Total organic carbon removal by the carbon samples increased proportionally with total pore volume as determined from N2 adsorption isotherms at −196 °C. The activated carbon produced by CO2 activation had similar removal levels for total organic carbon from the water (up to 70%) to those samples activated with H3PO4, but lower yields, due to losses during pyrolysis and activation. A method to increase the yield when using CO2 activation was proposed and consisted of recycling bio-oil produced from previous runs to the aspen wood feed, followed by either KOH addition (0.48%) or air pretreatment (220 °C for 3 h) before pyrolysis and activation. By recycling the bio-oil, the yield of CO2 activated carbon (after air pretreatment of the mixture) was increased by a factor of 1.3. Due to the higher carbon yield, the corresponding total organic carbon removal, per mass of wood feed, increased by a factor of 1.2 thus improving the overall process efficiency.Several samples of activated carbon were prepared by physical (CO As reported previously, the adsorption capacity of carbon depends on the properties of the adsorbing molecules  . Moreoveerature) ,2,3,4.i.e., heating in an oxygen deprived atmosphere), and results in a solid carbon (char), which can be further treated  at high temperature with oxidizing agents, such as steam, CO2 or air. Alternatively, activated carbon can be produced by chemical activation, in which the feedstock is impregnated with KOH, H3PO4, ZnCl2, FeCl3, etc. The removal of organic compounds from water by carbon samples with different properties  was compared previously [et al. [2CO3 and steam, and showed that K2CO3 activated carbon had higher methylene blue uptake due to larger surface area, pore sizes and higher content of surface oxygen groups. Girgis et al. [3PO4, ZnCl2, KOH and steam activation, and found the largest adsorption capacities for the H3PO4 activated carbon, while KOH activated carbon had the lowest uptake. In a recent study [3PO4 than by CO2, probably due to the larger mesopore volumes and surface areas obtained with H3PO4. Mestre et al. [2CO3. The uptakes of ibuprophen, paracetamol, acetylsalicylic acid, caffeine, and clofibric acid were higher by chemically activated carbon, while the uptake of iopamidol was higher by physically activated carbon. These results were attributed to the differences in micro and mesopore structures of the activated carbon.Both physical and chemical activation techniques have been applied to produce activated carbon for water treatment. Preparation by physical activation consists of pyrolysis of the raw material  and chemical (H3PO4) activation of almond shells, pecan shells and vetiver roots has been performed [3PO4 activation were offset by high activated carbon yields. In contrast, the capital and operational expenses for physical activation are lower but the yields are also lower. The production costs of activated carbon for these materials were estimated as 1.46–2.72 $·kg−1 [−1 [−1 [2 and H3PO4 activation, respectively. Low yields of physically activated carbon are attributed to carbon loss during both the pyrolysis and activation stages. Depending on the raw material and operating conditions, only 43%–63% of carbon in biomass is converted to char during pyrolysis while the remaining carbon is contained in bio-oil and pyrolysis gases [Cost analysis of carbon production by physical  and H3PO4  activation were used and the yield, TOC removal, and, ultimately, the process efficiency of both methods determined. In addition to as prepared activated carbon, acid washed and heat treated samples with reduced ash content and surface functionalities were prepared to determine the effect of these parameters [As stated above, the efficacy of activated carbon as an adsorbent for organic compounds is partially determined by its properties and these properties can be manipulated by the activation method ,6,7,8. Tng costs . The acti.e., biomass) for pyrolysis in order to increase char production. Bio-oil from biomass is a carbon-rich product with limited application due to acidity, corrosiveness, high oxygen and moisture content, and coking upon reheating [2 activation of char produced from a mixture of biomass and bio-oil. The influence of the char preparation conditions on yield, porosity and removal of organic compounds from SAGD water are discussed.The second objective of the current study was the investigation of a new method to increase the yield of activated carbon prepared by physical activation. This method is based on the recycling of bio-oil from previous runs to the raw feedstock  and H3PO4 (at 500 °C) activation were controlled by activation time and H3PO4:wood ratio, respectively. After H3PO4 activation, the acid residue was removed by washing with deionized water. In addition to as prepared activated carbon samples, acid washed and heat treated (at 900 °C for 1 h in nitrogen) samples with reduced ash content and surface functionalities were prepared to minimize their effect, if any, on the TOC uptake and clarify the role of porosity. The samples prepared by CO2 activation are denoted as AC—for activated carbon—followed by the activation time in hours. Samples activated with H3PO4 were denoted by the letter P followed by the H3PO4:wood ratio of 1:2, 1:1 or 2:1. The abbreviation “HT” denotes samples that were acid washed and heat treated (HT). The TOC removal by the activated carbon samples was compared with a non-activated char (Char-800-HT) prepared from the same feedstock (at 800 °C) and commercial activated carbon (as supplied ColorSorb and acid washed and heat treated ColorSorb-HT) specially designed for the removal of organic molecules and decolorization.Activated carbon samples prepared with COThe UV-vis spectrum of the SAGD water in 2 adsorption and desorption isotherms of the selected samples are shown in 2 activation time resulted in increased surface areas and micropore volumes determined by N2 adsorption (pore sizes up to 20 Å) from 540 m2·g−1 and 0.18 cm3·g−1 for 0.3 h treatment (AC0.3-HT) to 910 m2·g−1 and 0.30 cm3·g−1 for 3.6 h treatment (AC3.6-HT). The micropore volumes determined by CO2 (pore sizes less than ~7 Å) also increased with activation time from 0.23 to 0.32 cm3·g−1. The meso/macropore volume increased during the first hour of activation up to 0.22 cm3·g−1 and did not change significantly thereafter. As expected, the surface areas and pore volumes of CO2 activated carbon samples were higher compared to non-activated char (Char-800-HT). With H3PO4 activation, higher H3PO4:wood ratios resulted in higher surface areas and meso/macropore volumes, while the micropore volume determined by N2 adsorption was the largest in the sample produced with a H3PO4:wood ratio of 1:1. The micropore volume determined by CO2 adsorption did not change with an increase in H3PO4 concentration. Compared to CO2 activation, H3PO4 activation generally resulted in larger surface areas and micropore volumes (determined by N2 adsorption). Pore size distributions of four of the activated carbon samples are shown in 3PO4 activation produced the widest pore diameters. More specifically, the P2:1-HT sample contained pores with diameters up to 100 Å, while the pores in samples AC3.6-HT and ColorSorb-HT were smaller than 40 Å and 60 Å, respectively. In the non-activated Char-800-HT sample no pores larger than 15 Å were observed.The porous properties of the samples are listed in The percentages of TOC removed from SAGD water by the prepared activated carbon samples are shown in 2 and higher H3PO4:wood ratios increased the TOC removal, which could be attributed to the development of porosity during activation as discussed below. In contrast, non-activated Char-800-HT did not remove organic compounds from SAGD water, suggesting that activation was a necessary step for the development of the pores responsible for adsorption. These results are consistent with a previous study [i.e., ColorSorb).Longer activation times with COus study , in whic2  did not contribute to the adsorption of TOC, as these pores were present in all carbon samples, including the non-activated carbon (Char-800-HT) that had no TOC removal  to develop porosity. In the case of H3PO4 activation, higher yields of carbon from lignocellulosic materials, including wood, are attributed to crosslinking reactions between decomposing fragments via phosphate and polyphosphate bridges that prevent release of relatively small molecules from the solid phase [The TOC removal was comparable for COtivation  but the tivation . These yDi Blasi . AlthougDi Blasi ,22,28. Fid phase . After tid phase .2 activation. In the first approach, the mixture of wood and bio-oil undergoes air pretreatment at 220 °C for 3 h in order to stabilize the mixture and then the temperature is increased up to 600 °C for the pyrolysis , while the carbon prepared by air pretreatment of wood and bio-oil includes “Air” in the name.In both approaches water and organic compounds with boiling points below 150 °C were removed by evaporation at 150 °C . Otherwi2 activated carbon. The char yields produced by bio-oil recycling increased to 30.4% and 30.9%  compared to 23.7% for the Char-600 sample. The porous properties of Char-600-Air were similar to Char-600, while the porosity of Char-600-KOH was lower, likely due to pore blockage by potassium compounds as discussed in our previous study [2 in the presence of potassium compounds. Potassium compounds are known catalysts for char gasification by CO2 [2 activation [3·g−1, 0.38 cm3·g−1 and 0.35 cm3·g−1, respectively). However, the increase in porosity was also observed in AC1.0-Air-HT, prepared without potassium hydroxide addition , peroxides (500–550 °C), lactols, lactones and anhydrides (above 600 °C). CO evolution can be ascribed to anhydrides (350–400 °C), phenols, hydroquinones (600–700 °C), carbonyls, quinones and ether groups (700–800 °C). At temperatures higher than 900 °C, CO evolves from pyrone and chromene groups. There were differences between CO and CO2 profiles of AC1.0-Air carbon from those of AC1.0  were similar to those from AC1.0 , suggesting no change in the number of surface oxygen groups due to bio-oil recycling.To investigate whether the bio-oil addition influenced the number of surface oxygen groups, the activated carbon prepared with air pretreatment was characterized with temperature-programmed decomposition . Accordiof AC1.0  due to dThe porous carbon samples prepared from wood and bio-oil were then tested for TOC removal and the results are shown in −1) as shown in The results for the as prepared carbon have been normalized for their different yields and TOC capacities by expressing the TOC removal in terms of mass of organic carbon removed per mass of wood used to prepare the activated carbon (mg·g-woodPopulus tremuloides) wood chips provided by Alberta-Pacific Forest Industries Inc.  were used as the feedstock. According to the proximate analysis, the wood chips contained 18.5% fixed carbon, 80.9% volatile matter and 0.6% ash on a dry basis. The contents of carbon, hydrogen and nitrogen were 48.6%, 6.0% and 0.6%, respectively, and were reported previously [2. The second method involved pyrolyzing the wood with recycled bio-oil with either KOH addition or with air-pretreatment prior to CO2 activation, and