Hepatocellular carcinoma (HCC) is a complex biological process and is often diagnosed at advanced stages with no effective treatment options. With advances in tumor biology and molecular genetic profiling, several different signaling pathways and molecular mechanisms have been identified as responsible for initiating and promoting HCC. Targeting these critical pathways, which include the receptor tyrosine kinase pathways, the Ras mitogen-activated protein kinase (Ras/Raf/MAPK), the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), the Wnt/β-catenin signaling pathway, the ubiquitin/proteasome degradation and the hedgehog signaling pathway has led to the identification of novel therapeutics for HCC treatment. In this review, we elaborated on our current understanding of the signaling pathways involved in the development and initiation of HCC and anticipate the potential targets for therapeutic drug development.  Hepatocellular carcinoma (HCC) accounts for 7% of all the cancers worldwide, and is the most common primary liver malignancy . It is aCurrent treatment options, including liver transplantation or surgical resections, can cure patients at early stages of HCC ,6. HowevHCC is a complex multistep biological process that involves genetic and epigenetic alterations . There hReceptors that activate multiple downstream signals in the receptor tyrosine kinase pathway include the epidermal growth factor (EGF) receptor, the fibroblast growth factor (FGF) receptor, the hepatocyte growth factor (HGF/ c-MET), the stem cell growth factor receptor c-KIT, the platelet-derived growth factor (PDGF) receptor, and the vascular endothelial growth factor (VEGF) receptor. Ligand binding and phosphorylation of these receptors, including the activation of the Grb2/Shc/SOS adapter molecule complex, lead to downstream activation of MAPK and PI3K pathways. Ras/Raf/MEK/ extracellular signal regulated kinase (ERK) pathway activation results in the activation of AP-1 transcriptional activators, cJun and cFos, which induce the transcription of genes that drive cell proliferation ,20,21. IThe liver, being a highly vascular organ, depends on effective angiogenesis for cellular regeneration. Likewise, in HCC, efficient angiogenesis is critical for tumor growth vascular invasion and metastasis . VEGF isIGF-2 is correlated with increased cell proliferation in the HCC transgenic mice model [IGF-2 ablation reduced cell proliferation and increased apoptosis in SK-hep-1, Hep G2, and Hep 3B HCC cells [Mutations in the EGFR signaling pathway appear to be rare in HCC pathogenesis, but it plays a significant role in tumor angiogenesis and proliferation. The IGF signaling pathway is activated by the binding of ligands (IGF-1 and IGF-2) to the membrane bound receptor (IGF-1R) . The IGFce model , whereasCC cells .HGF is a multifunctional cytokine that has been implicated in the invasion of tumor malignancies . The HGFGiven the contribution of this pathway during liver carcinogenesis, different compounds have been investigated at multiple levels to inhibit receptor tyrosine kinases. This includes inhibitors of the downstream activators like Raf and protein kinase C, as well as antibodies that bind to the receptors blocking ligand-receptor interactions. One such compound is BAY-439006, which inhibit multiple pathway components, Raf kinase and VEGF receptor, and initial clinical studies with this compound have shown partial responses in HCC patients . Other iThe Raf/MEK/ERK is a ubiquitous signal transduction pathway that regulates crucial cellular processes, including cell proliferation, differentiation, angiogenesis and survival . This paPTEN gene or reduced PTEN expression is observed in approximately half of all HCC tumors, suggesting that inactivation of PTEN is involved in the pathogenesis of HCC [In the PI3K/AKT/mTOR signaling pathway, the family of enzymes are activated by binding the of multiple growth factors (IGF and EGF) and cytokines to the receptor . Upon acs of HCC ,71. Furts of HCC . In a mis of HCC . mTORC1 s of HCC . Furthers of HCC . In a res of HCC . Recentls of HCC . Studiess of HCC . In anots of HCC . Currents of HCC . In precs of HCC . In the s of HCC . Rapamycs of HCC ,82. TakeMYC, MYB, CJUN and CYD1) angiogenesis , anti-apoptosis and the formation of the extracellular matrix [Iqgap2 gene in mice, which is associated with Iqgap1 overexpression and the activation of β-catenin and cyclin D1, lead to the development of HCC. Breeding of Iqgap2-null mice with Iqgap1-null mice resulted in reduced HCC phenotype suggesting that Iqgap2 acts as a tumor suppressor and its loss can lead to β-catenin activation and HCC development [In the Wnt/β-catenin signaling pathway, abnormal regulation of the transcription factor, β-catenin , play a major role in the development of early events in HCC. The Wnt/β-catenin signaling pathway is composed of Wnts, which are secreted cysteine-rich glycoprotein ligands ,84. The r matrix ,83,84. Ir matrix . Importar matrix , promotielopment . The crielopment , and theelopment . Currentelopment . In a stelopment . In anotelopment . The WntThe JAK/STAT pathway is activated by multiple cytokines, hormones and growth factors . It is iHippo signaling is an evolutionarily conserved pathway that modulates organ size and development by regulating cell proliferation, survival, differentiation and apoptosis . In mammThe ubiquitin-proteasome system (UPS) is involved in cellular protein degradation and is highly conserved in eukaryotic systems . This paCurrently, the hedgehog (HH) signaling plays a crucial role in tissue pattern, cell differentiation and proliferation. In humans, the HH pathway is activated by three ligands: Sonic Hedgehog (SHH), Indian hedgehog (IHH) and Desert Hedgehog, of which SHH is the most studied ,119. HH Targeting the tumor microenvironment is another issue. Even though the cells with self-renewing capacity in HCC is well supported, the origin of these cells is not clearly understood. Recently, growing evidence supports the novel notion that tumor initiation can be driven by a subset of cells with stem cell features. These cancer stem cells (CSCs) are responsible for not only tumor initiation, but also for tumor persistence, relapse, metastasis, chemoresistance and radioresistance . This ma+T cells is associated with better outcomes, whereas presence of regulatory T cells (Tregs) is correlated with poor prognosis [Liver is a lymphoid organ that receives blood from both the systemic circulation and intestine via the hepatic artery and portal vein, respectively. Since the liver is constantly exposed to exogenous antigens it maintains a unique immunological tolerance against invading pathogens. In HCC, tumor microenvironment has a complex interplay of T cells, B-cells, macrophages and hepatic cells to evade immune surveillance by activating multiple cellular signaling cascades. In tumors, the presence of CD8rognosis . In a strognosis . Anotherrognosis . An alterognosis . A studyrognosis . Recentlrognosis . Immunohrognosis . Recentlrognosis . A deepeHCC evidently has a complex pathogenesis with many critical signaling pathways being involved in its development. In addition to their independent signaling conduits, there are multiple levels of crosstalk interactions among these signaling pathways to regulate each other. With advances in our understanding of the molecular mechanism of tumorigenesis and in the development of the targeted therapeutics, we are at the beginning of a new era of applications of targeted therapeutics for the treatment of liver cancer. Different strategies that target these pathways showed a varied amount of success in the clinic. In addition, novel strategies have been investigated and raised the potential for more efficient therapies ,137,138.


Notch signaling coordinates numerous cellular processes and has been implicated in many pathological conditions, including rheumatoid arthritis (RA). Although the role of Notch signaling in development, maturation, differentiation, and activation of lymphocytes has been comprehensively reported, less is known about its role in myeloid cells. Certainly, limited data are available about the role of Notch signaling in macrophages during inflammation and infection. In this review, we discuss the recent advances pertaining to the role of Notch signaling in differentiation, activation, and metabolism of macrophages during inflammation and infection. We also highlight the reciprocal interplay between Notch signaling and other signaling pathways in macrophages under different inflammatory and infectious conditions including pathogenesis of RA. Finally, we discuss approaches that could consider Notch signaling as a potential therapeutic target against infection- and inflammation-driven diseases. Drosophila to mammals -S-phenylglycine t-Butyl ester (DAPT) attenuated NF-κB activity in LPS-induced macrophages [Macrophages express a variety of PRRs including TLRs that allow them to recognize and respond to the invading pathogens and direct the innate and adaptive immune responses ,43. Notcpression . Foldi epression . In contpression . Hu et apression . The bidpression . They alpression . Notch srophages . These frophages ,75,76,78rophages .Cytokines are potent signaling molecules that primarily function to orchestrate local and systemic immune response during health and disease . HoweverEmerging evidence indicates the contribution of Notch signaling in M1 macrophage polarization, leading to overexpression of TNF-α, iNOS, IL-6, and MCP-1 ,77. UsinDespite the overwhelming data that support the pivotal role of Notch signaling in macrophage polarization, the mechanisms by which Notch signaling implicating its effect still far to be elucidated. Xu et al., clearly demonstrated that Notch-RBP-J signaling promotes M1 macrophage polarization by directly regulated the expression of transcription factor interferon regulatory factor 8 (IRF8) . BesidesCC locus in the RBP-J gene, a key canonical Notch signaling mediator as one of RA susceptibility loci [RA is an autoimmune inflammatory joint disorder, characterized by macrophages and lymphocytes infiltration, synoviocyte hyperplasia, and progressive joint destruction . The rolity loci . Furtherity loci ,92, vascity loci , and perity loci  in patieity loci , and accity loci . Additioity loci ,95. Moreity loci . On the ity loci ,97,98. Iity loci ,100. TheMacrophages play a pivotal role in RA pathogenesis, evident by the numerous numbers and clear activation state of macrophages in synovial tissue, which are significantly correlated with disease severity ,102. Machigh monocyte subset of CIA mice, associated with increased arthritis severity and bone erosion. Whereas, in vivo delivery of miR146-a mimic to LY6Chigh monocyte rescue the bone erosion in CIA mice [Given the implication of Notch signaling in the pathogenesis of RA and the crucial role of Notch signaling in the polarization of the macrophages, Notch signaling seems to play a causal role in M1/M2 imbalance in RA, which significantly implies in RA pathogenesis. Using (TNF-α)-transgenic/(Hes-1)-GFP mice as RA model bearing Notch reporter transgene, Sun et al. identified M1 macrophages derived from bone marrow (BM) as the main cells with activated Notch signaling in the inflamed joint of (TNF-α)-transgenic mice. Additionally, they reported that RA synovial tissue promotes the activation of Notch signaling in BM-derived macrophages, leading to M1 polarization. While thapsigargin (Notch inhibitor) reduces TNF-α induced M1 macrophage polarization and attenuates inflammation and joint bone loss by switching M1 to M2 macrophages  Figure . In the CIA mice . InteresCIA mice . This clNumerous evidence supports the involvement of Notch signaling in the pathogenesis of many inflammatory conditions ,11,12,13Notch signaling is essential in all cellular processes, and its dysregulation has been linked to many pathological disorders including RA. A growing body of literature confirmed the involvement of Notch signaling in immunity through regulation of immune cells development and function. Although much remains unclear about the role of Notch signaling in macrophages, numerous lines of evidence support the important role of Notch signaling in macrophages inflammatory response to various stimuli. Interestingly, the role of Notch signaling in macrophages is not seemed ON/OFF effect, but it operated in an oscillatory feedback loop to intensify the inflammatory response and define their final outcome. As Notch signaling is easily amenable to pharmacological manipulation, targeting Notch signaling could offer a powerful and promising strategy to ameliorate inflammation in many pathological settings. Targeting Notch signaling has been shown to ameliorate inflammation and minimize associated tissue damage in many pathological conditions, including RA. However, there are still many challenges for using Notch signaling as therapeutic targets. Further studies are needed to elucidate the mechanistic role of various Notch receptors and ligands in various inflammatory conditions, which will provide a better understanding of how Notch signaling drives immune response under these conditions and offer a new therapeutic target to inflammatory disorders such as RA.


