibits PTP1B activity in a concentration-dependent manner . As shown in Fig 2C and S2A Fig, the incubation of PTP1B with FITC elicited a dose-dependent increase in the fluorescent intensity of PTP1B with a concomitant decrease in the reactive cysteinyl thiol. Furthermore, to obtain definitive evidence that FITC binds to the cysteinyl thiol of PTP1B, we prepared S-carbamidomethylated PTP1B by alkylation of free cysteine residues with iodoacetamide. As shown in Fig 2D and S2B Fig, compared with intact PTP1B, incubation of S-carbamidomethylated PTP1B with FITC drastically reduced the fluorescent intensity of PTP1B. These results suggest that ITCs inactivate PTP1B by binding to the catalytic cysteinyl thiol.As shown in c center ,42. AccoC-Biotin . After ltRA-induced Ob-Rb expression [S3 Fig, we validated that treatment of SH-SY5Y neuroblastoma cells with 100 nM RA for 5 days induces increased expression of Ob-Rb and augments neurite outgrowth. Moreover, treatment of differentiated SH-SY5Y cells with leptin functionally stimulated the tyrosine phosphorylation of Ob-Rb, JAK2, and STAT3 . In good agreement with this response, POMC mRNA expression, determined by qRT-PCR, was significantly elevated by leptin stimulation, whereas both NPY and AgRP mRNA expression was reduced .To investigate the effect of PEITC on leptin intracellular signaling, we used differentiated neuronal SH-SY5Y cells, which represent a suitable cellular model with apression . As showFig 3A). To examine whether or not PEITC inactivates cellular PTP1B, we measured PTP1B activity in PEITC-treated differentiated SH-SY5Y cells using a phosphorylated peptide substrate. As shown in Fig 3B, the treatment of differentiated SH-SY5Y cells with PEITC for 30 min induced concentration-dependent inhibition of cellular PTP1B activity like the Na3VO4, a PTP1B inhibitor [Fig 3C and S5A Fig, we observed that the exposure of differentiated SH-SY5Y cells to PEITC for 30 min resulted in a concentration-dependent phosphorylation of Ob-Rb, JAK2, and STAT3 but had no influence on the protein levels of PTP1B. The densitometric analysis of the bands showed a significant increase in the level of phosphorylated Ob-Rb, JAK2, and STAT3 by treatment of differentiated SH-SY5Y with 1 μM PEITC for 30 min . The leptin-independent activation of Ob-Rb was induced from 30 to 60 min after the exposure with 1 μM PEITC . On the other hand, the phosphorylation of JAK2 and STAT3 was observed for up to 120 min after the treatment with PEITC. We also confirmed that Na3VO4 led to a ligand-independent activation of leptin signaling . Thus, our results indicate that PEITC can cause a leptin-independent activation of leptin signaling through PTP1B inhibition. Additionally, we also observed that PEITC potentiates the ligand-dependent activation of leptin signaling in differentiated SH-SY5Y cells .After establishing the model SH-SY5Y cell system we assessed cellular responses to PEITC treatment. First, we ascertained that PEITC showed no cytotoxicity against differentiated SH-SY5Y cells at a concentration up to 10 μM over a period of 24 h , treatment of differentiated SH-SY5Y cells with PEITC for 30 min resulted in a dose-dependent accumulation of p-STAT3 in the nucleus. This observation was further confirmed by determination of p-STAT3 levels in nuclear and cytosolic fraction by immunoblotting. As shown in Fig 4B and S5E Fig, our results clearly indicated that the exposure to PEITC increased the nuclear level of p-STAT3 in a concentration-dependent manner but had little effect on the cytosolic level. Furthermore, qRT-PCR analysis revealed that PEITC treatment significantly activates POMC transcription while inhibiting AgRP and NPY transcription . These data suggest that PEITC could reduce food intake and increase energy expenditure by leptin signaling-mediated transcriptional control by inhibiting PTP1B.We next examined whether PEITC-inducible activation of leptin signaling promotes nuclear translocation of activated STAT3. As demonstrated by an immunofluorescence microscopic analysis  or the vehicle, and further deprived of food for 1 h. Then, the mice were refed with a powdered diet (standard rodent chow). As shown in Fig 5A, the oral administration of PEITC significantly reduced 14- and 24-h food intake after refeeding, compared with vehicle control. The anorexigenic effect of PEITC was observed from 1 h to 14 h after refeeding . On the other hand, PEITC treatment did not significantly decrease body weight gain during 24-h refeeding, compared with vehicle treatment . We also observed that there was no significant difference in plasma leptin levels 2 h after the administration between the vehicle and PEITC groups . Our data demonstrated a rapid effect of feeding inhibition (as early as 2 h post-administration) by oral treatment of PEITC. Furthermore, we evaluated the phosphorylation levels of Ob-Rb, JAK2, and STAT3 in the hypothalamus 3 h after the oral administration by immunoblotting. As shown in Fig 6A–6D, PEITC administration caused significant increases in the phosphorylation levels of Ob-Rb, JAK2, and STAT3 in the hypothalamus compared with the vehicle control. These data suggest that PEITC may decrease food intake by stimulating the leptin signaling via PTP1B inhibition.To demonstrate Figs 4). Moreover, we also demonstrated that the oral administration of PEITC to mice induced enhanced phosphorylation of Ob-Rb, JAK2, and STAT3 in the hypothalamus, and significantly reduced food intake . Our current findings provide, for the first time, insight into the preventive implications of an orally available PTP1B inhibitor, PEITC, for obesity.Most obese subjects develop leptin resistance and hyperleptinemia, restricting the therapeutic efficacy of leptin treatment. Targeting molecules required for the development of leptin resistance is a potentially effective therapeutic approach for preventing the obesity epidemic. Accumulating evidence from animal models, clinical and cellular studies suggest that PTP1B could be involved in the pathways leading to leptin resistance as a major negative regulator of leptin signaling. A recent study revealed that hypothalamic PTP1B levels in rats increase coincident with the initial development of leptin resistance, and that intracerebroventricular administration of a pharmacological competitive PTP1B inhibitor restores leptin sensitivity, resulting in a dose-dependent inhibition of food intake . StudiesFig 7. PTP1B consists of 435 amino acids with a molecular weight of approximately 50 kDa, and is localized on the cytoplasmic face of the endoplasmic reticulum by means of a hydrophobic C-terminal sequence (35 residues) [Ka) (~5.4), making it a thiolate anion (Cys–S−) at a physiological pH [Ka enhances the