the third method involved treatment of the wood with phosphoric acid (H3PO4).Aspen . The reactor was heated at 4 °C·min−1 under N2 flow (space velocity 1 min−1) to 600 °C and pyrolyzed at this temperature and gas flow for 30 min to produce char (named Char-600). Bio-oil was collected in a flask attached to the outlet of the reactor. For the preparation of activated carbon in this study, the activation temperature was fixed at 800 °C, while the degree of activation and porosity were varied by activation time. Approximately 1.5 g of Char-600 was loaded into the reactor (bed volume 10 cm3), purged with N2 for 1 h and then heated at 10 °C·min−1 under this N2 flow to 800 °C. After 800 °C was reached, N2 was switched to CO2 (space velocity 10 min−1) for activation. The activation time with CO2 varied from 0.3 to 3.6 h. The char sample (Char-800) without CO2 activation was prepared by heating Char-600 in N2 flow for 1 h at 800 °C. All samples were crushed and sieved (US mesh 100) to obtain particle sizes less than 150 μm before further treatment, adsorption tests and/or characterization.Char production and COeviously . Approxi2 flow at 900 °C (maximum safe temperature for operation of the setup). To remove acid soluble ash components, approximately 2 g of sample was soaked in 0.1 M hydrochloric acid for 18 h, washed with deionized water until pH 5–5.5  and then dried at 105 °C for 24 h. To remove surface oxygen groups, the dried sample (~1.5 g) was loaded into a quartz boat, which was then placed into a horizontal furnace, purged with N2 (100 cm3·min−1) for 1 h and then heated at 10 °C·min−1 under N2 flow to 900 °C and held at this temperature for 1 h. To reduce the amount of ash and surface oxygen groups, some of the samples were washed with hydrochloric acid and then heat treated in N2, to remove the water and light organic compounds, and then heated in N2 at the conditions used for the preparation of Char-600. The char sample was then activated with CO2 for 1 h at 800 °C following the procedure used for the activation of Char-600.Wood impregnated with bio-oil generated in a previous pyrolysis run was also used as a precursor of activated carbon. Two methods were adopted from our previous work  to produ−1 in air (space velocity 1 min−1) first to 150 °C for 1 h, to evaporate water and light organic compounds, and then to 220 °C for 3 h. After the air pretreatment, samples were purged with N2 (space velocity 1 min−1) for 0.5 h and heated in N2 at the conditions used for the preparation of Char-600.For experiments with air pretreatment, 3.8 g of bio-oil and 7.5 g of wood were mixed in a beaker and then loaded into the reactor. The reactor was heated at 4 °C·min3PO4 activated carbon, dried wood chips were used as received. Approximately 60 g of wood chips were placed into a 1 dm3 beaker to which 300 cm3 of an aqueous solution of H3PO4  were added. The volume of solution was kept constant while the concentration of H3PO4 was varied to target H3PO4:wood ratios of 1:2, 1:1 and 2:1 by weight. The mixture was soaked for 1 d at ambient temperature (23 °C) and then water was evaporated from the surface of wood chips by heating at 100 °C on a hot plate. The prepared mixture of H3PO4 and wood was left for two more days at ambient temperature for impregnation and then dried in air at 110 °C overnight. Activation was carried out in a horizontal alumina reactor with an inner diameter of 8 cm. Approximately 30 g of the H3PO4:wood mixture was loaded into the alumina reactor and heated at a constant rate (4 °C·min−1) to 500 °C under N2 flow (space velocity 1 min−1). The temperature was maintained at 500 °C for 0.5 h and then the reactor was cooled under N2 to 70 °C for sample removal. The activated carbon samples were washed with deionized water until pH of 5–5.5, dried overnight at 110 °C and then crushed and sieved (US mesh 100) to obtain particles less than 150 µm. After washing, the samples were either used or subjected to a further heat treatment at 900 °C under N2 for 1 h.To prepare HA commercial activated carbon, ColorSorb G5 prepared by steam activation of wood was provided by Jacobi Carbons AB .2 activated carbon were calculated as mass of carbon after pyrolysis and activation, respectively, per mass of dried wood. The yield of H3PO4 activated carbon was calculated as mass of dried carbon, after H3PO4 removal by water washing, per mass of dried wood.Yields of char and CO2 and CO2 adsorption measured at −196 °C and 0 °C, respectively . Surface areas were calculated from N2 adsorption isotherms using the BET method [2 for the cross-section of the N2 molecule. Micropore volumes were obtained from N2 and CO2 adsorption isotherms using the t-plot with Carbon black STSA reference isotherm and Dubinin-Radushkevich methods [2 adsorption at relative pressures of 0.96–0.97. The meso/macropore volume was calculated as the difference between total pore volume and micropore volume determined by N2 adsorption. The non-local density functional theory  [Activated carbon samples were characterized with NT method . Samples methods ,37, respFL, USA)  was used2 at 5 °C·min−1 to 1000 °C. The evolution of CO2 and CO during heating was detected with an infrared gas analyzer  and recorded.The surface oxygen groups were quantified by temperature-programmed decomposition. Approximately 10 mg of sample was placed in a thermogravimetric analyzer  and heated in N−3, depending on the sample. After cooling, the samples were shipped to the University of Calgary and stored at 4 °C.Steam assisted gravity drainage (SAGD) water was collected from the process stream of an industrial SAGD facility  immediately following the water softening stage but prior to entering the boiler. This 80 °C water had a pH of 9.5–9.8 and total organic carbon (TOC) content ranging from 910 to 1162 mg·dm3 of SAGD water reheated to 80 °C. To measure the initial TOC content in the SAGD water, vials with 10 cm3 of the reheated water without carbon addition were prepared. The mixtures and pure SAGD water were shaken at 80 °C  and 175 rpm for 18 h before analysis. A series of kinetic measurements have been carried out, and 18 h treatment was sufficient to attain adsorption equilibrium on the carbon samples. After settling for 2–3 min, the supernatant (~9 cm3) was obtained with a syringe and filtered through a 0.45 μm nylon membrane filter . The cooled solutions were analyzed by a TOC analyzer . To avoid premature precipitation of the organic acids in the SAGD water  with BlueLine 56 pH electrode .3PO4) and physically (CO2) activated carbon samples removed similar percentages of total organic carbon from SAGD water. As CO2 activated carbon had lower yields compared to carbon prepared with H3PO4, a method to increase the yield, and the conversion efficiency of wood by CO2 activation was investigated. Specifically, bio-oil was recycled and added to the wood feed before CO2 activation. Air pretreatment of the bio-oil and biomass mixture (220 °C for 3 h) was required but increased the yield of as prepared activated carbon by 1.3 times and total organic carbon uptake from water per mass of utilized wood by 1.2 times.Conversion of wood to activated carbon for industrial water treatment was investigated. Both chemically (H


This toxin blocks sodium channels resulting in neuromuscular paralysis and even death. As a part of the risk assessment process leading to a safe seafood level for TTX, oral toxicity data are required. In this study, a 4-level Up and Down Procedure was designed in order to determine for the first time the oral lethal dose 50 (LD Pseudomonas and Vibrio spp. /LMBL8.−3 M. Immediately before administration, TTX was diluted in 0.9% saline solution to achieve each dose (toxin carrier concentration <2.5%).TTX used for all the experiments was obtained from CIFGA . The stock solution was initially solved in aqueous [AcOH] = 1 × 10ad libitum. The next morning they received a single oral dose of TTX by gavage (10 mL/kg) and were placed individually in metabolic cages for the following 2 h with free access to chow and water. A modified 4-level Up and Down Procedure  = 0.03 M (70:30 v/v) (standard dilution solvent) in polypropylene vials. A six point calibration was generated with variable concentrations ranging from 1 to 20 ng/mL.The analysis of TTX was carried out using an Acquity UPLC BEH Amide 100 mm × 2.1 mm × 1.7 µm column , equipped with a 0.2 μm Acquity UPLC in-line filter. Mobile phases were as follows; A: water and B: acetonitrile (95%


Epithelial-mesenchymal transition (EMT) is an early event in tumour invasion and metastasis, and widespread and distant metastasis at early stages is the typical biological behaviour in small cell lung cancer (SCLC). Our previous reports showed that high expression of the transcription factor E2F1 was involved in the invasion and metastasis of SCLC, but the role of E2F1 in the process of EMT in SCLC is unknown.Immunohistochemistry was performed to evaluate the expressions of EMT related markers. Immunofluorescence was used to detect the expressions of cytoskeletal proteins and EMT related markers when E2F1 was silenced in SCLC cell lines. Adenovirus containing shRNA against E2F1 was used to knock down the E2F1 expression, and the dual luciferase reporter system was employed to clarify the regulatory relationship between E2F1 and ZEB2.ZEB2 gene expression.In this study, we observed the remodelling of cytoskeletal proteins when E2F1 was silenced in SCLC cell lines, indicating that E2F1 was involved in the EMT in SCLC. Depletion of E2F1 promoted the expression of epithelial markers (CDH1 and CTNNB1) and inhibited the expression of mesenchymal markers (VIM and CDH2) in SCLC cell lines, verifying that E2F1 promotes EMT occurrence. Next, the mechanism by which E2F1 promoted EMT was explored. Among the CDH1 related inhibitory transcriptional regulators ZEB1, ZEB2, SNAI1 and SNAI2, the expression of ZEB2 was the highest in SCLC tissue samples and was highly consistent with E2F1 expression. ChIP-seq data and dual luciferase reporter system analysis confirmed that E2F1 could regulate ZEB2 gene expression in SCLC.Our data supports that E2F1 promotes EMT by regulating The online version of this article (10.1186/s12885-017-3701-y) contains supplementary material, which is available to authorized users. RB1  gene loss, which is a typical genetic characteristic of SCLC, leads to the deregulation of E2F1 [The cancer statistics for China in 2015 showed that there were 733,330 new cases of lung cancer , and 610,200 deaths  due to lung cancer. The incidence and mortality rate for lung cancer ranked first out of all tumours [E2F1 is a transcription factor that is involved in the cell cycle, proliferation, apoptosis and differentiation . Recent EMT is an early event in the process of tumour invasion and metastasis . E2F1 caZEB2 by binding to its promoter. Our results suggest that E2F1 promotes EMT by regulating ZEB2 in SCLC.In this study, we analysed the expression pattern of EMT-related proteins in SCLC tissue samples. We then examined the changes in cell morphology and cytoskeleton remodelling processes when E2F1 was knocked down by shRNA in SCLC cells. In these cells, epithelial markers were significantly increased and mesenchymal markers were significantly decreased. ChIP-seq and dual-luciferase