Tgr5+/+ mice but not in their Tgr5−/− littermates. This phenotype is recapitulated in vitro in differentiated adipocytes, in which TGR5 activation increases free fatty acid availability through lipolysis, hence fuelling β-oxidation and thermogenic activity. TGR5 signalling also induces mitochondrial fission through the ERK/DRP1 pathway, further improving mitochondrial respiration. Taken together, these data identify TGR5 as a druggable target to promote beiging with potential applications in the management of metabolic disorders.Remodelling of energy storing white fat into energy expending beige fat could be a promising strategy to reduce adiposity. Here, we show that the bile acid-responsive membrane receptor TGR5 mediates beiging of the subcutaneous white adipose tissue (scWAT) under multiple environmental cues including cold exposure and prolonged high-fat diet feeding. Moreover, administration of TGR5-selective bile acid mimetics to thermoneutral housed mice leads to the appearance of beige adipocyte markers and increases mitochondrial content in the scWAT of  White adipose tissue can undergo a process of beiging and acquire functional characteristics similar to brown adipose tissue, including the ability to dissipate energy via uncoupled respiration. Here, Velazquez-Villegas et al. show that activation of the bile acid membrane receptor, TGR5, leads to white adipocyte beiging by promoting mitochondrial fission. Such beige cells display functional characteristics similar to brown adipocytes, including the ability to convert chemical energy into heat via the expression of uncoupling protein 1 (UCP1), known to dissipate the mitochondrial electrochemical gradient through proton leak and to induce uncoupled respiration4. Through this process, beiging of WAT increases the utilisation of nutrients and may contribute to whole-body energy homeostasis7.Obesity and its associated metabolic comorbidities are not only posing an important medical problem but also have a significant societal impact. Novel preventive and therapeutic strategies to limit and/or reverse obesity are therefore receiving increasing attention from the biomedical research community. Recently, brown-like cells, also referred to as beige or brite cells, have been described within the white adipose tissue (WAT)9. Additional transcriptional regulators that orchestrate brown and beige adipose-related gene networks include the PR domain-containing protein-16 (PRDM16)12, that confers brown fat cell identity by promoting brown fat-specific gene expression through co-regulation of C/EBPs and PPARγ12 and the peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC-1α), which co-activates PPARγ and fine-tunes mitochondrial biogenesis, oxidative metabolism and thermogenesis13.In coherence with their functional similarities, beige and brown cell differentiation also involves a shared transcriptional cascade that mainly relies on the transcription factors peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPs)15, and promotes beige fat differentiation and thermogenic capacity through β3-adrenergic receptor (β3-AR) signalling16. Interestingly, cold exposure also induces changes in bile acid (BA) composition, including an increase in secondary BAs18.Cold exposure is one of the main triggers of adipose tissue beiging. Reduction in ambient temperature elicits the release of norepinephrine from sympathetic fibres19. BA signalling is mainly mediated by the nuclear farnesoid X receptor (FXR) and the membrane G protein-coupled receptor TGR522. TGR5 has a broad expression pattern and is responsible for many of the systemic metabolic actions of BAs including protection against diet-induced obesity24, insulin resistance26, liver steatosis26 and atherosclerosis27. Dietary supplementation of TGR5-selective BA mimetics, such as 6-ethyl-23(S)methyl-cholic acid , efficiently prevents body weight gain in mice on a high-fat (HF) diet26. Although it has been demonstrated that TGR5 activation increases energy expenditure and fat mass oxidation by stimulating the basal metabolic rate28, it is unknown whether beige cell formation within white fat depots contributes to this phenotype.BAs, beyond their classical role in dietary lipid absorption, are signalling molecules and exert significant systemic metabolic effects, including the promotion of energy expenditure in the brown adipose tissue (BAT) and muscleHere, we identify the BA–TGR5 axis as a novel molecular circuitry to enhance subcutaneous WAT (scWAT) beiging after cold exposure or HF diet feeding. More importantly, we demonstrate that TGR5 activation is sufficient to induce scWAT beiging at thermoneutrality, a condition in which the sympathetic drive to the fat is negligible. Furthermore, we show that TGR5-specific BA mimetics increase lipolysis and modulate the mitochondrial number and morphology in mature white adipocytes. The enhanced release of free fatty acids (FFAs) not only fuels β-oxidation, but also promotes UCP1-dependent thermogenesis and mitochondrial fission, the latter process being entirely dependent on the presence of an intact ERK1–DRP1 signalling axis. These results confirm the role of TGR5 as a target to modulate mitochondrial function and beige remodelling in the adipose tissue.30. Moreover, secondary BAs—potent endogenous ligands of TGR5— are elevated in plasma of mice maintained at reduced ambient temperature18. To study the importance of the BA–TGR5 axis in cold-induced beiging of scWAT, we used TGR5 wild-type (Tgr5+/+) and TGR5 germline knock-out (Tgr5−/−) mice and exposed them to an ambient temperature of 8 °C for 1 week with free access to food and water. Tgr5+/+ and Tgr5−/− mice at thermoneutrality (30 °C) were used as proper controls for this study. As expected, there was a significant difference in body weight between mice at 30 and 8 °C  and less body weight loss (P = 0.061) after cold exposure  levels of multiple brown fat-specific genes including Ucp1   and the tumour necrosis factor receptor superfamily, member 9 (Tnfrsf9 or Cd137) , a mitochondrial outer membrane marker, and mitochondrial complexes III and IV, was observed in the scWAT of Tgr5−/− mice  Fig. , Pgc1a, 37) Fig. . Unlike ice Fig. , supportice Fig. . Moreoveice Fig. , suggestice Fig.  and the ice Fig. . AlthougTgr5−/− model  by breeding mice carrying floxed Tgr5 alleles (Tgr5fl/fl mice) with the tamoxifen-inducible adiponectin (Adipoq)CreERT2 mouse line. Validation of the Tgr5Adipoq−/− mouse line was carried out to verify the selective deletion of TGR5 in the adipose depots. The Tgr5 gene was indeed deleted in the scWAT and epiWAT and, to a lesser extent, in the BAT of the Tgr5Adipoq−/− mice compared to their controls  and subjected to a daily administration of the TGR5 selective BA mimetic INT-777 or vehicle for 1 week. Interestingly, activation of TGR5 significantly reduced body weight gain  and the rate-limiting enzyme catalysing the synthesis of catecholamines, tyrosine hydroxylase (Th) were noted   Fig. . TogetheTgr5+/+ and Tgr5−/− mice and differentiated into adipocytes in the presence of INT-777 or DMSO for 7 days. Exposure to INT-777 induced several beige adipocyte markers in Tgr5+/+ cells both at the transcript    cells were isolated from subcutaneous fat pads of ipt Fig.  and protipt Fig. . DiffereTgr5−/− cells, but were significantly increased by TGR5 stimulation in Tgr5+/+ adipocytes. These findings support the previous results that TGR5 is required for mitochondrial biogenesis, a process intrinsically linked to the beiging phenotype. To evaluate if the increase in mitochondrial content also impacts mitochondrial function, we performed respirometry assays. TGR5 activation increased spare respiratory capacity of Tgr5+/+ INT-777 stimulated adipocytes after exposure to the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)  inhibitor, etomoxir  Fig. . We thenrol Fig.  and FFA rol Fig.  release.xir Fig. , which p36 and could be a key process to establish the functional effects of beiging. To study the role of TGR5 in modulating mitochondrial morphology, we analysed the mitochondrial architecture and evaluated mitochondrial circularity from fluorescent micrographs of Tgr5+/+ and Tgr5−/− preadipocytes and adipocytes stained with TOMM20 antibody after INT-777 exposure. Acute activation of TGR5 transformed the mitochondrial network composed of elongated mitochondria into fragmented mitochondria in both Tgr5+/+ preadipocytes adipocytes47, which are important for cell viability and senescence, and for mitochondrial health, bioenergetic function, and intracellular signalling59. One of the main findings emerging from this study is that TGR5 activation is critically required for driving the full remodelling process of beige fat differentiation. Our data showed that this occurs not only by coordinating the biogenesis and architecture of mitochondria, but also by enhancing their thermogenic capacity, and further consolidates the role of TGR5 as a key molecular target to modulate mitochondrial function.Mitochondrial remodelling comprises changes in mitochondrial morphology and architecture19. It was also reported that stimulation of the TGR5 signalling pathway activates mitochondrial oxidative phosphorylation and increases the ATP/ADP ratio in enteroendocrine L cells26. Despite these initial clues, this is the first time that TGR5 signalling is described to modulate mitochondrial morphology to increase cellular respiration. In the BAT, mitochondrial fission is used as a compensatory mechanism to activate thermogenesis under high nutrient supply by increasing fatty acid oxidation and promoting 'nutrient wasting' in the form of heat47. Our findings show that the activation of ERK/DRP1 signalling by TGR5 agonists induces mitochondrial fission as a mechanism to increase respiration in primary adipocytes. TGR5 signalling furthermore provoked a massive induction of lipolysis and FFA release in primary beige adipocytes. This observation, most likely the consequence of cAMP/PKA induction, is fascinating, especially because FFA not only serve as substrates for β-oxidation, but also promote thermogenesis through UCP1 activation61, and facilitate mitochondrial fission62. It is hence conceivable that lipolysis is an important process that contributes to the higher respiration rate and fragmented mitochondrial shape induced by FFA availability.The role of TGR5 in regulating mitochondrial function was previously studied in the BAT, where