catalytic function of the Cys-215 as a nucleophile but renders it susceptible to oxidation or electrophilic alkylation [via facilitated tyrosine phosphorylation of signaling proteins. With this reactivity in mind, we demonstrated that ITCs caused a dose-dependent modification of the reactive cysteinyl thiol in PTP1B . These results strongly suggest that ITCs inactivate PTP1B by covalent binding to the catalytic cysteinyl thiol. To determine the modification site by PEITC, the native and PEITC-treated PTP1B were digested with V8 and trypsin and then analyzed by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) [Ay mice. In the meantime, several quinone compounds including 1,2-naphthoquinone and pyrroloquinoline quinone have been reported to inhibit PTP1B activity through the oxidation and/or covalent modification of the active site cysteinyl thiol [3VO4 gives rise to the leptin-independent phosphorylation of Ob-Rb, JAK2, and STAT3 in differentiated SH-SY5Y cells . JAK2 associates with the cytoplasmic domains of Ob-Rb, and leptin-induced receptor rearrangement facilitates JAK2 trans-phosphorylation of activation-loop Tyr-1007 and Tyr-1008 in JH1 (the tyrosine kinase domain), leading to receptor phosphorylation and recruitment and phosphorylation of STAT3 [Fig 7.In the present study, we also characterized the molecular mechanism underlying PEITC-mediated inhibition of PTP1B as summarized in esidues) . Due to gical pH . The lowkylation ,49,52. T-TOF-MS) . However-TOF-MS) . Recentl-TOF-MS) . Safranayl thiol . These qyl thiol . In thisof STAT3 . Notablyof STAT3 . This obFigs 4). Accordingly, these observations indicate that oral administration of PEITC can result in effective concentrations in the brain. Indeed, we revealed that the oral administration of PEITC (25 mg/kg/body weight) stimulated hypothalamic leptin signaling, and significantly reduced food intake . Hence, PEITC administration including the high ingestion of cruciferous vegetables can be expected to be beneficial for preventing and improving obesity without adverse effects. However, long-term in vivo studies are required to establish the anti-obesity efficacy, efficient dosage, and safety profile of PEITC. In the future, we must adequately assess the long-term potency of PEITC ingestion for suppression of food intake and body weight gain, and improvement of leptin responsiveness in obese mice fed a high-fat diet or in obese human subjects.In humans, the amount of glucosinolates consumed is estimated to be about 300 mg/day from various cruciferous vegetables , and appS1 FigA) Concentration-dependent modification of cysteinyl thiol in PTP1B by PEITC. Recombinant PTP1B was incubated with the indicated concentration of PEITC in HEPES buffer (pH 7.5) at 37°C for 30 min. Then, the reactive thiol group and total PTP1B were determined by biotin-labeling assay and immunoblotting, respectively. (B) The structures of food-derived ITCs. (C) The concentration-dependent inhibition of PTP1B activity by ITCs. Recombinant PTP1B was incubated with the indicated concentration of ITC in HEPES buffer (pH 7.5) at 37°C for 30 min, and then PTP1B activity was measured. The results are shown as means ± S.E.M. (n = 3). (D) Concentration-dependent modification of cysteinyl thiol in PTP1B by ITCs. Recombinant PTP1B was incubated with the indicated concentration of ITC in HEPES buffer (pH 7.5) at 37°C for 30 min. Then, the reactive thiol group and total PTP1B were determined.((TIFF)Click here for additional data file.S2 FigA) Concentration-dependent modification of PTP1B by FITC. Recombinant PTP1B was incubated with the indicated concentration of FITC in HEPES buffer (pH 7.5) at 37°C for 30 min. (B) Cysteine-targeted modification of PTP1B by FITC. Intact and S-carbamidomethylated recombinant PTP1B were incubated with or without 10 μM FITC in HEPES buffer (pH 7.5) at 37°C for 30 min. (A and B) The reactive thiol group in PTP1B was determined by biotin-labeling assay. The FITC-modified PTP1B was detected by fluorescent imaging. The total PTP1B level was determined by immunoblotting.((TIFF)Click here for additional data file.S3 FigA) The expression level of Ob-Rb in atRA-exposed SH-SY5Y cells. The cells were differentiated by treatment with 0–100 nM atRA between 1 and 6 days after plating on a collagen-coated culture plate. Then, the levels of Ob-Rb, JAK2, STAT3, and GAPDH were determined by immunoblotting. (B) The mRNA expression levels of Ob-Rb in the undifferentiated and differentiated SH-SY5Y cells. The RT-PCR was performed on undifferentiated cells and differentiated cells following the exposure to 100 nM atRA for 5 days using PrimeScript One Step RT-PCR Kit Ver.2 (Takara Bio). Primers for the human OB-RB  was purchased from Thermo Fisher Scientific. Primers for human ribosomal protein S18 (RPS18) (Primer Set ID: HA067807) were purchased from Takara Bio. (C) Phase-contrast images of the undifferentiated cells (left panel) and differentiated cells following the exposure to 100 nM atRA for 5 days (right panel).((TIFF)Click here for additional data file.S4 FigA and B) The SH-SY5Y cells were treated with 100 nM leptin for 30 min in serum-free DMEM. (A) Activation of leptin signaling in leptin-stimulated differentiated SH-SY5Y cells. After leptin treatment, the levels of p-Ob-Rb, p-JAK2, p-STAT3, and GAPDH were determined by immunoblotting. (B) Levels of POMC, NPY, and AGRP mRNA in leptin-stimulated differentiated SH-SY5Y cells. After leptin treatment, mRNA levels of POMC, NPY, and AGRP were analyzed by qRT-PCR. The results are shown as means ± S.E.M. (n = 3). *p < 0.05, **p < 0.01 vs vehicle-treated control (Student’s t-test).((TIFF)Click here for additional data file.S5 FigA and B) Activation of leptin signaling in the differentiated SH-SY5Y cells by PEITC treatment. The SH-SY5Y cells were treated with the indicated concentrations of PEITC for 30 min (A) or 0–120 min (B) in serum-free DMEM. (C) Activation of leptin signaling in the differentiated SH-SY5Y cells by Na3VO4 treatment. The SH-SY5Y cells were treated with 100 nM leptin for 30 min or with 200 μM Na3VO4 for 60 min in serum-free DMEM. (D) Effect of PEITC on ligand-dependent activation of leptin signaling in the differentiated SH-SY5Y. The SH-SY5Y cells were treated with the indicated concentrations of PEITC in the presence or absence of 50 nM leptin for 30 min. (E) Nuclear accumulation of p-STAT3 by treatment of the differentiated SH-SY5Y cells with PEITC. The SH-SY5Y cells were incubated with the indicated concentrations of PEITC or 100 nM leptin for 30 min. Then, nuclear and cytoplasmic fractions were prepared. (A–E) The levels of p-Ob-Rb, p-JAK2, p-STAT3, PTP1B, histon H3, and GAPDH were determined by immunoblotting.((TIFF)Click here for additional data file.