reporter experiments indicated that E2F1 directly regulated the expression of Sixty SCLC biopsy tissue samples before treatment were obtained from the affiliated hospital of Binzhou Medical University from January 2014 to January 2015. All patients signed informed consent forms before providing tissue samples. This research was approved by the Medical Ethics Committee of Binzhou Medical University (No. 2013027). This study was performed according to the Declaration of Helsinki and to the relevant ethical guidelines for research on humans. The basic patient information is listed in Table All tissue sections were dewaxed in dimethylbenzene and rehydrated in an alcohol gradient ranging from 100% to 75%. Antigen retrieval was performed by placing samples in an EDTA antigen repair solution (ph = 9.0). Sections were then washed three times with PBS buffer (ph = 7.0). Next, the sections were incubated with a primary antibody at 4 °C overnight. The antibodies were used at the following dilutions: 1:100 for VIM , 1:150 for CDH2 , 1:250 for CLDN1 , 1:100 for CTNNB1 , 1:400 for CDH1 , 1:50 for E2F1 , 1:200 for ZEB2 , ZEB1 , SNAI1 , and SNAI2 . The sections were washed three times with PBS and incubated with HRP-conjugated secondary antibodies for 40 min at 37 °C. Then, the DAB (diaminobenzidine) reaction, hematoxylin staining, differentiation with hydrochloric acid alcohol, dehydration and transparency steps were conducted in turn. All images were captured by Leica Microsystems CMS (DFC365 FX). All staining was scored according to our previous reports .9 titers) containing shRNA against E2F1 was provided by Gene Pharma Company. H446 cells were conventionally cultured in six-well plates. At a confluence of approximately 70%–80%, 1 × 106 virus titers were added and mixed gently. After 12 h, the completed medium was added to cells and the previous medium was removed. After 72 h, puromycin (5 μg/ml) was added to the media. After 5 days, all cells were digested, diluted, and cultured in a 96-well plate to form monoclonal colonies. The medium containing puromycin (1 μg/ml) was changed every 3 days until the confluence was approximately 90%–100%. Cells were then digested and inoculated into 48-well, 24-well and 6-well plates. These cells were named H446-E2F1sh.Adenovirus , and cDNA was synthesized with the Prime Script RT reagent kit with gDNA Eraser . Real-time PCR was performed according to the instructions provided for the SYBR Fast qPCR Mix . The primers used are listed in additional file All cells were lysed with RIPA lysis buffer containing a protease inhibitor cocktail . The protein concentrations were measured, and 50 μg of protein was run on an SDS-PAGE gel and electrophoretically transferred onto NC membranes. The membranes were blocked with 5% fat-free milk and incubated overnight at 4 °C with primary antibodies, including VIM (1:1000), CDH2 (1:1000), CTNNB1 (1:1000), CDH1 (1:1000), E2F1 (1:500), ZEB2 (1:500), α-tubulin (1:1000), β-actin (1:1000) and GAPDH (1:2000). The membranes were then washed three times and incubated for 40 min with HRP-conjugated secondary antibodies. Protein bands were detected by the ECL system .Cells were placed on slides, washed three times with PBS buffer, and fixed with ice-cold methanol and acetone (1:1). Next, the slides were blocked for 30 min with goat serum, washed three times with PBS-TX (PBS containing 1% Tritonx-100), and incubated overnight at 4 °C with primary antibodies. The slides were then incubated with Rhodamine or FITC-labelled fluorescent secondary antibodies and DAPI. Laser scanning confocal microscope (leica-LM7000) was used to observe cell morphology and capture pictures.ZEB2 promoter was amplified by PCR and purified. The PCR fragment and pGL3-basic vector were digested with Nhe I and Bgl II enzymes, and T4 DNA ligase was used to ligate these two fragments together to construct the ZEB2 promoter reporter vector. Luciferase activity analysis was performed according to methods described in our previous report [Genomic DNA was extracted from H446 cells, and the t-test. Multivariate survival analysis was performed by Cox’s regression.SPSS.17.0 statistical software and GraphPad Prism 7 was used. The results from the IHC experiments were analysed by a Chi Square test. The expression differences among target genes were analysed by a r = 0.9985, (p < 0.001) and that both proteins were located on the cellular membrane and absent from the cytoplasm and nucleus. CTNNB1 and CDH1 were expressed in 90% (54/60) of small cell lung cancer cells  and clinical stage (p < 0.001) not age (p = 0.461), gender (p = 0.335), smoking (p = 0.224). CTNNB1 has been previously observed in the nucleus when CDH1 was deregulated [CDH1 and CTNNB1 expressions from the gene expression database of SCLC cell lines (https://sclccelllines.cancer.gov/) [r = 0.01671, p = 0.443). This told us that the expression pattern of CDH1 and CTNNB1 was different between SCLC tissues and SCLC cell lines.Distant metastasis in early stages is a typical feature of SCLC, and the occurrence of EMT is considered to be an early event in the process of tumour invasion and metastasis. Therefore, it is important to determine the expression pattern of EMT markers in SCLC tissue. We examined CDH1 expression and found that it was highly related with CTNNB1 expression   (Additiop < 0.001) [CLDN1 and CDH2 were not detected in any SCLC samples (0/60) Fig. . VIM was< 0.001) .RELA, MMPs and ADAM12 [Our previous paper reported that E2F1 was highly expressed and was an independent and adverse prognostic factor for SCLC. We also found that E2F1 could directly regulate the expressions of d ADAM12 , 9. NF-κd ADAM12 , indicatE2F1 gene was silenced by specific siRNAs in SCLC. To further study cellular morphology, stable E2F1 gene knockdown cells were generated using an adenovirus containing shRNA against E2F1 in H446 cells (H446-E2F1sh cells). Eight clones were randomly tested for E2F1 expression. E2F1 was efficiently knocked down in clone-4 , and the expressions of E2F2, E2F3, E2F5 and E2F8 mRNA was up-regulated, but not to statistically significant levels in H446-E2F1sh cells  and the mRNA levels of other E2F family members were almost no changed  were examined in SCLC cell lines. Real-time PCR showed that CTNNB1 was significantly increased, while VIM and CDH2 were significantly decreased in H446-E2F1sh cells. Additionally, CDH1 was decreased, but this change was not statistically significant . In A549-E2F1 cells, we found that CDH1 and CTNNB1 were decreased, and VIM and CDH2 were increased in mRNA and protein levels Additional file 2: Figure S1.The differential expression intensity of CDH1 and CTNNB1 in SCLC tissue samples. (TIFF 10260 kb)Additional file 3: Figure S1.The staining of CDH1 and CTNNB1 in bronchial epithelial cells. (TIFF 6069 kb)Additional file 4: Figure S3.CDH1 and CTNNB1 in differential SCLC cell lines. (TIFF 13906 kb)The expression levels of Additional file 5: Figure S4.E2F1 expression was quantified in H446-E2F1sh and H1688-E2F1si cells. * represents p < 0.05, ** represents p < 0.001. (TIFF 3949 kb)Additional file 6: Figure S5.The relative protein levels of E2F1, CTNNB1, VIM and CDH2 in H446-E2F1sh cells, and E2F1, CTNNB1 in H1688-E2F1si cells. (TIFF 533 kb)Additional file 7: Figure S6.CDH1 and CTNNB1, and promote the expression of CDH2. (TIFF 6963 kb)E2F1 overexpression in A549 cells could inhibit the expression of Additional file 8: Figure S7.The differential expression intensity of ZEB2 in SCLC tissue samples. (TIFF 7166 kb)Additional file 9:The target genes of E2F1 by ChIP-seq in H1688 cell line. (XLSX 431 kb)


Concurrent diabetes mellitus and tuberculosis represent a significant health problem worldwide. Patients with diabetes mellitus have a high risk of tuberculosis, which may be mediated by an abnormal innate immune response due to hyperglycaemia or low vitamin D levels.M. tuberculosis, including phagocytosis of M. tuberculosis, antimycobacterial activity, LL-37, human β defensin-2 and IL-10 gene expression and nitric oxide production, between type 2 diabetes mellitus patients (n = 51) and healthy volunteers (n = 38).In the present study, we evaluated inactive vitamin D serum levels and the monocyte response to infection with M. tuberculosis phagocytosis between monocytes were similar across groups according to microscopy. Intracellular mycobacterial growth was similar in infected monocytes from both groups. However, M. tuberculosis growth was significantly higher in monocytes obtained from type 2 diabetes mellitus patients and lower vitamin D levels after 1-h (D0) and 72-h (D3) post-infection (p ≤ 0.05). LL-37, human β defensin-2 and IL-10 mRNA expression were similar between monocytes across groups; vitamin D serum levels and LL-37, human β defensin-2 and IL-10 expression were not correlated. Nitric oxide production was significantly higher in healthy volunteers than in type 2 diabetes mellitus patients with low vitamin D serum levels at D3 post-infection (p ≤ 0.05).Twenty-seven type 2 diabetes mellitus patients had inadequate inactive vitamin D levels (<50 nM). The percentages of M. tuberculosis, which is not associated with significant decrease of LL-37 or human β defensin-2 expression. Vitamin D could be the link between diabetes and tuberculosis susceptibility.Our results show that monocytes from type 2 diabetes mellitus patients and low vitamin D serum levels show an impaired ability to control the intracellular growth of The online version of this article (10.1186/s12879-017-2705-1) contains supplementary material, which is available to authorized users. Vitamin D insufficiency is a risk factor for osteomalacia in adults and rickets in children and is associated with a variety of conditions, such as various cancers, type 1 and 2 diabetes, hypertension, multiple sclerosis, metabolic syndrome and infectious diseases, including tuberculosis .3), which is the predominant inactive form in plasma or serum and is the first hydroxylation product of vitamin D that is synthesized in the liver.The human body can synthesize vitamin D from the precursor molecule 7-dehydrocholesterol, which is generated by the skin upon sufficient exposure to solar ultraviolet radiation. Vitamin D is also obtained from natural food, particularly from fish. Since the final effector molecule of vitamin D is produced in humans, vitamin D is also considered a pro-hormone . Vitamin3 levels of 50 to 125 nM as sufficient, levels between 30 and 49.99 nM as inadequate, levels below 30 nM as deficient and levels above 125 nM as potentially harmful [Currently, there is a debate regarding the definition of vitamin D insufficiency. Although the majority of Working Group members nominated 75 nM as the appropriate target concentration for older individuals in the International Osteoporosis Foundation position statement . However harmful .2D3), influences pancreatic β-cells and insulin secretion [The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 ecretion . Althoug3 are associated with higher fasting glucose, insulin resistance and metabolic syndrome [In persons with established DM and in the general population, low levels of 25OHDsyndrome .2D3 [3) into the active 1,25(OH)2D3 form and the activation of the VDR that directly modulate gene expression that favours antimicrobial peptides production [Vitamin D plays a prominent role in innate immunity, and vitamin D receptors (VDR) are found on monocytes; these cells differentiate into macrophages under the influence of 1,25(OH)2D3 . The vit2D3 , 11. Vit2D3 . Vitamin2D3 . In macr2D3 . The antoduction .Mycobacterium tuberculosis replication via the induction of antimicrobial peptides such as cathelicidin (LL-37) and human β defensin-2 (HBD2) [2D3 induces differentiation of the human monocytic THP-1 cell line and enhances the chemotactic and phagocytic activity of macrophages [2D3 also plays a role in acquired immunity directly by acting on T cells and indirectly by regulating dendritic cells; it also restricts Th1/Th17 cell differentiation and favours Th2 response [Vitamin D restricts intracellular 2 (HBD2) , 16. Prerophages , 18; 1,2response .In addition, previous research has shown that DM triples the risk of tuberculosis (TB) . SeveralM. tuberculosis infection observed in persons with DM suggests an impaired immune response, and vitamin D could be the link between DM and TB susceptibility [The high prevalence of DM in Mexico results in a considerable proportion of TB cases attributable to this disease . The sustibility .Therefore, in this work, we evaluate the correlation between serum levels of vitamin D in type 2 diabetes mellitus (T2D) patients and antimycobacterial activity.Fifty-one T2D patients (17 man/34 woman) and 38 people healthy volunteers (12 man/26 woman) were enrolled in this study from 2008 to 2015. The T2D patients were invited to participate in the study at the Metabolic Syndrome Clinic of the National Institute of Respiratory Diseases in Mexico City. They were diagnosed according to the criteria of the American Diabetes Association (Diagnosis and Classification of DM) , and act3). Serum 25OHD3 levels were determined using a commercial serum vitamin D kit  according to the manufacturer’s instructions and reported as nM.Blood samples were drawn from all participants, and the serum samples were separated and stored at −80 °C until determination of the inactive form of vitamin D  .Monocytes were enriched by positive selection using MACS® magnetic beads coupled to anti-human CD14 antibodies , according to the manufacturer’s recommendations. The purity of monocytes was assessed by conventional flow cytometry using anti-human CD14 antibodies. The cell preparations were routinely >95% pure, and viability was evaluated using the trypan blue exclusion assay, which revealed 99% viability.2 atmosphere.Monocytes were cultured in RPMI 1640  supplemented with 50 μg/ml gentamicin sulfate, 2 mM L-glutamine and 10% heat-inactivated pooled human serum  at 37 °C under a 5% COM. tuberculosis strain H37Ra  was prepared in Middlebrook 7H9 broth medium  supplemented with 10% albumin-dextrose-catalase  and 0.2% glycerol. The mycobacteria were incubated for 21 days at 37°C with 5% CO2 on an orbital shaking incubator, and the M. tuberculosis suspension was harvested, centrifuged , resuspended in fresh 7H9 medium and aliquoted into one-millilitre samples that were stored at −70 °C until use. The mycobacteria stock concentration was determined by colony forming units (CFU) counts from ten-fold serial dilutions in 7H9 medium after 21 days of incubation on 7H10 agar. For infection assays, M. tuberculosis stock was thawed and centrifuged , and the supernatant was replaced by complete medium (supplemented medium with 10% non-heat-inactivated human pool serum). Then, mycobacteria were declumped by mixing and repeatedly forcing the sample out of a 27-G syringe needle, followed by a 1-min sonication  and centrifugation . The supernatant containing disaggregated mycobacteria was recovered and used in infection assays.The 5 monocytes/well were placed in a chamber slide  and were allowed to adhere for 1 h at 37 °C in 5% CO2. The cells were subsequently incubated with either culture medium  or infected with M. tuberculosis at multiplicity of infection (MOI) 10 (10 mycobacteria: 1 monocyte) and incubated for 1 h. Three washes with supplemented RPMI were performed to remove the extracellular mycobacteria, and the cells were fixed for 10 min with methanol. Then, the slide was stained with a TB stain kit-K  and analysed under a microscope  at 100X with immersion oil. We counted 300–400 monocytes, and the number of monocytes with intracellular mycobacteria was expressed as the phagocytosis percentage.A total of 1 × 105 monocytes/well were infected with M. tuberculosis at MOI of 10 in a 96-well culture plate . Subsequently, the infected monocytes for 1 h (D0), 24 h (D1) and 72 h (D3) and mycobacterial intracellular growth was evaluated by serial dilution and CFU counts.A total of 2 × 10M. tuberculosis was assessed by CFU. Briefly, the infected monocytes at D0, D1 and D3 were lysed as previously reported [2 atmosphere, the CFU were counted.The intracellular growth of reported . Ten-folM.tb) were lysed, and total RNA was extracted by column purification using a RNeasy Mini Kit  following the manufacturer’s instructions. RNA was eluted in DEPC-water and used for cDNA synthesis with a Superscript First-Strand-cDNA Synthesis System . Briefly, RNA with random hexamers and dNTPs was denatured , and a reverse transcription mix  was added. The synthesis was performed with Superscript II enzyme in a thermal cycler . Quantitative real-time-PCR (qPCR) was performed using Taqman assays for LL-37 , human IL-10  and ribosomal RNA 18S (rRNA 18S) as a housekeeping control was used for the normalization (Applied Biosystem). Briefly, a universal master mix (Applied Biosystem), TaqMan assay (LL-37-FAM or IL-10-FAM and rRNA18S -VIC), DEPC-water and cDNA were mixed and added to each well of a 96-well plate. The plates were sealed and centrifuged . Amplification was performed in a StepOne Plus Sequence Detection System (Applied Biosystem) starting with 2 min at 50°C, 10 min at 95°C and 40 cycles of 30 s at 95°C and 60 s at 62°C. Data were analysed with 7500 Fast System SDS software version 1.4 (Applied Biosystem), and the results are reported as fold changes respective to UN monocytes. A relative quantification method (∆∆Ct) was used for gene expression analysis.After a 1-h (D0) or 72-h (D3) incubation, the uninfected (UN) and infected monocytes (2 (3 mM), dNTPs (0.4 mM/each one), primers  or 60 °C (HPRT) and 1 min of extension at 72 °C. The amplification products were mixed with GelRed and analyzed on 2% agarose gel electrophoresis and the imagen captured in a ChemiDoc MP Imaging System . The amplification band fluorescence was analyzed with Image Lab software and results were reported as arbitrary units.A 102 bp fragment of human β defensin-2 (HBD2) was amplified using the primers 5′-CAT CAG CCA TGA GGG TCT-3′(forward) and 5′-AGG CAG GTA ACA GGA TCG-3′ (reverse) and a fragment of 496 bp from Hypoxanthine Phosphoribosyl Transferase (HPRT) as internal control using the primers 5′-TAT GGA CAG GAC TGA ACG TCT TGC- 3′(forward) and 5′-GAC ACA AAC ATG ATT CAA ATC CCT CA-3 r´(reverse). cDNA were mixed with PCR buffer, MgClM. tuberculosis, the supernatants were recovered at 1 h (D0) and 72 h (D3) post-infection, and the NO concentration was determined as previously reported [2 concentrations was included, and the optical density was determined at 550 nm with a plate microreader . The NO concentration was reported in μM.After monocytes were infected with reported . Brieflyp ≤ 0.05. We used GraphPad Prism Software package, version 5.04 (2008). All data are expressed as the median.Comparisons between T2D patients and healthy volunteers were made using nonparametric Mann-Whitney Rank Sum test, Wilcoxon signed rank tests or Fisher exact test. For rank correlations, we used Spearman’s rank correlation coefficient on nonparametric data. For PPD, gender and the recruitment of people in the two groups during years and seasons, we used proportion tests. Statistical significance was defined as p ≤ 0.05). Most T2D patients were taking metformin and glibenclamide, but none were prescribed angiotensin receptor blockers, insulin, or statins. Healthy volunteers did not exhibit any infections or inflammatory diseases at the time of blood donation for the experiments and did not take any medications during the study period. Complementary clinical characteristic and data base are presented in Additional file The clinical characteristics of the study groups are presented in Table Since the levels of vitamin D could have seasonal variations, we searched for seasonal differences among samples of T2D patients and healthy volunteers obtained at various times, and we did not find any differences (p>0.05). The serum levels of vitamin D in the T2D patients and healthy volunteers groups were significantly different. Our results show that 54% of T2D patients had an inadequate level of vitamin D (between 30 and 49.99 nM) Fig. .Fig. 1SeM. tuberculosis by monocytes from T2D patients and healthy volunteers. Our results showed that the percentages of monocytes that phagocytosed M. tuberculosis were similar (p>0.05) in T2D patients   after 1 h (D0) and 72 h (D3) post-infection (p ≤ 0.05)  Fig. . However05) Fig. , with a Vitamin D directly and indirectly regulates the expression of the antimicrobial peptides cathelicidin (LL-37) and human β defensin-2 (HBD2), which exert bactericidal activity , 16, BecThen, we measured IL-10 gene expression in monocytes from T2D patients with low serum levels of vitamin D and healthy volunteers. We found not significant difference on IL-10 expression comparing T2D patients to healthy volunteers neither correlation of IL-10 expression with the levels of vitamin D (p>0.05) Fig. .p>0.05)  Fig. .p ≤ 0.05). NO production was significantly higher in healthy volunteers than in T2D patients with low serum vitamin D levels (< 50 nM) (p ≤ 0.05)  and healthy volunteers post-infection (05) Fig. .M.tuberculosis. We wondered whether vitamin D could be involved in antimycobacterial activity in T2D patients. Thus, we explored the levels of vitamin D and its correlation with phagocytosis of M. tuberculosis and antimycobacterial activity in monocytes.T2D patients have increased susceptibility to TB, and previous research has shown that low vitamin D levels are associated with both DM and TB. Because vitamin D is a molecule that exert immunoregulatory activities and it has been described that vitamin D play an important role in the immunity against pathogens as M. tuberculosis in monocytes from T2D patients. Our results showed no significant difference in the percentage of monocytes that phagocytosed M. tuberculosis from T2D patients compared with those from healthy volunteers, as determined by light microscopy. These results are inconsistent with a recent report describing a reduced percentage of monocytes from T2D patients associated with M. tuberculosis; this report also demonstrated that phagocytosis was strongest under higher autologous serum concentrations (20% versus 5%), and the authors concluded that the association involved complement factors [M. tuberculosis in T2D patients did not correlate with serum levels of vitamin D.Our results showed that 54% of the study participants with T2D had inadequate sera levels of vitamin D, and these results agree with previous reports. Previous studies have shown that T2D patients are more susceptible to TB , 30. The