its activation increases deiodinase 2 expression to catalyse the bioactivation of thyroid hormone and to induce mitochondrial fat oxidation in brown adipocytes34, showed distinct features of beiging remodelling when differentiated in the presence of TGR5 activators, suggesting that our findings could be potentially translated to the clinic. Until now, the majority of human studies have focused on the modulation of circulating BAs to confer improvements in body weight control and glucose metabolism66 but did not evaluate the target tissues affected by altered BA signalling. One earlier study reported the use of the BA chenodeoxycholic acid (CDCA) to promote mitochondrial uncoupling and energy expenditure via TGR5 in human BAT28; however, a more detailed analysis of the WAT beiging in vivo in humans is still lacking and the role of the other BAs that could activate TGR5 is unclear. Future studies are warranted to evaluate the use of TGR5 agonists in humans to assess the potency of these molecules in improving mitochondrial dynamics and respiration rate in white fat.This study identified beiging of white adipose fat depots as a novel route by which BAs or TGR5 selective BA mimetics induce fat oxidation. As for all beiging pathways identified in mice, extrapolation of mouse studies to human biology remains challenging. A data set analysis performed in this study revealed that TGR5 is present in human adipocytes and its expression increases along the adipocyte differentiation process. Notably, SGBS cells, considered to be a representative in vitro model of human white pre-adipocytesTgr5+/+) and TGR5 germline knockout (Tgr5−/−) mice in addition to floxed TGR5 (Tgr5Adipoq+/+) mice and tamoxifen-inducible adipose tissue-specific Tgr5−/− mouse strain (Tgr5Adipoq−/−). Mice exposed to thermoneutrality (30 °C) were used as controls. Mice were randomised into the different groups according to the genotype. To explore the molecular mechanisms through which TGR5 can promote the development of beige adipocytes, we used an in vitro model of adipocyte precursors isolated from scWAT SVF of Tgr5+/+ and Tgr5−/− mice, as well as the 3T3-L1-mouse fibroblast cell line and the human SGBS cell line.The primary objective of this study was to investigate the role of TGR5 signalling as a novel pathway important for the induction of scWAT beiging. In vivo studies were performed in different mouse models of WAT beiging development, such as diet-induced obesity and prolonged cold exposure. We included cohorts of TGR5 wild-type , were killed and excluded from the data analyses. These criteria were established before starting the experiments. Experiments in cell models were performed at least three times. In order to focus on TGR5-specific signalling and avoid confounding effects of FXR activation, the selective TGR5 agonist INT-777 was used for all experiments instead of natural BAsTgr5−/− mice were generated by breeding mice carrying floxed alleles for the TGR5 gene (Tgr5fl/fl mice) with the tamoxifen-inducible adiponectin (Adipoq)CreERT2 Cre mouse line . For the validation, two groups of Tgr5Adipoq+/+ and Tgr5Adipoq−/− 8-week-old male mice (10 mice per group) were treated with 1 mg tamoxifen for 5 consecutive days to ensure recombination in adipocytes. Successful spatial knockout of TGR5 mRNA expression in WAT was carefully assessed.The inducible adipose tissue-specific Tgr5+/+ and Tgr5−/− mice or Tgr5Adipoq+/+ and Tgr5Adipoq−/−) were exposed to prolonged cold test  at the Phenotyping Unit in EPFL. The mice were housed in groups of three mice per cage with access to food and water. Rectal measurements of body temperature were performed daily during the week of cold exposure. Mice were killed on the 7th day of cold exposure in normal feeding conditions (9:00 a.m.). The mice were anaesthetised by isoflurane inhalation, blood was collected by cardiac puncture, and tissues were harvested, weighed, and flash-frozen in liquid nitrogen or fixed with 4% formalin to perform biochemical and histological analyses, respectively.Two groups of 10 mice (either Tgr5+/+ and Tgr5−/− mice or Tgr5Adipoq+/+ and Tgr5Adipoq−/−) were housed in a 30 °C cabinet for 1 week and used as controls for the prolonged cold exposed mice. Mice were killed on the 7th day of 30 °C housing in normal feeding conditions (9:00 am) anaesthesia protocol and organ collection was performed as mentioned above for the prolonged cold exposure mice.Four groups of 10 mice (either Tgr5+/+ and Tgr5−/− mice) were housed at thermoneutral conditions (30 °C) for 2 weeks. The first week was used for adaptation and blunting of the sympathetic response. During the second week of thermoneutrality, mice were gavaged once per day with 60 mg/kg of INT-777 or vehicle (carboxyl methyl cellulose). Body weight was measured before and after treatment. Mice were killed after 7 days of INT-777 or vehicle delivery in normal feeding conditions, blood and organs were collected as previously mentioned for the prolonged cold exposure and thermoneutrality control mice.Four groups of 10 mice (Tgr5+/+ (2 groups of 10 mice each) were fed with a HF diet  for 20 weeks, supplemented or not with the TGR5-specific agonist INT-777 (30 mg/kg). Body weight was monitored weekly. After 20 weeks of diet, mice were fasted overnight and were killed under isoflurane anaesthesia. Blood and metabolic tissues were collected as described above for the prolonged cold exposure and thermoneutrality mice models.Eight-week-old All the mice experiments were authorised by the Veterinary Office of the Canton of Vaud, Switzerland under the license authorisations no. 2614 and 3077.Tgr5+/+ and Tgr5−/− mice. Lymph nodes were carefully removed and the tissue was subsequently digested with collagenase type II (1 mg/ml) for 45 min at 37 °C with shaking. Digestion was stopped by adding 15 ml of complete medium (Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% foetal calf serum (FBS) and penicillin/streptomycin (PS—10 mg/ml)) followed by centrifugation at 700×g for 10 min. The pellet was resuspended, filtered through a 70 μm nylon mesh and centrifuged again. SVF cells were resuspended in red blood cell lysis buffer  for 10 min followed by centrifugation at 700×g for 10 min. Cells were resuspended, plated and cultured until confluence in DMEM, including glucose (4.5 g/l), 10% FBS and PS (10 mg/ml). Differentiation of precursor cells into adipocytes was induced for 3 days with DMEM, including glucose (4.5 g/l), 10% FBS, PS (10 mg/ml), insulin (6.063 μg/ml), dexamethasone (1 μM), and 3-isobutyl-1-methylxanthine (IBMX—0.5 mM). Cells were maintained for 4 consecutive days in DMEM, including glucose (4.5 g/l), 10% FBS, PS (10 mg/ml) and insulin (6.063 μg/ml). Cells were treated with the TGR5 agonist INT-777 (30 μM), the ERK inhibitor FR180204 (10 μM) or vehicle (dimethyl sulfoxide— DMSO) since the first day of differentiation. The medium was changed every 2 days.Subcutaneous WAT  was dissected from 8 to 12 weeks old For acute stimulation of TGR5 signalling pathway, low-glucose DMEM (1 g/l) FBS-free medium with 0.1% bovine serum albumin (BSA) was used for 4 h in pre-adipocytes and/or adipocytes to normalise cells to basal levels, followed by INT-777 or vehicle (DMSO) treatment for 30 min for protein collection or 1 h for TOMM20 immunocytochemistry to evaluate the mitochondrial network.For lipolysis assessment, differentiated adipocytes were stimulated acutely (60 min) with either DMSO or INT-777 and incubated in 1 ml of medium (DMEM GIBCO 21063 Invitrogen) containing 0.1% BSA. After 1 h of incubation at 37 °C, the medium was collected and glycerol or FFA concentrations were measured using a free glycerol determination kit (Sigma-Aldrich) or a non-esterified fatty acid assay (Wako), respectively, accordingly to the manufacturer’s instructions.Tgr5+/+ and Tgr5−/− adipocytes were pretreated with the CPT-1 inhibitor etomoxir (50 μM), for 3 h in the presence and absence of INT-777 or vehicle (DMSO). Basal respirometry was assessed as specified below.To evaluate the effect of TGR5 activation in fatty acid oxidation, primary The 3T3-L1 mouse fibroblast cell line was obtained from the American Type Culture Collection (CL-173). 3T3-L1 fibroblasts were cultured in DMEM, including glucose (4.5 g/l), 10% FBS and PS (10 mg/ml). Overexpression of mouse TGR5 (mTGR5) or silencing with sh-TGR5 or control (sh-Co) were performed by transfecting the cells using Lipofectamine 2000 (Invitrogen) with 2.5 μg DNA per well in a six-well plate. Transfection of the cells was performed twice, before differentiation and at day 4 of differentiation. Differentiation of 3T3-L1 cells into adipocytes was induced for 3 days in DMEM, including glucose (4.5 g/l), 10% FBS, PS (10 mg/ml), insulin (6.063 μg/ml), dexamethasone (1 μM), and IBMX (0.5 mM). Cells were then maintained for 4 consecutive days in DMEM, including glucose (4.5 g/l), 10% FBS, PS (10 mg/ml) and insulin (6.063 μg/ml). Cells were treated with INT-777 (30 μM), litocholic acid (LCA 10 μM) or dimethyl sulfoxide (DMSO) as vehicle since the first day of differentiation.SGBS human pre-adipocyte cell line from scWAT were a kind gift from Prof. Martin Wabitsch. SGBS cells were cultured until confluent in basal medium , 10 mg/ml penicillin/streptomycin and 10% FBS), quick-differentiated for 3 days in Quick-diff medium , and then differentiated for 2 more weeks in 3FC medium , following the protocol optimised in Prof. Wabitsch’s laboratory. INT-777 or DMSO were added from the beginning of differentiation and medium was changed every 2 days.Cells were tested for mycoplasma using MycoProbe , following the manufacturer’s instructions.36b4) and beta-2-microglobulin gene (B2m) for mice samples and to mean of hypoxanthine phosphoribosyltransferase 1 (Hprt1) for human cells (DDCt method). The average of at least three technical repeats was used for each biological data point.RNA was extracted from scWAT, epidydimal WAT, BAT or in vitro differentiated adipocytes using TRIzol and was transcribed to complementary DNA using QuantiTect Reverse Transcription Kit (Qiagen) following manufacturer’s instructions. Expression of selected genes was analysed using the LightCycler 480 System (Roche) and SYBR Green chemistry. All quantitative polymerase chain reaction (PCR) results were presented relative to the mean of acidic-ribosomal phosphoprotein PO gene  following manufacturer’s instructions. Then, quantitative PCR was performed using mitochondrial