Mycobacterium marinum or Mycobacterium tuberculosis, excess tumor necrosis factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here, we show that this necrosis pathway is not mitochondrion-intrinsic but results from an inter-organellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide that ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors, and the resultant mitochondrial calcium overload triggers cyclophilin-D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-type calcium channels as druggable targets to intercept mitochondrial calcium overload and necrosis of mycobacterium-infected zebrafish and human macrophages.Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis by releasing mycobacteria into the growth-permissive extracellular environment. In zebrafish infected with   •TNF induces mitochondrial ROS to cause necrosis of mycobacterium-infected macrophages•Mitochondrial ROS activate lysosomal enzymes that lead to BAX activation•2+ flow into the mitochondrionBAX activates ER ryanodine receptors to cause Ca•2+ overload prevent pathogenic macrophage necrosis in TBDrugs preventing mitochondrial Ca 2+ channels.During TB infection, the necrosis of infected macrophages involves an inter organelle signaling relay involving druggable targets such as ryanodine receptors and plasma membrane L-type Ca Mycobacterium tuberculosis (Mtb), the agent of human tuberculosis (TB), is fueled by its multiple interactions with host macrophages , a close genetic relative of Mtb (4)  . Humans le to TB . Among crticoids .The human relevance of the zebrafish findings provided the impetus to carry out a detailed mechanistic dissection of the necrosis pathway. In the zebrafish, we showed that susceptibility of the LTA4H-high state is due to the excessive production of the pro-inflammatory cytokine tumor necrosis factor (TNF) that, at optimal levels, is host protective . Excess 2+ overload, which is a likely trigger for cyclophilin D activation   Figure A. In thitivation . We findIn vitro, ceramide produced by acid sphingomyelinase activates lysosomal proteases, including cathepsins B and D  to create TNF-high animals see . TNF-higNext, we asked if BAX was involved. There are two zebrafish paralogs of human BAX, BAXA and BAXB, with BAXA more similar to human BAX  FiguresA and S1BBAX is thought to participate in mitochondrion-mediated apoptosis through recruitment and oligomerization on the mitochondrial outer membrane to form pores . It has Prior reports described a necrosis pathway where non-oligomerizing BAX interacts with the mitochondrial outer membrane to mediate necrosis in conjunction with cyclophilin D acting in the inner membrane . If this2+ release from the ER  TNF-mediated necrosis should be associated with mitochondrial Ca2+overload, (2) inhibiting mitochondrial Ca2+ overload should rescue necrosis, (3) mitochondrial Ca2+overload should be BAX-dependent as well as dependent on its upstream activators, (4) BAX’s action in the ER should be sufficient for mitochondrial Ca2+overload and necrosis, and (5) reducing ER Ca2+ stores should prevent necrosis.How might BAX facilitate cyclophilin D-dependent necrosis? In addition to its mitochondrial interactions, BAX is reported to regulate Cam the ER . Becausenecrosis , we wond2+overload was linked to necrosis. First, we visualized mitochondrial Ca2+ in animals expressing the genetically encoded Ca2+ indicator GCaMP3 specifically in mitochondria , the major route of Ca2+ entry into the mitochondrial matrix    . To test4 domain . Expressolvement A and 4B.olvement , did notnecrosis C and 4D.rs (RyR) . While Rrs (RyR) . To ask rs (RyR) . BCL-XL olvement A and 4E.necrosis C and 4D.necrosis   they should precede mitochondrial Ca2+ overload, (2) removing mitochondrial ROS should remove Ca2+ overload. Using two different fluorogenic probes to detect ROS specifically in the mitochondrion, MitoTracker Red CMH2Xros and MitoSOX, we found that mitochondrial ROS were induced within 40 min after TNF administration to the animals, the first time point that it was technically feasible to image them  located in the plasma membrane . We asked if nifedipine, diltiazem, and verapamil inhibit mitochondrial Ca2+ overload and macrophage necrosis in TNF-high animals and found that they did , a biosafety level 2 pathogen owing to its inability to grow in vivo . The zebrafish findings lead to four testable predictions for human macrophages: (1) TNF should increase death of infected but not uninfected macrophages, (2) TNF-induced death should occur through RIPK1-mediated necrosis , (3) it should be dependent on BID, RyR, and mitochondrial Ca2+ overload, and (4) it should be inhibited by inhibiting Ca2+ uptake by the cells. To test these predictions, we infected THP-1 macrophages with Mm or Mtb for 24 h, then added exogenous TNF to the culture medium to create a TNF-high state. 5 h of TNF treatment increased cell death in a concentration-dependent fashion in Mm-infected macrophages; this was specific to infected cells and spared uninfected cells in the same well and cells in uninfected wells   . These t channel . Specifihondrion . Recent hondrion  .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Lalita Ramakrishnan (Baxarr1 and Baxbrr10 mutant lines at the University of Washington). Zebrafish AB wild-type strain  and transgenic or mutant lines in the AB background were used, including Tg(mpeg1:YFP)w200 (with yellow fluorescent macrophages) (Tg(mpeg1:Brainbow)w201 (with red fluorescent macrophages) (Tg(BH:GFP-mfap4:Mito-GCaMP3)  (this work), baxarr1 (this work), baxbrr10 (this work) and ripk3rr7 (this work). All zebrafish lines were maintained in buffered reverse osmotic water systems and were exposed to a 14 hr light - 10 hr dark cycle to maintain proper circadian conditions. Fish were fed twice daily a combination of dry food and brine shrimp. Zebrafish embryos were housed in fish water (reverse osmosis water containing 0.18 g/l Instant Ocean) at 28.5°C. Embryos were maintained in 0.25 μg/ml methylene blue from collection to 1-day post-fertilization (dpf). 0.003% PTU  was added from 24 h post-fertilization (hpf) on to prevent pigmentation. Larvae  were anesthetized in fish water containing 0.025% tricaine (Sigma) and infected at 48 hpf via caudal vein (CV) injection using single-cell suspensions of Mm or Mtb of known titer (Zebrafish husbandry and experiments were conducted in compliance with guidelines from the UK Home Office (experiments conducted in the University of Cambridge) and with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Larvae using protocols approved by the Institutional Animal Care and Use Committee of the University of Washington (generation of ophages) , Tg(mpegophages) , Tg(BH:Gwn titer . Larvae Tg(BH:GFP-mfap4:Mito-GCaMP3): the plasmid pTol2-PhiC31LS-BH:GFP-mfap4:Mito-GCaMP3 was generated by PCR amplifying the mito-GCaMP3 cassette from the plasmid pME mitoGCaMP3    of PCR p5 macrophages per well. On day 3 post-differentiation, the resulting adherent cells were washed with fresh medium and incubated for 3-5 days before infection (protocol modified from (THP-1 cells (ATCC TIB-202) were maintained in RPMI 1640 culture media supplemented with 10% fetal bovine serum  and 1% L-glutamine (Sigma). THP-1 cells were differentiated into macrophages by stimulation with 200 nM phorbol 12-myristate 13-acetate (PMA) (Sigma) in 24 well black optical bottom plates (Perkin Elmer), at a density of 3 × 10ied from ).M. marinum M strains (ATCC #BAA-535) expressing tdTomato, tdKatushka, or EBFP2 under the msp12 promoter (WT promoter  were gropromoter .M. tuberculosis ΔleuD ΔpanCD double auxotroph expressing mCherry or GFP under control of the msp12 promoter were grown under hygromycin B (Formedium) and kanamycin selection in Middlebrook 7H9 broth (Difco) supplemented with oleic acid, albumin, dextrose, catalase, Tween-80 (Sigma), 0.05 mg/ml L-leucine and 0.024 mg/ml calcium pantothenate (Sigma) ( (Sigma) .m-cresol (4CmC) (10 μM) (Sigma), nifedipine (dihydropyridine) (3 μM) (Cambridge Bioscience), diltiazem (benzothiazepine) (5 μM) (Cambridge Bioscience) and verapamil  (10 μM) (Fischer Scientific) were administered 5 h prior and removed 24 h post-TNF administration. For experiments quantifying mitochondrial ROS production or mitochondrial Ca2+ overload, drugs were administered 4 h after infection or 5 h prior to TNF administration as detailed before and maintained during imaging.0.5 ng of recombinant zebrafish soluble TNF  or vehicCathepsin D-splice-blocking and BID-, cyclophilin D- and PGAM5-translation-blocking morpholinos see . The con4SG4SG4 followed by the MOM-targeting sequence from the protein ActA from Listeria monocytogenes  or DMSO  and incubated for 6 h at 28.5°C . Then al2+ overload was assayed by the increase in fluorescence intensity of the cell permeant mitochondrion-targeted Ca2+ indicator Rhod-2 AM (red fluorescence) (2+ reporter GCaMP3 (green fluorescence) targeted to the mitochondrion [Tg(BH:GFP-mfap4:Mito-GCaMP3)]. Tg(mpeg1:YFP)w200 or Tg(mpeg1:Brainbow)w201; Tg(BH:GFP-mfap4:Mito-GCaMP3) larvae were infected with 90-120 EBFP2-expressing Mm or 185-200 GFP-expressing Mtb. To calculate the percentage of macrophages positive for Rhod-2, 1 day post infection (dpi) Tg(mpeg1:YFP)w200 larvae were microinjected via CV with a solution containing TNF and 31.25 μg/ml Rhod-2 or a solution containing vehicle for TNF and Rhod-2 only. 50 μg Rhod-2 were diluted in 20 μL DMSO and stored in 1 μL aliquots at −20°C and protected from light. Then Rhod-2 was diluted 1:40 in PBS and added 1:2 to the TNF solution. To quantify increased mitochondrial Ca2+ concentration, 1 dpi Tg(mpeg1:Brainbow)w201; Tg(BH:GFP-mfap4:Mito-GCaMP3) larvae were microinjected via CV with TNF. After TNF or TNF and Rhod-2 administration, larvae were prepared for time-lapse confocal imaging. Time-lapse images were taken at different intervals for 5 hr. Mitochondrial GCaMP3 fluorescence was quantified as maximum fluorescence intensity per macrophage for a single time point using NIS-Elements. Mitochondrial Ca2+ overload quantified as total Rhod-2 positive macrophages per animal in the area indicated in Mitochondrial Caescence)   was used for global analysis without end-gap penalty of amino acid residue sequences. Percentage identity, percentage similarity and global/local score are shown in LALIGN (2-Xros (red fluorescence) w200 larvae were infected with 90-120 EBFP2-expressing WT Mm or 80 GFP-expressing Mtb. Larvae were microinjected 1 dpi via CV with a solution containing TNF and 50 μΜ MitoTracker Red CM-H2-Xros or 68 μΜ MitoSOX or a solution containing vehicle for TNF and MitoTracker Red CM-H2-Xros or MitoSOX only. After TNF administration, larvae were prepared for confocal imaging and maintained within a heated incubation chamber (Oko-labs) attached to the confocal. Time-lapse images were taken at different intervals for 2-3 hr starting 40–60 min after TNF administration. MitoTracker Red CM-H2-Xros or MitoSOX were diluted in DMSO and stored in 1 μL 5 mM and 2.5 mM aliquots, respectively, at −20°C and protected from light. Then both probes were further diluted in PBS and added to the TNF solution. Mitochondrial ROS production was quantified as MitoTracker Red CM-H2-Xros or MitoSOX maximum fluorescence intensity per macrophage using NIS-Elements. When not otherwise stated in the figure legend, mean of maximum MitoTracker Red CM-H2-Xros fluorescence was quantified only in Mm- or Mtb-infected macrophages. The use of the heating chamber allowed us to quantify mitochondrial ROS production under more physiological conditions: we had previously showed that ROS production occurred 3 h post TNF administration   (Fisher escence)   or 37°C (Mtb), then washed and incubated in fresh medium at 33°C (Mm) or 37°C (Mtb) overnight. The next day, human recombinant TNF (Sigma) in a solution of 5% trehalose/PBS (Sigma) was added to treatment wells for a final concentration of 10ng/ml and cells were then incubated for 5 h. In experiments with small molecule inhibitors 10 μM Necrostatin-1, 10 μM Nec-1 s (Cambridge Bioscience), 10 μM Q-VD-OPh (Source Bioscience), 10 μM Z-VAD-FMK (Cambridge Bioscience), 10 μM BI-6C9, 10 μM dantrolene and 2 μM Ru360), cells were pre-incubated with drug or 0.1% DMSO vehicle control for 1 hr prior to TNF addition. In experiments with LTCC inhibitors , drug or 0.1% DMSO vehicle control were added immediately after Mm infection and incubated overnight before TNF addition. To identify necrotic cell death, SYTOX® Green Nucleic Acid Stain (Life Technologies), a green-fluorescent stain that is impermeant to live cells, was added to culture medium at least 30 min prior to imaging. Macrophages were imaged using a Nikon Ti-E inverted microscope fitted with 20x objective and 2-5 arbitrary images per well acquired with NIS Elements (Nikon). Cell necrosis was quantified by counting SYTOX® Green fluorescence-positive infected and uninfected cells in each image using ImageJ or Imaris (Bitplane Scientific Software), and the total percentage per treatment well of SYTOX+ infected or SYTOX+ uninfected cells calculated by summing values obtained from all images per well. To calculate the ratios of Mm infected +TNF versus infected –TNF, infected versus uninfected dead within TNF-treated wells, and control +TNF versus control –TNF : One sample t test, one-way ANOVA with Bonferroni’s or Tukey’s post-test, Fisher’s exact test and Student’s unpaired t test. Error bars represent standard error of mean. Post-test The following software was used: NIS-Elements , Imaris , Corel Draw (figure preparation) and ImageJ ; see Materials and Data will be made available upon request.