Nicotiana tabacum plants were compared with AOX knockdown and overexpression plants under normoxia, hypoxia (near-anoxia), and during a reoxygenation period following hypoxia. Paradoxically, under all the conditions tested, the AOX amount across plant lines correlated positively with leaf energy status (ATP/ADP ratio). Under normoxia, AOX was important to maintain respiratory carbon flow, to prevent the mitochondrial generation of superoxide and nitric oxide (NO), to control lipid peroxidation and protein S-nitrosylation, and possibly to reduce the inhibition of cyt oxidase by NO. Under hypoxia, AOX was again important in preventing superoxide generation and lipid peroxidation, but now contributed positively to NO amount. This may indicate an ability of AOX to generate NO under hypoxia, similar to the nitrite reductase activity of cyt oxidase under hypoxia. Alternatively, it may indicate that AOX activity simply reduces the amount of superoxide scavenging of NO, by reducing the availability of superoxide. The amount of inactivation of mitochondrial aconitase during hypoxia was also dependent upon AOX amount, perhaps through its effects on NO amount, and this influenced carbon flow under hypoxia. Finally, AOX was particularly important in preventing nitro-oxidative stress during the reoxygenation period, thereby contributing positively to the recovery of energy status following hypoxia. Overall, the results suggest that AOX plays a beneficial role in low oxygen metabolism, despite its lower affinity for oxygen than cytochrome oxidase.Alternative oxidase (AOX) is a non-energy conserving terminal oxidase in the plant mitochondrial electron transport chain (ETC) that has a lower affinity for oxygen than does cytochrome (cyt) oxidase. To investigate the role(s) of AOX under different oxygen conditions, wild-type (WT)  Electro10.3.11) , 2018a.2–) and nitric oxide (NO) (One consequence of AOX respiration is that it can influence the generation of reactive oxygen species (ROS) and reactive nitrogen species by the mitochondrial ETC. For example, studies have shown that chemical inhibition or genetic knockdown/knockout of AOX can increase mitochondrial amounts of such species, including superoxide (Oide (NO) .Studies using different plant species and tissues have shown that AOX amount can change in response to hypoxia, anoxia, or reoxygenation after a low oxygen treatment . The majIn the current study, we compared wild-type (WT) tobacco plants to both AOX knockdown and overexpression plants under different oxygen conditions, including normoxia, hypoxia (near-anoxia), and reoxygenation following hypoxia. Our results show that AOX amount influences leaf carbon and energy metabolism under all these conditions. This indicates that AOX has role(s) in low oxygen metabolism, despite its lower affinity for oxygen than cyt oxidase.Nicotiana tabacum L. cv. Petit Havana SR1) WT, AOX knockdown  and AOX overexpression  plants were grown for 1 month in controlled environment growth chambers  with a 14 h photoperiod, temperature of 26°C/20°C (light/dark), relative humidity of 50%, and photosynthetic photon flux density of 150 μmol m–2 s–1. Plants were irrigated every other day and fertilized every 2 weeks with a 20-20-20 fertilizer containing 5.9% of nitrate nitrogen. The transgenic lines used in this study have been characterized earlier (2) growth conditions, all these plant lines  display similar growth, respiratory rates, photosynthetic properties, and maximal cyt oxidase activities and protein amounts (Tobacco ( earlier . Amongst amounts .–1 (Alphagaz 1 grade having ∼0.001% oxygen) at one opening and unidirectional air valves facing outward in the openings to maintain ambient pressure within the chamber while preventing ambient air from entering . The amount of superoxide anion (O2–) in the supernatant was then measured at 550 nm by reduction of cyt c using a method modified from Fresh leaf biomass (100 mg) was crushed using a mortar and pestle over ice in 1 mL of 8 M KOH and then centrifuged  was crushed using a mortar and pestle over ice, homogenized for 30 min in 6% trichloroacetic acid (TCA) and then centrifuged . H2O2 content in the supernatant was determined using a Pierce Quantitative Peroxide Assay kit (Thermo Fisher Scientific).For Hg, 5 min, 4°C). A 1 mL aliquot of supernatant was combined with 4 mL of 20% TCA containing 0.5% TBA. This mixture was incubated , quickly cooled in crushed ice, and centrifuged . The absorbance of the sample was measured spectrophotometrically at 532 and 600 nm. The non-specific absorbance at 600 nm was subtracted from the absorbance at 532 nm and the concentration of MDA was calculated using an extinction coefficient of 155 mM–1 cm–1.Lipid peroxidation was measured as malondialdehyde (MDA) content, using the thiobarbituric acid (TBA) method described earlier , with miS-nitrosylation was performed by reducing R-SNO to R-SH in the presence of ascorbate and then assaying free thiol groups using 5,5’-dithiol-bis (2-nitrobenzoic acid) (DTNB)  and proteins in the supernatant were precipitated by two volumes of ice cold acetone (-20°C) overnight. The protein precipitate was collected by centrifugation  and washed four times with chilled 70% acetone before resuspension in the same volume of extraction buffer. The protein solution was separated into two 0.9 mL samples. One sample  was combined with 50 μL of 100 mM ascorbate and the other sample (control) was combined with 50 μl of water. After incubating for 1 h at 25°C, 50 μL of 10 mM DTNB in 75 mM phosphate buffer (pH 7.0) was added to each sample, and each were then measured spectrophotometrically at 412 nm. Samples without protein were used as blanks, and the difference between experimental and control samples was used to calculate the quantity of R-SNO. The total concentration of protein was measured using Bradford reagent .The measurement of protein ) (DTNB) . Leaf big, 10 min, 4°C). ATP and ADP in the supernatant were then measured using a luciferase-based bioluminescent assay  and FB 12 Single Tube Luminometer by Titertek-Berthold .The extraction of ATP and ADP was modified from 2WO4). The leaves in buffer were then placed in an air tight chamber as described earlier with a constant inflow of nitrogen gas at 120 mL min–1 (0.001% O2). NO was measured by chemiluminescence detection  by a vacuum pump connected to an ozone destroyer. The ozone generator of the chemiluminescence detector was supplied with dry oxygen (99%). The inflowing gas (air or nitrogen) was made NO free by passing it through a NO scrubber . Calibration was routinely carried out with NO free air (0 ppt NO) and with various concentrations of NO (1–35 ppb) adjusted by mixing the calibration gas  with NO-free air. Flow controllers (Fisher Scientific) were used to adjust all gas flows.Leaves were detached, weighed and immediately placed in 20 mM Hepes buffer (pH 7.0) containing 50 mM sodium nitrate. When inhibitors were used, this buffer also contained either 2 mM potassium cyanide (KCN), 5 mM salicylhydroxamic acid (SHAM) or 300 μM sodium tungstate . The extract was centrifuged  and the supernatant was then centrifuged again . The pellet (containing mitochondria) was then resuspended in a buffer containing 50 mM Tris–HCl (pH 7.4), 2 mM trisodium citrate, 5 mM dithiothreitol and 100 mM MgCl2 and used to measure mitochondrial aconitase activity. The supernatant was also collected and used to measure cytosolic aconitase activity. Cross-contamination between the mitochondrial and cytosolic fractions was determined using marker enzymes , as described earlier (–1 cm–1 was used for cis-aconitate (Fresh leaf biomass (100 mg) was homogenized in 1 mL of cold extraction buffer . Samples were then freeze-dried for 48 h before being homogenized in heavy water (D2O) using a ceramic mortar. After incubation for 24 h at room temperature, the solutions were centrifuged and an aliquot of the supernatant (aqueous extract) with a volume of ∼0.6 mL was placed in a vial for NMR analysis. Subsequently, 140 μL of a standard buffer solution , 5.84 mM 2-chloropyrimidine-5 carboxylate, and 0.1% NaN3 in H2O) was added to the sample. The plant sample (350 μL) was then transferred to a 3 mm SampleJet NMR tube for subsequent spectral analysis (1H-NMR spectra were collected on a 700 MHz Avance III (Bruker) spectrometer equipped with a 5 mm HCN Z-gradient pulsed-field gradient (PFG) cryoprobe. 1H-NMR spectra were acquired at 25°C using the first transient of the NOESY pre-saturation pulse sequence (noesy1dpr), chosen for its high degree of quantitative accuracy. All FID’s (free induction decays) were zero-filled to 250 K data points. The singlet produced by the DSS methyl groups was used as an internal standard for chemical shift referencing (set to 0 ppm) and for quantification. All 1H-NMR spectra were processed and analyzed using the Chenomx NMR Suite Professional software package version 8.1 . The Chenomx NMR Suite software allows for qualitative and quantitative analysis of an NMR spectrum by manually fitting spectral signatures from an internal database to the spectrum. Typically, 90% of visible peaks were assigned to a compound and more than 90% of the spectral area could be routinely fit using the Chenomx spectral analysis software. Most of the visible peaks are annotated with a compound name.Metabolomics was performed using NMR analysis . Leaf tianalysis . All 1H-g, 20 min, 4°C). The residue was extracted twice again under the same conditions and the supernatants were combined.Soluble phenolics and flavonoids were extracted from leaves by homogenizing in 80% (v/v) acetone with 0.2% (m/v) formic acid in a ratio of 1:10. The homogenate was then shaken  and then centrifuged . The absorbance was measured at 725 nm. Total soluble phenolic content of each sample was determined using a gallic acid standard curve and expressed as gallic acid equivalents (GAE) per leaf fresh weight.Total soluble phenolic content was determined using Folin-Ciocalteu reagent as described by 2, 0.3 mL of 10% (m/v) AlCl3 (after 5 min) and 2 mL of 1 M NaOH (1 min later). The volume was adjusted with water to 10 mL. The absorbance was measured spectrophotometrically at a wavelength of 510 nm. Total flavonoid content was expressed as catechin equivalent (CE) per leaf fresh weight.Total flavonoid content was measured by aluminum chloride colorimetric assay . The aboAll experiments were repeated at least three