DNA (16S) and nuclear DNA- (hexokinase 2 (Hk2)) specific primers. The primers sequences used for qPCR are available in Supplementary Table To measure mtDNA content , genomic DNA was extracted from cultured differentiated adipocytes or from scWAT of rial DNA 6S and nu32.Heat map was generated using R and was derived from data deposited under GSE80614 Geo data sets repositorySamples were lysed in lysis buffer (50 mM Tris (pH 7.4), 150 mM KCl, 1 mM EDTA, 1% NP-40, 5 mM NAM, 1 mM sodium butyrate, protease and phosphatase inhibitors). Proteins were separated by SDS–PAGE and transferred onto nitrocellulose or polyvinylidene difluoride membranes. Blocking and antibody incubations were performed in 5% BSA or non-fat dry milk. PGC-1α (ab54481 1:1000), UCP1 , TBX1 (ab109313 1:1000) and VDAC1 (ab14734 1:1000) antibodies were from Abcam; anti-CREB (9197 1:1000), p-CREB Ser133 (9191 1:1000), ERK (4695 1:1000), p-ERK (4376 1:1000), DRP1 (4E11B11 1:1000), p-DRP1 Ser616 (3455 1:1000) and p-DRP1 Ser637 (4867S 1:1000) antibodies were from Cell Signalling; anti-MFF antibody (17090-1-AP 1:1000) was from Proteintech group; anti-PARP1 (sc-7150 1:1000), GAPDH (sc-47724 1:2000) and TOMM40 (sc-11414 1:1000) antibodies were from Santa Cruz Biotechnology. The MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (MS604-300 1:500) for mitochondrial subunits was purchased from MitoSciences. Antibody detection reactions were developed by enhanced chemiluminescence (Advansta) and imaged using the c300 imaging system (Azure Biosystems). Quantification was done using ImageJ software.68.scWAT, epidydimal WAT and BAT samples were fixed in buffered formalin (4%) overnight and embedded in paraffin. About 5 μm thick serial sections were made from paraffin-embedded tissue and then stained with haematoxylin and eosin. A Leica DM500 light microscope  was used for imaging. Quantification of adipocyte area was performed with Adiposoft automated software. Briefly, all the adipocytes were manually selected using ImageJ’s multipoint selection and were separated by using the Watershed transform plugin. Adipocyte area and number were measured as described2O2 in PBS 1×. Heat-induced epitope retrieval was performed in 10 mM citrate buffer pH 6 at 95 °C for 20 min. Blocking and antibody incubations were performed in 3% BSA. Blocking was for 1 h at room temperature (RT) and UCP1 primary antibody (U6382 from Sigma 1:500) was incubated overnight at 4 °C. Secondary antibody (anti-rabbit HRP-conjugated) was incubated for 1 h at RT. Enzymatic revelation was performed with 3,3ʹ-diaminobenzidine tetrahydrochloride (HRP-DAB) followed by nuclear staining using Mayer’s haematoxylin. A Leica DM500 light microscope  was used for imaging. Quantification of chromogen intensity of immunohistochemistry was performed via reciprocal intensity method as using ImageJ software. We split the image using the colour deconvolution tool and selecting the chromogen used for the staining (DAB). After measuring the intensity of the stained region of interest (ROI) in the DAB channel, we subtracted this value from 250, deriving a reciprocal intensity that is directly proportional to the amount of chromogen present. As previously reported69, standard colour images acquired from bright field microscopy have a maximum intensity of value 250 . Stained areas, as marked by DAB have lower intensity values, resulting in an inverse correlation between the amount of antigen and its numerical value.About 5 μm thick sections were made from paraffin-embedded scWAT, epiWAT or BAT. Sliced tissue was dewaxed, rehydrated and quenching was performed with 3% H70.Differentiated adipocytes were rinsed with 1× ice-cold PBS and fixed with 4% paraformaldehyde for 15 min at RT. After three washes, the cells were permeabilised with 0.1% Triton X-100 solution for 15 min at RT. Blocking and antibody incubation were performed in 3% BSA. Blocking was for 1 h at RT, and TOMM20 (sc-11415 1:200) or UCP1 (ab10983 1:500) primary antibodies were incubated overnight at 4 °C. Three washes were performed before incubation with secondary anti-rabbit Alexa Fluor488 (ab150077 1:250) antibody for 1 h at RT. Cells were washed, incubated with DAPI dye for 10 min and mounted. The imaging was performed with Zeiss LSM 700 confocal microscope  and analysis of the mitochondrial network and circularity was done using ImageJ software using macro designed by Ruben K. Dagda at the University of Pittsburgh (2010). Quantification of immunofluorescence was performed with ImageJ software. The ROI was delimited followed by splitting the image into the two-colour channels. We determine the average number of cells by measuring the integrated density value (IDV) for the blue channel (DAPI staining). Ten representative nuclei were selected to calculate the mean nucleus value and the IDV of the blue channel was divided by the mean nucleus value to obtain the average number of cells in each ROI. Finally, the IDV of the green channel was divided by the average number of cells represented as protein content per nucleusTgr5+/+ and Tgr5−/− mice were used to measure OCR using high-resolution respirometry . Cells were detached, counted, resuspended in DMEM  and loaded in the Oroboros chamber to measure basal respiration followed by titration of the protonophore agent FCCP (0.2 mM) to achieve maximum electron transfer flux.Differentiated adipocytes from t tests. Differences between more than two groups were assessed using one-way analysis of variance (ANOVA). To compare the interaction between two factors, two-way ANOVA tests were performed. ANOVA, assessed by Bonferroni’s post hoc test, was used when comparing more than two groups. GraphPad Prism 6 was used for all statistical analyses. All P-values < 0.05 were considered significant. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001.Differences between two groups were assessed using two-tailed Data supporting the findings of this study are available within the paper and its supplementary information files. Data are also available from the corresponding author at request.Supplementary InformationPeer Review File


Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn’s disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function. The epithelial cells lining the gastrointestinal tract provide a critical barrier to prevent damaging agents from entering the underlying tissue. There are various factors that contribute to barrier function, including Paneth and goblet cell secretions, but it is the tight junction between epithelial cells that provides a final physical barrier against paracellular movement. Decreased barrier function is a characteristic of inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis. Over 1 million Americans and 2.5 million Europeans are estimated to have IBD. These diseases incur substantial costs for health care and decrease the quality of life for those affected .While the etiology of these diseases is not completely understood, IBDs are thought to arise through an inappropriate immune response to commensal bacteria . CurrentThe tight junction is composed of a variety of transmembrane proteins such as occludin and members of the claudin family, and is connected to the cortical actin cytoskeleton via intracellular zona occludens (ZO) family proteins. A variety of signaling pathways can modify the structure of the tight junction and the cytoskeleton to alter barrier function. One avenue of regulation is through phosphorylation of myosin II regulatory light chain (MLC) by Rho-associated protein kinase (ROCK) or myosin light chain kinase (MLCK). These events induce reorganization and contraction of the actin cytoskeleton . The forIn addition to barrier regulation mediated by changes to cytoskeletal components, the tight junction can also be modulated through removal or insertion of tight junctional proteins at the membrane by vesicle trafficking ,8. PhospWhile several signaling proteins have been identified as modulators of barrier function through modification of phosphorylation, this level of regulation is incompletely understood as the reported effects are cell-line and tissue-dependent . In geneSignaling molecules shown to regulate tight junctions include kinases such as Src kinase, protein kinase C (PKC) isoforms, and extracellular regulated kinase (ERK), as well as phosphatases . Dextranet al. [Another kinase known to phosphorylate occludin is casein kinase 2 (CK2) . This seet al.  found thOne well-studied group of kinases known to mediate barrier function is the PKC family. This large family of kinases is divided into three groups: the classical, novel, and atypical PKCs . At the plasma membrane, the nPKC isoforms δ and θ, and the aPKCs ζ and λ are closely associated with TJ proteins . PKCζ biThe serine proteases trypsin and elastase have been shown to decrease permeability in human airway epithelial cells through a calcium-dependent mechanism, suggesting the possibility of a similar role in intestinal epithelial cells . PreviouWhile it is known that serine proteases modulate barrier function, the mechanisms behind these changes are incompletely understood. Previous work has indicated that the increase in TER in intestinal epithelial cell lines is independent of ion secretion and protease-activated receptor 2 (PAR2), but dependent on a functional tight junction, specifically occludin . HoweverPorcine pancreatic trypsin (trypsin-1) was purchased from Sigma-Aldrich Canada  and diluted in sterile Dulbecco’s Phosphate Buffered Saline (DPBS) . The catalytically active subunit of matriptase was purified as previously described . The actγ and TNFα were purchased from R&D Systems and dissolved in sterile PBS.Forskolin was purchased from Alfa Aesar  and diluted in anhydrous ethanol. Soybean trypsin inhibitor (SBTI) was purchased from Sigma-Aldrich Canada and diluted in sterile DPBS. Aprotinin was purchased from Boehringer-Mannheim  and diluted in sterile water. Epidermal growth factor (EGF) was purchased from R&D Systems  and reconstituted in sterile DPBS. Bradykinin acetate salt was purchased from Sigma-Aldrich Canada and diluted in 5% acetic acid. The following inhibitors were used: PD153035 hydrochloride , U0126 , PP1 , LY294002 , wortmannin  , bradykinin B(2) receptor antagonist , GM6001 , marimastat , and TAPI-1 acetate salt . The CK2 inhibitor, tetrabromocinnamic acid (TBCA), was purchased from Cayman Chemicals and dissolved in DMSO. Canine IFNAntibody sources