Refractory diseases, including bacterial infections, are causing huge economic losses in dairy farming. Despite efforts to prevent and treat those diseases in cattle, including the use of antimicrobials, it is not well controlled in the field. Several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), play important roles in disease progression; thus, blocking these cytokines can attenuate the acute and sever inflammation and may be a novel strategy for treatment. However, biological drugs targeting inflammatory cytokines have not been used in cattle. Therefore, in this study, bovine sTNFR1 and sTNFR2 IgG1 Fc-fusion proteins (TNFR1-Ig and TNFR2-Ig) were produced, and their anti-inflammatory functions were analyzed in vitro, to develop decoy receptors for bovine TNF-α.Both TNFR1-Ig and TNFR2-Ig were shown to bind with TNF-α, and TNFR2-Ig showed higher affinity toward TNF-α than TNFR1-Ig. We next stimulated murine fibroblast-derived cells (L929 cells) with TNF-α to induce cell death and analyzed cell viability in the presence of TNFR-Ig proteins. Both TNFR1-Ig and TNFR2-Ig suppressed TNF-α-induced cell death, significantly improving cell viability. In addition, cell death induced by TNF-α was suppressed, even at low TNFR2-Ig concentrations, suggesting TNFR2-Ig has higher activity to suppress TNF-α functions than TNFR1-Ig. Finally, to examine TNFR2-Ig’s anti-inflammatory, we cultured peripheral blood mononuclear cells from cattle with TNF-α in the presence of TNFR2-Ig and analyzed the gene expression and protein production of the inflammatory cytokines IL-1β and TNF-α. TNFR2-Ig significantly reduced the gene expression and protein production of these cytokines. Our results suggest that TNFR2-Ig inhibits inflammatory cytokine kinetics by blocking TNF-α to transmembrane TNFR, thereby attenuating excessive inflammation induced by TNF-α.Collectively, the findings of this study demonstrated the potential of TNFR2-Ig as a novel therapeutic for inflammatory diseases, such as bovine clinical mastitis. Further investigation is required for future clinical application. In dairy farming and the livestock industry worldwide, numerous infectious diseases threaten animal health and productivity. There are effective controlling strategies, such as vaccination, for some diseases. However, various refractory diseases, including bacterial infections such as paratuberculosis, mycoplasmosis, and mastitis remain serious issues , 2. In tAs mentioned, inflammatory cytokines are critical factors in bacterial infection progression. Thus, inhibiting these cytokines may be promising as part of an overall control strategy. TNF-α is produced by fibroblasts and epithelial cells as well as activated immune cells. Immediately upon microbial invasion, it serves various functions, such as activation of immune cells and cytotoxicity to tumor cells , 9. TNF-On the contrary, soluble TNFRs (sTNFRs) might reduce inflammation as “decoy receptors” by competitively inhibiting TNF-α/membrane TNFR (mTNFR) interactions . sTNFRs In human inflammatory diseases, such as rheumatoid arthritis, inhibiting TNF-α with anti-TNF-α antibodies or sTNFR-fusion proteins has been gaining increasing attention as a promising treatment strategy. For example, sTNFR2-fusion proteins are widely used for rheumatoid arthritis, with high therapeutic performance –24. HoweTo evaluate the TNF-α decoy receptor’s efficacy for bovine inflammatory diseases, we first prepared recombinant bovine TNFR-Ig, consist of the extracellular domains of bovine TNFR1 or 2 and the Fc domain of bovine IgG1. In addition, as a negative control, Cont-Ig, consists of the signal peptide domain of bovine TNFR2, and the Fc domain of bovine IgG1, was generated using the Expi293 Expression System. Figure The binding of TNFR1-Ig and TNFR2-Ig to bovine TNF-α was analyzed by enzyme-linked immunosorbent assay (ELISA). TNF-α-dose dependent increases in absorbance were observed with TNFR1-Ig and TNFR2-Ig application, whereas changes in absorbance were not detected when using Cont-Ig, indicating that both TNFR1-Ig and TNFR2-Ig can capture bovine TNF-α  were cultured in low glucose Dulbecco’s Modified Eagle Medium (D-MEM)1 containing 10% heat-inactivated fetal bovine serum (FBS)2. Bovine peripheral blood mononuclear cells (PBMCs) were isolated from the venous blood of healthy cattle  by density gradient centrifugation in Percoll solution.Expi293F™ cells5 treatment for 10 min at 65 °C. The cDNA was synthesized using 1 μg of RNA with PrimeScript Transcriptasehttp://www.cbs.dtu.dk/services/SignalP/) and SOSUI (http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html) respectively, based on the sequences registered in the GeneBank database (BC113278 and NM001040490) , and the recombinant bovine TNF-α was added (0.098 ng/mL-25 ng/mL).13 for 1 h. After washing for five times with PBS-T, peroxidase-labeled Pierce™ NeutrAvidin™ Protein1 was added and incubated for 1 h. Finally, the mixture was washed five times with PBS-T and reacted with the TMB one component substrate2SO4 and the absorbance was measured at 450 nm. Each incubation was performed at room temperature. The assay was performed in triplicate.The binding of TNFR-Ig to bovine TNF-α was analyzed using the anti-bovine TNF-α antibody. Briefly, TNFR-Ig or Cont-Ig was fixed on 96-well plates for ELISA4/0.2 mL) were pre-cultured for 24 h on LIMITRAC® 96-well plates.4 and TNFR1-Ig or TNFR2-Ig or Cont-Ig at varying concentrations . Cell viabilities were measured using the RealTime-Glo™ MT Cell Viability AssayL929 cells (3 × 106/0.2 mL) from 6 healthy cattle were cultured for 4 h with recombinant bovine TNF-α [300 μg/mL (17.2 nM)], actinomycin-D (1 μg/mL)4 and TNFR1-Ig or TNFR2-Ig or Cont-Ig at varying concentrations . As a calibrator sample, PBMCs from each cattle were cultured in the absence of TNF-α, actinomycin-D, and TNFR2-Ig or Cont-Ig. Total RNA from cultured PBMCs was extracted and used to synthesize cDNA as described above. Real-time quantitative RT-PCR was performed using the LightCycler480® SystemII7 according to the manufacturer’s instructions. The primers and target genes are shown in Table -ΔΔCT method [PBMCs  from 6 healthy cattle were cultured for 24 h with recombinant bovine TNF-α [300 μg/mL (17.2 nM)], actinomycin-D (1 μg/mL)4 and TNFR1-Ig or TNFR2-Ig or Cont-Ig at varying concentrations . IL-1β and TNF-α in the cultural supernatant were measured by ELISA using IL-1beta ELISA Reagent Kit, bovine1 and TNF-α Bovine TNF alpha Do-It-Yourself ELISA13, respectively, according to the manufacturer’s instructions.PBMCs (5 × 10p-value less than 0.05 was considered statistically significant.The differences between the groups were examined using the Wilcoxon signed rank test. For multiple group comparisons, the Steel-Dwass’ test or Tukey’s test was performed depending on the sample numbers. A


A sandwich-type detection strategy with a secondary antibody anti-TNF-α provided chronoamperometric analyses in 10 s for each sample. Linearity, precision, limit of detection, and selectivity of devices were investigated. Interferences were evaluated by analyzing solutions containing other cytokine produced during the acute stage of inflammation. The immunosensor showed good performance within the clinically relevant concentration range, with a precision of 8%, and a limit of detection of 0.3 pg/mL. Therefore, it may represent a promising tool for monitoring HF in a non-invasive way.Tumor necrosis factor-α (TNF-α) is a biomarker of inflammation that occurs in patients suffering from heart failure (HF). Saliva can be sampled in a non-invasive way, and it is currently gaining importance as matrix alternative to blood in diagnostic and therapy monitoring. This work presents the development of an immunosensor array based on eight screen-printed gold electrodes to detect TNF-α in saliva samples. Two different functionalization strategies of electrodes were compared. In the first, anti-TNF-α antibodies were chemically bonded onto the electrode by functionalization with 4-carboxymethylaniline. The other functionalization procedure involved the binding of antibodies onto polymer-coated magnetic microparticles, which were then deposited onto the electrode by pulsed chronoamperometry. Finally, the chronoamperometry technique was applied to characterize the modified SPEAu. The use of a secondary antibody anti-TNF-α (Ab-TNF-α-HRP) labelled with horseradish peroxidase (HRP, 2 µg·mL Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine, considered as a central mediator of a broad range of biological activities, especially in immune and inflammatory related diseases whose severity may be determined by TNF-α improper balance . DysreguCompared to blood or urine, saliva is a relatively simpler matrix that can be used for disease monitoring due to its non-invasive sampling from people at any age and even by multiple collections, as well as for the stability of several analytes in this medium ,12,13,14TNF-α is usually quantified in saliva by enzyme-linked immunosorbent assay (ELISA) ,16,17. HThis paper describes the fabrication of a novel immunosensor array based on a screen-printed gold electrode (SPEAu) for the detection of the TNF-α in saliva samples. Two different SPEAu functionalization strategies were developed and compared: the first with a two dimensional (2D) SPEAu with anti-human TNF-α antibody (Ab-TNF-α) directly attached onto the electrode surface, and the second with a three-dimensional (3D) SPEAu with Ab-TNF-α linked to a new structure of magnetite magnetic microparticles (MMPs) coated with poly(pyrrole-co-pyrrole-2-carboxylic acid) (Py/Py-COOH) previously attached onto the SPEAu by pulsed chronoamperometry (PCA). Py/Py-COOH/MNPs were used to increase the surface are, and thus the number of antibodies, as the innovative aspect of this work. Antibody Ab-TNF-α was covalently bonded