times. Statistical analyses were performed using Prism 5.0 (GraphPad Software). Two-way ANOVA analyses were followed by a Bonferroni post-test to compare plant lines within a treatment.Wild-type, AOX knockdown  and AOX overexpression  tobacco plants were compared in an experiment in which potted plants were subjected to either a 4 h normoxia  treatment, a 4 h hypoxia  treatment, or a 4 h hypoxia treatment followed by a reoxygenation  treatment for 15 min or 120 min. Following treatment, leaf tissue was collected for a number of analyses, the results of which are described below.2– amount was similar between WT and overexpression plants, but significantly higher in the knockdowns  of reoxygenation. However, the AOX overexpressors also had higher H2O2 amounts than WT following hypoxia and after 15 min of reoxygenation (The hypoxia treatment increased Hreatment . Followigenation .S-nitrosylation (R-SNO) was significantly lower in the AOX overexpressors, compared to the WT and knockdowns  was lowest in the normoxia-treated plants. Under this condition, WT and overexpression plants displayed similar amounts, while the knockdowns displayed significantly higher amounts . The hypUnder normoxia, the ATP/ADP ratio was similar between the WT and AOX knockdown plants, but slightly higher in the overexpressors . In respInterestingly, following the hypoxia treatment, the overexpression plants were more visibly upright, healthy and robust than the WT and knockdown plants, an observation that may relate to some of the above factors (such as energy status) but which requires further investigation .Nitric oxide emission rates were measured from leaves incubated in nitrate solution and in an atmosphere containing 0.001% oxygen. Under these hypoxic conditions, NO emission rate was significantly higher in AOX overexpressors and significantly lower in AOX knockdowns, compared to WT . FurtherWhen leaves were pre-treated with the AOX inhibitor SHAM, the rates of NO emission decreased in all plant lines . However2. The absolute rates (in units of ppb NO g–1 FW h–1) were: B7, 5.69; WT, 5.65; and RI9, 3.87. Overall, these rates are much lower than measured at 0.001% O2  treatment. Under normoxia, the mitochondrial  and organic acids  in leaves were similar between the WT and AOX overexpressor (B7)  were identified and quantified using a sor (B7) . In mostsor (B7) . The AOXsor (B7) .Under normoxia, the amount of most amino acids measured was similar between the WT and AOX knockdown, but serine and glycine amounts were significantly higher in the knockdown . The amo2) treatment. Under normoxia, phenolics  a normoxia  treatment; (2) a short-term (3–4 h) hypoxia (near-anoxia) treatment; (3) a reoxygenation period (of 15 or 120 min) following a hypoxia treatment. The discussion below deals with each of these treatments in sequence.2– . One possibility is that the aberrant amount of ROS generation by the mitochondrial ETC can signal changes in the capacity of ROS-scavenging systems within the cell, leading to unexpected changes in H2O2 amount. This was reported before in tobacco plants with altered AOX amount. In one study, overexpression of AOX dampened the induction of scavenging systems, resulting paradoxically in an increase in H2O2 amount (2–) rather than H2O2, the amount of which will depend upon the secondary processes associated with its formation and scavenging. Interestingly, the overall pattern of H2O2 amount across plant lines mirrored the pattern of antioxidants , which also tended to be higher in both RI9 and B7, relative to WT  did differ across the plant lines under normoxia  of the different plant lines. Paradoxically, the ATP/ADP ratio was significantly higher in the AOX overexpressors than WT . It was 2–, 2– amount to the WT  could be explained in this manner. Inhibition of AOX in these plants by SHAM would increase O2– generation and hence NO scavenging.While aberrant amounts of NO emission in the knockdown and overexpression plants  is consioxidases . Since A hypoxia , then th hypoxia  could beAs discussed earlier, under normoxia, knockdown of AOX altered the abundance of the majority of the metabolites measured in this study (9 out of 16 metabolites differed significantly from WT), while overexpression of AOX had relatively little effect (2 out of 16 metabolites differed significantly from WT) . HoweverOne site of disruption of carbon metabolism in the overexpressor following hypoxia was at mitochondrial aconitase. The overexpressor retained only 5% of this activity following hypoxia (relative to normoxia), while the WT and knockdown retained 20 and 61%, respectively . AconitaIn summary, AOX clearly remains relevant at very low oxygen concentrations, despite its much lower affinity for oxygen than cyt oxidase. AOX activity under these conditions supports energy generation, at a time when mitochondria-generated NO is inhibiting cyt oxidase. AOX prevents excessive nitro-oxidative stress under these conditions.2– amounts remained low in the WT and overexpressors (particularly the overexpressors), while the knockdown plants showed high amounts of O2–. In fact, the differences in O2– amount across plant lines during the reoxygenation period were as great as, or greater, than seen under hypoxia. As a result, the differences in oxidative damage (MDA amount) across plant lines was greater following 120 min of reoxygenation than with any other treatment  of all plant lines improved relative to hypoxia, suggestive of a rapid recovery of ETC activity . Nonethereatment . Overallreatment . FurtherS-nitrosylation that was specific to the AOX knockdowns (Reoxygenation resulted in a spike in protein ockdowns . These rockdowns .In summary, reoxygenation represents a condition in which AOX appears particularly important in preventing excessive nitro-oxidative stress. In doing so, AOX improved the ability of leaf tissue to rapidly recover its energy status.The results show that AOX amount influences leaf carbon and energy metabolism under normoxia, hypoxia, and during a reoxygenation period following hypoxia. Under all these conditions, an optimal AOX amount was necessary to support metabolism and to prevent excessive nitro-oxidative stress.The datasets generated for this study are available on request to the corresponding author.JJ performed the experiments on normoxia, hypoxia, and reoxygenation including NO measurements, participated in discussion of results, and contributed to writing the manuscript. DC determined the aconitase activity, prepared the samples for metabolomics analysis, analyzed the results, and contributed to writing the manuscript. PV determined the phenolic and flavonoid content, and prepared the samples for metabolomics analysis. NB planned and supervised the experiments, analyzed the results, and contributed to writing the manuscript. GV developed the transgenic lines, analyzed the results, and contributed to writing the manuscript and shaping its final version. AI supervised the design, execution, and interpretation of the experiments, and prepared the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite the advancing treatments, female breast cancer is one of the most common cancers and a leading cause of cancer deaths in women. To help broaden the therapeutic spectrum of breast cancer, we identified the natural compound ursolic acid (UA) as a potentiator that enhances the oncolytic activity of measles virus (MV) against breast cancer cells through the induction of apoptosis. In addition, to increase clinical applicability, we further generated UA nanoparticles that achieved improved solubility. UA nanoparticles similarly synergized with MV in killing breast cancer cells by triggering apoptosis, and this synergistic anticancer effect was also observed in various breast cancer cell types. This study demonstrates for the first time that UA and its nanoparticles enhance MV’s oncolytic activity in breast cancer cells, suggesting that such combinations may be worth further exploring as an anticancer strategy against breast cancer.Oncolytic viruses (OVs) and phytochemical ursolic acid (UA) are two efficacious therapeutic candidates in development against breast cancer, the deadliest women’s cancer worldwide. However, as single agents, OVs and UA have limited clinical efficacies. As a common strategy of enhancing monotherapeutic anticancer efficacy, we explored the combinatorial chemovirotherapeutic approach of combining oncolytic measles virus (MV), which targets the breast tumor marker Nectin-4, and the anticancer UA against breast adenocarcinoma. Our findings revealed that in vitro co-treatment with UA synergistically potentiated the killing of human breast cancer cells by oncolytic MV, without UA interfering the various steps of the viral infection. Mechanistic studies revealed that the synergistic outcome from the combined treatment was mediated through UA’s potentiation of apoptotic killing by MV. To circumvent UA’s poor solubility and bioavailability and strengthen its clinical applicability, we further developed UA nanoparticles (UA-NP) by nanoemulsification. Compared to the non-formulated UA, UA-NP exhibited improved drug dissolution property and similarly synergized with oncolytic MV in inducing apoptotic breast cancer cell death. This oncolytic potentiation was partly attributed to the enhanced autophagic flux induced by the UA-NP and MV combined treatment. Finally, the synergistic effect from the UA-NP and MV combination was also observed in BT-474 and MDA-MB-468 breast cancer cells. Our study thus highlights the potential value of oncolytic MV and UA-based chemovirotherapy for further development as a treatment strategy against breast cancer, and the feasibility of employing nanoformulation to enhance UA’s applicability. Female breast cancer is the most commonly diagnosed adenocarcinoma globally , accountNanoparticle drug formulation is a popular strategy as it efficiently improves in vivo drug solubility, bioavailability, and bioactivity, especially for phytocompounds , a safe The use of OVs as cancer nanomedicine, oncolytic virotherapy, is another promising therapeutic strategy that selectively targets and destroys cancer cells , via dirDespite excellent safety profile and preclinical anticancer potential, many oncolytic viruses including MV exhibit modest efficacy as a single agent . Combina50) value of UA was 16.67 ± 1.10 µM for the 5-day treatment. As for oncolytic MV, which