factors . We eval factors . Our resM. tuberculosis between the groups. Since the percentage of infected monocytes was comparable between the groups, the magnitude of intracellular growth was similar. These results are also inconsistent with previous reports showing that monocyte bactericidal function was decreased in patients with poorly controlled non-insulin-dependent DM [Next, we evaluated antimycobacterial activity in T2D patients and healthy volunteers and observed similar intracellular growth of ndent DM . Althougndent DM , 34.M. tuberculosis in monocytes from T2D patients and associated it with the levels of vitamin D, we found that the mycobactericidal activity in T2D patients was significantly lower in monocytes from patients with inadequate levels of vitamin D. These data indicate that low levels of vitamin D are associated with the decreased antimycobacterial activity observed in T2D patients. Since vitamin D induces the production of LL-37 and HBD2, antimicrobial peptides with antimycobacterial activity [M. tuberculosis-infected monocytes from T2D patients with low serum vitamin D levels. Out of its participation in the regulation of antimicrobial peptides, vitamin D also regulates other human genes trough VDR-mediated response elements (Vitamin D Response Elements) and vitamin D inhibits the production of pro-inflammatory cytokines trough MKP-1 pathway [M. tuberculosis by monocytes from T2D patients with low serum levels of vitamin D could be associated with another mechanism regulated directly or indirectly by vitamin D other than LL-37, HBD2 or IL-10.In addition, we analysed the intracellular growth of activity , 35, 36, pathway . Other a pathway . TherefoM. tuberculosis infection in monocytes from T2D patients with serum low levels of vitamin D was significantly lower than that in monocytes from healthy volunteers at D3. Although lower NO production is in agreement with the decreased antibacterial activity observed in T2D patients with low serum levels of vitamin D, these results are inconsistent with a previous report showing that vitamin D-induced inhibition of NO release [NO is an innate metabolite that exhibits antibacterial activity, and previous reports have indicated that vitamin D affects the expression of mRNAs encoding inducible NO synthase (iNOS), iNOS protein expression and NO production , 41. TheM. tuberculosis (even though vitamin D levels are not associated with significant expression of LL-37 and HBD2), other mechanisms directly or indirectly regulated by vitamin D could be involved in the inability of monocytes from T2D patients to control M. tuberculosis growth.The results of our study demonstrate that vitamin D could be the mediator between DM and TB susceptibility. Since monocytes from T2D patients and low serum levels of vitamin D show an impaired ability to control the intracellular growth of


We further examined the phophatidylglycerol (PG) inhibition effects on inflammation. PG supplementation resulted in a 358-fold inhibition of COX-2 mRNA expression. PG(18:1)2 and PG(18:2)2 were incorporated into CLs to considerably alter the CL profile. The decreased CL and increased monolysocardiolipin (MLCL) quantity resulted in a reduced CL/MLCL ratio. KLA-activated macrophages responded differentially to PG(18:1)2 and PG(18:2)2 supplementation. Specifically, PG(18:1)2 induced less changes in the CL/MLCL ratio than did PG(18:2)2, which resulted in a 50% reduction in the CL/MLCL ratio. However, both PG types rescued 20–30% of the mitochondrial activity that had been affected by KLA activation.Chronic inflammation and concomitant oxidative stress can induce mitochondrial dysfunction due to cardiolipin (CL) abnormalities in the mitochondrial inner membrane. To examine the responses of mitochondria to inflammation, macrophage-like RAW264.7 cells were activated by Kdo2-Lipid A (KLA) in our inflammation model, and then the mitochondrial CL profile, mitochondrial activity, and the mRNA expression of CL metabolism-related genes were examined. The results demonstrated that KLA activation caused CL desaturation and the partial loss of mitochondrial activity. KLA activation also induced the gene upregulation of cyclooxygenase  Multiple enzyme types, such as phospholipases and cyclooxygenases (COXs), can be activated in macrophages to produce proinflammatory prostaglandins, followed by conversion to anti-inflammatory lipoxins2. Some studies have suggested that unresolved antigens may induce autoimmune disease and chronic inflammation4; in turn, chronic inflammation and concomitant oxidative stress damage mitochondrial DNA and functions, which promote aging5.External infections induce the activation of toll-like receptors (TLRs) or nucleotide-binding oligomerization domain-like receptors (NLRs) to trigger inflammation6. Macrophage activation repurposes the mitochondria from adenosine triphosphate (ATP) synthesis to ROS production7, but mitochondrial defects can further increase oxidative stress and promote inflammation8. Mitophagy is a selective pathway to prevent mtROS accumulation and excessive inflammation9. The promotion of cellular respiration of blood vessels during the macrophage-mediated engulfment of steroids increases oxidative stress, which leads to cardiolipin (CL) oxidation and mitochondrial defects10. The reduction of oxidative stress in the mitochondria alleviates atherosclerosis and chronic inflammation11.Mitochondria are the main source of mitochondrial reactive oxygen species (mtROS) production, which activate NLRP3 to produce proinflammatory factors12. In addition, CLs are critical in the stabilization of protein complexes on the mitochondrial inner membrane, such as electron transfer chain (ETC)I, III, and IV, as well as ATP synthase (which ensures ETC efficiency)13. CLs are prone to oxidization because of their location in the mitochondria, leading to reduced ATP production16. A lack of CL remodeling and excessive CL oxidation reduces CL symmetry, which can be crucial in certain mammalian organs18. Oxidized CLs can reduce their affinity toward cytochrome c, and result in cytochrome c release into the cytosol and subsequent apoptotic pathway activation23.CLs are negatively charged phospholipids that are specifically localized on the mitochondria and account for 15–20% of mitochondrial lipids. Because of their unique structure with four acyl chains, CLs are responsible for the curvature formation of the mitochondrial inner membrane24. CL remodeling alters the CL profile depending on the availability of fatty acyl chains in the environment, which can be manipulated by nutrient deprivation and lipid supplementation26. TAZ gene mutations on the X chromosome also cause mitochondrial abnormalities in patients with Barth syndrome27. Phospholipase (PL) A2 is a CL remodeling and degradation enzyme that can hydrolyze fatty acyl chains to form monolysocardiolipins (MLCLs) or dilysocardiolipins (DLCLs)28. According to our review of the relevant literature, eicosanoid production has never been reported to be associated with CL degradation in the mitochondria.CL synthase combines phosphatidylglycerol (PG) and cytidine diphosphate diacylglycerol (CDP-DAG) to synthesize CLs in eukaryotic mitochondria. These nascent CLs are further remodeled with the acyl chains from phosphatidylcholine or phosphatidylethanolamine to produce mature CLs through tafazzin (TAZ)Scenedesmus and contains a glycerol backbone-linked glycerol headgroup and two fatty acyl chains29. PG is distributed in the animal cell mitochondria as a precursor for CL synthesis30, and when located in lung surfactant, is critical for innate immunity regulation33. PG competes with lipopolysaccharides to disrupt the activation of the TLR pathway, thus inhibiting the formation of downstream inflammatory molecules35. However, PG-induced anti-inflammatory properties in the mitochondria have not been thoroughly studied. Therefore, we supplemented RAW264.7 cells with PG(18:1)2 and PG(18:2)2 to evaluate the effects of PG on CL synthesis and remodeling, mitochondrial functions, and the mRNA expression of CL metabolism-related genes after Kdo2-Lipid A (KLA)-induced macrophage activation.PG was discovered in 4 was partially hydrolyzed by secretory PLA2 to produce MLCLs and DLCLs, followed by a MS analysis with reverse phase chromatography 3 (m/z 1029.6) and DLCL(14:0)2 (m/z 819.2) were 25 and 11 min, respectively. Fatty acyl hydrolysis increased the polarity of CL metabolites; therefore, MLCL and DLCL traveled faster in the C18 column.To quantify the concentration of CLs, MLCLs, and DLCLs in a complex biological sample, we established a liquid chromatography–mass spectrometry (MS) detection method. CL14:0)4:04 was CLs and MLCLs are major phospholipids of the inner mitochondrial membrane. To access the integrity of the mitochondrial membrane structure, we identified and quantified the concentration of CLs and MLCLs in RAW264.7 cells, but DLCL was too scarce to be detected. The chromatographic analysis of CLs and MLCLs in the lipid extract from RAW264.7 cells demonstrated delayed retention times compared with that of the internal CL standard Fig. ; longer 26. In our measurement, Raw 264.7 cell contains 55 CL species with 42 types of molecular weights, and each species are identified and shown in Table 3 appeared by around 24 min. The retention time of MLCL in samples, which contain more long-chain MLCL, was eluted around 26–30 min. The results showed that MLCL species shows five clusters in m/z range of 1100–1300 and 33 MLCL species with 16 molecular weights 2-supplemented cells were then analyzed through MS 4 was the most abundant CL species in RAW264.7 cells, which suggests the efficient incorporation of the 18:1 fatty acyl chain from PG to CL in the first and second supplementation. PG(18:1)2 supplementation remarkably increased the percentage of symmetrical CLs. Furthermore, we evaluated the effects of PG(18:1)2 supplementation in fresh media to RAW264.7 cells during cell passage 2 in mitochondria increased 9.6 folds, indicating the supplemented PG(18:1)2 successfully entering mitochondria. The concentrations of PC(18:1)2 and PS(18:1)2 increases 2.4 folds and 1.4 folds respectively. These results indicated that the imported PG(18:1)2 in mitochondria stimulated either the conversions among PG, PC and PE phospholipids or the remodeling of PC and PE in mitochondria.We further examined the two-time PG supplementation effects on phosphatidylglycerol (PG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) Fig. . After P17. As indicated in the preceding section, we supplemented RAW264.7 cells with 50 μM of PG(18:1)2 and PG(18:2)2 twice to alter CL compositions  and CL(72:4) increased from 1.4% to 12.1% and from 3.6% to 28.1%, respectively, which were the species with the highest increase percentage. By contrast, CL66 and CL68 were the groups with decreasing percentages, most evident within CL(66:4) and CL(68:4) . Additionally, CL70, C72, and C74 showed a significant reduction in CL saturation.CLs are mainly composed of 18:1 and 18:2 fatty acyl chains in animal cells. CL symmetry is also critical for CL maturation2 supplementation, the shift of CL68 and CL70 to CL72 and CL74, respectively, was significant. In addition to the increase in chain length, CL saturation decreased drastically. The