to Py/Py-COOH/MNP-modified gold WEs through amide bonding. The prepared immunosensor has been successfully applied to increase the sensitivity of Ag-TNF-α antigen in complex matrices such as saliva. Both SPEAu electrodes were designed as a substrate for a “sandwich-type assay” by using a secondary antibody anti-TNF-α labelled with horseradish peroxidase (Ab-TNF-α-HRP) and by employing tetramethylbenzidine (TMB) as electrochemical substrate. TNF-α standard solutions and artificial saliva samples spiked with different TNF-α concentration have been analyzed to test the final device. The immunosensor specificity was proven by analyzing different standard solutions containing other molecules, such as interleukin-6 (IL-6) and interleukin-10 (IL-10), that may represent possible salivary interference due to their production during acute stage of inflammation .2, purity ≥ 97.0%), N-hydroxysuccinimide , 4-aminophenylacetic acid , and N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride , pyrrole , povidone (PVP), pyrrole-2-carboxylic acid , iron chloride hexahydrate , purchased from Acros Organics . Potassium chloride , phosphate buffer solution (PBS) tablets, sodium phosphate dibasic , anhydrous calcium chloride , potassium hexacyanoferrate (III) (K3Fe(CN)6, purity 99%), potassium hexacyanoferrate (II) trihydrate (K4Fe(CN)6⋅3H2O, purity ≥ 99.95%), pure ethanol, mucin, urea, interleukin 6 (IL-6), and interleukin 10 (IL-10) were purchased from Sigma Aldrich . Sodium nitrite (NaNO2) has been purchased from Merck, whereas hydrogen peroxide (H2O2), sulfuric acid (H2SO4), and ethanolamine were from Fluka .Sodium chloride , sodium nitrite  has been acquired from Ademtech S.A. . The image was processed for grain size measurement using ImageJ software. The average grain size is 1.07 μm.Ab-TNF-α, Ab-TNF-α-HRP, TNF-α, and tetramethylbenzidine (TMB) were purchased from Abcam . Millipore Milli-Q nanopure water (resistivity > 18 MΩ cm) was produced by a Millipore Reagent Water System . SPEAus (250BT) have been purchased from Metrohm .The 16 channels VMP3 potentiostat  controlled by EC-Lab software was used to carry out all the measurements at room temperature (20 ± 2 °C) in a Faraday cage. Auxiliary electrode (platinum) and reference electrode (Ag/AgCl) are designed to be used in flow injection analysis obtaining inlet and output flow perpendicular to the working electrodes surface, and amperometric measurements were performed in a flow-cell for 8X format SPEAu working electrode. Eight rubber seals delimit the volume of the eight independent electrochemical cells. Cyclic voltammetry (CV) measurements have been performed to investigate the optimum reduction potential for TMB substrate, to functionalized SPEAu electrodes and to characterize the SPEAu after CMA deposition.3·6H2O into the reactor, and the whole emulsion was kept under stirring for 12 h at room temperature (20 ± 2 °C).MNPs coated with poly (pyrrole-co-pyrrole-2-carboxylic acid) (Py/Py-COOH/MNPs) were obtained by the seeded polymerization technique described by Tenório-Neto et al.  and Hass−1 Ab-TNF-α, and 33.5 µg·mL−1 Ab-TNF-α-HRP were then obtained by dilution. To promote the adhesion of the antibodies, CMA molecules has been electrochemically deposited onto SPEAu using CV in a 10 mM CMA aqueous solution containing 20 mM sodium nitrite. The potential was scanned from 0 V to –1.4 V vs. Ag/AgCl and five cycles were performed at a scan rate of 80 mV s−1. In order to confirm the electrodeposition, CV analysis was performed on both bare and functionalized electrodes by immersing the device into a 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] in a PBS (pH 7.4) solution as redox couple. The voltammogram was recorded with a scan rate of 80 mV·s−1 and the potential was scanned between –0.4 to 0.6 V. In order to immobilize the Ab-TNF-α onto the electrode surface, carboxylic groups of the CMA molecules have been activated using 0.4 M EDC/0.1 M NHS coupling in ethanol solution for 1 h at room temperature (20 ± 2 °C). The device was then washed with HCl 0.1 M to remove the excess of EDC/NHS and then incubated for 1h at 4 °C in a 100 µg·mL−1 stock solution containing Ab-TNF-α  according to the protocol provided by the supplier. Stock solutions of 125 µg·mLNF-α see . Finally1 = −1.5 V for t1 = 0.5 s, induces a cathodic electrodeposition process, while the second potential E2 = 1.5 V for t2 = 0.5 s oxidizes the electrode surface, and in particular, cleans it from eventual extraneous deposited species. The cycling process was performed 10 times. Then, the SPEAu were rinsed with distilled water. It is interesting to note that the amplitude of the charge-discharge process remains practically unchanged during the prolonged cycling process, indicating that the electrode surface exhibits a homogenous Py/Py-COOH/MNPs deposition. The activation of carboxylic groups present in the Py/Py COOH/MMPs was carried out by the mixture of EDC/NHS (0.4 M/0.1 M) prepared, as described above. The blocking step was performed by incubating the immunosensor in an ethanolamine solution/PBS (0.01% V/V). Finally, the immunosensor was rinsed with PBS and used for the detection of TNF-α (This preparation was performed by electrodepositing Py/Py-COOH/MMPs onto SPEAu by using pulsed chronoamperometric technique (PCA). Briefly, two continuous repetition potentials have been applied: the first potential Eof TNF-α C. The ho2. The cantilever had a length of 125 μm, a width of 35 μm and a thickness of 4.5 μm. The tip radius was lower than 10 nm and the tip height (H) = 14.16 μm, with a frequency ranged between 200−400 kHz and a tip strength of K= 25.75 N·m−1. The scanning images were performed at 5 V amplitude, set point at 8 V and tip direct current at 477 nV. Measurements were carried out in a ‘tapping-mode’ (TM-AFM) which was used with a speed of 0.5 line·s−1 and a resolution of 1024 lines.Atomic force microscopy (AFM) was used to characterize the surface of 3D-SPEAu covered with Py/Py-COOH/MNPs. The analysis was performed using a Nano observer microscope  with a maximum area of scan is 110 μmThe morphology of Py/Py-COOH/MNPs electrodeposited onto the SPEAu was studied by scanning electron microscopy (SEM), performed using a JSM-5400 .−1 has been then obtained by dilution. The latter was then further diluted in PBS to have standard solutions with 1, 5, 10 and 15 pg·mL−1 of analyte.TNF-α have been reconstituted in PBS buffer  according to the protocol provided by the supplier, and a stock solution of 100 µg·mLStock and standard solutions of IL-6 and IL-10 were prepared with the same procedure and at the same concentrations to investigate the immunosensor specificity.−1) and to spike three aliquots of AS to obtain samples with known concentration (5 pg·mL−1) of TNF-α, successively used to simulate a real saliva sample analysis by performing the standard addition method.Artificial saliva (AS) was prepared according to a procedure previously described ,23. The −1 of TNF-α were analysed and the LoD was calculated in accordance with IUPAC guide-lines /K4[Fe(CN)6] caused by the CMA blocking layer.CV was used for CMA electrodeposition and to confirm the electrode surface complete coverage. As shown in OX1 = +0.30 V, EpOX2 = +0.48 V, EpRED1 = +0.32 V, and EpRED2 = +0.157 V, respectively) are present, which correspond to two reversible one-electron couples of TMB, observed at the bare gold electrode. In the presence of a peroxidase such as HRP, the oxidation of TMB is catalyzed, and the subsequent reduction can be recorded by applying a fixed voltage. The potential chosen was 0 V vs. Ag/AgCl. This potential was not chosen at the oxidation peaks so that the current change would not be due to the oxidation of any solution of TMB in the cell, nor to the reduction peaks, because the TMB is already reduced. In this way, we ensured that the current change in each chronoamperometric measurement was the result of the oxidation between the TMB substrate and the antibody conjugate Ab-TNF-α-HRP.The CV technique was also used to investigate the behavior of TMB  with a S−1, but the immunosensor with the 3D-SPEAu showed a better linearity, with an R2 of 0.992. Moreover, the device precision, expressed as CV% on triplicate measurement for each concentration level, resulted in a 22% working rate with the 2D-SPEAu, whereas it was 8% with the 3D-SPEAu. LoD resulted 0.3 pg·mL−1 for both devices.−1, but significant reduced current values were found by analyzing IL-6 (with a sensitivity of –0.0149 µA·pg·mL−1) and IL-10 (with a sensitivity of −0.0116 µA·pg·mL−1) standard solutions. All the equations and correlation coefficients (R2) of the obtained calibration curves are reported in To study the sensitivity of both 2D-SPEAu and 3D-SPEAu devices, the immunosensor was tested by analyzing standard solutions containing possible interferences, such as IL-6 and IL-10. These analytes were chosen because they may represent other cytokines secreted in the acute stages of inflammation. As seen in −1). Linear fitting of current values recorded after 10 s gave y = −0.0313x − 0.0968 as the equation of the corresponding calibration curve, with an R2 of 0.998.The linear response of the 3D-SPEAu immunosensor was investigated also by analyzing AS samples spiked with increasing TNF-α concentration. −1) were analyzed to simulate the 3D-SPEAu application on a real sample performing the standard additions method as already described in −1, which was in complete agreement with the expected result of 5.0 pg·mL−1.Finally, three AS samples spiked with a known amount of TNF-α . The immunosensor with the 3D-SPEAu allowed us to obtain very promising results also in analyzing AS samples spiked with TNF-α. Thus, further implementations of the proposed immunosensor would make it possible to obtain a useful tool for the diagnosis and monitoring of HF by saliva analysis.