is known to highly infect MCF-7 cells due to their upregulated Nectin-4/PVRL4 expression [50 indices (≤10 µM for UA and MOI 0.1–0.01 for MV) for the subsequent experiments devised for exploring their potential synergistic activities.We first examined the individual anticancer effect of UA and oncolytic MV on human breast cancer cell line MCF-7 by testing a range of drug concentrations or viral multiplicity of infection (MOI) for 5 days and evaluating their effects on cell viability. UA was prepared in DMSO solvent as it is insoluble in water. Following UA treatment, cell viability decreased gradually to 75% as UA concentration increased from 2 to 15 µM, and 20 µM UA sharply reduced cell viability to 20% A. The 50pression , cell viTo determine whether UA and oncolytic MV would exert stronger potency when used in combination, both agents were concurrently added to MCF-7 cells. Data obtained from the cell viability analysis in the ensuing incubation was then assessed by the Chou–Talalay method , where tWhile UA and MV co-treatment demonstrated synergy, precaution was taken to further examine whether UA would interfere with the oncolytic MV infection by evaluating its effect on the viral infectivity through a series of experiments, each focused on a specific stage of MV infection in MCF-7. For the early viral entry stages, three assays were performed to assess the impact of UA on (i) free oncolytic MV particles A, (ii) vCell cycle and apoptosis analyses were next performed to study the mechanism underlying the anticancer activity of UA and oncolytic MV combinatorial treatment on MCF-7 cells. Compared to cells treated with UA or MV alone, the combinatorial treatments caused synergistic elevation in the population of sub-G1 phase cells to approximately 25% and 60% for MOI 0.01 and MOI 0.1 of oncolytic MV, respectively A. IncreaAs UA is a triterpenoid compound with poor water solubility and low bioavailability in vivo, which limit its applicability, we next employed nanoemulsification using the nonionic polymer polyvinylpyrrolidone (PVP) to generate water soluble PVP-based UA nanoparticles (UA-NP) as a strategy to improve these issues . Physico50 value was found to be 36.52 ± 1.02 µM , in which it is not soluble, was also tested in MCF-7 cells. As shown in  1.02 µM B. The po 1.02 µM . Their c 1.02 µM D, indicaFinally, cell cycle analysis showed that the combinatorial treatments of UA-NP and oncolytic MV, at MOIs 0.01 and 0.1, similarly enriched sub-G1 phase populations to 46% and 69%, respectively A. With tUA has been reported to induce autophagy in MCF-7 cells at the range of concentrations that we used , and intLastly, we examined whether the combined treatment using oncolytic MV and UA-NP could also exert enhanced killing effect on other breast cancer cell lines. To this end, we tested each agent, alone or their combination, in the human breast cancer BT-474 and MDA-MB-468 cells. While MCF-7 represents luminal type A breast cancer , BT-474 represents luminal type B  , and MDAAltogether, our results demonstrated that UA-NP and oncolytic MV could be used in combination to induce an enhanced cell death in the breast cancer cells.Treatment toxicities and limited efficacy against metastases in the current standard breast cancer therapies, particularly the chemotherapeutics, have raised longstanding concerns. Most first-line drugs for breast cancer are known to cause severe adverse effects, such as taxane-induced peripheral neuropathy, anthracyclines- or trastuzumab-induced cardiotoxicity, drug-induced alopecia, everolimus-induced mucositis, CDK4/6 inhibitor-induced neutropenia and diarrhea, PARP inhibitor-induced neutropenia and anemia, and receptor tyrosine kinase inhibitor-induced diarrhea . TherefoIndividually, both oncolytic MV and UA have significant potential against breast cancer. The newly discovered oncolytic MV receptor and tumor marker Nectin-4 is selectively overexpressed and identified as an important therapeutic target for primary and metastatic triple-negative breast cancers as well as for lung, bladder, and pancreatic cancers ,39,40,41−4 µM [50 concentration (16.67 ± 1.10 µM) against MCF-7 cells when completely dissolved in DMSO. In view of the caveats of poor water solubility and, hence, low bioavailability of UA, we have successfully generated nanoformulated UA (UA-NP) with improved drug dissolution profile. Biophysical examination by FESEM showed micrometer needle-shaped UA crystals that were rendered into the nanoscale UA particles  was obtained from Dr. Roberto Cattaneo  and propagated in marmoset B lymphoblastoid cells (B95a) as previously described [50) assay was used to determine the viral titer, and virus concentrations were represented by MOI. Ursolic acid (UA) and polyvinylpyrrolidone (PVP) were purchased from Sigma-Aldrich Chemicals Co. . All other experimental reagents were of analytical grade.Human breast adenocarcinoma cell lines MCF-7 , BT-474 , and MDA-MB-468  were maintained in Dulbecco’s Modified Eagle medium , supplemented with 10% fetal bovine serum , 100 U/mL of penicillin G, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin B (GIBCO-Invitrogen) in a 5% COescribed . The 50%4 cells per well) were treated with various concentrations of the test agent over a 5-day incubation at 37 °C. Cell viability was then determined according to the manufacturer’s protocol, and optical densities (OD) were recorded at 550 nm using an ELISA plate reader and calculated as follows: Cell viability (%) = (Absorbance test compound/Absorbance control) × 100%. The 50% cytotoxic concentration (CC50) value was then calculated using the GraphPad Prism 7 software . Additional analysis using lactate dehydrogenase (LDH) cytotoxicity detection kit  is described in the The impact of treatment on breast cancer cell viability was assessed using the 3--2,5-diphenyltetrazolium bromide (MTT) cell viability assay kit . Briefly, cells seeded in 96-well plates (104 cells per well) were infected with MV (MOI 0.01 or 0.1) and concurrently treated with drug. The MV infection was performed for 1.5 h at 37 °C, and cells were washed with phosphate buffered saline  before and after viral challenge. Subsequently, cells were incubated in fresh media containing the drug at 37 °C for 5 days. Cell viability was determined via the MTT or LDH assay as described above. The impact of drug-virus co-treatment on the cancer cell viability was determined by calculating the combination index (CI) value using the Chou–Talalay method with the equation CI = (D)1/(Dx)1 + (D)2/(Dx)2, where (Dx)1, (Dx)2 are the respective concentrations of drug 1 and drug 2 used in their single treatments that decrease the cell viability by x%, and (D)1, (D)2 are the respective concentrations of drug 1 in combination with drug 2 that together decreased the cell viability by x% [To determine the impact of drug-virus co-treatment, cells seeded in 96-well plates  for 1.5 h. Cells were then washed with PBS and incubated in fresh 2% FBS DMEM for 3 days at 37 °C. (ii) The virus-drug inoculum containing MV (MOI 0.1) and varying concentrations of UA  were prepared and added to pre-chilled (at 4 °C) MCF-7 cells seeded in 96-well plates (104 cells per well) for 1.5 h at 4 °C, before removing the virus-drug inoculum, washing with PBS, and further incubating for 3 days at 37 °C in fresh 2% FBS DMEM. (iii) MCF-7 cells seeded in 96-well plates (104 cells per well) were first pre-bound with MV (MOI 0.1) at 4 °C for 1.5 h, which allows for virus binding but precludes internalization [The impact of UA treatment on the early MV viral entry steps, including (i) free virus particles, (ii) viral attachment, and (iii) viral penetration was examined as previously described  with somlization . This wa4 cells per well of 96-well plates). In pre-treatment analysis, cells were pre-treated with UA  for 24 h after which the supernatants were removed and cells were challenged with MV (MOI 0.1) for 1.5 h at 37 °C. Viral inoculum was later removed and replaced with fresh 2% FBS DMEM for 3 days’ incubation at 37 °C. In co-addition analysis, MV (MOI 0.1) and UA  were concurrently added to the cells for 1.5 h before the virus-drug inoculum was discarded and replaced with fresh 2% FBS DMEM for 3 days’ incubation at 37 °C. In post-infection analysis, cells were infected with MV (MOI 0.1) for 1.5 h, followed by removal of viral inoculum before incubating the cells with 2% FBS DMEM containing UA  for 3 days at 37 °C. For all assays, supernatants were collected at the end of the 3-day incubation for viral titration using TCID50.Drug addition at different time-points  were performed as previously described  to inves5 cells per well of 6-well plates) were treated with MV (MOI 0.01 or 0.1) and the drug, individually or concurrently, for 1.5 h at 37 °C. The supernatants were subsequently removed and the treated cells were refreshed with 2% FBS DMEM with or without the drug for 5 days’ incubation at 37 °C. The cells were then trypsinized, collected into 15 mL tubes, washed twice with ice-cold PBS by centrifugation, and finally fixed with 70% ethanol overnight at 4 °C. After fixation, the cells were washed twice with PBS by centrifugation prior to 30 min incubation in PBS solution containing 10 mg/mL ribonuclease A from bovine pancreas  at 37 °C. Propidium iodide  was subsequently added to the cells for 15 min incubation in the dark at 37 °C before subjecting to flow cytometric cell cycle analysis using the Beckman Coulter FC500 flow cytometer .Cells (3 × 105 cells per well) seeded in 6-well plates were treated with MV (MOI 0.01 or 0.1) and the drug, individually or concurrently, for 1.5 h at 37 °C. The supernatants were then removed before refreshing the cells with 2% FBS DMEM with or without the drug for 5 days’ incubation at 37 °C. Subsequently, the cells were trypsinized for collection into 15 mL tubes, ice-cold PBS-washed twice by centrifugation, and finally resuspended in binding buffer containing 1 µl/mL PI and 1 µl/mL allophycocyanin (APC)-conjugated Annexin V . Apoptosis detection via flow cytometry was then performed using the Beckman Coulter FC500 apparatus (Beckman Coulter Inc.).Cells (3 x 105 cells per well) seeded in 6-well plates were treated with MV (MOI 0.01 or 0.1) and the drug, individually or concurrently, for 1.5 h at 37 °C. The virus-drug inoculum was then discarded and the cells were refreshed with 2% FBS DMEM with or without the drug for a 5-day incubation at 37 °C. The cells were subsequently lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing protease inhibitor , and protein concentrations were measured by bicinchoninic acid (BCA) protein assay kit . Protein samples were then subjected to standard western blot analysis and proteins were probed using primary antibodies for PARP  and β-actin  followed by anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies . For autophagy analysis, cells seeded in 12-well plates (2.5 × 105 cells per well) were treated with UA-NP (30 μM), MV (MOI 0.1), or concurrently treated with both agents for 48 h before being harvested and analyzed for LC3 , p62 , and β-actin  expression using western blotting. Bafilomycin A1  was added to the indicated groups 4 h before harvesting the cells. MV H protein was probed to indicate MV infection using a rabbit anti-MV H serum H606 [For PARP analysis, cells  to remove aggregates. The filtrate containing UA-NP was collected and stored at 4 °C for immediate use, or lyophilized and then stored at −20 °C in a moisture-proof container for longer period storage.The UA loaded nanoparticles (UA-NP) were prepared using the emulsion-solvent diffusion technique as previously described . UA was Mean size of UA-NP was measured by the Malvern Zetasizer . The temperature was maintained at 25 °C and the test sample was diluted 50-fold using deionized water before measurement. Each determination was performed in triplicate.