used throughout the study are as follows: phospho-PKCζ (threonine 560) , β-actin (Sigma-Aldritch A5441), and pan-myosin light chain . The following antibodies were from Cell Signaling Technology: phospho-Akt (serine 473) (9271), Akt (9272), phospho-p44/42 MAPK (4370), p44/42 MAPK (9102), phospho-Src family (tyrosine 416) (2101), Src (36D10) (2109), and phospho-myosin light chain (Thr18/Ser19) (3674).2. Cells were seeded at 1 x 105 cells/well on 1.1 cm2, 0.4 μM pore-size polycarbonate Snapwell™ or transwell semi-permeable supports and grown for 5 days. Media was replaced on the basolateral side of each well every two days. Confluence was determined by ability of the cell monolayer to hold back fluid.The canine epithelial cell line, SCBN, was provided by Dr. André Buret . SCBNs were grown in Dulbecco’s Modified Eagle Medium  supplemented with 5% fetal bovine serum  and 2% L-glutamine (Thermo Scientific), 1% penicillin-streptomycin (Thermo Scientific), and 5 μg/ml Plasmocin . Cells were maintained in a 37°C humidified incubator with 5% COSC) and transepithelial electrical resistance (TER). Cells were bathed in Krebs buffer solution (pH 7.4) consisting of 115 mM NaCl, 2 mM KH2PO4, 2.4 mM MgCl2*6H2O, 25 mM NaHCO3, 8 mM KCl, 1.3 mM CaCl2, and supplemented 10 mM glucose and 10 mM mannitol on the basolateral and apical sides, respectively. Experiments with TBCA were performed in Krebs buffer without MgCl2. Tissue was voltage clamped to 0 V and unclamped every 20 seconds and a 5 mV potential difference applied using a voltage-clamp apparatus . Change in current was measured using a digital data acquisition system  and TER calculated using Acquire and Analyze software (Physiologic Instruments). Cell viability was determined at the end of each Ussing chamber experiment through measurement of the change in short circuit current (Isc) induced by 10 μM forskolin .Confluent cells grown on Snapwells were mounted in Ussing chambers  to measure short circuit current . SCBN cells mounted in Ussing chambers were incubated apically with SBTI for 30 minutes then stimulated with trypsin to confirm a requirement for proteolysis by trypsin in initiating protease-induced increased TER. The involvement of catalytic activity by trypsin or matriptase to sustain the protease-induced increased TER was investigated via the addition of SBTI post-trypsin stimulation or aprotinin post-matriptase stimulation. SCBNs mounted in Ussing chambers were treated apically with SBTI or aprotinin 10 minutes (at the beginning of the sustained phase) after trypsin or matriptase stimulation. The ability of SBTI to inhibit the response to serine proteases was also determined 30 minutes (when the sustained phase had been established) after trypsin stimulation.Percent peak TER was determined as an indicator of the ability of the cells to increase TER in the initiation phase, and percent change in TER was used to determine the effect of an inhibitor on the sustained phase, usually 15–30 minutes after the addition of trypsin. Effects of inhibitors on the protease-induced increase in TER were determined by measuring the change in TER from baseline with inhibitor, and taking it as a percentage of change in TER in cells treated with vehicle.SCBNs mounted in Ussing chambers were stimulated apically with increasing doses of EGF to confirm the growth factors’ ability to increase TER in intestinal epithelial cells. To ensure SBTI was selectively inhibiting the catalytic activity of trypsin, the effect of SBTI pre-treatment on an unrelated molecule that increases TER was investigated.® RNeasy Mini Kit Protocol (Qiagen Inc.) as per manufacturer’s instructions. cDNA was made using Invitrogen Superscript™ III Reverse Transcriptase  according to manufacturer’s protocols. Primers for the ErbB receptor family  were designed using Primer-BLAST. Primers were designed to span exon-exon junctions and are as follows: EGFR forward: GGT GGT CCT GGG GAA TTT GG, EGFR reverse: CAG CGC CTT GTA GTA TTT CAT GC (product 266 bp); ErbB2 forward: GCA CCC AAG TGT GCA CCG, ErbB2 reverse: TAG CCT CTG CAG TGG GAT CT (product 233 bp); ErbB3 forward: TAA AGT CCT TGG CTC AGG CG, ErbB3 reverse: GGC CAG CAT ATG ATC CGT CA (product 151 bp); ErbB4 forward: ATG GAC CGG GAC CTG ACA A, ErbB4 reverse: TGA CTA GTG GGA CCG CTA CA (product 184 bp); Bactin forward: CGT GGG CCG CCC TAG GCA CCA, Bactin reverse: TTG GCC TTA GGG TTC AGG GGG (product 260 bp).SCBNs grown on transwell inserts were lysed using the Qiagen Quick-Start Protocol . mRNA was isolated using the Qiagen® HotStarTaq™ Master Mix, according to the manufacturer’s protocols. RT-PCR was performed using the Bio-Rad T100™ Thermal Cycler with the following conditions: 95°C for 15 minutes, followed by 35 cycles of 95°C for 1 minute, 57°C for 1 minute, and 72°C for 1 minute. A final elongation step is performed at 72°C for 10 minutes. PCR samples were combined with 5x loading dye  and run on a 1.5% agarose gel containing 0.004% ethidium bromide. Sizes of bands were determined by comparing to the 1kb Plus DNA Ladder (Invitrogen). The gel was imaged using the Bio-Rad Chemidoc, and analysed using QuantityOne software.RT-PCR was carried out using QiagenSelected cell signaling proteins were investigated for their role in the initiation phase of the serine-protease induced increase in TER. Inhibitors were added apically to the cells for 15–30 minutes prior to the apical challenge of 45 BAU/mL trypsin. The EGFR inhibitor PD153035 or the CK2 inhibitor TBCA were added 20 or 15 minutes (respectively) prior to trypsin challenge, while the MMP inhibitors GM6001 and marimastat, the TACE inhibitor TAPI-1, the Src inhibitor PP1, PI3-K inhibitors LY294002 and wortmannin, the MEK1/2 inhibitor U0126, the bradykin B(2) antagonist, or the PKCζ pseudosubstrate inhibitor were added 30 minutes prior to the addition of trypsin. The concentration of PD153035 was determined based on the ability of the inhibitor to block the EGF-induced increase in TER. The concentration of bradykinin B(D) antagonist was chosen based on the ability of the inhibitor to block the basolateral bradykinin-induced increase in chloride secretion.To determine the role of these signaling proteins in the sustained phase of the serine protease mediated increase in TER, selected inhibitors were also added apically to the cells 10 minutes post apical trypsin treatment.3VO4, and 50 mM NaF. Protease inhibitor cocktail was also added, producing a final concentration of 40 μM AEBSF, 280 nM E-64, 2.6 μM bestatin, 20 nM leupeptin, 6 nM aprotinin, and an additional 20 μM EDTA. Inserts were scraped, and cells were vortexed and frozen at -80°C overnight. Lysates were then centrifuged at 20,000 x g for 10 minutes at 4°C to pellet cell membrane debris, and the supernatant was collected. Whole protein quantification was done using the colorimetric DC™ Protein Assay Kit .Confluent SCBN monolayers were washed with DPBS then serum starved for 60 minutes using SCBN media without FBS. When used, inhibitors were added during the serum starvation period. Monolayers were then stimulated apically with 45 BAU/ml trypsin, 100 ng/ml EGF, or Krebs buffer solution for control wells for 5 minutes. DMSO was used as a vehicle control for inhibitor experiments. Monolayers were washed with ice cold DPBS and 300 μl of lysis buffer were added to each well. Lysis buffer contained 100 mM NaCl, 20 mM Tris-HCl (pH 8), 0.5% Triton-X 100, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 2 mM Na™ (Bio-Rad) and 15 μg of each protein sample were loaded into a Criterion™ 4–12% gradient, Bis-Tris polyacrylamide pre-cast gels (Bio-Rad). Following separation, proteins were transferred to 0.22 μM nitrocellulose membrane .Protein lysates were combined with DPBS and 5X loading buffer  and were boiled for 5 minutes. Precision Plus Protein Kaleidoscope™ Western Enhanced Chemiluminescent Substrate (Bio-Rad). Blots were visualized using the Kodak Image Station 4000 mm Pro and Kodak MI SE Software  or Bio-Rad ChemiDoc. Densitometry for visualized bands was performed using QuantityOne® 1D analysis software (Bio-Rad).Membranes were washed three times for 5 minutes each in 0.1% Tween and Tris-buffered saline containing 50 mM Tris-HCl, 137 mM NaCl, and 2.7 mM KCl (TTBS). Blots were blocked in 5% non-fat dry milk or bovine serum albumin  in TTBS for 60 minutes at room temperature. Blots were incubated with primary antibody at 4°C with shaking overnight. Blots were washed and appropriate secondary antibody  in 5% milk at 1:5000 dilution was added for 60 minutes shaking at room temperature. Blots were washed and incubated in Clarity2+-phosphate binding tag, which separates proteins based on their phosphorylation state. The method has previously been optimized for MLC [3VO4, and 12.5 mM NaF. Positive controls include SCBN cells treated for 30 minutes apically with 3 μM of the phosphatase inhibitor Calyculin A (Santa Cruz Biotechnology sc-2400). Protein concentration was determined and samples made as previously described.To determine the phosphorylation of myosin regulatory light chain (MLC), Phos-tag gel SDS-PAGE was utilized. This method uses a polyacrylamide-bound Mn for MLC . SCBN ceN,N’,methylenebisacrylamide (30:1 acrylamide-bisacrylamide), 0.1% SDS, 125 mM Tris HCl pH 6.8, 0.06% ammonium persulfate, and 0.001% TEMED . The resolving gel was composed of 11.8% (wt/vol) acrylamide, 0.34% (wt/vol) N,N’,methylenebisacrylamide (30:1 acrylamide-bisacrylamide), 40 μM Phos-tag acrylamide , 80 μM MnCl2, 0.1% SDS, 375 mM Tris HCl pH 8.8, 0.07% ammonium persulfate, and 0.0007% TEMED. Samples were made to 1 μg/μL and 35 μg/lane were run on 12-well gels or 15 μg/lane on 26-well gels.Gels were made in Bio-Rad Criterion Empty Cassettes (1 mm thick). The stacking gel was composed of 4.5% (wt/vol) acrylamide, 0.12% Gels were run in buffer containing 0.1% SDS, 25 mM Tris, and 192 mM glycine at 20 mA/gel with the BLUeye Prestained Protein Ladder . Gels were removed and incubated in transfer buffer with 4 mM EDTA for 20 minutes to chelate the Phos-tag. Gels were transferred to PVDF membrane overnight at 28 mV at 4°C. The membrane was fixed in 0.5% gluteraldehyde in PBS for 20 minutes at room temperature. Membranes were washed and blocked for 1 hour in 5% milk, and incubated in primary antibody for two hours at room temperature. Blots were washed and incubated in secondary antibody for 1 hour at room temperature, and bands visualized as described previously.Data analysis was