Worldwide morbidity and mortality from acute myocardial infarction (AMI) and related heart failure remain high. While effective early reperfusion of the criminal coronary artery after a confirmed AMI is the typical treatment at present, collateral myocardial ischemia-reperfusion injury (MIRI) and pertinent cardioprotection are still challenging to address and have inadequately understood mechanisms. Therefore, unveiling the related novel molecular targets and networks participating in triggering and resisting the pathobiology of MIRI is a promising and valuable frontier. The present study specifically focuses on the recent MIRI advances that are supported by sophisticated bio-methodology in order to bring the poorly understood interrelationship among pro- and anti-MIRI participant molecules up to date, as well as to identify findings that may facilitate the further investigation of novel targets. Among clinical emergency events, ST-segment elevation (STE) or the non-STE electrocardiogram diagnosis of acute myocardial infarction (AMI) is particularly common worldwide, with a staggering number of annual first episodes as well as recurrent ones . The mos2+ overload, and mitochondrial permeability transition pore (mPTP) opening. It is important to understand how these mechanisms are dynamically regulated by pivotal molecular targets and potentially reversed in the context of cardioprotection [2S) donor AP39 and its auxiliary targets have recently been identified as critical participants in H2S synthesis for modulating the postischemic cardiomyocyte survival in a manner independent of classical cytosolic signaling mechanisms [in vitro and in vivo [This coupled comorbidity of pathological ischemia and therapeutic reinjury of infarcted myocardium, namely, myocardial ischemia-reperfusion injury (MIRI), is particularly refractory to treatment , 5. Tradotection . For inschanisms , 8. In a in vivo , the sub in vivo , 12. HowTo facilitate comprehension of the discordance between the many positive animal outcomes and the inconsistent findings of the clinical data, focusing on significant and universal cellular mechanisms contributing to MIRI in order to identify more novel cardioprotective targets will be necessary , 13. TypWe herein review several novel MIRI-driven agents and antagonistic targets for cardioprotection, mainly from the biochemical levels to the potential therapeutic implications.2S suggests that interfibrillar mitochondria (IFM) play an important role in cardioprotection against MIRI [2S donor, against MIRI using substrates for complex I  and complex II  in both IFM and subsarcolemmal mitochondria (SSM) showed that AP39 could inhibit mito-ROS generation and mPTP opening via a cyclophilin D-independent mechanism without influencing mitochondrial respiration [2O2 production may be related to Mg2+-dependent NADH generation by malic enzyme, and it can be blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone in ROS reduction [MIRI develops when the effective myocardium-supporting circulation is decreased and subsequently restored, and cardiomyocyte mitochondrial breakdown during MIRI is the common denominator stemming from the aberrant functioning of the controller that maintains homeostasis between oxidative and reductive stress, which are typically dual dynamic phases experienced by the cells adapting towards endogenous or exogenous noxious stimuli , 21. In nst MIRI . An explnst MIRI , a mitocpiration , 23. Onepiration . During eduction .+/H+ exchanger subsequently excretes excess hydrogen ions to buffer this accumulation of hydrogen ions, which creates a large influx of Na+, as well as Ca2+. In the process of MIRI, Ca2+ overload in cardiomyocyte is accompanied by the activation of intracellular proteases that damage myofibrils and induce hypercontracture and contracture band necrosis [During myocardial ischemia, anaerobic metabolism is predominant due to energy failure, thereby reducing the intracellular pH. The Nanecrosis , 25. Recnecrosis –28. The necrosis . After rnecrosis . Based onecrosis , 28. Hownecrosis .Based on the hypothesis that the onset of the mitochondrial permeability transition may be a prominent mechanism in MIRI, the emerging role of mitochondrial translocator protein (TSPO) in cardioprotection has recently been targeted . TSPO isMore than 60% of mitochondrial proteins contain acetylation sites involved in energy regulation, such as the inhibition of mitochondrial metabolism and ATP synthesis , which iMIRI is often initiated as an adaptive response to primary ischemia injury , 10, 13 α [Under physiological conditions, ischemia activates FUN14 domain containing 1 (FUNDC1), a receptor that mediates mitophagy caused by hypoxia and mitochondrial stress through interaction with Microtubule Associated Protein 1 Light Chain 3 Alpha (LC3), to selectively remove the damaged mitochondria and confine Cyt-c effusion, thus inhibiting cardiomyocyte apoptosis . Accordiα  inactivaα –48. An iα . Throughα , dual-spα , Bax inhα , and melα , 54  and hexokinase 2 (HK2) can be recovered by melatonin and therefore blocking mitophagy-associated pathology by inhibiting mitochondrial fission through mPTP opening prevention. The decreased level of mitochondrial fission during MIRI may be associated with the inactivation of Drp1 triggered by AMPKSince myocardial infarction is strictly, at least to a large extent, associated with atherosclerosis, apart from the mechanical blockage of the criminal coronary vessels, ischemia plus rescuing reperfusion usually triggers local sterile inflammatory responses as well as myocardial cell apoptosis , 57. How2BR found in myocardium during MIRI may exert an infarct-limiting effect in the reperfused heart at an early stage not by targeting cardiomyocytes but possibly by affecting macrophages via the PI3K/Akt pathway, which is believed the vigor to switch cardiac macrophage phenotypes to an anti-inflammatory M2 subset [α and IL-6, loss-of-function studies have suggested that TLR5 may play an important role in cardioprotection against MIRI [The spontaneously elevated adenosine receptor A2 subset –63. Furtnst MIRI , 65. In nst MIRI . Howevernst MIRI , 65.in vitro or in vivo, which might reinforce the hypothesis that the direct supply of exogenous MIFs may not be beneficial in terms of infarct size reduction due to undetected mechanisms [In contrast, the spontaneously upregulated protein OPHN1 has been suggested to play a potential role as a cardioprotective inflammation regulator, which may inhibit macrophage migration and cardiomyocyte apoptosis in MIRI mice . Intereschanisms .α-induced protein 3; TNFAIP3) may somehow be involved in such cardioprotection by limiting NF-κB signaling-guided inflammation, although the mechanisms underlying the activation of A20 in cardiomyocytes require further investigation [κB kinase IKKɑ has also been identified as a novel mediator capable of attenuating the outcomes of MIRI, possibly by the negative control of macrophage polarization towards the M1 phenotype [When profiling the inflammatory response mediated by macrophages, chemerin15  was identified as a novel effector that can ameliorate MIRI by decreasing cardiomyocyte apoptosis, reducing neutrophil infiltration, and, more interestingly, inducing alternative M2 macrophage polarization . While atigation , 73. Thehenotype .