® Purospher® STAR; Merck KGaA; Darmstadt, Germany). The mobile phase consisted of acetonitrile and 0.1% phosphoric acid . The analytical process was carried out under a flow rate of 1 mL/min for 15 min with UV detection at 210 nm. The calibration curve was linear over the concentration range of 0.45–90 μg/mL with a coefficient estimate of 0.999.HPLC was performed using the Hitachi D-7000 HPLC system  with a reverse-phase C18 column  × VUA-NP (ml)/WUA (μg)) × 100Yield (%) = (CUA (μg/mL): concentration of UA detected in UA-NPCUA-NP (ml): final volume of UA-NPVUA (μg): quantity of UA used in the preparation for nanoformulationWThe morphological characteristics of the UA-NP were imaged using FESEM as previously reported . UA-NP sAn X-ray diffractometer with Cu-Kα radiation and Ni filter  was used to examine the crystalline properties of UA, PVP, UA-PVP physical mixture (UA-PM), and lyophilized UA-NP powder. UA-PM was prepared by thoroughly mixing UA and PVP with a mortar in the same composition ratio as for UA-NP, which was 1:1 in weight. Prior to XRD analysis, all samples were dried overnight to remove moisture. XRD patterns were obtained at 40 kV and 25 mA, with a scanning rate of 4°/min, over the diffraction angle (2θ) range of 2° to 50°.n = 6). During the experiment, 1 mL of sample was withdrawn at successive time intervals . The concentration of UA was analyzed by HPLC and the data acquired were calculated and converted into “percent of dissolved amount” to represent the drug dissolution property.The dissolution test was performed based on the United States Pharmacopeia (USP) apparatus II (paddle) method  in accorp < 0.05 was considered statistically significant.All data are expressed as means ± the standard error of means (SEM) unless otherwise indicated. Statistical significance was evaluated using GraphPad Prism (GraphPad Software), by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison, and a In conclusion, we demonstrated for the first time that UA and nanoformulated UA-NP can work synergistically with oncolytic MV in combinatorial treatments against breast cancer cells, by effectively enhancing induction of apoptotic cell death, thus facilitating improved anticancer efficacy as compared to mono-treatment with either agent alone. Our results also showed that combination of UA-NP with MV exerts synergistic killing effect against various breast cancer cell types, including luminal type A, luminal type B, and TNBC. This observation indicates that oncolytic MV chemovirotherapy using UA-based combinations merit further investigation and development as a treatment strategy for the management of breast cancers. Future studies should also explore whether such combinations would also be effective against other Nectin-4-positive adenocarcinomas.


Biased signaling is a concept that has arisen in the G protein-coupled receptor (GCPR) research field, and holds promise for the development of new drug development strategies. It consists of different signaling outputs depending on the agonist’s chemical structure. Here we review the most accepted mechanisms for explaining biased agonism, namely the induced fit hypothesis and the key/lock hypothesis, but we also consider how bias can be produced by a given agonist. In fact, different signaling outputs may originate at a given receptor when activated by, for instance, the endogenous agonist. We take advantage of results obtained with adenosine receptors to explain how such mechanism of functional selectivity depends on the context, being receptor-receptor interactions (heteromerization) one of the most relevant and most studied mechanisms for mammalian homeostasis. Considering all the possible mechanisms underlying functional selectivity is essential to optimize the selection of biased agonists in the design of drugs targeting GPCRs. Biased signaling consists of different signaling outputs depending on the agonist chemical structure. The concept has taken hold in the field of G protein-coupled receptor (GPCR) research and has opened up new perspectives for therapeutic drug development. The underlying idea is that a given agonist biased towards a particular signaling may be therapeutic while another agonist biased towards activating an alternative pathway may not be helpful, and may even be harmful.2AR antagonist, istradefylline , has been approved for chronic use in the therapy of Parkinson’s disease  whereas beta-adrenergic blockers/antagonists are used for a variety of diseases in both acute and chronic regimes. In the purine field, adenosine is used in bolus administration to combat paroxysmal tachycardia whereas the adenosine A disease .Two complementary points of view are needed to underline the mechanisms underlying differential signaling arising from a given GPCR. In a previous paper we already made a distinction between biased signaling and biased functionality . Here weThe resolution of the structure of various GPCRs and the molecular dynamics of macromolecules in aqueous solutions give indications as to how GPCR-mediated signaling occurs. Binding of the agonist to the orthosteric site leads to significant structural rearrangements that are transmitted to the coupled G protein and allow signaling .It is not necessary to be very specific with the details to explain the basis of the most accepted mechanism underlying biased signaling. In fact, assuming that GPCRs have a loose orthosteric center, the binding of structurally different chemicals to the site can result in different conformations  up. SaidFollowing the analogy with substrate/enzyme interaction, there is another point of view which is that the cell surface receptor is in different conformational states while waiting for the arrival of the agonist. Similar to the key/lock idea  center, In summary, in the classical view each agonist favors a specific signal transduction and that this may be due to two conceptually different mechanisms. One is by assuming different receptor states due to pre-coupling to signaling mechanisms and each chemical structure preferentially binding to a given state, thereby preferentially engaging such particular signaling pathway. The second is by assuming the GPCR in a given state that, after agonist-induced conformational changes, would lead to a receptor prone to interact to (and engage) a particular signaling machinery.Biased agonism fits into a more general framework, called functional selectivity. A given GPCR may provide different signaling outputs depending on the context. In other words, functional selectivity may be afforded using a single agonist. Yet another way to express the idea is that the endogenous agonist (hormone/neurotransmitter) will give rise to different signals depending on the cell/tissue and the general pathophysiological state.We argue, as suggested elsewhere , that bi1 and D2 receptors are, respectively, Gs and Gi  signaling pathway. Surprisingly, it appears that D1 receptors within this heteromeric context bind to Gi rather than its cognate G protein, Gs.  pathway. Also ß-arrestin recruitment may lead to receptor internalization and intracellular signaling  receptor function  what is the signaling pathway to target 2) how to reliably measure the pathway output and 3) what is the status of the target GPCR. Status means identifying the proteins/receptors that interact with the target GPCR and how that macromolecular complex is specifically coupled to the signaling machineries.The hopes placed on the biased agonism to give an extra boost to the drug discovery are not being fulfilled. For the above reasons, a biased agonist may provide a benefit in a given setting but provide a detrimental effect in other receptor settings and thus not be useful in therapy. Also relevant is how to reliably measure the output of the signaling pathway one wants to target; in fact, different assays claiming to evaluate the same pathway may produce a different result, some suggesting bias, some not. One wonders if it would be more proactive to skip The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.RF planned the contents and discussed them with RR‐S, IR-R, and GN. RF, and GN wrote a first draft. RR‐S, and IR-R made significant additions to the draft. IR-R made the figure. All authors have approved the submitted version.This research was funded by the Spanish Ministry of Economy and Competitiveness  and the Alzheimer’s Association (grant: AARFD-17-503612). The laboratory of the University of Barcelona is considered of excellence (grup consolidat #2017 SGR 1497) by the Regional Catalonian Government, which does not provide any specific funding for reagents or for payment of services or for Open Access fees.