performed using GraphPad Prism 6 software . All analyses represent at least three independent experiments and are expressed as mean ± standard error of the mean (SEM). Data from Ussing chamber experiments were analyzed using one-way ANOVA without repeated measures when comparing more than two groups; Dunnett post-tests were used when comparing all treatments to control value only, and Tukey’s post-tests were used when comparing treatments to control and other treatment values. One-sample t-tests were used when comparing a treatment value to a hypothetical control value (100%), student t-tests were used to compare two groups. For the western blotting timecourse experiment, ratios of phosphorylated protein to total protein densitometry values were calculated and analyzed using one-way ANOVA and Dunnett post-test. For western blotting inhibitor studies, ratios of phosphorylated protein to total protein densitometry values were calculated then normalized to total fluorescence of blot to account for variation between experiments. A p value of less than 0.05 (p<0.05) was considered to be a significant difference.In order to examine whether proteolytic activity was required to induce the serine protease-induced increase in TER, serine proteases were inhibited by SBTI or aprotinin during either the initiation or sustained phase of the response. Pretreatment of cells in Ussing chambers with 60 μg/mL SBTI prevented the increase in TER induced by trypsin, as demonstrated previously  Fig 1A)Fig 1A). Similar to trypsin, apical addition of EGF induces an increase in TER in SCBN cells, although the change in TER is less robust. We initially validated the expression of EGFR, ErbB2, and ErbB3 mRNA in SCBN cells by RT-PCR . ErbB4 wTo determine whether trypsin induces a change in TER through EGFR activation, the EGFR tyrosine kinase inhibitor PD153035 was used. First, the lowest concentration of PD153035 that could block the EGF-induced increase in TER was determined . The apiWe hypothesized that serine proteases may be cleaving growth factors from the cell surface that could then act on the cell to induce an increase in TER. Activation of EGFR can occur through extracellular cleavage of a membrane-bound precursor ligand to release a soluble ligand that can then bind to and activate its cognate receptor . MMPs haApical treatment with GM6001, an inhibitor of MMP-1, -2, -3, -8, -and -9, or marimastat, an inhibitor of MMP-1, -2, -3, -7, -8, and -14, for 30 minutes had no effect on the trypsin-mediated increase in TER at all doses investigated . A disinSrc kinase has been shown to play a role in the regulation of barrier function ,14. SincEGFR is known to activate PI3-K, and therefore the involvement of EGFR in the protease-mediated increased TER was investigated. PI3-K activates Akt, a serine/threonine kinase known to regulate barrier function through modulation of tight junctional proteins, as well as the modulation EGFR signaling –35. To dIn Ussing chambers, pretreatment with the PI3-K LY294002 at 20 μM decreased both the peak and sustained TER responses to trypsin . HoweverERK1/2 is downstream of EGFR and modulates barrier function through phosphorylation of tight junctional proteins . Since tIn Ussing chambers, SCBN cells were treated with 0.1, 0.5, 1, 5, 10, or 20 μM U0126 for 3 minutes then stimulated with 45 BAU/mL trypsin. 1, 10, and 20 μM U0126 significantly decreased the peak TER . AnotherPrevious work has determined that CK2 can modulate barrier function in epithelial cells through effects on phosphorylation of tight junction proteins ,36. SCBNSince EGFR inhibition was shown to only partially reduce the change in TER induced by serine protease, it suggests other cell surface targets are involved. The bradykinin B(2) receptor can be directly activated by tissue and plasma kallikreins, cathepsins, and trypsin . BradykiIt was previously determined that the serine proteases trypsin and matriptase cannot reverse disruption of the tight junction induced by the inflammatory cytokines IFNγ and TNFα . The inaTo determine MLC phosphorylation, two methods were employed. The first was the measurement of phosphorylated MLC using specific antibodies to the Thr18/Ser19 phosphorylation site of MLC. SCBN cells were treated for 48 hours with 2.5 ng/mL IFNγ and 2.5 ng/mL TNFα, as this dose was previously found to induce barrier disruption without apoptosis ,38. ThisTo verify these results, MLC phosphorylation was also investigated using Phos-tag western blotting. Using this technique, MLC protein resolves into 3 bands, representing MLC that is phosphorylated at 0, 1, or 2 residues. Some higher phosphorylated bands were also observed. SCBN cells were treated for 15 minutes apically with trypsin (45 BAU/mL) or matriptase (0.5 BAU/mL) and phosphorylation pattern assessed . No chanRecent literature suggests an important role for the endogenous epithelial serine proteases matriptase and prostasin in establishing and maintaining the intestinal epithelial barrier ,24,39,40We found that the trypsin- and matriptase-induced increases in barrier function were dependent on catalytic activity of the protease. Pretreating cells with SBTI prevented the increase in TER induced by trypsin. Interestingly, inhibition of trypsin or matriptase during the sustained phase of the response induced an immediate and rapid decrease of TER. This suggests that ongoing cleavage of a receptor or ligand from the surface is needed to induce and maintain the effect. There is no significant delay in the decrease, which suggests any signal transduction pathways induced by serine proteases are rapidly switched on or off.The apical addition of EGF significantly increased TER in SCBN monolayers compared to non-treated controls. The fact that EGF exhibited similar effects on TER as trypsin suggested that serine protease-mediated barrier enhancement could be occurring through EGFR signaling. EGF-mediated epithelial barrier enhancement has been documented in other cell lines, namely MDCK ,42, and We investigated the effect of EGFR tyrosine kinase inhibition on the protease-mediated increase in TER using PD153035. Inhibiting EGFR prior to trypsin stimulation altered the overall response; although TER increased with the same slope and in the same time frame after trypsin treatment, the maximal increase was blunted and the sustained phase was significantly decreased in inhibitor-treated cells, suggesting some requirement for EGFR activation in the persistence of the protease-mediated TER response. The addition of the EGFR inhibitor PD153035 10 minutes after trypsin stimulation, when the initiation phase was tapering to a plateau, significantly decreased the sustained phase of the response. Unlike the immediate decrease in TER seen following SBTI treatment, PD153035 treatment did not elicit a decrease until approximately 20 minutes after its addition. This is likely due to the requirement for PD153035 to get through the cell membrane and into the intracellular receptor domain prior to having an effect . InitiatAlthough matriptase has been shown to directly cleave the extracellular domain of EGFR , the EGFIntracellular, Src-mediated EGFR transactivation was hypothesized to be one potential mechanism by which trypsin increases TER in SCBNs. We showed that pre-treatment with the Src inhibitor, PP1, decreased trypsin-induced increased TER in a concentration-dependent fashion. The addition of PP1 10 minutes after trypsin stimulation did not decrease trypsin-induced sustained TER. Thus Src is involved in the initiation, but not sustained phase of the serine protease induced increase in TER. Src phosphorylation was not changed by serine protease treatment, but the levels of endogenous phosphorylation were high, as we have observed for several signaling proteins in unstimulated SCBN cells (unpublished observations). It may be that subtle changes in phosphorylation status following addition of pharmacological inhibitors are sufficient to elicit and change in TER, but which may not be detected by western blotting.EGFR, ErbB3, and ErbB4 contain PI3-K-specific tyrosine residues, and ErbB signaling is known to activate PI3-K . PI3-K aERK1/2 signaling has been shown to modulate barrier function. The previously observed ouabain-induced increase in TER occurs through EGFR and ERK1/2 signaling in MDCK cells . IFNγ me2. Pretreatment with this CK2 inhibitor significantly reduced the trypsin-induced increase in TER. In the study by Raleigh et al, CK2 inhibition by TBCA in Caco-2 cells induced an increase in TER. However, we observed a decrease in TER in SCBN cells treated with TBCA. One reason for this is that SCBN cells do not express claudin-2 [CK2 is a kinase previously shown to regulate binding of occludin with ZO-1 and claudin family members through phosphorylation of occludin and to affect barrier function ,36. We ilaudin-2 , which iBecause cytokines have been shown to induce both the internalization of tight junctional proteins and a phosphorylation of myosin light chain to increase permeability, we examined whether trypsin and matriptase impact the dephosphorylation of MLC. Dephosphorylation through myosin light chain phosphatase has been demonstrated in smooth muscle tissues to induce relaxation . While eEGFR inhibition did not fully block the response to trypsin, suggesting some other cell surface receptor is the initial protease target that mediates initiation of increased TER. One potential target was the bradykinin B(2) receptor, which has been shown to modulate tight junction permeability through an ADAM17-EGFR transactivation mechanism that results in ZO-1 rearrangement . KallikrTaken together, our results show that the serine protease-induced increase in TER can be divided into two phases, the initiation of the change in TER and the sustained phase where the TER remains elevated. The initiation phase is dependent on ERK1/2, Src, PI3-K, CK2, and the sustained phase is dependent on EGFR . We also