species with more than four double bonds increased in percentage, which strongly indicates the incorporation of the 18:2 fatty acyl chain to replace the fatty acids with 0–1 double bonds. CL(72:6), CL(72:7), and CL(72:8) in CL72, and CL(74:7), CL(74:8), and CL(74:9) in CL74 showed a significant increase in percentage. Conversely, CL(68:4) decreased by 10.2%, which is the most remodeled species.Following PG(18:2)2 and PG(18:2)2 supplementation. PG(18:1)2 supplementation produced CLs with 2–4 double bonds, and PG(18:2)2 supplementation produced CLs with 5–8 double bonds. We also confirmed that the PG(18:1)2 and PG(18:2)2 supplementation increases the abundance of 18:1 and 18:2 fatty acyl chain in CL respectively.In short, the CL70, CL72, and CL74 groups showed a high variation in CL species after PG(18:1)2, MLCL(54:3) increased drastically from 10.16% to 34.84% to become the major MLCL species. In addition, MLCL(56:6) showed a significant percentage increase of 6.6%. However, the percentages of the short chain MLCL50 and MLCL52 species decreased. PG(18:2)2 supplementation resulted in a significant increase of 10.6%, 8.2%, 4.3%, and 8.3% in MLCL(54:5), MLCL(54:6), MLCL(56:5), and MLCL(56:6), respectively. PG(18:1)2 supplementation specifically increased the percentage of MLCL(18:1)3, which is different from the effects of PG(18:2)2 supplementation that increased the percentage of multiple unsaturated species. These effects on MLCLs are in accordance with those on CLs, supporting the conceptualization that MLCLs are the hydrolysis products of CLs.MLCLs are considered to be the hydrolyzed products of CLs because the biological synthesis of MLCLs has yet to be reported; nevertheless, it is suspected that the species and concentrations of MLCLs are strongly related to those of CLs. In the present study, we examined the changes in MLCLs after PG supplementation Fig. . After tHowever, a 50-μM CDP-DAG supplementation of RAW264.7 cells resulted in nonsignificant changes in both CLs and MLCLs 2 supplementation and KLA activation, the CL quantity was 1.29 fmole/cell, and the MLCL quanitity was 0.14 fmole/cell. Therefore, the CL/MLCL ratio did not fluctuate and was maintained at 9.5.In the RAW264.7 inflammation model, KLA-induced TLR4 activation altered the phospholipid composition and CL saturation2 supplementation for 48 hours, the CL and MLCL quantities were 1.02 and 0.11 fmole/cell, respectively, with a CL/MLCL ratio of 9.4. This is similar to the quantity changes that occurred after PG(18:1)2 supplementation. However, KLA activation of PG(18:2)2-supplemented cells resulted in a drastic decrease in the CL/MLCL ratio to 5.4. Although supplementation with both PG types decreased the CL concentration and increased the MLCL concentration, PG(18:2)2-supplemented cells tended to increase the MLCL percentage of the total CL species after KLA activation, indicating that CL(18:2)4 is an unfavorable CL species after KLA activation.After PG(18:2)2 to alter CL compositions and subsequently activated by KLA 2 and KLA 2 supplementation; however, it decreased.KLA triggered differential changes in the CL composition of PG-supplemented RAW264.7 cells Fig. . After tKLA Fig. , most ofKLA Fig. , CL satuAfter PG supplementation and KLA treatment, the mitochondrial activity in RAW264.7 cells was examined using a Seahorse XF-24 extracellular flux analyzer Fig. . The ove2-supplemented cells did not exhibit significant changes in the control group up Figs. . Howeverlls Fig. . For the2-supplemented cells, KLA reduced the OCRs of the mitochondria in RAW264.7 cells 2-supplemented cells exhibited a slightly alleviated the mitochondrial activity, which is probably due to the original reduction in PG(18:2)2 supplementation control.In PG(18:2)lls Fig. ; specifiPtgs1, Ptgs2, and Alox5, were evaluated 2 supplementation alone in RAW264.7 cells resulted in 23% and 45% reductions in the mRNA expression of Ptgs1, respectively; however, no apparent effects were observed on the mRNA expression of Ptgs2 and Alox5. In RAW264.7 cells treated with KLA, the mRNA expression of Ptgs1 and Alox5 decreased by 66% and 77%, respectively, but that of Ptgs2 increased significantly by 358 folds, indicating significant macrophage activation.To further investigate the gene regulations associated with CL and MLCL profile changes in response to KLA activation and PG supplementation, we analyzed the mRNA expression of CL-metabolism-related genes through reverse transcription quantitative real-time polymerase chain reaction Fig. . Three rted Fig. . PG2 or PG(18:2)2, compared with cells without PG supplementation. However, both PG(18:1)2 and PG(18:2)2 drastically reduced the KLA-induced mRNA expression of Ptgs2 by 27.5 folds.After PG supplementation, KLA-induced inflammation reduced drastically. KLA did not affect the mRNA expression of Alcat1) and Taz are crucial in mitochondrial CL remodeling , which is associated with fatty acid desaturation, which is an important step during fatty acid elongation. The mRNA expression of Fads2 decreased by 49% after KLA treatment, and decreased by 64% and 40% after PG(18:1)2 and PG(18:2)2 supplementation, respectively. Additionally, a 40% downregulation of Fads2 mRNA expression was observed in cells with KLA and PG(18:2)2 supplementation compared with those with only PG(18:2)2 supplementation. This phenomenon was not observed in cells with PG(18:1)2 supplementation alone.The mRNA expression of CL synthesis-related genes did not exhibit significant changes after KLA treatment and PG supplementation Fig. . Acyl-Coing Fig. . When RAPld decreased by 64% after KLA activation in PG(18:2)2-supplemented cells, and Pla2g6 mRNA expression increased by 65% after KLA activation in PG(18:1)2-supplemented cells.KLA treatment and PG supplementation did not significantly change the mRNA expression of CL hydrolysis-related genes Fig. . Howeverc (Cycs), BH3-interacting domain death agonist (Bid), and phospholipid scramblase 3 (Plscr3) were the genes associated with apoptosis regulation 2 were selected to compare the saturation effects. The incorporation of both PG types was exceptionally efficient 2-supplemented cells did not exhibit significant difference. Notably, PG supplementation did not affect the mRNA expression of CL metabolism-related genes, except to inhibit Δ-6 desaturase. The mRNA expression of CL metabolism-related genes was mostly unaffected by PG supplementation; however, a slight downregulation of Ptgs1 mRNA expression was observed. Altogether, although PG incorporation significantly altered the mitochondrial phospholipid components, the cellular mitochondrial functions remained affected.On the basis of the fatty acid compositions of CLs in cells, 18:1 and 18:2 were the two most common fatty acyl chains. PG(18:1)ent Fig. , and proPtgs2 mRNA expression after PG(18:1)2 and PG(18:2)2 supplementation, which was not observed in Ptgs1 or Alox5 mRNA expression. Although KLA activation of PG(18:1)2-supplemented cells did not change the CL/MLCL ratio, the ratio was significantly reduced in PG(18:2)2-supplemented cells. Because abnormal MLCL accumulation can trigger the aggregation of mitochondrial cristae in people with Barth syndrome46, PG(18:1)2 was a more favorable PG type for medical purposes than PG(18:2)2. One study reported that the Taz gene defects associated with Barth syndrome can reduce the in vitro remodeling of PG(18:2)2 and CL(18:2)4, but not PG(18:1)247. Furthermore, the highly unsaturated forms of CLs and MLCLs can undergo oxidation during KLA-induced inflammation. Therefore, macrophage activation significantly increases CL saturation. After PG supplementation, KLA-triggered desaturation effects were ineffective, probably because desaturation was nonessential in the inflammation-suppressed conditions. KLA-induced macrophage activation drastically reduced the mitochondrial activity; however, the effects of KLA were alleviated after supplementation with both PG types. In addition, Δ-6 desaturase was inhibited after KLA addition in PG(18:2)2-supplemented cells.Evidence from the present study supports the idea that PG supplementation suppresses inflammation, particularly the 358-fold reduction in 4, phosphatigylglycerol standard PG(18:1)2, PG(18:1)2 and CDP-DAG(18:1)2 standard were purchased from Avanti Polar Lipids . 3-deoxy-d-manno-octulosonic acid-lipid A , (S)-Bromoenol lactone (S-Bel), (R)- Bromoenol lactone (R-Bel) were bought from Cayman . Bio-Rad protein assay, iScriptTM cDNA synthesis Kit and iQ™ SYBR® Green Supermix were purchased from Bio-Rad . MTT -2,5-diphenyltetrazolium bromide), oligomycin and FCCP were purchased from Sigma-Aldrich . Rotenone was acquired from Merck EMD Millipore . Mitochondria isolation kit for cultured cells was purchased from Thermo Fisher Scientific, US.Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco . Tetramyristoyl cardiolipin standard CL(14:0)4 standard in chloroform was air-dried with N2 gas. The dried CL was resuspended in 498 μl of buffer  and sonicated for 10 times under the condition of 125 watt, 5 sec cycle, amplitude 80% to make CL vesicles. The hydrolysis reaction was initiated by adding 2 μl of purified sPLA2 and incubated at 40 °C water bath28. After 3 hours of incubation, the reaction was stopped by adding 2 ml of methanol (MeOH). Fatty acids were extracted by Bligh-Dyer methods and subjected to mass spectrometry analysis.The 50 nmole CL(14:0)2 at 37 °C. While cells were grown to 80% confluency in culture dishes, the cells were removed from dishes by treatment with 0.05% trypsin-EDTA solutions and washed with PBS before cell seeding. For activation, the adherent RAW264.7 cells were treated with 100 ng/ml of KLA for 24 hours.Mouse leukaemic monocyte macrophage cell line (RAW264.7) was cultured in Dulbecco’s modified eagle medium with 10% fetal bovine serum, which was heat-inactivated for 30 minutes at 56 °C, 0.5% penicillin-streptomycin and 25 mM HEPES in 5% CO2 at 37 °C. The collected 6 × 105 cells were transferred to a 6-cm culture dish and treated with 50 μM of CDP-DAG or PG(18:1)2 or PG(18:2)2 twice at 0 and 12 hours at 37 °C before harvest at 48 hours. In the KLA activation experiments, 100 ng/ml of KLA was added at 24 hours and harvested at 48 hours. In the multiple supplementation treatment, 50 μM of PG(18:1)2 were added four times to cells at 0, 12, 24 and 36 hours. The cells were then harvested at 48 hours, washed with PBS and stored in −20 °C until lipid extraction. CDP-DAG or PG(18:1)2 or PG(18:2)2 were dissolved in ethanol. The final concentration of ethanol in medium was 0.16%.RAW264.7 cells were cultured in Dulbecco’s modified eagle medium with 10% heat-inactivated fetal bovine serum, 0.5% penicillin-streptomycin and 25 mM HEPES in 5% CO7 RAW264.7 cells treated with PG(18:1)2 were harvested for mitochondria isolation following the protocol of mitochondria isolation kit . In brief, 800 µL of Mitochondria Isolation Reagent A. was added to cells. The samples were incubated ice for 2 minutes. Then, add 10 µL of Mitochondria Isolation Reagent B. and incubate the samples on ice for 5 minutes. After added 800 µL of Mitochondria Isolation Reagent C, the samples were gently mixed and centrifuge at 700 × g for 10 minutes at 4 °C. The samples were centrifuged at 12,000 × g for 15 minutes at 4 °C to collect the isolated