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The vascular barrier between blood and tissues is a highly selective structure that is essential to maintain tissue homeostasis. Defects in the vascular barrier lead to a variety of cardiovascular diseases. The maintenance of vascular barriers is largely dependent on endothelial cells, but the precise mechanisms remain elusive. Recent studies reveal that primary cilia, microtubule-based structures that protrude from the surface of endothelial cells, play a critical role in the regulation of vascular barriers. Herein, we discuss recent advances on ciliary functions in the vascular barrier and suggest that ciliary signaling pathways might be targeted to modulate the vascular barrier. Blood vessels are found throughout the body to deliver oxygen and nutrients to body tissues, and remove metabolic waste and carbon dioxide from tissues. In some organs in mammals, there are distinct boundaries between blood and tissues. These barriers separate blood from tissues and are composed of endothelial cells, pericytes, other vascular mural cells, and intercellular connections. In contrast to the common vascular barriers, there are extremely strict vascular barriers in some important organs, such as the blood-brain barrier , the bloThe vascular barrier has been shown to be regulated by a number of signaling pathways, such as Mfsd2a, lipolysis stimulated lipoprotein receptor, angiopoietin-2, Wnt, and Norrin pathways . In addiVascular endothelial cells play an important role in vascular barrier function. Recent studies demonstrate that primary cilia are present on vascular endothelial cells with typical “9 + 0” axonemes . Primary2+ is an important second messenger that regulates many signaling pathways and regulates vasoconstriction and vasodilation through NO in vascular endothelial cells. A number of previous studies have shown that fluid shear stress triggers ciliary calcium signaling, and this process is mediated by polycystin 2 (PC2)  . Recentl of Ca2+ . Thus, tDifferent blood flow or velocity has different effects on endothelial cells, resulting in changes in blood vessel phenotypes and functions, a process called hemodynamic stimulation . The priIft172 mutation  and Wnt signaling . The modmutation . Normal mutation . Therefomutation . IFT is mutation .Ift172 and Ift122 mutants show cranial neural tube defects and bleeding (Ift88 mutant mice with polycystic kidney disease (PKD). Compared with wild-type cells, Ift88 mutant endothelial cells have reduced stress fibers and focal adhesion, and higher permeability to dextran, indicating that cilium defects cause damage to the vascular barrier. The expression of heat shock protein 27 in Ift88 mutant endothelial cells is inhibited by approximately 90%, and the phosphorylation level of its downstream target, focal adhesion kinase, is significantly reduced  mutations cause autosomal dominant PKD, and vascular abnormalities are usually observed in these patients. Due to vascular leakage and hemorrhage, PKD1 knockout mice are embryonic lethal at embryonic day 15.5 (PKD2 (the gene encoding PC2) mutations demonstrate no bleeding phenotype , is another protein related to cilia that show a correlation with the vascular barrier. PC1 usually forms a channel-receptor complex with PC2. These transmembrane proteins regulate cell activity by detecting external stimuli and transmitting calcium-mediated signals . PC1 is day 15.5 . Howeverhenotype , suggestHedgehog signaling is critical for vascular development, maturity and integrity , and is The zebrafish model study shows that when the HH signaling is inhibited due to ciliary defects, the possibility of intracranial hemorrhage increases. Conversely, activation of this pathway reduces the risk of cerebral hemorrhage , indicatIft88 knockout mice could reduce Notch activity, resulting in thickening of corneal epithelial cells (The Notch pathway is the main signaling pathway that coordinates angiogenesis with angiogenin signaling and cell metabolism . Notch sal cells . Limitedal cells .In vitro studies show that glucocorticoids improve the integrity of the blood-brain barrier in patients with multiple sclerosis, but this method alone can cause serious adverse reactions (Due to the high selectivity of the vascular barrier, it is challenging to deliver drugs to certain tissues. It is therefore important to modulate the vascular barrier. Some solutions have been documented. For example, in the blood-brain barrier, the use of mannitol or focused ultrasound can temporarily open the junction between endothelial cells, so that molecules unable to pass through the barrier can temporarily enter the brain tissue. However, simultaneously, substances such as plasma proteins in blood vessels that may cause harm to brain tissue, are able to enter . In vitreactions . Stimulaeactions .Maintenance of the vascular barrier depends on the regulation of various signaling pathways. Cilia play an important role in some of these signaling pathways, especially in HH signaling, and indirectly regulates the formation and maintenance of the vascular barrier. A number of studies have shown that there is a certain relationship between cilia and the integrity of the vascular barrier. In addition, the target of certain drugs, such as cytochalasin D, is part of the signaling pathway regulated by cilia . Thus, tThe vascular barrier plays a key role to maintain tissue homeostasis, while cilia are involved in the generation and maintenance of the vascular barrier. Although more evidence is available, several aspects have not been resolved. For example, it is not clear whether cilia play different roles in different tissues and how cilia regulate various parts of the vascular barrier through different signaling pathways. The molecules responsible for regulating the formation and maturation of the vascular barrier, the method used to assess the vascular barrier and how to target cilia to regulate the vascular barrier with treatments still need to be investigated. Further research will provide new strategies for the treatment of vascular diseases caused by ciliary defects.Destruction of cilium-related proteins can damage the vascular barrier, which may be mediated by HH, Notch, Wnt, and other signaling pathways. It is possible that cilium defects cause damage to intercellular junctions and paracellular transport, such as tight junctions and adherens junctions, which are important in vascular barriers . AlternaNM wrote the manuscript and drew the figures. JZ conceived the study and revised the manuscript. Both authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therapeutic targeting of Wnt has long been suggested for gastrointestinal (GI) cancer treatment because deregulation of Wnt signaling is associated with GI cancers. However, therapeutic targeting of Wnt is still challenging because of the pleiotropic roles of Wnt signaling in the human body. Thus, targeting strategies of Wnt signaling are continuously evolving. The current flows of targeting Wnt signaling for cancer treatment are focused on increasing the specificity of drugs and combinatory treatment with other cancer drugs that minimize side effects and increase efficacy. Additionally, increased knowledge about the β-catenin paradox has expanded the cases that can be treated with Wnt targeting therapy, not strictly considering Wnt upstream and downstream mutations. Here, we discuss these evolving views of targeting Wnt signaling and describe examples of current clinical trials.Wnt signaling governs tissue development, homeostasis, and regeneration. However, aberrant activation of Wnt promotes tumorigenesis. Despite the ongoing efforts to manipulate Wnt signaling, therapeutic targeting of Wnt signaling remains challenging. In this review, we provide an overview of current clinical trials to target Wnt signaling, with a major focus on gastrointestinal cancers. In addition, we discuss the caveats and alternative strategies for therapeutically targeting Wnt signaling for cancer treatment. Drosophila (Wingless) and the mammalian system (Int-1) . W. WDrosop (Int-1) ,4,5. Not (Int-1) . Therefo (Int-1) ,8.Although it has been confirmed in in vitro and in vivo cancer studies that targeting Wnt signaling has drastic tumor-suppressing effects, no targeted drugs have been successively advanced to clinical applications to date ,8,9. ThiWnt signaling is an autocrine and paracrine signal-transducing module that is activated by lipid-modified WNT ligands and their receptors ,11. In h2+ signaling pathway . In. InSFRP2The palmitoylation of WNT ligands by the protein-serine O-palmitoleoyltransferase porcupine in the endoplasmic reticulum  is essenVantictumab (OMP-18R5) is a monoclonal antibody that binds to FZD 1, 2, 5, 7, and 8 and inhibits Wnt signal transduction . A phase2+ signaling by stimulating intracellular Ca2+ flux in zebrafish and frogs amino] benzoate (MSAB)  binds toWith its increased fold change, nuclear β-catenin replaces the transducin-like enhancer protein corepressor with coactivators by forming the β-catenin–TCF/LEF complex ,120. ThiTRAF2 and NCK-interacting protein kinase (TNIK) phosphorylates the serine 169 residue of TCF7L1 and the serine 154 residue of TCF7L2, acting as an activating kinase of the β-catenin-TCF/LEF transcriptional complex ,126,127.CREB-binding protein (CREBBP), histone acetyltransferase EP300, pygopus homolog (PYGO), and B-cell CLL/lymphoma 9 protein (BCL9) are coactivators that interact with the β-catenin–TCF/LEF complex . PRI-724The major caveat in Wnt targeting strategies is their detrimental side effects on normal cells in which Wnt signaling plays pivotal roles in tissue homeostasis and regeneration ,4,5. ForAPC and β-catenin/CTNNB1) [Targeting the downstream effectors of Wnt signaling, e.g., β-catenin and TCF/LEF, might maximize Wnt signaling inhibition on the basis of signaling convergence into downstream gene regulation. However, targeting downstream Wnt signaling might also generate severe side effects by disrupting Wnt signaling in normal tissues. Conversely, targeting the upstream molecules of Wnt signaling, e.g., ligands and receptors, was initially considered ineffectual in cancer cells carrying mutations in Wnt signaling downstream  . IntriguTargeting cancer type- or tissue-specific Wnt signaling components or modulators may overcome the side effects of Wnt signaling blockade on normal tissues. For instance, specifically targeting the constitutively active form of β-catenin mutants may be ideal. A recent study found that small-molecule enhancers of mutant β-catenin and its E3 ligase (β-TrCP) interaction potentiate the ubiquitination-mediated degradation of mutant β-catenin , suggestThere are also several promising