Shigella flexneri, a gram-negative bacterium responsible of bacillary dysentery, uses multiple strategies to overcome host immune defense. We have decrypted how this bacterium manipulates host-cell chromatin binders to take control of immune gene expression. We found that OspF, an injected virulence factor previously identified as a repressor of immune gene expression, targets the chromatin reader HP1γ. Heterochromatin Protein 1 family members specifically recognize and bind histone H3 methylated at Lys 9. Although initially identified as chromatin-associated transcriptional silencers in heterochromatin, their location in euchromatin indicates an active role in gene expression. Notably, HP1γ phosphorylation at Serine 83 defines a subpopulation exclusively located to euchromatin, targeted to the site of transcriptional elongation. We showed that OspF directly interacts with HP1γ, and causes HP1 dephosphorylation, suggesting a model in which this virulence effector “uses” HP1 proteins as beacons to target and repress immune gene expression ). OspF alters HP1γ phosphorylation mainly by inactivating the Erk-activated kinase MSK1, spotlighting it as a new HP1 kinase. In vivo, infectious stresses trigger HP1γ phosphorylation in the colon, principally in the lamina propria and the intestinal crypts. Several lines of evidence suggest that HP1 proteins are modified as extensively as histones, and decrypting the impact of these HP1 post-translational modifications on their transcriptional activities in vivo will be the next challenges to be taken up. Shigella bacterial species, a causal agent of bacillary dysentery in humans, delivers through the type III secretion system (T3SS) the virulence effector OspF to directly inactivate MAPK signaling in the nucleus of infected epithelial cells. OspF inactivates MAPKs through a phosphothreonine lyase activity. This enzymatic activity irreversibly inactivates the dual-phosphorylated host MAPKs (pT-X-pY) through beta elimination of the phosphate group, converting the phosphothreonine residue required for MAPK activity into a dehydrobutyrine (Dhb). We previously showed that OspF binds host chromatin in a gene-specific manner and, by intercepting with nuclear MAPK signaling, installs repressive chromatin marks on a subset of proinflammatory chemokines, rendering chromosomal sites inaccessible to both NF-κB and the RNA polymerase II (RNAPII). Therefore, OspF acts as an epigenetic regulator reprogramming the host transcriptional response by irreversibly inactivating nuclear MAPKs, which results in an immunosuppressive environmentfavoring life of the bacterium at the mucosal surface.Shigella T3SS, modulates the HP1γ phosphorylation state in infected cells by targeting the nuclear MAPK signaling pathway. More precise decrypting of the pathway showed that MSK1, a kinase downstream of Erk, is a HP1 kinase and that OspF injection inactivates the Erk-MSK1 signaling cascade leading to HP1γ phosphorylation .As a chromatin-binding protein-kinase inhibitor, OspF provides an interesting tool for decrypting the interface between MAPK and chromatin. We showed that OspF directly interacts with the chromatin reader HP1γ and, once injected by the OspF reveals a new interplay between nuclear MAPK signaling and chromatin. Various evidences suggest that kinases interact with chromatin as integral component of transcription activation complexes at promoters and with elongating RNAPII at transcribed regions. Importantly, we identified a tight molecular complex between MSK1 and HP1γ in living cells, an interaction that possibly stabilizes MSK1 on the chromatin template, and facilitates HP1γ phosphorylation .HP1 are extensively modified proteins. For example, SUMO modifications promote HP1 targeting to pericentric heterochromatin while phosphorylation at Ser 83 defines a subpopulation exclusively located to euchromatin, interacting with the elongating form of the RNAPII. At these intragenic positions, HP1γ may stabilize ongoing transcription and/or participate in both alternative splicing and co-transcriptional RNA processing, presumably by favoring the recruitment of the splicing machinery. The role of phospho-modifications on these HP1γ transcriptional activities remains poorly characterized; one report indicates that Ser 83 phosphorylation abrogates HP1γ silencing activity in a transcriptional reporter assay. Since OspF precisely targeted this Ser 83 residue, we questioned the role of HP1γ and of this phospho-event in gene expression. Using HP1γ null cell lines rescued with either WT HP1γ or with a S83A mutant, we found that upon MAPK activation, WT HP1γ was required for the activation of pathways involved in cell cycling and proliferation, purine metabolism, DNA replication and repair. These data spotlight HP1γ as a chromatin integrator for the MAPK-driven proliferative gene expression program. Conversely, at immune genes, WT HP1γ was required for fine tuning of the transcriptional activity, reducing the amplitude of the response to cell stimulation at a selective set of OspF target genes, such as CCL2, CCL20, and others. Unexpectedly, a single S83A mutation interfering with Ser 83 phosphorylation reduced but did not abolish the impact of HP1γ on transcription, suggesting the implication of additional phospho-modifications. Overall, these results suggested that HP1γ signaling promotes a transcriptional program aimed at cell proliferation and repair while dampening inflammation.in vivo model of rectocolitis, we showed that the non-invasive - albeit proinflammatory - mxiD Shigella mutant promoted pronounced HP1γ phosphorylation in the colon when compared to wild type Shigella. PAMPs (pathogen-associated molecular patterns) such as LPS were shown to induce major dynamic changes in the cell phosphorylation state, with multiple phosphorylation sites on HP1γ, including Ser 83. Therefore, it remains possible that bacterial challenge directly -through LPS release - or indirectly initiates proinflammatory signaling cascade(s) leading to increased HP1γ phosphorylation at multiple residues, including the Ser 83 residue monitored in our study. In turn, triggering of HP1γ signaling may be an important "checkpoint" in the control of inflammatory gene activation, aimed at preventing excessive or deregulated immune gene expression . Thus, the development of in vivo approaches targeting HP1γ in the intestine will be essential to unravel these important physiological issues.The physiological function of HP1γ remains poorly investigated since the HP1γ null mutation induces a high rate of embryonic lethality. Furthermore, only 1% of animals reach adulthood with severe hypogonadism characterized by a meiotic defect in spermatocytes and oocytes, suggesting a role in facilitating centromere clustering in early meiotic prophase. Triggering of HP1γ activation by infectious stresses remained totally unexplored. In an


D) in the low nanomolar range determined using surface plasmon resonance. FELIAP is the first FXIa-inhibitory aptamer to be described and constitutes a lead compound to develop related aptamers for in vivo use.Factor XIa (FXIa) is a serine protease that catalyzes the activation of Factor IX (FIX) in the blood coagulation cascade. FXIa and its precursor FXI are emergent therapeutic targets for the development of safer anticoagulant agents. Here, we sought a novel DNA-based agent to inhibit FXIa. Towards this goal, an 80 base, single-stranded DNA aptamer library (containing a 40 base randomized core) was screened for FXIa-binding candidates, using ten rounds of positive and negative selection. After selection, 6 of 89 different sequences inhibited FXIa-mediated chromogenic substrate S2366 cleavage. The most active anti-FXIa aptamer had a hypervariable central sequence 5′-AACCTATCGGACTATTGTTAGTGATTTTTATAGTGT-3′ and was designated Factor ELeven Inhibitory APtamer (FELIAP). FELIAP, but not a scrambled aptamer control (SCRAPT), competitively inhibited FXIa-catalyzed S2366 cleavage, FIX activation, and complex formation with antithrombin. No effect of FELIAP on FXI activation was observed. FELIAP inhibited plasma clotting and thrombin generation assays to a significantly greater extent than SCRAPT. Immobilized FELIAP bound FXIa with strong affinity and an equilibrium binding constant (K Thrombosis is responsible for one in four deaths worldwide4. Therefore, there is a need for effective and safe anticoagulants to prevent and treat thrombotic disorders.The coagulation system can function in a protective or pathological manner. Haemostatic blood clots prevent excessive blood loss at sites of vascular injury5, whereas dabigatran inhibits thrombin and rivaroxaban, apixaban and edoxaban inhibit activated factor X (FXa)6. The direct oral anticoagulants are at least as effective as warfarin, but produce less bleeding, particularly less intracranial bleeding6. Nonetheless, serious bleeding can occur even with the direct oral anticoagulants7. Therefore, the search for safer anticoagulants continues.Currently available anticoagulants include vitamin K antagonists, such as warfarin, and direct oral anticoagulants; dabigatran, rivaroxaban, apixaban and edoxaban. Warfarin attenuates clotting by reducing the hepatic synthesis of multiple coagulation factors9. FXI is a 160 kDa homodimer comprising two identical disulphide-linked polypeptide chains; specific proteolysis of the Arg369-Ile370 bond, mediated either by FXIIa or thrombin, converts FXI from an inactive precursor to enzymatically active FXIa10. FXIa catalyzes the conversion of FIX to FIXa10, which leads to FXa and thrombin generation. Basic and epidemiological studies indicate that FXI is important in thrombosis16. In contrast, FXI has little role in hemostasis because patients with congenital FXI deficiency rarely have spontaneous bleeding and only bleed with surgery or trauma17. Consequently, inhibition of FXI has the potential to attenuate thrombosis without impairing hemostasis. In support of this concept, knockdown of FXI in patients undergoing elective knee replacement was more effective than enoxaparin, the current standard of care, at preventing postoperative venous thromboembolism and did not increase the risk of bleeding18. Therefore, there is a push for development of FXI inhibitors.FXI has emerged as a promising target for safer anticoagulantsin vitro selection procedure called systematic evolution of ligands by exponential enrichment (SELEX)19. SELEX selects for ssDNA or ssRNA molecules able to adopt stable three-dimensional structures and bind molecular targets from a pool of ~1014 unique strands20. Although aptamers against numerous coagulation factors have been developed, to our knowledge none have targeted FXIa27. Here, we describe the selection and characterization of a DNA aptamer that binds the active site of FXIa and inhibits its enzymatic action on both artificial and natural substrates.DNA and RNA ligands, or aptamers, are short single-stranded