κB signaling [Another novel component of the acute inflammatory response in the process of MIRI is nucleotide-binding oligomerization domain-like receptor (NLR) family –76, an uignaling , 78. In ignaling , 80. Takignaling –79. Furtignaling , 81.in vitro and in vivo experiments, several protective cell types of the immune system have been found to actively respond to MIRI, including pericytes, Ly6Chigh monocytes, M2 macrophages, and CD8+/AGTR2+ T cells [Following the early inflammation stage, pathophysiological changes in MIRI might next cause endogenous signals that may trigger anti-inflammatory phases dominated by the innate immune response , 83, the T cells –89.+/CD25+ subset of lymphocytes) in fighting MIRI [Increasing evidence support the role and potential therapeutic value of regulatory T lymphocytes  , paracri pathway . A studyhway [β1 . Therefoirection , 99.β as well as the regulation of mPTP [As immunotherapy of post-MIRI patients has been actively explored, a recent study regarding sphingosine-1 phosphate (S1P) and its analogue FTY720 showed that postconditioning with FTY720 was able to mitigate MIRI in patients as effectively as splenectomy but with improved long-term outcomes . One pos of mPTP . Similar of mPTP . Intrigu of mPTP , encouraOther emerging targets of autoimmune response in MIRI treatment include TRAF3 interacting protein 2  , 105, plα)-binding motifs on the promoter of renalase (an enzyme elevated during MIRI that can metabolize catecholamine) are novel loci involved in cardioprotection against MIRI [2+-binding motifs, as well as Annexin-5 (a member of the Annexin family containing Ca2+-dependent phospholipid binding motifs) [To investigate the mechanisms underlying potentially crucial molecular targets in MIRI for corresponding treatment, their expression in the myocardium at a genome-wide level must be determined , 111. Benst MIRI . S100A1 nst MIRI  and S100 motifs) , might r motifs) , GSTP [1 motifs) , SGLT1 [ motifs) , BCL2 [1 motifs) , and SER motifs) .MIRI can also genetically activate intracellular signaling pathways, like mitogen-activated protein kinases (MAPKs), emerging subfamilies of which include p38MAPK and JNK that might trigger the inflammation-apoptosis chain during MIRI by regulating the expression of extracellular regulated kinase (ERK1/2) , 120. Inin vitro and ex vivo [When starting MIRI therapy with rapamycin (another therapeutic molecule with mTOR inhibition), the transcription of ERK1/2 was surprisingly found to have increased, but with p38 deactivation, and subsequent genetic evidence which confirmed the selective activation of ERK with the parallel inhibition of p38  as a novel therapeutic strategy . Another ex vivo , suggest ex vivo , 111.Epigenetics refers to the study of cellular heritable modifications involving PTM  of histone or nonhistone protein, DNA methylation, and chromatin architecture without changing its DNA sequence, which is considered an important reader-writer-eraser mechanism in gene expression in response to rapid and drastic events, such as cardiovascular injury or disease , 134. Si+-) dependent and strictly conserved protein deacetylase family (SIRT1-7) has been well studied [2S postconditioning was able to improve the cardiac function and reduce the infarct size via the PGC-1α signaling pathway [in vitro and in vivo experiments suggested that the natural accumulation of trimethylation of lysine 9 of histone 3  at the proximal SIRT1 promoter by SUV39H1 (a H3K9 tri-methyltransferase) in myocardium suffering from MIRI might be the epigenetic mechanism responsible for the downregulation of SIRT1 in this scenario (In a recent study of the epigenetics of MIRI, the role of the nicotinamide adenine dinucleotide- (NAD studied . The upr studied . Based o studied , while a pathway . Furtherscenario  [141–143Evidence also suggests that methylation/demethylation of histone may be crucial for the treatment of MIRI, as well as for the further study of cardioprotection. In a study exploring the epigenetic response mechanism of cardiac IPC , researcin vitro showed that the H3K9 demethylase KDM3A binding to NOX promoter interacted with BRG1 to activate NOX transcription, the suppression of which was paralleled by the local reappearance of H3K9me2 (in vivo MIRI experiments [in situ promotes neutrophil infiltration and exacerbates MIRI. These well-described findings suggest the switching of gene profiling via the regulation of key epigenetic enzymes might be practical for the study and treatment of MIRI and cardioprotection [Another recent study  characte H3K9me2  as well eriments  have shootection , 154. Fuin vivo and in vitro protocols must be proposed in order to resolve all scientific issues concerning the MIRI mechanism and beyond [Despite early reperfusion into the coronary artery with PPCI and/or other advanced treatment, the clinical morbidity and mortality of MIRI remain significant , 23, 84 d beyond . In spitd beyond , 9, 155.


Collagen expression was measured with RT-PCR, apoptosis was measured by using either the APOPercentage assay kit  or the Caspase-Glo 3/7 assay  and Ca2+ signaling by conventional epifluorescence imaging. The effect in vivo was tested in bleomycin-induced lung fibrosis in mice. DPPC and PG did not affect collagen expression, which was downregulated by PE. Furthermore, PE promoted apoptosis and induced a dose-dependent Ca2+ signal. PE-induced Ca2+ signal and apoptosis were both blocked by phospholipase C, endoplasmic reticulum pump and store-operated Ca2+ entry inhibition. PE-induced decrease in collagen expression was attenuated by blocking phospholipase C. Finally, surfactant enriched with PE and PE itself attenuated bleomycin-induced lung fibrosis and decreased the soluble collagen concentration in mice lungs. This study demonstrates that PE strongly contributes to the surfactant-induced inhibition of collagen expression in NHLF through a Ca2+ signal and that early administration of Beractant enriched with PE diminishes lung fibrosis in vivo.Lung surfactant is a complex mixture of phospholipids and specific proteins but its role in the pathogenesis of interstitial lung diseases is not established. Herein, we analyzed the effects of three representative phospholipid components, that is, dipalmitoilphosphatidylcoline (DPPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), on collagen expression, apoptosis and Ca A common pathological feature in acute lung injury and diverse interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF), is the migration of fibroblasts and myofibroblasts from the interstitium to the alveolar spaces ,2,3. Whe2+ concentration ([Ca2+]i) that was initiated by the recruitment of phospholipase C (PLC). Intriguingly, neither DPPC nor PG were able to induce an increase in [Ca2+]i, which suggests that other surfactant components induce apoptosis and regulate gene expression in normal lung fibroblasts (NHLF).The lipids of lung surfactant mainly include phospholipids (PL) ~90–95 wt %) and a small amount of neutral lipids (~5–10 wt %), such as cholesterol. Phosphatidylcholine (PC) accounts for ~80% of the total PL content, whereas the remaining 20% consists primarily of unsaturated anionic PL ), which account for ~15% of the total PL and by small amounts of non-PC zwitterionic PL )  wt % and. In a pr2+]i and that the pharmacological blockade of this Ca2+ signal attenuates type I collagen expression and apoptosis. Finally, we also show that Beractant enriched with PE ameliorates lung fibrosis in bleomycin-induced lung injury in mice.The aim of this work was to test the effect of three representative phospholipids on the expression of collagen in NHLF: DPPC (PC class), PG (anionic PL) and PE (non-PC zwitterionic PL) and to test this effect in vivo in an experimental model