preclinical and clinical studies evaluating antibodies against RSPOs and LGRs, Wnt signaling amplifiers . Since RAn alternative method of overcoming limitations in Wnt signaling targeting strategies is to identify a safe dose that is highly effective but does not disrupt normal physiologic processes. A specific dose of LGK794 had lower severity of side effects with effective pharmacologic outcomes in a phase I clinical trial , supportIn general, combination therapy is considered to result in more AEs. However, it does not always induce more AEs than does monotherapy. The incidences and degrees of AEs depend on various factors, such as the doses of single drugs, the timing of administration, the period of treatment, the supportive treatment, and the heterogeneity of the patients themselves. Thus, certain drug dose combinations may be more effective, with fewer AEs. Furthermore, monotherapy targeting one pathway does not guarantee complete anticancer activity because of multiple crosstalks and compensations by other signaling pathways. Although its efficacy may be counterbalanced by correspondingly increased toxicity, combination therapy that simultaneously targets several pathways might be more efficient. In addition, combination therapy is the most common approach to achieving survival benefits in clinical practice, and most promising phase III Wnt targeting trials use combination therapy.ICG-001 and PRI-724 inhibit Wnt target gene expression by antagonizing CBP, a β-catenin coactivator ,151. PRIAPC or β-catenin/CTNNB1 [APC mutations [APC [APC mutations [The β-catenin paradox was introduced on the basis of heterogeneous Wnt signaling activity in CRC cells, carrying homogenous genetic alterations in n/CTNNB1 . This obn/CTNNB1 ,164,165.n/CTNNB1 ,166,167,n/CTNNB1 ,167. Morutations ,116. In ons [APC ,110. A rutations . TherefoAberrant Wnt signaling is crucial for the potential clonal source of tumor cells and is considered an environmental and metastatic niche for tumor progression. Indeed, LGR5+ colon cancer cells are required for the formation of metastatic colonization in the liver . A studyAPC-mutated CRC by promoting β-catenin deubiquitination [FZD5 is feasible in the mouse models [Tmem9 or Paf did not display any discernible phenotypes, suggests that blockade of cancer-related Wnt signaling activators or amplifiers minimizes side effects in Wnt signaling targeting approaches.Several cancer-specific Wnt signaling regulators were identified in GI cancers. Amplification of USP21 deubiquitinase promotes pancreatic cancer cell growth and stemness via Wnt/β-catenin signaling . RNF6, atination . Transcrtination . BCL9 antination ,174,175.tination . Given te models ,177, it e models ,179. As e models ,166. Of Additionally, recent technological advances in organoids made it feasible to perform high-throughput chemical screening  and genetic screening (gene knock-out or knock-down) of tumor organoids ,181,182.To date, many studies have reported the marked impact of molecular targeting of Wnt signaling on tumor suppression in preclinical settings. Despite the ongoing clinical trials, it is still imperative to overcome recurring pitfalls—catastrophic adverse effects on tissue homeostasis and regeneration. Like the sword of Damocles, targeting Wnt signaling poses a high risk but has significant potential in cancer therapy. With evolving concepts in Wnt signaling deregulation and manipulation, new and improved approaches, including molecular targeting of upstream signaling modules or cancer-specific regulators and combination therapy, are expected to open a new window of opportunity in the treatment of Wnt signaling-associated cancer.


Neuronal Signaling, Aideen M. Sullivan, reflects on the journal’s journey so far and welcomes the new Editor-in-Chief, Clare Stanford, as she shares some of the exciting initiatives and plans for its future.The inaugural Editor-in-Chief of  Neuronal Signaling was launched in 2017 by the Biochemical Society, through its publisher Portland Press, to address the need for a dedicated journal for research and reviews on the molecular, cellular and systematic signaling processes involved in the development, functioning and disorders of the nervous system. Over the past couple of decades, there have been significant advances in our understanding of the mechanisms by which cells communicate within the nervous system. The mission of Neuronal Signaling is to provide an interdisciplinary home for neuroscientific research, covering all aspects of in vitro and in vivo signaling, from cell to brain in health and disease. Neuronal Signaling journal cover images, representing some of the excellent and innovative work that has been published in the journal.Neuronal Signaling during its first 4 years. I have had the pleasure of working with an excellent Editorial Board, the members of which hold international reputations and expertise in many aspects of nervous system development and physiology, as well as neurodegenerative, neurological and psychiatric disorders. Over the past 4 years, the Editorial Board has continued to expand, achieving growth and diversity in terms of both expertise, geographic location and gender.It has been an honor to act as Editor-in-Chief of Neuronal Signaling. Founded in 1911, the Biochemical Society is the U.K.’s largest single-discipline learned society and its mission is to advance the molecular biosciences. Portland Press is committed to open scholarship and to open access publishing. It works in collaboration with the Publications Committee of the Biochemical Society to ensure the delivery of high-quality, peer-reviewed content in their suite of journals. Portland Press supports the scholarly and charitable activities of the Biochemical Society and enhances its global reach and service to the international research community. As a Portland Press journal, Neuronal Signaling has benefitted from the vast experience and success of its sister journals in the portfolio, including the Biochemical Journal and Clinical Science.The partnership between the Biochemical Society and Portland Press is key to the success of Neuronal Signaling launched a collaboration with ScienceOpen, a platform committed to open science via facilitating open and public communication between academics. This collaborative platform has further strengthened the impact of the journal on the neuroscientific research community and will be enhanced by the ongoing work of the expert Editorial Board.In March 2019, Neuronal Signaling achieved the significant milestone of being indexed in PubMed and PubMed Central, a crucial step for the journal’s growth and discoverability. PubMed Central indexing, alongside Google Scholar and the Directory of Open Access Journals (DOAJ), provides a measure of the quality and importance of the articles that are published in Neuronal Signaling, and enhances their availability to, and discoverability by, the scientific community and the general public.In May 2020, Neuronal Signaling as of January 2021.To reflect on her outlook for the journal’s future, I am delighted to hand over to Professor Clare Stanford, the incoming Editor-in-Chief of Neuronal Signaling has achieved a great deal, over the last 4 years, and we are keeping up the momentum in planning the further development of the journal. Some initiatives are already underway, while others are on the horizon, but they will all need conscientious support from the scientific community.Although Impact Factors attract considerable scepticism and even overt criticism, it is inevitable that they are regarded as a benchmark for a journal’s perceived status in the scientific community. Our successful application for listing in PubMed Central means that achieving indexing on Web of Science (and achieving an Impact Factor) is now our next objective, through sustaining our efforts to encourage submission of high-quality papers.Read & Publish’ agreements with several influential institutions, which will certainly help us to achieve our objectives. These ‘Read & Publish’ agreements enable authors, from participating institutions, to publish open access in Neuronal Signaling without needing to pay any author-facing article publishing charges (APCs). This is part of the Biochemical Society’s open scholarship strategy and will provide a real incentive for authors to put Neuronal Signaling on their list of first-choice journals when deciding where to send their manuscripts.The Biochemical Society and Portland Press have set up ‘Neuronal Signaling is also increasing its journal profile though collaborations with other groups. The Biochemical Society is a partner society for the forthcoming British Neuroscience Association Festival of Neuroscience, and Neuronal Signaling will be sponsoring a symposium on ‘From stem cells to whole animals: the scope and appraisal of research models in vitro and in vivo’. It is a pity that this will have to be a virtual meeting, but the quality of the science will not be affected. The Biochemical Society will be hosting a virtual booth for Neuronal Signaling at that meeting, so please do visit to find out more about the scope of the journal, seek advice for submission of articles and learn about our plans for its development. We hope to arrange more collaborations of that kind, in the future.Neuronal Signaling is planning a series of themed collections of original and review articles in 2021. We will be inviting submissions for a topical collection on olfaction, including themes such as the mechanisms behind the loss of sense of smell (hyposmia) in relation to COVID-19, Parkinson’s disease and Kallmann syndrome, as well as nasal drug delivery and sexual dimorphisms in olfactory signaling. We are also excited to launch a collection based on the sponsored BNA symposium, mentioned above, which will be entitled ‘Emerging technologies for research models of neuronal human disorders in vivo and in vitro’. Dr Tom Cunningham , one of my fellow speakers at the symposium, has kindly agreed to act as Co-Guest Editor for this Neuronal Signaling collection. We encourage readers to offer their own suggestions, for more themed topics to be featured in Neuronal Signaling, especially if they would like to act as a Guest Editor for the collection.In addition to these projects, Neuronal Signaling will be looking to recruit additional Editorial Board members, especially from the other continents, to ensure we maintain