oligonucleotides (ssDNA or ssRNA) that can be isolated from complex combinatorial libraries of nucleic acids using an iterative 40 different DNA molecules. As shown in Fig. in vitro aptamer selection protocol was employed. Initially, we employed only positive selection to enrich for aptamers binding to FXIa. After 4 and 10 rounds of selection, we noted no inhibition of FXIa-mediated amidolysis when the selected aptamer pool was introduced into the reaction (data not shown). Accordingly, we modified the selection protocol by the addition of alternating positive and negative selection steps and rescreened the initial library. The modified protocol included negative selection of aptamers binding to any component of the FXIa-antibody-bead assemblies except the FXIa active site, by introducing the FXIa active site-binding, small protein inhibitor KPI28, after Round 4. In contrast to our initial results, after Round 10, a small but reproducible reduction in amidolysis was observed in the presence of the selected aptamer pool.Our objective was to select FXIa-inhibiting aptamers from a large library of ssDNA molecules 80 nucleotides in length containing an internal randomized 40 nucleotide region flanked by primer binding sites. Such a library theoretically contains 4Sequencing of the pool after Round 4 indicated that the number of unique sequences had been reduced to 289; Table 29 showed that the portion of FELIAP corresponding to the variable part of the aptamer library likely adopted an extended stem-loop structure containing a bulge separated by two small loops  were closely related, differing in at most two of 36 positions; the fifth (Apt10_E) was only distantly related by virtue of T-rich areas . The aligned sequences are shown in Fig. ops Fig. . Three oi for FELIAP of 29 µM by non-linear regression.The mode of inhibition of FELIAP was investigated by fixing the concentrations of chromogenic substrate and FXIa and increasing the concentration of FELIAP and measuring the rate of substrate cleavage. A dose-dependent reduction in the reaction rate was observed as FELIAP was increased from 0 to 20 µM, while the same concentrations of SCRAPT had no inhibitory effect Fig. . Next, tPrior to examining the inhibitory effects of FELIAP on FXIa-mediated interactions with specific macromolecular substrates, we examined the capacity of FELIAP to inhibit FXIa-induced clotting in plasma. FXIa was pre-incubated with buffer, FELIAP, or SCRAPT, and combined with FXI-deficient plasma and APTT reagent containing kaolin and cephalin, and recalcified. As shown in Fig. 145-Ala146, and the other at Arg180-Val181 of the FIX polypeptide30. This reaction liberates a glycosylated activation peptide , and a disulphide-linked γ-carboxylated light chain  and heavy chain 32. An intermediate product comprised of the activation peptide linked to the heavy chain  may also be detected31. Figure FXIa activates FIX by cleaving two peptide bonds, one at Arg370-Val607) is joined to antithrombin residues His1-Arg393 via an acyl linkage33. Figure FXIa also reacts with its natural inhibitor, antithrombin, in a reaction that is accelerated by heparin, to form a denaturation-resistant complex between the FXIa active site and the reactive centre of antithrombin, in which the light chain of FXIa (Ile34. No inhibition of this reaction was noted in the presence of either excess FELIAP (lane 4) or SCRAPT (lane 5), but activation of FXI was abrogated in the presence of excess hirudin, a potent thrombin inhibitor (lane 6). FELIAP therefore inhibited FXIa-dependent but not FXI-dependent reactions.Having demonstrated that FELIAP inhibited the action of FXIa on two macromolecular substrates, FIX and antithrombin, we next asked if it had any effect on the activation of FXI by thrombin. In the presence of cofactor dextran sulphate Fig. , lane 3,35. As shown in Fig. Recalcified dilute normal human pooled plasma, in which the contact pathway of coagulation was activated by micronized silica, was employed to assess the effects of FELIAP on thrombin generation. The thrombin generation assay (TGA) provides information on the timing and kinetics of thrombin generation in plasma using a thrombin-specific fluorogenic substrate and calibrators unaffected by fibrin clot formation, using standardized analytic methods and parametersThe partial anticoagulant activity observed for SCRAPT in silica-activated TGA using normal pooled plasma was also noted for other single-stranded oligonucleotides of similar length  at equimolar concentrations. To ascertain whether or not this phenomenon was related to FXIa inhibition, we repeated TGA, substituting FXI-depleted plasma for normal plasma and tissue factor for silicates as activators Fig. . SCRAPT d) and the association rate constant (Ka), permitting calculation of the equilibrium binding constant KD, which is Kd divided by Ka. Ka values of 5.2 ± 0.1 × 104 M−1 S−1, Kd values of 9.5 ± 0.1 × 10−5 S−1, and KD values of 1.8 ± 0.1 × 10−9 M were obtained (mean ± SD of three determinations).FELIAP and SCRAPT were biotinylated at their 3′ ends and immobilized on a streptavidin-coated gold chip. Immobilized SCRAPT served as the reference cell. When increasing concentrations of FXIa from 0 to 500 nM were flowed over these surfaces, increasing response unit binding isotherms were generated when the difference between immobilized FELIAP and immobilized SCRAPT binding was plotted Fig. . It shouTo determine the minimum active sequence of FELIAP required for inhibition of FXIa, several truncated forms of FELIAP were synthesized, using the predicted structure of the aptamer to delete predicted loops and stems progressively. The truncated sequences ranged from 32 to 64 nucleotides long (compared to 74 for full-length FELIAP), were designated FELIAP_X , and constituted 5′ and 3′ deletion mutants of FELIAP see Fig. . As showp-nitroanilide) which must, by virtue of its small size, enter the interior of the FXIa active site pocket to be cleaved and liberate the coloured product nitroanilide. Secondly, it inhibited two different FXIa-dependent reactions: FXIa-mediated activation of FIX; and FXIa-mediated formation of FXIa-antithrombin complexes. While this observation does not per se exclude an allosteric effect of FELIAP on FXIa, it renders it less likely than active site binding. Thirdly, FELIAP had no effect on FXI activation, further indicating specificity for FXIa, one of whose cardinal features is the active site. Taken together, these data suggest that FELIAP binds specifically to FXIa at or near its active site, with high affinity consistent with the observed nanomolar KD.Aptamers from combinatorial libraries can be selected to bind virtually any protein of interest. Here, we describe the selection and characterization of FELIAP, a FXIa-binding DNA aptamer. Our protocol favoured the selection of aptamers binding at or near the active site of FXIa over those binding to other portions of the enzyme. Several lines of indirect evidence support the conclusion that this strategy was successful. Firstly, FELIAP acted as a competitive inhibitor of chromogenic substrate S2366, which is a tripeptide nitroanilide compound  secreted from α-granules on platelet activation. The 57 amino acid KPI domain accounts for all of the FXIa-inhibitory activity of APPWhile to our knowledge no aptamers to FXI or FXIa have been previously described in the biomedical literature, several aptamers to other coagulation factor targets have been isolated.D values of 10–13 nM, inhibiting the enzyme by disrupting tissue factor-FVII(a) complex assembly22. A truncated form of a selected RNA aptamer designated 9.3t was found to bind FIXa with a KD of 0.58 nM38, specifically at an extended substrate binding position, or exosite39. RNA11F7t aptamer bound FXa with a reported KD of 1.1 nM and inhibited prothrombinase activity by interfering with the interaction between FXa and FVa21, while R4CxII-It bound FXII or FXIIa with KD values of 8.9 and 0.4 nM, respectively, by interfering with FXII and anionic binding24. RNAR9D-14T bound thrombin or prothrombin with KD values of 1 or 10 nM via anion binding exosite I23. With respect to DNA aptamers, the only ones described to date that target coagulation factors are HD1 and HD22, which bind prothrombin with KD values of 7.1 and 2.4 nM, via exosites I and II, respectively41. All previously described aptamers targeting coagulation factors, therefore, act at sites distinct from the active site of these serine proteases. Our discovery of FELIAP, which acts at or near the active site of FXIa, may have been facilitated by the use of KPI in negative selection, as screening protocols employed in previously published studies used only positive selection. Although the time- and labour-intensive nature of aptamer library screening precluded a systematic analysis, anecdotally we did observe FELIAP selection within ten rounds of screening with positive and negative steps, but not until twenty rounds of screening with positive steps alone.Among RNA aptamers, RNA 16.3 bound FVII or FVIIa with K20. We observed many examples of such artifacts using high throughput sequencing after Round 4, some of which persisted to Round 10, as well as the selection of aptamers with barely detectable affinity for FXIa (e.g. Apt10_E). Our results are therefore consistent with the known limitations of SELEX.Although SELEX and SELEX-related aptamer screening protocols have been employed to isolate aptamers that bind to their targets with high affinity, numerous limitations need to be overcome for successful application of this biotechnological approach. These include interference from non-degenerate sequences flanking the variable regions in the aptamer library; non-specific retention of sequences not binding the desired target; and accumulation of amplification artifacts and artifactual sequences arising from library regenerationD of 1.8 nM), in the general nanomolar range of previously reported aptamers against other coagulation factors, its potency as an inhibitor of FXIa-mediated reactions was less impressive. Its Ki for inhibition of FXIa-mediated amidolysis of S2366 was 29 µM, and µM concentrations of FELIAP were required to impair nM concentrations of FXIa with respect to its reactions with FIX or antithrombin or to elicit substantial reduction of thrombin generation in plasma systems. This pattern is consistent with FELIAP binding with high affinity to a location