To study pitavastatin's effects on nuclear factor-kappa B (NF-μM/L) and high-dose (1μM/L) pitavastatin calcium were administered as a frontline therapy in human saphenous endothelial cell culture, followed by induction of inflammation by TNF-α and determination of mRNA level alterations of ICAM-1 and NF-κB genes of endothelial cells using the qRT-PCR method. Additionally, immunofluorescence method was used to show the expression of NF-κB and ICAM-1. Finally, LDH levels were determined by the ELISA method to quantify cytotoxicity. Low-dose . The low-dose pitavastatin+TNF-α group had a similar NF-κB mRNA expression with TNF-α and high-dose pitavastatin+TNF-α groups. ICAM-1 mRNA expression in the low-dose pitavastatin+TNF-κB. Novel studies are needed in this field. Pitavastatin increases ICAM-1 mRNA expression in saphenous vein endothelial cells. Furthermore, the effect of pitavastatin on adhesion molecules appears independent of NF- Pitavastatin the recent clinically approved HMG-CoA is a reductase inhibitor [κB activation [In the etiology of atherosclerosis, multiple factors are known to play a role and the pathogenesis of atherosclerosis is known to be extremely complex . As an u30 years . NF-κB a plaques , 12. NF-eactants . In respd VCAM-1 . Studiestivation , 15. Thenhibitor . In the nhibitor . One of tivation , 17. CurκB and ICAM-1 together with VCAM-1 appear to be on the inflammation pathway. That is why in our study we aimed to investigate the effects of pitavastatin on NF-κB and the important ICAM-1 expression on the endothelial cell culture being induced by daily used TNF-α and inflammation.In the recent studies on the patient who had CABG with saphenous vein harvestmen, the usage of statins led to a better reduction on LDL and an extra time to remain open. In addition to that, besides the lipid-reducing properties of statins, data showing the improvement of the endothelial function, reducing vascular inflammation and oxidative stress, and improving saphenous vein graft disease were also presented. However, the effect of pitavastatin on the adhesion molecules in human saphenous vein graft endothelial cells is yet not clear. In the pathogenesis of saphenous graft disease, adhesion molecules such as NF-α to form the inflammation model, followed by pitavastatin application, the isolation, and dying of the mRNA for the endothelial cells were performed in the Neurodegenerative Diseases Research Laboratory. cDNA synthesis, determination of LDH levels by ELISA, and gene expression assays were performed in Pharmacogenetic Research Laboratory of Medical Pharmacology Department of Our Medical Faculty.This endothelial culture study, PromoCell C-12231, was carried on human saphenous vein endothelium. The endothelial cells were provided and were replicated after being dissolved according to the protocol. The steps of our study as the preparation of saphenous vein endothelial cell cultures and the stimulation of endothelial cells by TNF-α  pitavastatin doses and experimental groups were formed as follows:Control group: endothelial cells without any type of treatment was defined as the untreated control groupμM/L of pitavastatin calcium application for an hour were defined as the low-dose pitavastatin group.Low-dose pitavastatin group (PTV1): endothelial culture cells with 0.1 μM/L of pitavastatin calcium application for an hour were defined as high-dose pitavastatin group.High-dose pitavastatin group (PTV2): endothelial culture cells with 1 α group: endothelial culture cells with solely 10 ng/mL of TNF-α application for six hours.TNF-α group (PTV1 + TNF-α): endothelial culture cells with prior 0.1 μM pitavastatin calcium application for an hour followed by TNF-α administration for 6 hours.Low-dose pitavastatin + TNF-α group (PTV2 + TNF-α): endothelial culture cells with prior 1 μM of pitavastatin calcium application for an hour followed by TNF-α administration for 6 hours.High-dose pitavastatin + TNF-α was dissolved in 5μM of DMSO ∗DMSO group: defined as the group pitavastatin, calcium, and TNF-Human saphenous vein endothelial cells used in our study were supplied with the catalog number of Promo Cell C-12231 . Pretreatment was performed to all the experimental groups prior to the experimental procedure applied in the saphenous vein endothelial cell culture. Pitavastatin (Santa Cruz Biotech SC-208176A) was applied to all endothelial cell culture groups for an hour. Procedure was followed by TNF-μM; thus the highest doses' cytotoxicity was calculated and, for any lower doses, no extra groups were formed.For DMSO application, the highest level of DSMO in PTV2 group was taken into consideration and the concentration was prepared as 5κB proteins with ICAM-1, a dual staining procedure was planned.In order to perform immunofluorescence staining of NF-μl PBS for 10 minutes. After the bathing stage, endothelial cells in 0.01% normal goat serum  were prepared in PBS containing 0.01% Triton X-100  and were incubated for an hour. After incubation, the endothelial cells were reconstituted with Anti-ICAM-1  primary antibody and 30% goat serum prepared in 0.01% T-PBS with a dilution of 1:50 and left overnight at + 4°C. At this stage, 30% goat serum prepared in 0.01% T-PBS without primary antibody was applied to the negative controls and the first day of the staining procedure was completed.Firstly, the cultured endothelial cells were fixed in a 3,7% paraformaldehyde  for 30 minutes. After fixation, endothelial cells were washed 5 times with 2000 μl of PBS for 10 minutes. Then 1.5% goat serum in 0.01% T-PBS with goat anti-mouse IgG, H & L (Alexa Fluor 488)  was diluted in secondary antibody as 1: 100 in a dark medium was incubated for an hour at room temperature. After incubation steps, endothelial cells were washed 3 times in dark medium at room temperature for 10 minutes with 2 ml PBS.On the second day of the staining procedure, the cultured endothelial cells were washed 5 times with 2000 κB  in a ratio of 1: 100 with 10% goat serum prepared in 0.01% T-PBS and were incubated for 2 hours. 10% goat serum without primary antibody was administered. After two hours of incubation, endothelial cells with goat anti-rabbit IgG Alexa Fluor 568 (Red)  secondary antibody diluted 1: 100% in 0.01% T-PBS in 1.5% goat serum was incubated for an hour. Then the process of closing the knitting of the dying process was done using the closing medium.In the next stage, endothelial cells were kept at room temperature for 30 minutes in a 10% normal goat serum that was prepared in 0.01% T-PBS. The endothelial cells were then reconstituted with primary antibody NF-In the next step, the endothelial cells were examined in an inverted fluorescence microscope followed by the photo shooting by fluorescent camera system using the TR, I3, and A3 filters . After photos were taken, the matching of the photographs of the same area with different filters and using the special software  was completed. Then, the pixel density of 10 photographs taken from randomly determined regions of each petroleum was determined using Image J software.In the last stage, the numerical data of each photograph were determined separately. Thus, statistical analysis of numerical data was obtained and the differences between the pixel intensities reflecting the expression of ICAM-1 and NF-presyonB proteins were determined.RNA isolation of human saphenous vein endothelial culturing cells was performed according to the protocol of RNA isolation kit (Life Tech AM1728 Cells-To-CT). After cDNA synthesis was complete, by qRT-PCR method, the amounts of mRNA fluctuation were determined. The qRT-PCR method is available with the Applied Biosystems 7500 Fast , TaqMan Probes, and FG TaqMan Cells-To-CT  kit.-TaqMan Gene Expression Test for VCAM-1 Human VCAM-1 Hs01003372_m1 - 4331182 Life Tech., Carlsbad, CA, 92008, USA.For ICAM-1, -TaqMan Gene Expression Test Human ICAM-1 Hs00164932_m1 - 4331182 Life Tech., Carlsbad, CA, 92008, USA.κB, - TaqMan Gene Expression Assay Human NF-7B Hs00765730_m1 - 4331182 Life Technology, Carlsbad, CA, 92008, USA.For NF--TaqMan Gene Expression Test Human ACBT Hs01060665_g1 - 4331182 Life Tech., Carlsbad, CA, 92008, United States.μl of the sample to determine the level of LDH for ELISA was taken and was maintained at -80°C until the day of study. To measure the LDH amount being released to the media, kit of Cytotoxicity Detection (LDH)  was used.400 The data obtained from the study were analyzed with SPSS software version 22.0. Mean, median, lowest, highest, and standard deviation values were used in the descriptive statistics. U test. Statistical significance was accepted as p <0.05.The distribution of the data was tested by one-sample Kolmogorov-Smirnov test. And the data did not show a normal distribution; Kruskal-Wallis test was used with Mann-WhitneyThe ICAM-1 relative mRNA expression levels of the experimental groups were summarized in κB relative mRNA expression levels of the experimental groups were summarized in NF-κB Pixel Density Results of the experimental groups are summarized in ICAM-1 Pixel Density Results of the Experimental Groups summarized in α, low-dose pitavastatin + TNF-α, high-dose pitavastatin + TNF-α and DMSO groups  (Comparison results of mRNA expression for ICAM-1 levels between groups are summarized in = 0.001) .α was significantly lower than Low-Dose Pitavastatin + TNF-α, high-dose pitavastatin + TNF-α, and DMSO groups    Figure . However= 0.001) .α, Low-Dose Pitavastatin + TNF-α, high-dose pitavastatin + TNF-α, and DMSO groups   .α group, the ICAM-1 mRNA expression level was significantly higher than the TNF-α group (P = 0.001)      . Similar= 0.001) . However= 0.001) .α group the level of ICAM-1 mRNA expression was found to be significantly higher compared to solely TNF-α and DMSO groups (P = 0.001 for both) (And in high-dose pitavastatin + TNF-or both) .α group were compared the TNF-α showed a significantly higher level of ICAM-1 mRNA expression than in DMSO group (P = 0.001).Lastly, when the DMSO group and the TNF-κB mRNA expression between the groups are summarized in Comparative tables showing NF-κB are compared control group showed higher levels of mRNA expression than both low and high doses of pitavastatin groups   (When mRNA expression levels of NF-ctively) . On the ctively) . FurtherκB mRNA expression was lower than the groups including high-dose pitavastatin, TNF-α, Low-Dose Pitavastatin + TNF-α, high-dose pitavastatin + TNF-α, and DMSO   .κB was lower compared to TNF-α, Low-Dose Pitavastatin + TNF-α, high-dose pitavastatin + TNF-α, and DMSO groups   .α group, the level of NF-κB mRNA expression was significantly not different from the TNF-α and high-dose pitavastatin + TNF-α groups  (In low-dose pitavastatin + TNF-= 0.673) .κB mRNA expression in low-dose pitavastatin + TNF-α, was significantly higher than in DMSO group (p = 0.001) (But the level of NF-= 0.001) .α group and TNF-α alone showed no significant difference in the term of NF-κB mRNA expression level (P = 0.205) (High-dose Pitavastatin + TNF-= 0.205) .α group, the level of NF-κB mRNA expression was higher than in DMSO group (P = 0.001) (For TNF-= 0.001) .The control group and the low-dose pitavastatin group showed no significant difference in terms of the intensity of the pixel (P = 0.133) .α, Low-Dose Pitavastatin + TNF-α, high-dose pitavastatin + TNF-α, and DMSO groups   .Low-dose pitavastatin group and the DMSO group showed no difference in terms of the pixel density of the ICAM-1 (P = 0.565) .