mitochondria. The ingredients of the Reagent A, B and C are not disclosed by Thermo scientific. The isolated mitochondria were added 1 ml of MeOH for sonication for 60 sec. The phospholipids were extracted by Bligh-Dyer’s method. The extracted lipids were N2 dried and then re-suspended in 200 µL of ACN(acetonitrile)/IPA(isopropanol)/DDW(Distilled and deionized water) (65/30/5) for LC-MS/MS analysis.The 2 × 1048. The internal standard, 0.2 nmole tetramyristoyl cardiolipin CL(14:0)4, was added to the cell pallets in 2 ml MeOH. After 3 times of pulse sonication at the condition of 125 watt, 20 sec, amplitude 80% on ice, 1 mL of dichloromethane was mixed with samples, which were further vortexed for 10 min. Then, 1 ml of dichloromethane and 1 ml of distilled deionized water were added to samples and further vortexed for 10 min. The lower phase in the glass test tube was collected by centrifugation at 2000 rpm for 5 min and the lipid sample was stored at −20 °C.The total lipid in the collected cells was extracted according to the Bligh-Dyer’s method as our previous experiments2O (65:30:5). The samples were capped and stored in −20 °C to prevent evaporation until LC/MS Ion-Trap analysis (Bruker Corporation). Reverse phase HPLC contained solution A: ACN:H2O (60:40), 10 mM ammonium formate, 0.1% formic acid and solution B: IPA:ACN (90:10), 10 mM ammonium formate, 0.1% formic acid for the elution gradient from 60% solution A/40% solution B to 100% solution B in 25 min and maintained 100% solution B until 45 min. The elution was in an Acclaim RSLC 120 C18 2.1mm × 100 mm 2.2 μm column (Thermo) at a flow rate of 0.2 mL/min at 55 °C. Data were further analyzed by Bruker DataAnalysis (ver.4.1). The extract ion current (XIC) of each cardiolipin species was quantitated by their area ratio of XIC to internal standard. The total CL is the sum of all quantitated cardiolipin species. The percentage of CL was the ratio of XIC of each cardiolipin species to total XIC. Standard deviations were calculated by the STDEV function in Microsoft Excel for the error bars of the histograms and t-tests are applied to all triplicated dataThe extracted total lipid was dried under nitrogen gas and immediately re-dissolved with acetonitrile/2-propanol/H2 at 37 °C. The 1 × 104 cells were transferred to a 24-well culture plate and treated with 10 μM, 25 μM, 50 μM, 100 μM, 250 μM and 500 μM PG(18:1)2 for 24 hours at 37 °C, washed with PBS, and added medium containing 0.5 mg/ml MTT-2,5 diphenyl tetrazolium bromide). After 4 hours of incubation at 37 °C, MTT turned into purple formazan crystal. The medium was replaced with 200 μl of DMSO for 5 min. The absorbance of the dissolved cells in DMSO was measured at the wavelength of 550 nm.RAW246.7 cells were cultured in DMEM with 10% heat inactivated fetal bovine serum in 5% CO4 RAW264.7 cells were seeded into each well of XF-24 cell culture microplate (Seahorse Bioscience) and allowed to adhere. The cells were then supplemented with 50 μM PG(18:1)2 or PG(18:2)2 twice at 0 and 12 hour. At 24 hour, 100 ng/ml KLA was added and the cells were incubated for another 24 hours. The media was replaced with DMEM pH 7.4 for 1 hour at 37 °C. The cell oxygen consumption rate (OCR) was analyzed by Seahorse XFe Extracellular Flux Analyzer . The optimized concentrations of the mitochondrial inhibitors in the experiments were 10 μM oligomycin, 1 μM FCCP and 5 μM rotenone.A total of 5 × 10After seahorse mitochondrial assay, the protein concentration in each well of the XF24 cell culture microplate was measured by Bradford protein assay. The microplate was added 50 µl of 0.1% triton in PBS to each well and placed on ice for 5 min, and then 450 µl of DDW was added to each sample. The Bradford assay was started by mixing 10 µl of the prepared sample with 190 µl of 1X solution of Bradford protein-binding assay (Bio-Rad) in a 96-well plate. The absorbance at wavelength 595 nm of the plate was measured by SpectraMax M Series Multi-Mode Microplate Readers (Molecular Devices). The protein concentration was calculated based on the standard curve of BSA.RAW264.7 cells were washed with cold PBS, added 1 ml of TRIZOL reagent and then placed on ice for 5 min. The cells were transferred to a 1.5 ml eppendorf tube and added 200 μl of chloroform. The upper phase in the tube was collected after centrifugation at 12000 rpm for 15 min at 4 °C. Equal 500 μl of supernatant and isopropanol were mixed gently and kept at −20 °C for 20 min. The sample was then placed at room temperature for 10 min and collected the precipitate by centrifugation at 12000 rpm for 15 min at 4 °C. The precipitate was washed with 500 μl of ethanol and centrifuged at 9000 rpm for 5 min at 4 °C. After removing the supernatant, the sample was added 20 μl of DEPC water and heated to 50 °C for 5 min to restore the mRNA sample. The cDNA synthesis Kit from Bio-Rad was utilized for the mRNA reverse transcription. Briefly, 1 μg mRNA was mixed with 4 μl iscript reaction mix and 1 μl iscript reverse transcriptase, and then DEPC treated water was added to 20 μl of total volume. The sample was kept at room temperature for 5 min, and then heated to 42 °C for 30 min and 85 °C for 5 min. After DNA quantitation, the sample was stored at −20 °C.The quantitative PCR mix contains 50 ng of cDNA, 10 pmole of forward primer, 10 pmole of reverse primer and 10 μl of iQ™ SYBR® Green Supermix (Bio-Rad) in a total 20 μl volume. The sequences of the primers are provided in Supplemental Fig. Supplementary Information


The PIP2 sensitivity of Kv channels is well established for all five members of the Kv7 family and for Kv1.2 channels; however, regulation of other Kv channels by PIP2 remains unclear. Here, we investigate the effects of PIP2 on Kv2.1 channels by applying exogenous PIP2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP2 rescued Kv2.1 channels from rundown and partially prevented the shift in the voltage-dependence of inactivation observed in inside-out patch recordings. Native PIP2 depletion by the recruitment of FKBP-Insp54P or M1R activation in whole-cell experiments, induced a shift in the voltage-dependence of inactivation, an acceleration of the closed-state inactivation, and a delayed recovery of channels from inactivation. No significant effects were observed on the activation mechanism by any of these treatments. Our data can be modeled by a 13-state allosteric model that takes into account that PIP2 depletion facilitates inactivation of Kv2.1. We propose that PIP2 regulates Kv2.1 channels by interfering with the inactivation mechanism.Phosphatidylinositol 4,5-bisphosphate (PIP They open and close in response to changes in transmembrane voltage, and are involved in numerous physiological processes, for example, in the generation of action potentials1.Voltage-gated potassium (Kv) channels are integral membrane proteins that enable the passage of potassium ions (K2. Transitions between these three states (gating) of Kv channels can be modulated by different stimuli. For instance, phosphorylation3, SUMOylation5 polyunsaturated fatty acids6, accessory subunits7 and different natural and synthetic compounds9, are well known modulators of Kv channel gating. Phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PIP2), have also been shown to modulate the gating mechanism of several Kv channels12, although, some of these results have been debated14.Almost all Kv channels share a similar mechanism of operation. Upon depolarization, Kv channels transition from a resting (closed) to an activated (open) state, however, during prolonged depolarizations, Kv channels switch to an inactivated (open non-conductive) state2 is a minor phospholipid found in the inner leaflet of the plasma membrane and plays an important role in modulating several ion channels16. While it is well established that some members of the Kv channel family, specifically Kv1.2 and Kv7, are regulated by PIP216, studies in other Kv channels have shown contradictory results. Oliver et al. (2004) first reported the modulation of Kv1.1/Kβ1.1, Kv1.4 and Kv3.4 channels by PIP2. Application of PIP2 micelles to the cytoplasmic side of membrane patches abolished the fast (N-type) inactivation of these channels10. However, Kruse et al.13 using whole-cell recordings and enzymatic methods to deplete native PIP2 in intact cells, did not report PIP2 sensitivity for several Kv channels, including channels previously reported to be PIP2 sensitive . Kv2.1 is another ion channel that was first reported to be PIP2 sensitive. Hilgemann et al.17 reported that Kv2.1 channels were rescued from rundown in excised inside-out patches by applying exogenous PIP2. However, experiments using intact cells failed to demonstrate the PIP2 sensitivity of Kv2.113. The reasons why some Kv channels (e.g. Kv2.1) show PIP2 sensitivity in excised patches, but not in intact cells still remains poorly understood and deserves further investigation.PIP2 regulation of Kv2.1 channels expressed in HEK293 cells by employing different strategies to manipulate PIP2 levels in excised patches and intact cells. In excised inside-out patches, PIP2 recovered the rundown of Kv2.1 channels and prevented the shift in the voltage dependence of inactivation. In whole-cell experiments, native PIP2 depletion by membrane translocatable enzymes or M1R activation facilitated the inactivation of Kv2.1 channels. Our data and quantitative analysis suggest that PIP2 depletion increases the transitions from closed- and open-states to the inactivated-states.In this work, we explored the PIP2 from the plasma membrane, and alterations of channel function that accompany patch excision can reflect PIP2 dependence. For instance, rundown and changes in the voltage-dependence of Kv1.2 channels in inside-out patches can be reduced by manipulations that slow the loss of PIP2 and can be reversed by application of exogenous PIP2 to the intracellular face of the patch11. To test PIP2 dependence on Kv2.1 channels, we first recorded Kv2.1 currents in excised inside-out patches. Following patch excision into symmetrical K+, Kv2.1 currents activated by a test pulse to + 60 mV from a holding potential of −80 mV, successively decreased in amplitude (rundown) to ~20% of the initial current  to the excised inside-out patch significantly reversed the current rundown to ~76% of the initial current , to limit the known PIP2 dephosphorylation in inside-out patches slowed the rundown of Kv2.1 currents; the rundown rate (τrundown) in normal external solution was 105.7 ± 9.2 s compared to 298.4 ± 39.8 s in FVPP , whereas it was −76.9 ± 1.6 mV (k = 5.3 ± 0.3 mV) in the inside-out patch configuration . From the curve in the inside-out patch mode it can be seen that there is significant inactivation at −80 mV, in contrast to when the patch is held at −120 mV. Thus, it is likely that the rundown observed in the inside-out patch recordings at a holding potential of −80 mV was due to an accumulation of inactivation of Kv2.1 channels, as rundown was not observed at a holding potential of −120 mV, where inactivation does not take place. Because PIP2 could recover the current from rundown, we tested if this effect was related to the inactivation of Kv2.1. As anticipated, application of PIP2 partially prevented the hyperpolarizing shift in the inactivation curve