of lung fibrosis. Evidence is presented that physiological concentrations of PE downregulate collagen expression and induce apoptosis in NHLF. We also provide evidence that PE elicits an increase in i similar to that evoked by Beractant -dependent Ca2+ release from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) [2+ (0Ca2+) PE induced a transient increase in [Ca2+]i which lacked the plateau or oscillation phase , a rather selective PLC blocker i was prevented by cyclopiazonic acid , a specific inhibitor of Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA) activity, which depletes the ER Ca2+ content in NHLF i [2+ release [2+]i that was patterned by the interplay between InsP3-dependent ER [Ca2+]i release and SOCE [2+]i. Herein, we demonstrate that PE elicits an intracellular Ca2+ signal in lung fibroblasts that mimics the Ca2+ response to Beractant. Accordingly, PE induced different patterns of intracellular Ca2+ signals, including a transient Ca2+ spike, a biphasic Ca2+ elevation and intracellular Ca2+ oscillations. Moreover, the onset of PE-induced intracellular Ca2+ signals was prevented by inhibiting the PLC/InsP3 signaling pathway with U73122 or 2-APB and was sustained by SOCE. Therefore, these findings lend credit to the notion that PE is the major phospholipid component that underlies the Ca2+ response to Beractant. Furthermore, the effect of PE on collagen expression was attenuated by U73122. This is in accordance with our findings reported previously with Beractant and confirms that PE regulates gene expression in NHLF through an increase in [Ca2+]i [2+ signaling represents an established mechanism to control gene expression in mammalian cells [2+]i has been shown to regulate collagen expression also in cultured human cardiac i ,8. Type 2+]i ,17. In t release . Furtheran cells ,20,21. M cardiac  and pulm cardiac  fibrobla cardiac . Finally2+ signaling may recruit both Erk i was measured by calculating the ratio of the average fluorescence emitted at 510 nm when the cells were excited alternatively at 340 and 380 nm (ratio F340/F380). An elevation in [Ca2+]i leads to an elevation in the Ratio F340/F380. Ratio measurements were carried out and plotted every 3 s by custom-made software. All recordings were carried out at room temperature (21–23 °C). In some experiments, U73122 (a widely employed PLC inhibitor) at 10 µM in dimethyl sulfoxide (DMSO) was used. Cells were pre-incubated for 15 min with this substance before adding the phospholipids and the corresponding controls had the same amount of DMSO as the experiments. The analysis of the Ca2+ data was performed by using ImageJ software .The technique for intracellular Calsewhere . Briefly−1/10 mg·Kg−1) and a single intratracheal instillation of saline  or saline containing bleomycin sulfate  in a volume of 30 μL was administered.C57/BL mice  were used. All the experiments were performed according to protocols approved by the Animal Care and Use Committee of Animals of the Benemerita Universidad Autonoma de Puebla College of Medicine. Mice were anesthetized with Ketamine/Xylazine (100 mg·KgAll treatments were administered via aerosol as described elsewhere . Brieflyw/v). Samples were filtered and soluble collagen content was measured using Sircol assay (Biocolor Ltd.), according to manufacturer specifications. Briefly, the contents of all tubes were adjusted to 100 µL with distilled water; a collagen standard was prepared with 100 mL aliquots containing 5, 10, 25 and 50 µg. 1 mL of Sircol Dye reagent was added and the content was mixed by inverting. Tubes were shaken mechanically for 30 min; during this time, collagen-dye complex precipitate out of solution. Samples were centrifuged 10,000× g for a 10 min period. The unbound dye solution was removed by carefully inverting and draining the tubes. One mL of Alkali reagent was added and vortexed. Samples were read at 540 nm in a spectrophotometer. Results are reported as µg of collagen/µg of lung tissue.Lungs were weighted, minced and agitated for 24 h in acetic acid 0.5 M 1:10 (p < 0.05).Lung sections from bleomycin-injured and control mice were stained, coded and scored blindly for percent of fibrosis as described elsewhere . The presn-glycero-3-phosphorylcholine (DPPC), l-α-Phosphatidyl-dl-glycerol (PG), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (PE) were purchased from Sigma-Aldrich . DPPC and PG were sonicated  in Ham F12 medium at 4° C for 2 min. PE was re-suspended in chloroform at a concentration of 20 mg mL−1 and stored at −70° C. Prior to use, the desired amount of PE was evaporated with nitrogen, Ham F12 medium with 1% albumin was added to the pellet and sonicated as specified above.1,2-Dipalmitoyl-t-test was used; a p < 0.05 was considered significant. The software used was R version 3.3.3 with the multcomp package version 1.4-6 . The data for all the experiments presented in this work, can be found at Unless otherwise stated, results are expressed as the mean ± SD of triplicates of two or three pooled experiments. For statistical analysis ANOVA or


It is possible that diabetic cardiomyopathy differently affects men and women, as the latter present higher risk to develop heart failure and a higher plasmatic level of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα), than men. However, the gender-dependent regulation of Ca2+ signaling in diabetes and its relationship with TNFα signaling are still unclear. Here, we analyzed TNFα signaling pathway and its role in Ca2+ signaling dysfunction in male and female rodent models of type 2 diabetes linked to obesity (db/db mice) using confocal microscopy in freshly isolated cardiomyocytes. TNFα increased [Ca2+]i transient amplitude and accelerated its decay without affecting SR Ca2+ load or Ca2+ spark frequency in cells from control mice. All TNFα effects on Ca2+ handling were prevented by the inhibition of the ceramidase and the phospholipase A2 (PLA2). While the plasmatic level of TNFα was similar in male and female db/db mice, only male db/db hearts over-expressed both TNFα converting enzyme (TACE) and the protective TNFα receptors 2 (TNF-R2). TNFα receptor 1 (TNF-R1) expression, involved in negative inotropic response of TNFα, was unchanged in both male and female db/db mice compared to controls. We found that male db/db mice cardiomyocytes presented a decrease in [Ca2+]i transient amplitude associated to a drop of sarcoplasmic reticulum Ca2+ load, not seen in female db/db mice. Interestingly, sustained incubation with TNFα did not restored Ca2+ signaling alteration observed in male db/db mice but still induces an increase in Ca2+ spark frequency as seen in control littermates. In cardiomyocytes from female db/db mice, TNFα had no visible effects on Ca2+ handling. In conclusion, our study shows that the alteration of Ca2+ signaling and TNFα, seen in db/db mice, is gender specific presenting an increase in TNFα cardio-protective pathway in male mice.Cardiovascular complications are the primary death cause in type 2 diabetes, where inflammation can play a role. We, and others, have previously shown that, in diabetic cardiomyopathy, cardiac dysfunction is associated with Ca This release of Ca2+ by the RyR  activates contractile myofibrils to generate cardiomyocyte contraction. After the contraction, the Ca2+ is re-uptaken into the SR by the SERCA pump and extruded outside the cardiomyocytes mainly by the Na+/Ca2+ exchanger, resulting in cardiomyocyte relaxation. We and others have shown that, in animal models of type 2 diabetes linked to obesity, contractile dysfunction is