α, Low-Dose Pitavastatin + TNF-α, and high-dose pitavastatin + TNF-α groups were significantly lower and lower   .α, Low-Dose Pitavastatin + TNF-α, and high-dose pitavastatin + TNF-α groups , the difference between high-dose pitavastatin + TNF-α group and DMSO group was not statistically significant (P = 0.123) (Even though ICAM-1 pixel density in the high-dose pitavastatin group was significantly lower than TNF-= 0.123) .α group and TNF-α and high-dose pitavastatin + TNF-α groups; ICAM-1 pixel density did not show any significant difference  (α group showed higher ICAM-1 pixel density than DMSO group (P = 0.001) (Another important result was between the Low-Dose Pitavastatin + TNF-= 0.624) . But low= 0.001) .α group and TNF-α group showed no significant difference in terms of ICAM-1 pixel density (P = 0.814) (α group was higher than the DMSO group (P = 0.001)   . But ICA= 0.001) . Lastly,= 0.003) .α group, the pixel density of NF-κB was statistically higher than the control group (P = 0.006) (κB pixel density in low-Dose Pitavastatin + TNF-α group was significantly higher than in control group (P = 0.034) . The comparison of the groups showed no significant difference in terms of NF-κB pixel density  (In the TNF-= 0.006) . Similar= 0.034) . Also, Nctively) .κB was applied to the human saphenous vein endothelial culture that previous pitavastatin was applied, followed by TNF-α application and 6 hours of incubation. Images of immunofluorescence staining were given in Figures Immunofluorescent staining for ICAM-1 and NF-μM pitavastatin, weak immunostaining was observed for ICAM-1. In 0.1 μM pitavastatin and TNF-α group, a strong immune positive reaction was observed and this immune positivity was significantly localized in the cell membrane. In the group with 1 μM pitavastatin application, weak immune positivity was observed for ICAM-1. The group where pitavastatin and TNF-α were applied together showed a very strong immunopositivity compared to the group of control and 1 μM pitavastatin. Similarly, in group where 0.1 μM pitavastatin was applied and the group where 1 μM pitavastatin and TNF-α coadministered together were compared with the other groups immune positivity was found to be stronger. A strong ICAM-1 immune positive reaction was observed in the TNF-α group.In the control and DMSO groups, weak immune positivity was observed for ICAM-1 in the cells. In the group treated with 0.1The endothelial cells in this group showed different appearance from the other groups and showed a diffuse morphology.In the control group and in the DMSO group, immunoblotting was observed in the cytoplasm and in the nucleus.κB immunopositivity was decreased slightly in the 0.1 μM pitavastatin treated group compared to the control group.NF-μM pitavastatin and TNF-α coadministrated group, NF-κB immunopositivity was found to be increased compared to the control group and mostly was localized in the nucleus.In 0.1 κB was found to be weaker than the control group.NF-μM pitavastatin and TNF-α coadministration, control for NF-κB and only 1 μM were denser and mostly localized in the nucleus, and some in the cytoplasm.In the group with 1 κB was observed only in the TNF-α treated group. There was also a prominent cytoplasmic presence.A strong immune positive reaction localized in the nucleus for NF-α group, the LDH level was significantly lower than the high-dose pitavastatin group (P = 0.010)  (α were found to be significantly higher than the low-dose pitavastatin + TNF-α group (P = 0.016)   . Similar= 0.025) . In addi= 0.016) . Similar= 0.010) . No stat= 0.010) .α to form inflammation model, expression of ICAM-1 was increased. Secondly, in the inflammation models that were formed by TNF-a stimulation being pretreated with low doses or high doses of pitavastatin, NF-κB expression was not altered significantly. As far as we know our study is the first to examine mRNA expression of ICAM-1 and NF-κB in endothelial cell culture of saphenous vein grafts induced by TNF-a with the treatment of pitavastatin. It is known that saphenous vein grafts are widely used in CABG operation but also show problems due to development of saphenous graft occlusions as saphenous graft disease develops. There are data showing that statin group antihyperlipidemic drugs can be useful in the treatment of saphenous vein graft disease [α induced ICAM-1 by the pretreatment of pitavastatin is suspected to be an interesting and unexpected result, similar findings in the literature support our results [Our study has reached two important results. Firstly in the human saphenous vein grafts endothelium pretreated with pitavastatin and stimulated by TNF- disease . However results , 20.α-induced ICAM-1 by statins appears to be the inhibition of generalization which is very important for the function of small G proteins [α-induced ICAM-1, VCAM-1, and E-selectin expression compared to the TNF-α alone group [α-induced endothelial cells in concordance with our results [The potential molecular mechanism of increased expression of TNF-proteins  while thproteins . By the ne group . Another results . Another results .As a result of these findings, atorvastatin was found to reduce the adhesion of monocytes to endothelial cells by about 42% as an effect . Althougα induction due to experimental design and its protocols. In our experiment pitavastatin was applied for the short term; it is possible that chronic administration just like in the clinical practice of pitavastatin can alter these results whereas the HMG-CoA reductase inhibitors are lifelong drugs being used. Another possible explanation for the ICAM-1 overinduction is due to the specific molecular structure of the pitavastatin. In this context, Niwa S et al. in one of the HUVEC culture studies showed that fluvastatin did reduce the ICAM-1 expression, whereas another HMG-CoA reductase inhibitor, Pravastatin, did not affect ICAM-1 expression [There might be other possible reasons for TNF-pression . Therefopression . In addiα induced ICAM-1 is the inhibition of synthesis of isoprenoids due to inhibition of mevalonate synthesis among possible theories and new studies are needed to clarify this issue.As the fourth reason, treatment with pitavastatin may be acutely inducing ICAM-1 expression as in some cases anti-inflammatory molecules can act as proinflammatory. In addition, the inhibition of pitavastatin on ICAM-1 expression may increase the expression of ICAM-1 as a reply to rebound response. As the fifth reason, the anti-inflammatory effect, which is one of the pleiotropic effects of pitavastatin, may not be playing a role in inhibiting the expression of ICAM-1. In endothelial cells, pitavastatin might induce ICAM-1 expression in the acute phase duration and cause a proinflammatory effect. It is also known that there may be negative effects of HMG-CoA reductase inhibitors. In heart failure studies including patients with ischemic heart failure, the positive effects of statins on mortality have not been demonstrated . In partα increases the expression of ICAM-1 on the endothelial cell surface, while high-dose pitavastatin pretreatment further enhances the expression of ICAM-1 on the TNF-α-induced cell surface. But this increase has not gained statistical significance. This difference might be a consequence of qRT-PCR method in which method for detection of mRNA expression by immunofluorescent staining is based on different foundations. On the other hand, since each mRNA molecule synthesized may also require some postsynthesis modifications, and this process cannot be successfully completed; the mRNA molecule's inability to turn into the target protein may also explain this statistical nonsignificance.Also, when the density of pixels of immunofluorescence staining of ICAM-1 adhesion protein was evaluated among the groups; administration of TNF-κB and TNF-α expression were significantly induced in saphenous endothelial cells, low- and high-dose pitavastatin pretreatment on NF-κB expression induced by TNF-α were not statistically significant. However, immunofluorescent staining of NF-κB pixel density shows that NF-κB protein, which is being expressed as a result of NF-κB induction after being treated with different doses of pitavastatin, is reduced and with the high doses of pitavastatin pretreatment NF-κB pixel density lost its significance. Thus, it may be caused by difference in protocols and expression of every single mRNA molecule does not mean they are going to be transformed into target protein as many posttranscriptional and translational processes are required; additionally, our findings of mRNA expression were relative results. But results suggest that reduction in NF-κB pixel inhibition is because the VCAM-1 expression was inhibited by high-dose pitavastatin pretreatment; so it can be thought that NF-κB plays an active role on VCAM-1 expression.Another important point of our study is that although NF-κB is a rat model of cerebral aneurysm where pitavastatin was found to inhibit NF-κB expression [κB activation [α. The reason might be the complex process of cytokine-induced ICAM-1 expression, where transcription proteins play a role in regulation of adhesion molecules expression besides taking a role in cytokine-induced ICAM-I expression. In this context, a nuclear receptor and Peroxisome proliferator-activated receptor (PPAR-γ) are also important in ICAM-1 expression [One of the important studies among the limited studies on NF-pression . Similartivation . In our pression .γ in human endothelial cells reduces the expression of adhesion molecules [κB as well as other nuclear transcription factors and PPAR. Thus, in our study, ICAM-1 expression induced by pitavastatin pretreatment might be not only due to NF-κB, but also due to the effects on different nuclear receptors such as PPAR. To support this, data on PPAR receptors are also available but still more is required [Wang N et al. demonstrated that activation of PPAR-olecules . In addiolecules , 28. As required –31.α group because of inflammation induced cytotoxicity. LDH levels were significantly decreased, especially in high-dose pitavastatin pretreatment + TNF-α group. This result strongly supports that the high-dose pitavastatin prophylaxis has markedly increased the protective effect of endothelial cells in the environment of inflammation. Thus, it may be concluded that the use of high-dose pitavastatin in the treatment of saphenous vein graft disease and coronary artery disease may be more beneficial.Finally, when the LDH levels were measured for the evaluation of the cytotoxicity of pitavastatin applied groups, lower LDH levels were obtained in the group of high-dose pitavastatin compared to low-dose pitavastatin. This suggests that high-dose pitavastatin is more cytoprotective. In the same way, the LDH level was increased in TNF-As a result, especially high-dose pitavastatin may be used in the prevention and treatment of saphenous vein graft disease. However, new experimental studies are needed to clarify the molecular basis of the beneficial effect of pitavastatin in the treatment of saphenous graft disease. On the other hand, in order to evaluate the success of pitavastatin treatment in maintaining saphenous vein graft patency, it is clear that clinical studies are needed to evaluate saphenous graft patency with long-term angiographic follow-up in patients with CABG operation and saphenous vein grafts, because, in some cases, the results obtained in daily clinical practice with experimental results may be different.