of Kv2.1 channels , as the voltage dependence of inactivation is shifted to hyperpolarized potentials. The recovery of Kv2.1 from rundown induced by PIP2 is in turn due to a shift of the inactivation curve towards more depolarized potentials.Patch excision leads to a gradual loss of PIPent Fig. . Bath apent Fig. , while dion Fig. . Thus in2 dependent by means of their responses to exogenous PIP2 application in excised inside-out patches, were shown to be insensitive to PIP2 depletion in intact cells13. A number of reasons have been proposed to explain these disparities; among them is the incorporation of large amounts of PIP2 to the inner leaflet of the plasma membrane (in inside-out patches), generating unphysiological protein conformations14. Therefore, we turned to whole-cell recordings to study modulation of Kv2.1 channels following native PIP2 depletion in intact cells. For this purpose, we expressed Kv2.1 channels together with a rapamycin-translocatable lipid 5-phosphatase (FKBP-Inp54p) and the membrane anchor LDR-CFP. The FKBP and LDR domains dimerize when rapamycin is added to cells, leading to the dephosphorylation of PIP2 on the 5′ position to PI4P21. In all experiments, we used 1 μM rapamycin. We also expressed Kv2.1 channels together with the M1 muscarinic receptor (M1R). M1R is a Gq-coupled receptor that activates phospholipase C (PLC) leading to PIP2 depletion22. In all experiments, we used 10 μM acetylcholine (ACh) to activate the M1R.Several ion channels reported to be PIP2 depletion by several experimental approaches13. In this work, the amplitude of Kv2.1 currents was barely affected by PIP2 depletion; however, no effects on other Kv2.1 biophysical parameters were studied. In our hands, we observed that the recruitment of the 5-phosphatase (FKBP-Inp54p) to the plasma membrane by rapamycin had small but significant effects on the amplitude of Kv2.1 currents  and 27.4 ± 4.5% . We also expressed Kv2.1 channels together with M1R. Upon activation of M1R, Kv2.1 peak- and end-pulse currents were inhibited 23.7 ± 5.3% (p < 0.01) and 42.1 ± 7.2% .Recently, Kv2.1 channels were reported to be insensitive to PIPnts Fig. . The pea2 depletion in intact cells shows smaller effects on the ion conduction of Kv2.1 channels than in excised patches. Thus, we next turned to study the effects of PIP2 depletion on the voltage-dependent gating of Kv2.1 channels.From these results, we concluded that PIP2 depletion on the voltage-dependence of activation of Kv2.1 channels, we determined the conductance-voltage relations (G-V curves) recorded from cells studied in the whole-cell mode before (control) and after PIP2 depletion induced by the application of rapamycin  was 13.8 ± 0.9 mV (k = 15.8 ± 0.6 mV) under control conditions, and was not significantly modified after rapamycin application   under control conditions and 12.8 ± 2.2 mV (k = 17.9 ± 1.3 mV) after M1R activation (p > 0.05).To examine the effect of PIPcin Fig. . Figure 05) Fig. . Similarion Fig. . The V1/2 , while it was −41.3 ± 2.0 mV (k = 5.6 ± 0.2 mV) following rapamycin application . Activation of M1R also resulted in a ~8 mV shift of the inactivation curve to hyperpolarized potentials , while it was −31.9 ± 1.3 mV (k = 6.6 ± 0.4 mV) following M1R activation (p < 0.001). These results also demonstrate modulation of the inactivation gating of Kv2.1 channels by PIP2 in whole cells.We have already seen modulation of the voltage-dependence of inactivation of Kv2.1 channels in excised inside-out patches that could be prevented  by exogenous PIP2 Fig. . Thereforol Fig. . The V1/als Fig. . The V1/20. Because the V1/2 of inactivation is altered by PIP2 depletion, is conceivable that the inactivation of Kv2.1 channels is also modified from the closed-states. To test this hypothesis, we isolated closed-state inactivation by examining the development of inactivation at a non-activating voltage (−40 mV), before (control) and after PIP2 depletion by rapamycin . The closed-state inactivation was also accelerated by the activation of M1R .An important pathway of inactivation in Kv2.1 channels originates from the closed statescin Fig. . In thescay Fig. . For insM1R Fig. , the tim2 depletion . Similar effects on the recovery kinetics were observed after M1R activation .Next, we examined the time course of the recovery from inactivation of Kv2.1 channels. Recovery from inactivation was measured using the protocol shown in the inset of Fig. ion Fig. . The twoion Fig. , The two2 alters the function of Kv2.1 channels, we constructed a kinetic model. Figure 2 depletion induced by rapamycin on Kv2.1 channels. As previously proposed by others20, in the building of a Kv2.1 channel model we assumed a sequential activation of the four channel subunits in the closed state  the time course of the potassium currents at different membrane voltages  theges Fig. , (c) theges Fig. , and (d)ges Fig. . Figure ons Fig. . On the 24. The sets of rate constants are listed on Table 2 becomes depleted. Additional studies will be necessary to resolve these differences.To make sure that the model captured steady-state properties of Kv2.1 channels, we simulated the currents using IonChannelLab2 sensitive15. However, differences between intact cells and excised patches have been observed regarding the modulation of Kv channels by this lipid14. Kv2.1 channels were originally reported as PIP2 sensitive, as PIP2 was able to rescue Kv2.1 currents from rundown in excised patches17. However, later reports showed that native PIP2 depletion in intact cells barely affected Kv2.1 function13. Here, we have further investigated the regulation of Kv2.1 channels by PIP2 using different approaches. We found that inactivation of Kv2.1 channels is sensitive to PIP2 levels.Numerous ion channels have been reported to be PIP2 or Mg-ATP to excised inside-out patches  the changes in inactivation that occurred following patch excision  to the plasma membrane, or the activation of a Gq coupled receptor (M1R) with the consequent activation of phospholipase C (PLC) and PIP2 hydrolysis, confirmed the modulation of the inactivation gating of Kv2.1 channels by PIP2.Depletion of PIP20. PIP2 depletion did not modify the U-shaped inactivation curve, suggesting that preferential inactivation from closed-states was conserved  in pancreatic β-cells29. Opening of Kv2.1 channels repolarizes β-cell action potentials, limiting Ca2+ entry and, thus, insulin secretion31. Interestingly, the covalent attachment of small ubiquitin-like modifiers proteins to Kv2.1 channels (SUMOylation) modulates pancreatic β-cell excitability. SUMOylation modulates the inactivation gating of Kv2.1, which resulted in the widening of β-cell action potential and a decrease in firing frequency5, leading to an increase in the glucose-dependent insulin secretion. In this context, the modulation of Kv2.1 channels inactivation by PIP2 could result in similar effects. For instance, PIP2 levels in β-cells are dynamically regulated32. An important mechanism of such regulation is the stimulation of muscarinic receptors by acetylcholine, which is the major parasympathetic neurotransmitter released within pancreatic islets during food intake33. Thus, muscarinic signaling could be influencing the functioning of Kv2.1 channels, influencing β-cell excitability.What are the physiological implications of our findings? Kv2.1 channels are expressed in a wide variety of cells, such as central neurons2 modulates the inactivation gating of Kv2.1 channels, which could have an influence on the excitability of Kv2.1 expressing cells.In summary, our data indicate that PIP2, natural from porcine brain) was purchased from Avanti Polar Lipids,  and prepared as previously described34.Rapamycin ready-made solution (2.74 mM in DMSO) and Acetylcholine (ACh) chloride were purchased from Sigma-Aldrich . ACh was dissolved in water to make a stock solution of 25 mM. The stock solutions were diluted in bath solution to the final concentrations required for patch-clamp recordings. L-α-phosphatidylinositol-4,5-bisphosphate (PIP® CRL-1573TM) cells were grown in 60-mm tissue culture dishes  in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum  and 1% Antibiotic Antimycotic solution (Sigma-Aldrich) in a humidifier incubator at 37 °C (5% CO2). HEK-293 cells were transiently transfected with cDNAs encoding rKv2.1 subcloned in the pXoom vector, LDR (Lyn11-targeted FRB)-CFP and FKBP (FK506-binding protein)-Inp54p , and the human M1 muscarinic receptor  with the use of Lipofectamine 2000 reagent  according to the manufacturer´s specifications. As a marker for successfully transfected cells, cDNA encoding the enhanced green fluorescent protein (EGFP) was co-transfected with the cDNAs of interest.Human embryonic kidney 293 [HEK-293] . The pipette solution contained (in mM): 110 KCl, 5 MgCl2, 5 K4BAPTA, 5 K2ATP, and 10 HEPES (pH adjusted to 7.2 with KOH). Inside-out recordings were performed using symmetrical potassium concentrations (in mM): 120 KCl, 5 K2EDTA, 7 KH2PO4, and 8 K2HPO4 (pH was adjusted to 7.4 with KOH). Solutions were applied using a Fast-Step Perfusion System .Electrophysiological recordings were performed at room temperature (22–25 °C) 24 h after transfection, by using the patch-clamp technique in the whole-cell and inside-out configurations. Micropipettes were pulled from borosilicate glass capillary tubes  on a programmable puller  and had resistances of 1.5–2.5 MΩ when filled with the internal solution. Currents were recorded using an Axopatch 200B amplifier . Data acquisition and generation of voltage-clamp protocols were performed using a Digidata 1440 A interface (Molecular Devices) controlled by the pCLAMP 10 software (Molecular Devices). For whole-cell recordings, the external solution contained (in mM): 135 NaCl, 4 KCl, 1 MgClPatch-clamp data were processed using Clampfit 10 (Molecular Devices) and analyzed in Origin 8.6 .Ip is the peak current amplitude at the test potential V, and Vrev is the potassium reversal potential. Normalized conductance was then plotted against V to determine the voltage dependence of activation. The activation curves were then analyzed using the Boltzmann equation:V represents the test potential, V1/2 is the potential at which the conductance was half-maximally activated, and K is the slope.Conductance (G) was calculated using the following equation:1), after a brief repolarization to the holding potential, a 6-s conditioning pulse to potentials between −120 and + 80 mV was applied (P2) followed by a final pulse to + 80 mV (P3). The normalized current was calculated dividing the P3/P1 current and plotted versus the conditioning potential (P2). The data were fitted with the Boltzmann equation:V is the conditional potential, V1/2 is the potential at which the conductance was half-inactivated, and K is the slope.The voltage dependence of Kv2.1 channel steady-state inactivation was determined using a three-step protocol. From a holding potential of −120 mV, a 100 ms depolarizing step to + 80 mV was applied . Statistical comparisons were made using paired or unpaired Student´s t-test where applicable. Statistical significance was set at p < 0.05.The datasets generated during and/or analysed during the current study are available from the corresponding authors upon request.Supplemetary information