associated with a decrease in the Ca2+ transient amplitude. This lower Ca2+ transient amplitude is associated to reduced L-type Ca2+ current density combined with downregulation of RyR expression , and their control littermates (db/+). Mice were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/kg). Cardiac ventricular myocyte isolation was performed by standard enzymatic methods  using the Langendorff perfusion as previously described .2+ handling was recorded in freshly isolated ventricular adult cardiomyocytes loaded with the fluorescent Ca2+ dye, the Fluo-3 acetoxymethyl ester  at 5 μM diluted in a mixture of DMSO-pluronic acid 20%. A line scan across the longitudinal axis of the myocyte was performed to measure cardiomyocyte shortening. Cardiomyocyte shortening corresponds to the difference between cardiomyocyte length at rest and cardiomyocyte length during contraction , as previously described  in line scan mode (1.54 ms) along the longitudinal axis of the cell. Ca2+ transients were evoked by field stimulation (1 Hz) applied through two parallel platinum electrodes. Spontaneous Ca2+ sparks were recorded in quiescent cells after Ca2+ transient recording. Ca2+ transient decay time corresponds to the kinetic of the relaxation phase due to the re-uptake of Ca2+ into the SR by the SERCA pump as well as the extrusion of Ca2+ by the Na2+/Ca2+ exchanger. Ca2+ transient decay time is calculated using a mono-exponential function to fit the Ca2+ transient decline phase. SR Ca2+ load was assessed by rapid caffeine application (10 mM) after 1 min pacing to reach the steady state. Parameters were studied with or without TNFα (1 h to 1 h 30 min) supplemented or not with a ceramidase inhibitor n-oleoylethanolamine  and a phospholipase A2 (PLA2) inhibitor  (Sigma-Aldrich). Fluo-3 AM was excited with an Argon laser (λex = 488 nm), and emission was collected at wavelengths >505 nm. Image analysis was performed using homemade routines in interactive data language (IDL).Caescribed . Ca2+ trAdult ventricular homogenates were quickly frozen in liquid nitrogen and then placed in Tris solution  containing proteases and phosphatase inhibitors . Homogenization was performed on ice using a Politron. Homogenate was centrifuged at 18,925 g for 10 min at 4°C. Proteins were resuspended in Laemmli (5%) sample buffer, boiled (90°C for 5–10 min), and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels. After separation, proteins were transferred to polyvinylidene fluoride membranes (Amersham Biosciences), and non-specific binding sites were blocked overnight at 4°C in 5% dried milk and Tris-buffer saline  and 0.01% Tween 20. Membranes were incubated overnight (at 4°C) for the rabbit polyclonal anti-TACE  and the rabbit polyclonal anti-TNFR2 (H-202) , at room temperature for 1 h 30 min for the rabbit polyclonal anti-TNFR1 (H-271) . A secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham Biosciences) was used in combination with an enhanced chemiluminescence detection system  to visualize the primary antibodies. Band densities were determined with a laser-scanning densitometer (HP-3970) and Quantity One software (BioRad SA). Protein loading was controlled by probing all Western blots with anti-GADPH antibody  (Ambion).t test or non-parametric Mann-Whitney test. Data involving more than two groups were analyzed using either one-way ANOVA or two-way ANOVA as appropriate. We used GraphPad Prism 7 (GraphPad) for statistical comparison. Differences with values of p < 0.05 were considered significant.Results were expressed as mean ± SEM. Significance between two groups was determined using unpaired Student’s 2+ signaling regulation is quite controversial, which is probably due to protocol differences. Therefore, we first studied, in our experimental settings, the effect of sustained activation (1–1 h 30 min) of TNFα on Ca2+ handling parameters such as Ca2+ transient, Ca2+ spark frequency, and SR Ca2+ load . Moreover, TNFα significantly accelerated the Ca2+ re-uptake into the SR as shown by the faster SR Ca2+ transient decay time (p < 0.01). This acceleration of Ca2+ re-uptake did not modified SR Ca2+ load  and a PLA2 inhibitor (10 μM ATK). NOE fully prevented the increase of Ca2+ transient amplitude  is correlated with a drop in SR Ca2+ load (p < 0.01), which could explain the smaller  cardiac cell shortening  (p = N.S.)  ,F. In fetermates . Indeed,mplitude , Ca2+ sprequency , SR Ca2+ and db/db   . In female db/db . Therefo2+ mishandling, resulting from a decrease in the Ca2+ channels involved in the Ca2+-induced Ca2+ release process (RyR and L-Type Ca2+ channels)  . Althoughannels) , little 2+ signaling are quite important in the literature with reported positive or negative ionotropic effect. For instance, in cat cardiomyocytes, short time exposure of TNFα reduced Ca2+ transient amplitude in response to a disruption of Ca2+ influx via L type Ca2+ channels leading to cellular shortening, supporting, then, a negative ionotropic effect of TNFα  and PLA2 (for arachidonic acid production). Sphingosine is commonly associated to short-term (within minutes) negative inotropic effect of TNFα  through different mechanisms such as PLA2 or sphingosine signaling pathway . Here, w of TNFα . Howevermyocytes . Those ritor NOE ,C. Moreonock out . This PKhe PLA-2 . In addinhibitor ,C.db/db mice, a model that recapitulates, in that sense, the human pathology. Indeed, the leptin receptor mutation of db/db mice impairs the satiety feeling and leads to obesity around 4–5 weeks of age, which is followed by diabetic state with hyperglycemia and insulin resistance , TNFα is not as effective as in C57Bl6 strain. Indeed, genetic background, such as between C57BL6/J and C57BL6/N, has been shown to influence cardiac phenotype and propensity to develop cardiomyopathies (db/db mice (db/db, the dramatically reduced SR Ca2+ load would prevent the high Ca2+ systolic release induced by TNFα probably due to the phosphorylation of the RyR via the activation of PLA2. Indeed, we found in the presence of TNFα an increase in Ca2+ spark frequency in both db/+ and db/db mice reflecting an elevated diastolic RyR opening resulting from RyR phosphorylation by PKA previously described in male db/db (db/db mice, the TNF-R2 was overexpressed, which is known to exert cardio-protective effects via the activation of NF-κB (2+ spark frequency in male db/+ cardiomyocytes treated with TNFα (2+] overload was prevented and Ca2+ transient increased leading to an increase in inotropic response. The over-expressed TNF-R2 in a male db/db appears as an attempt to counteract the already present Ca2+ mishandling to protect from cardiac dysfunction. Indeed, prolonged activation of the TNF-R2 pathway in the db/db male cardiomyocytes could then activate phosphorylation of excitation-contraction coupling key proteins, such as phospholamban, to restore Ca2+ transient and cardiomyocytes contraction.To our knowledge, plasmatic TNFα level parallels the degree of cardiac dysfunction in diabetic patients. In the d injury , TNFα diBl6 mice ,C. One eopathies . This co/db mice . Althoug/db mice . Anotherle db/db . Interesof NF-κB . Indeed,of NF-κB . This acith TNFα . Moreoveith TNFα . As a re2+ and TNFα signaling are altered only in male type 2 diabetic mice, whereas female does not seem to be affected. Although this study has several limitations in the interpretation such as non-comparable hormonal state between female db/db mice and diabetic women, lower effect of TNFα in db/+ than C57BL6 control, we still clearly show that male db/db mice develop Ca2+ mishandling leading to impaired contraction already at a young age, while woman seemed to be protected. Moreover, we found that male db/db mice put into place a protective mechanism to counteract those negative effects by over-expressing TNF-R2 cardio-protective signaling pathway.In conclusion, we found for the first time that both CaThe datasets generated for this study are available on request to the corresponding author.The study was carried out in accordance to the ethical principles of the French Ministry of Agriculture and the European Parliament on the protection of animals. The protocol was approved by the French Ministry of Agriculture and Bioethical Committee of the CSIC following recommendation of the Spanish Animal Care and the European Parliament on the protection of animals.CD and AG conceived and designed the project, supervised the data acquisition and participated in analysis. LP and GR performed most of the experiments and analyses. LP interpreted the data and wrote the first draft of the manuscript. MS participated in the figure preparation. All authors have edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The presence of natural organic matter (NOM) in drinking water sources can stabilize toxic antimony (Sb) species, thus enhancing their mobility and causing adverse effects on human health. Therefore, the present study aims to quantitatively explore the complexation of hydrophobic/hydrophilic NOM, i.e., humic acid (HA), salicylic acid (SA), and L-cysteine (L-cys), with Sb in water. In addition, the removal of Sb species and total organic carbon (TOC) was evaluated with ferric chloride (FC) as a coagulant. The results showed a stronger binding affinity of hydrophobic HA as compared to hydrophilic NOM. The optimum FC dose required for Sb(V) removal was found to be higher than that for Sb(III), due to the higher complexation ability of hydrophobic NOM with antimonate than antimonite. TOC removal was found to be higher in hydrophobic ligands than hydrophilic ligands. The high concentration of hydrophobic molecules significantly suppresses the Sb adsorption onto Fe precipitates. An isotherm study suggested a stronger adsorption capacity for the hydrophobic ligand than the hydrophilic ligand. The binding of Sb to NOM in the presence of active Fe sites was significantly reduced, likely due to the adsorption of contaminants onto precipitated Fe. The results of flocs characteristics revealed that mechanisms such as oxidation, complexation, charge neutralization, and adsorption may be involved in the removal of Sb species from water. This study may provide new insights into the complexation behavior of Sb in NOM-laden water as well as the optimization of the coagulant dose during the water treatment process. Antimony (Sb) is a semi-metallic element widely found in the environment that is introduced into water bodies as a result of natural processes and anthropogenic activities . In receAntimony mobility in the environment is regulated by various advanced technologies including coagulation, membrane separation, ion exchange, adsorption, phytoremediation, and electrochemical processes . As compThe presence of NOM in Sb-rich water may provide a potential route for human exposure, particularly if the source is being used for drinking water. Previous studies ,14,22 haAccordingly, the present work aims to quantitatively determine the complexation of Sb with hydrophobic/hydrophilic NOM under different Sb concentrations. Moreover, the current study also intends to evaluate the complexation behavior of Sb with NOM following FC coagulation. This study also assesses Sb and TOC removal under varying FC doses and NOM concentration by the coagulation–flocculation (C–F) process. Finally, this study also investigates the binding and removal mechanism of Sb in the heterogeneous aquatic environment.2O3), potassium hexahydro-antimonate (KSb(OH)6) and NOM  were purchased from Sigma-Aldrich . The ferric chloride hexahydrate (FeCl3.6H2O), nitric acid (HNO3), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were obtained from Samchun . The stock and synthetic water solutions were prepared with deionized (DI) water produced using a Milli-Q water purification system . All glassware and polyethylene bottles were washed with 15% HNO3 solution and rinsed with DI water prior to use. In addition, glass vessels were used to store supernatant.All chemicals used in the experiments were of reagent grade. The antimony (III) oxide (Sb2O3 and KSb(OH)6 into 2M HCl and DI water, respectively. The coagulant stock solution of 0.1 M FC was prepared by dissolving FeCl3.6H2O into DI water. The remaining stock solutions of each NOM, i.e., humic acid (HA), salicylic acid (SA), and L-cysteine (L-cys), were prepared by adding 100 mg powder into 0.1 L DI water. The pH of the HA stock solution was adjusted to 11 by adding 0.1 M NaOH, then stirred at 600 rpm for 24 h so as to improve the solution stability. The suspension was filtered using a 0.45 µm glass fiber filter in order to remove insoluble particles, then the pH was adjusted to 7.0 ± 0.1, and the stock solution was stored in the dark at 5 °C [The stock solutions of 100 mg/L Sb(III) and Sb(V) were freshly prepared by adding Sb at 5 °C . These m at 5 °C ,24,26. TThe interactive behavior of Sb(III) and Sb(V) with each NOM (10 mg/L) in water was studied prior to and after FC coagulation. To this end, complexation experiments were conducted with different Sb concentrations (0.1 to 10 mg/L) at neutral pH (7.0 ± 0.1). During the experiments, pH was maintained using 0.1 M HCl or 0.1 M NaOH solutions. The reaction vessels were placed into a 360⁰ rotator  for 120 h in darkness with a rotating speed of 27 rpm at room temperature [The coagulation experiments were conducted using a jar tester  with six blades at room temperature. Prior to the experiments, a 100 mL solution was added to the beaker and had its pH adjusted to 7.0 ± 0.1 using 0.1 M HCl or 0.1 M NaOH. The coagulation experiments followed three sequential steps as reported in our previous studies ,29: (a) For all coagulation experiments, the concentrations of Sb species were maintained at 1 mg/L. This set of experiments was performed to investigate the influence of the FC dose (0.1 to 0.3 mM) on Sb and TOC removal in the presence of each NOM (10 mg/L). Another set of experiments was also conducted under optimum FC doses to study the effect of each NOM (0 to 20 mg/L) on Sb and TOC removal, respectively. The average values were reported for triplicate analysis. e (mg C/g) and qm (mg C/g) are the equilibrium and maximum adsorption capacity of organic ligands on FC surface sites, respectively; Ce (mg C/L) is the equilibrium concentration of organic ligands in solution; KL (L/mg) in the Langmuir equation represents the Langmuir constant related to adsorption energy; and KF (mg C/g) (L/mg)n in the Freundlich equation represent the constants related to the adsorption capacity and intensity of heterogeneity, respectively.Adsorption data were calculated assuming that the removal of NOM was due to the adsorption onto Fe precipitates. The two most commonly used adsorption isotherm (Langmuir and Freundlich) models were applied to fit the experimental data using non-linear Equations (1) and (2):The pH of the solution was measured using a pH meter  and ion selective electrode (ISE) Multi-Parameter Meter, Hach Company, Loveland, CO, USA). The residual concentration of Fe and total Sb  were analyzed by inductively coupled plasma optical emission spectrometry . For the measurement of free Sb species, a