The current treatment regimen for this debilitating disease focuses on controlling symptoms and preventing further joint damage through the use of therapies which affect different areas of the immune system at the synovium. One of the main therapies involves Tumor Necrosis Factor alpha (TNF-α) inhibitors. In the RA immune-environment, TNF-α has been shown to have an influential and extensive but as yet poorly understood effect on Treg function in vivo, and undoubtably an important role in the treatment of RA. Interestingly, the high levels of TNF-α found in RA patients appear to interfere with the mechanisms controlling the suppressive function of Tregs.The autoimmune disorder rheumatoid arthritis (RA) causes chronic inflammation and destruction of joints. T cells are a predominant component of the synovial environment in RA, however the functional role of these cells is not yet fully understood. This is in part due to the fact that the balance and importance of the relation of TRelevance for patients: This review focuses on the conflicting literature available regarding the role played by Tregs in RA and the impact of TNF-α and anti-TNF-α therapies on Tregs in this scenario. Individuals suffering from RA can benefit from better insight of the treatment mechanisms of the immunologic processes which occur throughout this disease, as current treatments for RA focus on several different areas of the immune system at the synovial compartment. RA is cA hyperplastic synovium is the major contributor to the cartilage damage in RA. The loss of the protective effects of the synovium result in the alteration of the protein-binding characteristics of the cartilage surface, promoting synoviocytes (FLS) adhesion and invasion. These processes lead to the destruction of the surface . Bone erStudies in Europe have shown that there is a gradient in the prevalence of RA, starting from a low prevalence in the South  , to a hiThe use of different case definitions makes the estimates vary as widely as 25 to 115 per 100,000 . The annCurrently, treatment focuses on controlling symptoms and preventing further joint damage. Medications used in the treatment of RA include non-steroidal anti-inflammatory drug (NSAIDs), disease-modifying anti-rheumatic drug (DMARDs), TNF-α inhibitors, IL-6 inhibitors, T-cell activation inhibitors, B-cell depletors, Janus kinase (JAK) inhibitors, and steroids  Figure . Since a2.Several of the risk alíeles linked to RA consistently map functionally with immune regulation such as the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB)-dependent signaling, T-cell stimulation, activation, and functional differentiation. This suggests that these immunologic pathways are amongst the key modulators of the development of the autoimmune inflammation in RA -13.The costimulation-dependent interactions among dendritic cells, T cells, and B-cells are thought to occur primarily in the lymph node, generating an autoimmune response to citrulline-containing self-proteins . The infA variety of innate effector cells, including macrophages, mast cells, and natural killer cells, are found in the synovial membrane, while neutrophils reside mainly in synovial fluid. The main role of macrophages in this scenario is that of releasing cytokines , reactive oxygen intermediates, nitrogen intermediates, production of prostanoids and matrix-degrading enzymes, phagocytosis, and antigen presentation . These fIt has become more apparent from various reports in the literature that cytokines play an integral role in the activation and maintanence of the innate immune pathway. Cytokine production that arises from numerous synovial cell populations is central to the pathogenesis of RA . TNF-α i3.TNF-α is an inflammatory cytokine consisting of a trimeric protein encoded within the major histocompatibility complex. It’s first identified form was the 17 kDa secreted form, but further research then showed that a noncleaved 27 kDa precursor form was also present in transmembrane form . TNF-α a4.Even though T cells are a predominant component of the synovial environment in RA, the functional role of T cells is not yet fully understood. This is mainly due to the fact that lymphocytes, including T cells, act and react according to the presence and numbers of other subsets of lymphocytes, and this systematic approach to immunity has only recently started to be investigated in detail . Therefo+ T cells stimulate monocytes, macrophages, and synovial fibroblasts to produce the cytokines IL-1, IL-6, and TNF-α [+ T cells also stimulate B-cells and these produce immunoglobulins, including the RF. The precise immunologic role of RF is still unknown, but it may involve the activation of complement through the formation of immune complexes [+ T cells also express RANKL, and as explained previously, this stimulates osteoclast differentiation. Thus these activated T cells give rise to cartilage erosion caused by excessive osteoclasts [Activated CD4nd TNF-α . Activatomplexes . Activateoclasts .RA is conventionally considered to be a disease mediated by type 1 helper T cells, however there is increased attention on the role of type 17 helper T cells (Thl7). Thl7 is a subset of T cells that produces interleukin-17A, 17F, 21, and 22 and TNF-α ,26. Otheregs), thus shifting T-cell homeostasis toward inflammation rather than autoregulation [regs are critically involved in immune tolerance and homeostasis. Tregs that are detected in tissues from patients with RA seem to have limited functional capability, as inferred via Forkhead box P3 (FoxP3) transcript levels, which are lower in the synovial membrane compared to those in peripheral blood or synovial fluid [It is interesting to note that IL-6 suppresses the differentiation of regulatory T cells  [regs in the peripheral blood between healthy individuals and RA patients, varying from reports of decrease to an increase in Tregs, comparatively [regs themselves seems to be observed. FoxP3+ Tregs sampled from the SF of RA patients are able to suppress the proliferation of effector T cells [regs from RA patients do suppress proliferation, they are defective in their ability to suppress pro-inflammatory cytokine production [regs obtained from peripheral blood rather than SF. On the other hand, a conflicting recent study also performed using Tregs obtained from peripheral blood, shows that there is no significant difference between the suppressive effects of FoxP3+ Tregs on certain cytokines and the proliferation of effector T cells, between Tregs obtained from healthy individuals and from RA patients [In RA, there are two distinct classes of Tuid (SF) -31. Diffratively -36. In moduction , and thupatients .+ FoxP3+ Tregs cells play an indispensable role in maintaining immune homeostasis and in suppressing deleterious excessive immune responses [regs, with various effects on effector T cells in the autoimmune scenario [regs will result in an upregulation of T-effector cells and any other cell type under suppression by Tregs Thus it is important to understand better how TNF-α, being one of the most present and influential cytokines in the synovial immuno-environment, affects the function of Tregs.Various studies provide compelling evidence that CD4esponses . There ascenario . Any dis5.regs [regs function [+ CD62L~ Tregs population, which was able to suppress cytokine production of effector T cells via a TGF-β and IL-10 pathway [+ Tregs remained defective in infliximab-treated patients clearly showed that TNF-α was responsible in promoting the development of a new dysfunctional subset of FoxP3+ Tregs. Another study highlighting the importance of TNF-α in RA vis-á-vis FoxP3+ Tregs reported that overexpression of TNF-α in human TNF-α transgenic mice led to the development of arthritis, with an increased number of Tregs expressing the TNF receptor II (TNFRII) [regs, however the Tregs still failed to control inflammation. When TNF-α was blocked, a further increase in the frequency of Tregs was observed, and these Tregs had upregulated CTLA-4 expression, resulting in enhanced suppressor activity [- Treg population as observed in the previous study [TNF-α and IL-7 are two cytokines which act against the suppressive activity of human Tregs . High lefunction . Treatme pathway . The fac(TNFRII) . TNFRII (TNFRII) . The TNFactivity . The TNFus study .+ Treg suppressive function, particularly in suppressing inflammation. Further to this, it has been shown that TNF-α signaling via TNFRII downregulates FoxP3 expression in humans in both naturally-occurring Tregs and adaptive Tregs, and this results in the inhibition of Treg suppressive activity [regs via TNF-α signaling of the TNFRII receptor, the TNF-α activated the canonical NF-κB pathway and induced a pro-inflammatory phenotype [reg suppressive activity could be reversed by treatment with anti-TNFRII antibody. This shows that TNF-α signaling via TNFRII may be one mechanism which leads to Treg defects in RA. This study was performed using both Tregs from peripheral blood and synovial fluid and the results showed similar trends in both types of Tregs.These studies suggest that TNF-α plays an important role in the inhibition of FoxP3activity . Anotherhenotype , howeverreg suppressive activity is by influencing the formation of the immunological synapse between Tregs and antigen presenting cells (APCs) [+ Tregs, PKC-9 is concealed from the immunological synapse. A study was conducted in which a model system on supported planar bilayers containing the mobile fluorescently labelled intercellular adhesion molecule-1 (ICAM-1), anti-CD3 antibodies and CD4+CD25+ effector T cells or CD4+ CD25+ Tregs  was devised, in order to emulate the immunological synapse. Addition of TNF-α to this model increased the PKC-9 recruitment to the Treg immunological synapse, and this inhibited their suppressive activity. This contrasts with the fact that after blocking PKC-θ, Treg function was enhanced [regs from inactivation by TNF-α and this restores the suppressive function of defective Tregs in RA patients. Disc large homolog 1 (Dlghl) is another identified molecule involved in the immunological synapse formation which regulates Treg function independently of PKC-θ [regs than in effector T cells. It was also found that Tregs from RA patients with active disease had defective Dlghl recruitment to the immunological synapse. This defective recruitment resulted in reduced suppressive activity of Tregs. Exposing healthy control Tregs to TNF-α decreased the Dlghl recruitment and thus also reduced Treg suppressive activity [+ Tregs, PKC-θ-mediated negative and Dlghl-mediated positive pathways seem to regulate suppressive function independently, and in RA, one or both of these pathways may be defective as a possible con4equence of TNF-α [Another way TNF-α might inhibit Ts (APCs) . Althougenhanced . This stof PKC-θ . Dlghl wactivity . These fof TNF-α .FOXP3 gene in Tregs [regs.TNF-α was also found to interact directly with the DNA-binding activity of the in Tregs . In this+ Tregs purified from lymph node expressed remarkably higher surface levels of TNFRII than effector T cells in vitro [regs was initially observed after exposure to TNF-α, however longer exposure to TNF-α restored the suppressive effects. Furthermore, TNF-α expanded Treg populations in this study, and these TNF-α-expanded Tregs had up-regulated expression of CD25 and FoxP3, enhancing the suppressive activity of these Tregs. Thus in this study the stimulatory effect of TNF-α on Tregs resembled the reported costimulatory effects of TNF-α on effector T cells. Another study to determine the effect of TNF-α on Tregs showed that when Treg cells were cultured for 20 h with or without IL-2 before the suppression assays, the presence of TNF in the pre-culture had no effect on their suppressive function in any assay condition [regs in a 3-day culture of whole CD4+ T cells resulted in an increased proportion of Tregs and the upregulation of FOXP3 expression. A suggestion for the slower response of Tregs to TNF-α could be a delayed immunosuppressive feedback effect [regs present in the synovial fluid of RA patients is also up-regulated [+ FoxP3+ Tregs are more functionally suppressive , and TNF-α which is cleaved from the membrane and released as a soluble cytokine (sTNF-α) . Moreove6.regs in the RA scenario. TNF-α seems to interfere with the mechanisms controlling Treg suppressive function, and therefore it is plausible to predict that anti-TNF-α therapies would counter this effect. In fact, studies show that anti-TNF-α therapy has a regulatory effect on the immune system of RA patients by promoting an increase in the proportion of Tregs and suppressing effector T cells [regs from RA patients by binding to monocyte membrane bound TNF, enhancing its expression and its binding to TNF-RII expressed on Tregs [+ Tregs able to suppress Thl7 cells through an IL-2/STAT5-dependent mechanism. This study demonstrated that a therapeutic antibody thought to act by blocking TNF-α can also enhance the regulatory properties of this pro-inflammatory cytokine.It is clear from the conflicting literature above, that we are still far from deducing the exact role and effect of TNF-α on T T cells . One sucon Tregs . This reHowever, clinical trials have accumulated evidence that anti-TNF-α therapies might promote rather than suppress certain forms of autoimmunity. In RA, anti-TNF-α therapy is sometimes associated with adverse events, such as multiple sclerosis and lupus . Cases oreg function in vivo in the RA immune-environment. Although anti-TNF-α therapies have been widely used to treat RA with significant clinical result, more research is still needed to understand better the total effect of such therapies on all cell types involved in the synovial immune-environment. Anti-TNF-α therapies might exhibit serious side effects, and the mechanisms leading to such side effects can be investigated further to find methods of suppressing them. Alternate routes to suppress the over-reactive effector T cells as well as activate and enhance the Treg subsets can be investigated, whilst working to obtain a broader and clearer picture of the effect TNF-α has on Tregs and RA in general.TNF-α without a doubt has an important role in the treatment of RA, and it has been shown to have a powerful, varied and yet poorly understood effect on TThe authors declare no conflict of interest.