close enough to the active site to yield a competitive inhibition, but in a manner that does not fully block access to the active site for either small molecule or macromolecular substrates. As noted above, an alternative hypothesis would involve high affinity binding to an allosteric site that elicited a partially inhibitory change in the reactive site. FELIAP may therefore be regarded as a lead compound for the development of more potent FXIa-inhibiting aptamers, in the same way as some monosulphated benzofurans have been reported to be lead compounds for the design of small molecule FXIa inhibitors42.While FELIAP was found to bind FXIa with high affinity . The forward (A) and reverse primers (B) were 5′-GAATTCTAAT ACGACTCACT ATA-3′ and 5′-GCGTCCAACAC ATCG-3′ respectively. These and all other oligonucleotides employed in this study were purchased from Integrated DNA Technologies . FXI, FXIa, FIX and FXIIa were bought from Enzyme Research Laboratories . Biotinylated goat anti-human Factor XI (FXI) antibody was purchased from Affinity Biologicals . Dynabeads Biotin Binder was bought from Thermo Fisher Scientific . For thrombin generation assays (TGA), TGA substrate and TGA calibrator sets were obtained from Technothrombin GmbH . Activated Partial Thromboplastin Time (APTT) reagent was from Diagnostica Stago . Chromogenic substrate S2366 was purchased from Instrumentation Laboratory .The aptamer library comprised a ssDNA template with sequence 5′-GAATTCTAAT ACGACTCACT ATA-N19 with some modifications, as portrayed schematically in Figure 40-GCGTCCAACA CATCG-3′, N40 represents a 40 nucleotide randomized region. Before selection, streptavidin-coated magnetic beads (5 μL packed volume) were prewashed five times in Aptamer Folding Buffer . All reactions took place at room temperature; washes were performed using a magnet to concentrate the beads at the bottom of the reaction tube. Next, biotinylated goat anti-human factor XI antibody  was added at quadruple the FXIa concentration to be employed, and incubated for 45 minutes prior to washing. FXIa (60 nM) was then added to the antibody-coated beads and incubated for 1 hour. Five washes were carried out prior to the addition of 1000 picomoles of aptamer library which had been diluted to 4 nM in AFB and heated to 90 °C for 5 minutes before being cooled on ice. The folded library was then added to the immobilized FXIa and incubated for 1 hour with end-over-end rotation on a Barnstead Thermolyne Labquake. Unbound aptamers were removed by five washes in AFB. Bound aptamers were extracted from protein/antibody/bead assemblies using phenol: chloroform: isoamyl alcohol  and precipitated with 2/3 vol/vol absolute ethanol to conclude the selection round. The pool of selected aptamers was then PCR-amplified using a high-fidelity heat-stable DNA polymerase  in an asymmetric PCR protocol in which 13-fold more primer A than primer B was employed; the ssDNA sense strand was then purified by preparative agarose gel electrophoresis using a 2% (w/vol) agarose gel and an Ultrafree-DA centrifugal filter unit  to produce the amplified aptamer library and conclude the first selection round (Round 1). After Round 3, selection stringency was increased by switching from AFB to stringent wash buffer  and gradually reducing the incubation time of the library with FXIa such that it was only 15 min in Round 10.Solution-based SELEX was performed as previously describedPichia pastoris yeast and purified exactly as described28, at a concentration of 6.8 µM, to block the FXIa active site. Washing and incubations were as described above for positive selection, except that aptamers from the amplified library not binding to the KPI-FXIa-antibody-bead assemblies were magnetically separated and combined with fresh FXIa, anti-FXI antibodies and magnetic beads to start the next round of selection. Rounds six through ten of SELEX thus combined positively selecting aptamer candidates with unblocked FXIa and negatively selecting against aptamer candidates binding to KPI-blocked FXIa or anti-FXI antibodies or magnetic beads.After five rounds of positive selection, the resulting amplified library was combined with beads, biotinylated anti-FXIa, and FXIa as described above, and negatively selected using recombinant His-tagged Kunitz Protease Inhibitor domain of human protease nexin 2  expressed in 44.High-throughput  sequencing was employed to characterize the selected aptamer populations following four and ten rounds of SELEX. Single-stranded aptamers generated by asymmetric PCR were PCR-amplified using forward primer 5′-AATGATACGGC GACCACCGAG ATCTACACTA GATCGCACAC TCTTTCCCTA CACGACGCTC TTCCGATCTN NNNGAATTCT AATACGACTC ACTATA-3′ and reverse primer 5′-CAAGCAGAAG ACGGCATACG AGATTCGCCT TAGTGACTGG AGTTCAGACG TGTGCTCTTC CGATCTCGAT GTGTTGGACA AGCAGAAGAC GGCATACGAG ATTCGCCTTA GTGACTGGAG TTCAGACGTG TGCTCTTCCG ATCTCGATGT GTTGGACGCC GC-3′. The resulting amplicons were sequenced using an Illumina Miseq DNA sequencer at the Farncombe Metagenomics Facility, McMaster University. The raw sequencing data was processed using Illumina’s Basespace online NGS platform for tagged sequence pool sorting, and to ensure sequence data output was converted to FASTQ format. Further data processing was as describedThe full length FELIAP aptamer sequence was determined to be 5′-GAATTCTAAT ACGACTCACT ATAAACCTAT CGGACTATTG TTAGTGATTT TTATAGTGTG CGTCCAACAC ATCG-3′. A control aptamer of the same length but scrambled DNA sequence (SCRAPT) was synthesized for comparative purposes, with sequence 5′-TTCTAATACG ACTCACTATA AGGGAGGGCA GTGGGATGGC GTTAGTGAGG GAGGGTGTGG GGCGTCCAAC ACAT-3′. To generate truncated versions of FELIAP, nucleotides from both the 3′ and 5′ ends were sequentially removed as depicted schematically in Fig. Assays were performed in a 96-well flat bottom microtiter plate  at 37 °C in reaction buffer PPNE. Reactions (200 μL) contained 1 nM FXIa, 90 μM chromogenic substrate S2336 and aptamer concentrations ranging from 0.78 to 10 μM. The rate of substrate hydrolysis was recorded at 405 nm on an ELx808 Absorbance Microplate Reader .2 at 37 °C. Samples were incubated for 30 min. Following the 30 min incubation, the reactions were quenched using Sodium Dodecyl Sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer containing dithiothreitol and electrophoresed on a 12% SDS-polyacrylamide gel. Protein bands were visualized by staining with Coomassie Brilliant Blue.FIX (6.2 μM) was incubated with 2 nM FXIa in the presence of 10 μM FELIAP, 10 μM scrambled DNA or 2.6 μM KPI in TBS supplemented with 5 mM CaClFXIa (200 nM) was pre-incubated with 10 μM FELIAP or SCRAPT at 37 °C for 5 minutes, and then combined with 2 μM purified human antithrombin in the presence of 2 U/mL sodium heparin (Sigma-Aldrich) for a further 1 or 5 minutes. At the end of the antithrombin incubation time, samples were quenched and subjected to electrophoresis as described above.45. At the conclusion of the reaction samples were quenched and subjected to electrophoresis as described above.FXI (700 nM) in PPNE buffer was reacted with 70 nM thrombin in the presence or absence of 1 mg/L dextran sulphate (molecular weight 500 kDa) at 37 °C for 30 minutes in the presence or absence of 10 µM FELIAP or SCRAPT or 1.5 µM recombinant His-tagged hirudin variant 3 (HV3)Pichia pastoris45 served as a positive control for TGA inhibition. In the third protocol, 1 µM FELIAP, SCRAPT, or KPI were combined with 0.25 nM FXIa (10 μL) and pre-incubated for 5 min at room temperature prior to combination with FXI-DP and activation with APTT reagent as in the first protocol. In all protocols, thrombin generation was then followed at 37 °C for 60 min at 1 min intervals using a Fluoroskan Ascent plate reader (Thermo Scientific) set to a wavelength of 380/460 nm. Data was imported into a Microsoft Excel evaluation spreadsheet (www.technoclone.com) for analysis and derivation of TGA test parameters.Thrombin generation assays (TGA) were performed using either human normal pooled plasma (NPP) or FXI-depleted plasma (FXI-DP),  with or without supplementation with FXIa. Three variations of TGA were performed, using: human normal pooled plasma (NPP) activated with silicates; FXI-DP activated with tissue factor; and FXI-DP supplemented with FXIa. All TGA reactions were performed in black flat bottom 96-well microtiter plates (Greiner Bio One). In the first protocol, 40 μL of NPP  was mixed with 10 μL of 300 μM FELIAP or SCRAPT) or PBS, 10 μL of APTT reagent , 25 μL of 2 mM TGA substrate solution containing calcium chloride, and 15 μL of PBS, to comprise a total reaction volume of 100 μL. In the second protocol, the APTT reagent was substituted with the prothrombin time reagent Innovin  and NPP was substituted with FXI-DP. His-tagged recombinant hirudin variant 3 purified from 2 and the clotting time was determined.APTT assays were performed using a STart 4 coagulometer (Diagnostica Stago) with some modifications. For the reactions, 5 μL of aptamer or PBS buffer control  was heat-denatured and combined with an equal volume of varying concentrations of purified human FXIa at 37 °C for 3 min. APTT reagent  was separately preincubated with 45 μL FXI-deficient human plasma under the same conditions, and then clotting was initiated by mixing both sets of components with 50 μL of 25 mM CaClN-ethyl-N′-(dimethylamino-propyl) carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) (Sigma) solution, followed by binding of streptavidin . FELIAP and SCRAPT (3′ biotinylated) in 100 mM HEPES (pH 7.4), 150 mM NaCl, 0.01% Tween 20 were immobilized on the streptavidin-coated chips at a flow rate of 10 μL/min to 100 response units (RU). Flow cells were regenerated with 0.2% SDS (w/vol). FXIa at concentrations varying from 15.625 nM to 500 nM was injected at a flow rate of 50 μl/min for 180 s to monitor association, and HEPES buffer for 1800 s to monitor dissociation. The signal from the SCRAPT-immobilized flow cell was used as a reference and subtracted from the signal arising from FXIa binding to the FELIAP-immobilized cell. Binding of FXIa and aptamer was quantified by global analysis of on and off rates using the Langmuir 1:1 binding model, as determined with the instrument’s software provided by the manufacturer (Biacore). All experiments were done in triplicate.Kinetic measurements of aptamer binding were analyzed by SPR on a Biacore T200 . To immobilize the aptamer, a CM5 sensor chip  was activated with a 0.2 M