high-performance liquid chromatography and then inductively coupled plasma mass spectrometry  were used. The NOM bound Sb was determined by calculating the difference between total and free Sb. The detailed procedure can be found elsewhere . The eleThe relative percentage distributions of carbon (C), hydrogen (H), nitrogen (N), oxygen (O), and sulfur (S) of the studied hydrophobic/hydrophilic NOM are presented in −1 were assigned to the partial N–H and broad OH stretching vibrations [−1 were ascribed to the asymmetric and symmetric stretching vibrations of the aliphatic C–H and C–H2 moiety, respectively [−1 corresponded to the S–H and N–H stretching vibrations [−1 were attributed to the symmetric and asymmetric stretching vibrations of C=O (COO−) [−1 indicates the presence of ketones with C=O stretching vibrations [−1 were subjected to the symmetric stretching vibrations of COO−, CH3, S=O, and C–O, and the anti-symmetric stretching vibration of C–O, respectively [−1 indicate the presence of C–O stretching along with OH deformation in the COOH and phenolic group, C–O–C stretching in carbohydrate, C–O–C stretching in the ethers Ph–O–C group, and C–O stretching in the carboxylic acid group, respectively [−1 and 555 cm−1 correspond to C–H bending and C–O–C stretching vibrations, respectively [The FT-IR spectra for the studied NOM prior to and after interacting with Sb species are presented in brations ,28. The ectively . The twobrations . The banO (COO−) . Moreovebrations . The peaectively . The othectively ,34. Furtectively .−1 generally indicates the formation of the Sb-HA complex through C=O stretching of COOH and other carbonyl groups [−1 indicates the oxidation of Sb(III) to Sb(V) caused by the bending vibrations of N–H, COOH, and CH groups [−1, and a broad band appeared at ~1153 cm−1 as well, which indicated the interaction of the Sb species with a C–O group to form a metal ion complex [−1 in the case of L-cys indicates the formation of covalent bonds between thiols and Sb species [−1, which is ascribed to the vibration of phenolic groups in the Sb-NOM complex [−1 in each NOM further suggests the formation of a Sb-NOM complex through C=C stretching in the aromatic ring and OH bending in phenols [−1 in −1 in l groups . MoreoveH groups . The int complex . The dis species . In addi complex . The pre phenols . The peaIn order to further explore the complexation of Sb with each NOM, free and bound Sb were analyzed, and the results are presented in 3 and Sb(OH)6¯, respectively [− radicals in solution [The complexation of Sb with NOM has been associated with various functional groups in HA, SA, and L-cys through mechanisms such as ligand exchange and the formation of a negatively charged complex. Moreover, the stabilization of bound Sb occurs via chelation, H-bridges, or cationic metals . The cheectively . The presolution . Moreovesolution ,44,45. Hsolution . In addisolution ,47. Anotsolution . Our finsolution ,48,49, wBased on the experimental results, it may be suggested that the hydrophobic/hydrophilic NOM may form a complex with Sb(III) and Sb(V) with different binding affinities. Such complexation of Sb species with NOM could facilitate Sb mobility to drinking water sources, and may thus increase the associated health risks as well. Therefore, it is essential to investigate the effect of hydrophobic and hydrophilic NOM on Sb removal at environmental relevant conditions by FC coagulation.The TOC removal was also monitored for both the Sb(III) and Sb(V) systems, as shown in 3O4 surface. In addition, the correlation coefficient (R²) indicated better Langmuir fitting of all NOM on Fe precipitates in both systems, thus suggesting that there is monolayer adsorption onto Fe precipitates. In general, these results suggest that the characteristics of NOM should be considered for the treatment of heavy metal-polluted water.In order to further confirm the adsorption phenomena of each NOM onto precipitated Fe, an adsorption isotherm study was conducted at varying NOM concentrations (0–20 mg/L) in the Sb system A–D. As sIt is worth noting that the complexation abilities of Sb with NOM  could play a significant role in determining the underlying mechanism involved in the interactions among the ternary system . Moreover, the competitive inhibition of Sb adsorption onto Fe precipitates could be expected in the presence of NOM with stronger inhibition in the case of HA, thus leading to the release of more Sb free ions in the aqueous environment A,D. Ther−1 present in all flocs corresponds to the stretching vibrations of Fe hydrolyzed products, which form complexes with NOM molecules and Sb species [−1 in both Sb systems was subjected to covalent bonds between thiols and Sb species onto Fe surface [−1 were attributed to the C=O stretching of amide groups and CH2 stretching vibrations, respectively [−1 in the presence of HA and L-cys suggests the involvement of carboxylic, phenolic, and thiol groups in complexation with Sb via ligand exchange [−1, which is near ~682 cm−1 and assigned to Sb(V)–O stretching vibrations [−1 (corresponding to Sb(V) species), while a small peak was observed at ~474 cm−1 that was ascribed to Sb(III)-O stretching vibrations [−1 were observed in the cases of SA and L-cys, respectively, which correspond to Sb(III)-O stretching vibrations. Consistent with our observation, a previous study [ species ,57. The  surface . The peaectively . This coexchange . As showbrations . It shoubrations . On the us study  also fouThus, the significant shift and disappearance of distinct peaks in the FC composite flocs confirm the complex formation of various contaminants in the ternary system. The oxidation phenomena may also be responsible for the interaction of Sb species with NOM, followed by charge neutralization and adsorption onto active Fe surface sites. The characteristics of flocs supported the removal mechanism of Sb species in the ternary environment well, and can be proposed to be the combination of oxidation, complexation, charge neutralization, and adsorption. However, the removal mechanism may vary with the nature and type of NOM and Sb species present in water.In this study, we investigated the complexation behavior of Sb species with different NOM, i.e., hydrophobic (HA) and hydrophilic (SA and L-cys) in water. Furthermore, the removal, as well as the binding affinity of Sb, was also quantitatively evaluated in the presence of each NOM following FC coagulation. Our results demonstrated that hydrophobic ligands could form stronger complexes with Sb than hydrophilic ligands, with higher affinity for Sb(V) than Sb(III) species. The optimum FC dose was found to be higher for the removal of Sb species in the presence of HA. In addition, the TOC removal was also found to be higher than hydrophilic ligands, thus indicating increased competition of HA molecules with Sb for Fe binding sites. The adsorption of NOM onto the Fe surface indicated better fitting with the Langmuir model with the maximum adsorption potential for HA molecules. Moreover, the complexation capability of Sb with NOM was remarkably suppressed due to the adsorption of contaminants on Fe precipitates. Furthermore, the removal mechanism involved in Sb removal during the C–F process may be the combination of oxidation, complexation, charge neutralization, and adsorption, as indicated by the FT-IR analysis of FC composite flocs. These results suggest that the characteristics of NOM may influence the fate, mobility, and coagulation performance of Sb species in the drinking water treatment process.