Infrared (IR) thermography, where temperature measurements are made with IR cameras, has proven to be a very useful and widely used tool in biological science. Several thermography parameters are critical to the proper operation of thermal cameras and the accuracy of measurements, and these must usually be provided to the camera. Failure to account for these parameters may lead to less accurate measurements. Furthermore, the failure to provide information of parameter choices in reports may compromise appraisal of accuracy and replicate studies. In this review, we investigate how well biologists report thermography parameters. This is done through a systematic review of biological thermography literature that included articles published between years 2007 and 2017. We found that in primary biological thermography papers, which make some kind of quantitative temperature measurement, 48% fail to report values used for emissivity , which is the minimum level of reporting that should take place. This finding highlights the need for life scientists to take into account and report key parameter information when carrying out thermography, in the future. It is a key influence on living organisms –8, and tInfrared thermography is a valuable tool for biologists and has been widely applied for temperature measurements ,26,29,342.2.1.All objects of a temperature above absolute zero emit electromagnetic radiation. Increased temperature leads to increased levels of radiation ,36. ThisWobj) is dependent on the object's temperature  in accordance with the Stefan–Boltzmann formula )2.3.Tref) is an estimate of the level of background radiation reflected off the thermography target object  AND [thermograph* OR thermal imag* OR camera]' .Our literature search was carried out using the agriculture dairy animal science, agriculture multidisciplinary, agronomy, behavioural sciences, biology, biophysics, ecology, entomology, evolutionary biology, fisheries, forestry, horticulture, marine-freshwater biology, ornithology, physiology, plant sciences, psychology, psychology applied, psychology biological, psychology experimental, psychology multidisciplinary, veterinary sciences and zoology. Full texts of all search results were searched for using University of Bristol library subscriptions and through Google Scholar. If publications could still not be found, and the paper could not be excluded based on the information in the abstract provided by Web of Science or linked sites alone (see exclusion criteria), the corresponding authors (where contact details provided) were contacted for copies of publications. Any publication that was not obtained through these methods was excluded. A summary of the Web of Science search history used in our literature search can be found in electronic supplementary material, S1.The search was then refined further to include only publications in at least one of the following 23 Web of Science Categories: 4.2.Search results were examined in a chronological order by a biological scientist and qualified thermographer . Publications were checked for any criteria for exclusion (criteria detailed below), a process which left only primary biological science research papers that reported work using IR thermography in some way. These papers' methodology, how thermographic tools were employed, and the inclusion of thermographic parameters were assessed. Non-English language journals were assessed with aid of a native speaking translator if the journal could not be excluded based on the abstract alone . After completion of the full review process, all search results were worked through and assessed a second time to ensure confidence and consistency in our assessment.4.3.Web of Science search results, were not primary biological science papers that used thermal imaging. The exclusion criteria applied to our search results are summarized in The search criteria used in this systematic review was deliberately broad to allow for the many ways thermal cameras might be described in publications, such as ‘thermal camera’ and ‘infrared camera'. This was done to minimize the chance of accidently excluding papers that genuinely use IR thermography. This accidental exclusion of relevant papers has been identified as a major issue in systematic reviews . This haOnly publications which carried out thermography and reported data or images collected by IR thermography were included in our review, everything else was excluded as ‘not thermography'. These excluded works included those using non-thermal IR technologies, such as triggers and sensors –63, IR rThis review aims to assess the use of IR cameras in the life sciences area. Thus, if the application of IR thermography did not appear to be biological, publications were also excluded as ‘not biological'. Such application treated as non-biological included the industrial preparation of baked goods , materiaAny isolated abstracts from conferences were excluded, as such summary articles typically do not normally provide detailed information on their methodology. Published conference reports were not excluded if they featured a methods section. Any retracted articles, at time of the search, were also excluded.Lastly, review articles that either discussed IR thermography or thermography-dependent results were excluded, albeit for reference review articles were filed separately from other exclusions .4.4.Tref, Tenv, rh and d), indication that these were used in calculations was required. The method of Tref measurement was also monitored, and could either be a single quoted value used for the parameter at measurements or a continuous measurement alongside the thermography measurements, as both are acceptable [Included publications were assessed to obtain data on how IR thermography was used and has been reported. The information extracted from each publication can be found in ceptable . The infTenv, rh and d) in the sampling area were known, it was assumed they were input into the camera. This could be simply mentioning that these parameters were measured in the thermography sampling area. If the camera was mounted in a fixed position relative to the target it was assumed that distance had been measured and input. As several thermography parameters can be referred to by various names (listed previously), any of these were acceptable. As reflected temperature, Tref, is sometimes referred to as ‘ambient temperature' [Throughout the review, we aimed to give authors the benefit of the doubt where possible. If a study indicated at any point in the paper that the environmental factors  were deemed to use quantitative temperature measurements in some way, leaving 31 (5.52%) wholly qualitative studies. The frequency of quantitative and qualitative papers in each biological field is presented in 2 analysis revealed a significant association between the biological field and reporting of emissivity (p = 0.01). This association is largely due to papers in the ‘birds and poultry', ‘insects' and ‘earth and soil' biological fields reporting emissivity more frequently than expected and the ‘plants’ and ‘humans/medical' biological fields reporting emissivity less frequently than expected. 2 analysis. Of the 276 papers that provided emissivity values, 45.2% (126 publications) provided a source for that value choice and a further 5.4% (15 publications) measured the value within the study. A summary of emissivity values used in studies measuring similar targets, targets of the same research field, is given in Of the 531 quantitative papers, where camera parameter inputs are necessary for accurate temperature measurements, 52.0% of all quantitative studies provided emissivity values (276 publications) and 48.0% of all quantitative studies failed to provide emissivity values (255 publications). Reflected temperature was only reported in 26.7% (142 publications) of all quantitative papers. Within papers that gave emissivity values, reflected temperature was reported in 41.7% (115 publications) of papers. However, in 52.2% (60 publications) of these papers reflected temperature information was not explicitly given but ‘assumed to be ambient'. In papers that failed to give emissivity, reflected temperature was reported in only 10.6% of papers (27 publications).Environmental parameters associated less directly with IR thermography tended to be reported more frequently than emissivity and reflected temperature. With environmental temperature, environmental humidity and camera distance being reported in 81.2%, 51.6% and 66.7% of all quantitative papers respectively. Environmental temperature, environmental humidity and camera distance were reported more frequently in papers that gave emissivity values  than those that did not , but this difference between papers that report emissivity and those that did not was less stark than that seen in reflected temperature.A list of the 1219 papers found in our search categorized into primary papers, reviews and exclusions as well as the data extracted from each paper can be found in electronic supplementary material, S2. A summary of the frequency of parameter reporting, broken down further by biological field, can be found in 6.Infrared thermography parameters are an important part of making accurate thermography measurements –25. FailTenv), relative humidity (rh) and camera distance (d) have little influence on the accuracy of temperature measurements [Environmental temperature  appear to give information on reflected temperature when emissivity information is not given –95, or mWe found a significant association between research field and emissivity reporting, although the level of reporting was not high in most research fields . ResearcWhile our systematic review suggests that an issue exists with thermography parameter reporting in biology, it does not necessarily give a full representation of how well biologists carry out thermography. Successfully reporting parameters such as emissivity does not guarantee thermography was carried out correctly. Other operation issues can still occur when parameter settings are input correctly. Furthermore, it was beyond the scope of our review to evaluate in each instance how applicable the values used for emissivity actually were, and instead our focus was upon whether such appraisals can be done based on the information reported. Consequently, it is possible that the values chosen were still inappropriate and result in inaccurate temperature measurements. However, most often biological tissues have an emissivity of approximately 0.9 ,25, and Our review treats all quantitative thermography as equally important to studies; we made no evaluation of how critical the temperature measurements were to the paper's findings . It is possible that some papers may use thermography in such a minor way that authors felt parameter detail unnecessary. However, reporting parameter information represents a small addition to the methods. Furthermore, in such instances where the accuracy of measurements is less important, papers should still give the information on parameters, but perhaps need not worry for a precise estimate of emissivity or monitor environmental parameters with every measurement. Our review process did not penalize papers for applying these less accurate approaches if they reported the necessary information, consequently a less precise approach for less critical measurements was acceptable within our review.Frequently, emissivity and other parameter values were provided within a thermograph figure with no mention of it in the main text –105. Our7.This study has highlighted a common tendency for biologists to omit information on critical thermographic parameters such as emissivity and reflected temperature in published primary literature. This omission suggests a lack of understanding of thermographical methods. More care should be taken to include parameter information in publications. This will improve clarity and confidence in measurements but also allow the assessment of the limitations of thermography in different types of biological studies. Fortunately, the addition of parameter information represents a small effort which can significantly improve the evaluation of reported research and awareness of the correct use of thermal cameras in biological studies. It is recommended as a minimum that the emissivity values should be given, preferably with sources or measurements supporting the parameter choice. Additionally, the method of assessing reflected temperature should be included as well.


Here, we connect behavioural assays with survival trials and a modelling approach to quantify changes in antipredator escape performance of a larval freshwater fish following exposure to an environmental oestrogen, and predict changes in population abundance. We quantified the effects of short-term (21 days) exposure to 17β-oestradiol (E2) on the antipredator escape performance of larval fathead minnows  and the probability of predation by a natural predator, the bluegill sunfish (Lepomis macrochirus). Compared with unexposed minnows, minnows exposed to environmentally relevant concentrations of E2 that approach total oestrogenic activity of wastewater-dominated environments (38 and 103 ng l−1) had delayed response times and slower escape speeds, and were more susceptible to predation. Incorporating these data into a stage-structured population model demonstrated that enhanced predation mortality at the larval stage can result in population declines. These results indicate that subtle, sub-lethal shifts in the behaviour of individuals due to human-mediated environmental change can impact species interactions with measurable population-level effects. Such changes have the potential to alter higher-order trophic interactions and disrupt aquatic communities.Understanding population-level effects of environmental stressors on aquatic biota requires knowledge of the direct adverse effects of pollutants on individuals  Human-mediated environmental change, including habitat alteration or loss, the introduction of invasive species and influxes of aquatic contaminants have well-documented adverse effects on the health and viability of aquatic biota . These e−1) environmental concentrations. The diversity of oestrogenic EDCs in many wastewater-impacted environments results in aquatic organisms being exposed to a total oestrogenic activity much greater than that of the concentration of any one pollutant. For example, total oestrogenicity  in Boulder Creek, CO, USA has been estimated to be between 31 and 54 ng l−1 , repA similar level of effort aimed at assessing more complex, behaviourally mediated effects of exposure to contaminants of emerging concern (CEC) on higher levels of biological organization has not been made, despite recognition that an animal's behaviour is an integrated expression of its physiological response to the environment  and thatPimephales promelas). Previous work has shown that environmentally relevant concentrations of single oestrogens and oestrogen mixtures alter the evasive performance of larval minnows ..a,b) [17ImageJ . Two anatomical landmarks, the anterior tip of the snout and the posterior tip of the tail, were digitized on each video and used to calculate standard body length (BL). Two additional landmarks were digitized on the grid at a 1 mm distance to account for scale. Latency was recorded as the length of time to the induction of movement (in ms). Velocity was calculated during the first 40 ms after the initiation of movement, and adjusted to BL per ms. The total escape response of each subject was calculated as BL/latency (ms + 40 ms); we included this parameter because it simultaneously takes into account changes in both velocity and latency.We quantified latency to the induction of the escape response, escape velocity, turning angle and total escape response from the videos using the software program 2.6.c). Arenas were drained and scrubbed between trials to remove residual chemical cues. Water quality parameters  were recorded before each trial using the methods described above.Trials were conducted in opaque-walled PVC arenas measuring 114 cm in diameter and 15 cm in height, buffered from disturbance by the addition of shade cloth blinds. Each arena contained 83 l of clean, conditioned well water and an airstone c. ArenasLOW or E2HIGH) were simultaneously introduced to the arena. The sunfish was permitted to feed upon the 20 minnows until approximately 50% of the minnows were no longer visible , as assessed via visual scans of the arena by a single observer every 2 h. Once 50% of the larvae were eaten, the test was stopped and the predator removed via a hand net. Surviving larvae were removed from the arena by glass pipette and transferred to a glass beaker. We calculated the proportion of paired exposed versus control minnows in each trial that survived .Up to four trials were conducted simultaneously. To begin a trial, one sunfish  was introduced to the arena and given 48 h to acclimate to the set-up. The sunfish was fed 10 non-exposed minnow larvae on both acclimation days to condition it to the food source. At the end of the acclimation period, one group of 10 control larvae and one group of 10 exposed larvae . Subjects to be stained were placed in E2-free, aerated water 12 to 18 h before testing. These subjects were marked through static immersion in the dye for 6 h, at a concentration of 250 mg l−1. Pilot trials confirmed that florescence persisted for at least 2 days post-treatment. No mortalities or abnormal behaviours were observed during staining. We counted the number of larvae recovered from each trial using a SE-MARK detector to illuminate fluorescently marked fish.To facilitate the identification of survivors in the predation assay, subjects from either the control or the exposed group were marked before the trial using a fluorescent SE-MARK calcein dye  in accordance with the Investigational New Animal Drug (FDA INAD 10-987) protocol. To prevent mark-associated bias, we randomly selected the treatment to undergo staining in each trial d,e. Subj2.7.LOW, E2HIGH) subjects recorded from the escape behaviour assay using a one-way ANOVA, followed by pairwise least significant difference (LSD) post hoc tests. Preliminary analysis indicated that the escape performance data were normally distributed  and satisfied assumptions of linearity. We used one-way ANOVAs to compare the mean response latencies and escape velocities among control and exposed subjects, as well the total escape response.For the predator escape assay, we compared the mean BL of control and exposed (E2LOW or E2HIGH) subjects in predation trials using non-parametric Wilcoxon sign rank tests. We compared the survival of larvae from the E2LOW and E2HIGH treatments using a Mann–Whitney U test. All analyses were two-tailed and were conducted using SPSS v. 24 .Because the relative survival of control or exposed fish in any given predation trial was potentially non-independent, we compared the survival of paired control and exposed  varies positively with juvenile abundance (N) according toλ is a parameter that defines the sensitivity of M to N. This equation belongs to a family of Ricker stock-recruitment models in which recruitment decreases at higher densities, such that the resultant curve resembles a positively skewed, normal curve [λ to 14 777 948 so that the model produced an equilibrium abundance of 2000 adult females (or 4000 adult fatheads assuming a 1 : 1 sex ratio). This is a reasonable population estimate for the 34 ha experimental lake in [Our model includes life-stage-specific rates of background instantaneous mortality . Daily mength3.13 and thenght−0.288. We repeght−0.288, and to al curve . We cali lake in .The model generates 1000 estimates of mean adult abundance over the last 100 years of a simulation for two scenarios: stable conditions in the absence of oestrogen, and a scenario that includes oestrogen effects by incorporating the empirically estimated, 1.66 ± 2.90-fold increase in hourly larval mortality. Fecundity, and non-adult mortalities and stage durations are sampled annually to reflect uncertainty in parameter values as well as environmental variability . To avoi3.3.1.−1 (n = 3) for E2LOW and 103 ± 17 ng l−1 for E2HIGH (n = 4). The control treatment did not contain quantifiable concentrations of E2 . Mean (±s.d.) water quality parameters recorded during the exposure period were as follows: 22 ± 1°C; 5.6 ± 1.3 mg l−1 dissolved oxygen; 240 ppm CaCO3 alkalinity; 0.83 ± 0.10 mS cm−1 conductivity; and 8.4 ± 0.2 pH. Mean (±s.d.) water quality parameters in the predation trial arenas were 21.8 ± 1.24°C; 5.6 ± 1.3 mg l−1 dissolved oxygen; 240 ppm CaCO3 alkalinity; 0.83 ± 0.10 mS cm−1 conductivity; and 8.44 ± 0.23 pH.Mean (±s.d.) concentrations of E2 during the exposure period were 38 ± 8 ng l3.2.F2,753 = 7.42, p = 0.001; figure 2a). Mean (±s.d.) BLs on day 22 post-hatch were 10.51 ± 2.11, 10.40 ± 1.97 and 9.89 ± 1.75 mm for control, E2LOW and E2HIGH subjects, respectively. Post hoc tests (LSD) indicated that E2HIGH subjects were significantly shorter than subjects in either the E2LOW (p = 0.003) or control treatments (p < 0.001). No difference in BL was observed between control and E2low individuals (p = 0.524).ANOVA revealed a significant effect of E2 on BL . Mean (±s.d.) escape latencies for control, E2LOW and E2HIGH subjects were 63.25 ± 65.48, 89.46 ± 84.16 and 91.69 ± 81.65 ms, respectively. Post hoc tests (LSD) indicated that subjects from both the E2LOW (p < 0.0001) and E2HIGH (p < 0.0001) treatments had significantly longer escape latencies than control subjects. There was no difference in the escape latency of subjects in the two E2 treatments . We also observed a significant effect of E2 on escape velocity . Escape velocities of subjects in the E2HIGH  and E2LOW treatments  were significantly slower than those in the control (0.025 ± 0.016 BL ms−1). However, subjects exposed to E2HIGH and E2LOW treatments did not differ in escape velocity (p = 0.299). We observed a similar effect of E2 on total escape performance . Total escape performances of control, E2LOW and E2HIGH larvae were 0.008 ± 0.008, 0.006 ± 0.007 and 0.006 ± 0.012 BL ms−1, respectively. The total escape performances of E2HIGH (p = 0.01) or E2LOW treatments (p = 0.03) were significantly reduced compared to control subjects. Escape performances of E2HIGH and E2LOW subjects did not differ statistically from each other (p = 0.750).ANOVA revealed a significant effect of E2 on escape latency  and E2HIGH  treatments was significantly lower than that of paired control subjects . The model conservatively assumed that the increase in the hourly mortality rate due to oestrogen was present for six hours each day because predatory fishes tend to feed sporadically during daylight [−2 ± 3.3 × 10−2 for larvae in the presence of oestrogen. This elevated rate of mortality was projected to cause a 60 ± 14% reduction in mean adult abundance in the presence of oestrogen, relative to the pristine condition (A reduction in mean (±s.d.) larval survival from 55.4 ± 12.9% to 44.5 ± 12.85% over two hours translated into a 1.34 ± 0.08-fold increase over the baseline, hourly instantaneous mortality rate estimated from the literature  during the embryonic stage was sufficient to delay the onset of the fast-start evasive response in 12-day-old post-hatch minnows. Alvarez et al. [Exposure to E2 altered the escape performance of larval fathead minnows in response to a simulated  predator. Compared to unexposed fish, exposed larvae demonstrated significantly delayed reaction times and reduced swimming velocities. Overall, exposure resulted in a 25% reduction in the total escape performance of larvae compared with unexposed fish. These findings are consistent with the results of previous studies examining the effects of EDCs on larval fishes ,65, inclpromelas , that de [et al.  examinedz et al.  similarlExposure to EDCs probably affects larval predator evasion behaviour via modulation of the hypothalamus–pituitary–gonadal (HPG) axis, which regulates cellular signalling . Endocri−1 was reduced by 11% or 10%, respectively, compared to unexposed subjects. Together with findings from the escape performance assays, our data suggest that both a delayed onset of the evasive response and a slower burst-speed swimming speed contributed to this result; Murphy et al. [et al. [Because the behavioural performance of an individual strongly influences the outcome of a predator–prey interaction, exposure to contaminants can be expected to alter the response of an individual in ways that directly impact the probability of surviving a predatory attack ,70, incly et al.  found th [et al.  reported [et al. .et al. [Salvelinus namaycush) following the collapse of forage species in response to EE2, probably due to the reduction in the availability of prey. Concentrations of E2 in the current study matched the total oestrogenic activity of EE2 used in Kidd et al. [−1 E2 used here. The prevalence of wastewater-dominated aquatic environments in urban settings suggests that many fish populations are adversely affected by oestrogenic EDC exposure, especially in habitats already degraded by multiple stressors.Incorporating empirically derived, exposure-induced mortality rates into a stochastic, stage-structured model suggests that altered predator–prey interactions resulting from the sub-lethal behavioural effects of EDC exposure during early ontogeny can lead to population declines. Early-life-stage mortality rates are important regulators of recruitment and year-class strength . Therefoet al.  documentd et al. ,12. It iAlthough our results are consistent with the literature, we highlight three caveats to our results. First, a limitation of this study was that predators were not exposed; thus, an assessment of the effects of exposure on the predator is beyond the scope of the study. However, larval fish are often more site-bound than roaming predators , and theWe have at present only a limited understanding of how the initial behavioural responses of individual organisms to environmental change translate into effects at higher levels of biological organization. However, as knowledge is gained regarding the mechanisms underpinning shifts in population abundances and distributions, it is apparent that disrupted interactions among species are primary drivers of human-mediated extinction, with declines in food availability being the most common cause . Biotic