a truly international profile. We are proud that 60% of the Editorial Board are women and we shall strive to maintain a gender-balanced team through judicious recruitment of new members.We are aware that all these activities will generate more work for everyone involved with running the journal. To ensure that the processing of manuscripts remains timely and efficient, we shall be looking to expand the Editorial Board. We already have distinguished editors from the U.K., Ireland, Australia, mainland Europe and U.S.A., all of whom have made invaluable contributions to the journal’s success. ‘Developments in Neuroscience’, where we heard from four gifted ECRs who shared some of their innovative current work in neuroscience with the biosciences community. Full details on past webinars and the exciting programme for 2021 are available on the Biochemical Society webinars page.The Biochemical Society is proud to support early-career researchers (ECRs) through many initiatives including their new Biochemistry Focus webinar series dedicated to ECRs. I was delighted to take part in chairing the successful first webinar of 2021 on As well as all these initiatives, we need to keep abreast of developments in the scientific community, more generally. As a first step, we shall be revising our guidelines to authors, to offer some advice on the reporting of experiments that use animal models of complex, human neuronal disorders, with the aim of trying to improve their successful translation into humans. We shall also keep abreast of developments in research practice, particularly in respect of integrity and open science. As part of that process, we shall be reviewing the ARRIVE 2.0 guidelines  and highAs Professor Aideen Sullivan demits as Editor-in-Chief, I must thank her, the Editorial Board and everyone at Portland Press for all their efforts and resounding success with this fledgling journal. Fortunately, Aideen will be carrying on as an Associate Editor and I look forward to working with this outstanding team of professionals and building on their progress over the next 4 years.Neuronal Signaling [Finally, we hope this editorial will encourage you to read some of the excellent work being published in ignaling . Of cour


Our analyses clearly indicated that AOX proteins at certain stages in the appendices are degraded at 30°C, which is close to the maximum appendix temperature observed during thermogenesis. Interestingly, such temperature-dependent protease activities were specifically inhibited by E-64, a cysteine protease inhibitor. Moreover, purification and subsequent nano LC–MS/MS analyses of E-64-sensitive and DCG-04-labeled active mitochondrial protease revealed an ∼30 kDa protein with an identical partial peptide sequence to the cysteine protease 1-like protein from Phoenix dactylifera. Our data collectively suggest that AOX is a potential target for temperature-dependent E-64-sensitive cysteine protease in the appendices of A. maculatum. A possible retrograde signalling cascade mediated by specific degradation of AOX proteins and its physiological significance are discussed.Cyanide-resistant alternative oxidase (AOX) is a nuclear-encoded quinol oxidase located in the inner mitochondrial membrane. Although the quality control of AOX proteins is expected to have a role in elevated respiration in mitochondria, it remains unclear whether thermogenic plants possess molecular mechanisms for the mitochondrial degradation of AOX. To better understand the mechanism of AOX turnover in mitochondria, we performed a series of  Araceae, Aristolochiaceae, Annonaceae, Cycadaceae, Cyclanthaceae, Magnoliaceae, Nelumbonaceae and Nymphaeaceae [Arum maculatum, a thermogenic plant in Araceae, exhibit cyanide-insensitive respiration, significant progress has been made in establishing the structure-function relationships of cyanide-insensitive alternative oxidase (AOX). Currently thermogenesis in plants is attributed partially to the activity of AOX in the mitochondria [A. maculatum [Symplocarpus renifolius [Nelumbo nucifera [Dracunculus vulgaris [Cycas revoluta [Thermogenesis in reproductive organs occurs in various seed plants including haeaceae . Since thaeaceae  that mitchondria . AOX is chondria ,10. AOX chondria ,9,11,12.aculatum , Symplocnifolius , Nelumbonucifera , Dracuncvulgaris  and Cycarevoluta .AOX is an interfacial membrane protein oriented towards the matrix side of the inner mitochondrial membrane ,20, and 2−) from oxygen [AOX gene expression, thereby maintaining cell homeostasis [The level of AOX expression varies among non-thermogenic plant species. In potato tuber mitochondria, for example, the AOX protein level is much lower than that found in thermogenic plants. In general, AOX is considered to be expressed when the main respiratory chain is inhibited or the plant is exposed to extreme environmental stresses such as drought, excessive salinity or low temperatures . Moreovem oxygen ,26. AOX m oxygen ,28. Accoeostasis ,29.AOX has also been shown to be regulated in a retrograde manner . For insAOX gene expression under various environmental conditions and in various signalling pathways, little is known regarding the quality control of the AOX protein, such as its protein degradation in mitochondria. For instance, LC–MS/MS analyses using progressive 15N labelling of Arabidopsis cells have shown that different mitochondrial proteins turnover at widely divergent rates, with a >50-fold variation in protein degradation [c oxidase 15 and NAD (P) H dehydrogenase B2, showed no appreciable turnover during 7 days of measurements.Thus, although there is a wealth of information on radation , althougA. maculatum is developmentally regulated [AOX transcripts (AmAOX1a–AmAOX1g) in the appendix [A. maculatum [AmAOX1e gene transcripts is highly specific to thermogenic tissues in A. maculatum[AOX gene expression (i.e. levels of its transcripts and proteins) and the quality control of AOX proteins in the context of developmentally regulated thermogenesis.Thermogenesis in the appendix of egulated , and theappendix . The expaculatum . Becausemaculatum, we hypoAOX gene expression in thermogenic appendices is associated with specific turnover of the AOX protein in the mitochondria, we employed a series of in organello protein degradation assays using mitochondria purified from the appendices of A. maculatum.To investigate whether co-ordinated A. maculatum plants at β-, δ-, and γ/δ periphery stages were collected on the campus of the University of Sussex, U.K..The appendices of Thermal images were captured with an infrared colour LSD camera  and images were analyzed for temperature determination as previously described ,44.AmAOX1a-1g were AmAOXallFW (5′-ggaggccatccactcctaca-3′) and AmAOXallRV (5′-acgacgtcgcgcagggtg-3′), and amplification conditions were as follows: an initial denaturing step at 95°C for 30 s; 45 amplification cycles of 95°C for 15 s, 61°C for 15 s and 72°C for 20 s; and a dissociation curve analysis at 95°C for 15 s, 60°C for 30 s and 95°C for 15 s. Cycleave PCR was performed to quantify the transcripts of ENV-, QNT- or QDT-type AOX genes as described in our previous study [VDAC and ubiquitin were as follows: AmVDAC_FW (5′-caagttcgacaccctaacaac-3′) and AmVDAC_RV (5′-agaagctgtgcgtcaactc-3′) for VDAC; and AmUB_FW (5′-caggtggaatcctctgacacg-3′) and AmUB_RV (5′-cgtcctccagctgcttcc-3′) for ubiquitin. The same amplification protocol was used for VDAC and ubiquitin as follows: an initial denaturing step at 95°C for 30 s; 45 amplification cycles of 95°C for 15 s, 60°C for 15 s and 72°C for 20 s; and a dissociation curve analysis at 95°C for 15 s, 60°C for 30 s and 95°C for 15 s.Real-time qRT-PCR was performed with a Dice Thermal Cycler  as described previously . BrieflyA. maculatum as described previously [g at 4°C for 15 min, the supernatants were recovered (termed SUP), and precipitants (termed PPT) were resuspended in a buffer containing 0.4 M mannitol, and 10 mM MOPS-KOH (pH 7.2). Respiratory analysis was performed as described previously [Mitochondria were purified from the thermogenic appendices of eviously ,45. IsolA. maculatum were incubated in a buffer containing 200 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 mM CaCl2, 50 mM ATP and 0.1% Triton X-100 [Mitochondria purified from on X-100  at eitheMitochondrial proteins were separated on SDS–PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes as described previously ,45. MembCarbonylated proteins were detected using Oxidized Protein Western Blot Kit  in accordance with the manufacturer's instructions. Densitometric analysis of the signals was carried out using the ImageJ Software (version 1.51).Activity profiling of cysteine proteases was performed with DCG-04, a biotinylated analogue of the irreversible cysteine protease inhibitor E-64 . BrieflyTo purify the DCG-04-reactive proteases from mitochondria prepared from non-thermogenic appendices, 20 µl of magnetic streptavidin beads  was added to each reaction mixture and incubated for 16 h at 4°C. The magnetic beads were then washed with 300 µl of TBS buffer using a magnetic stand (Promega) to remove non-specifically bound proteins. Biotin-labelled proteins were recovered by boiling the magnetic beads in Laemmli SDS loading buffer and subjected to SDS–PAGE.Nano LC–MS/MS analysis was performed using a LTQ orbitrap XL system  as described previously ,45. The Modelling and computer simulation for AOX turnover was carried out using Excel spreadsheet program .P value <0.05.Results were presented as mean values ± SD. Differences between the means were tested by one-way factorial ANOVA and a Tukey's honest significant difference post hoc test using SPSS software . Significant differences between means were indicated by a A. maculatum show intensive thermogenesis at the δ stage during the development of inflorescence, and that the maximum temperature reached ∼30°C [AmAOXs in the appendices of this plant in our current study, the apparent δ stages of the thermogenic appendices with open but not wilted spathes were collected in the field together with the β stages of pre-thermogenic appendices with tightly closed spathes. All sampled appendices were analyzed in the laboratory with an infrared thermal camera to evaluate the level of thermogenesis , which amplified all AmAOX transcripts and cycleave PCR, which specifically amplified the ENV, QNT + QDT and QDT types of AmAOX transcripts. AmAOX transcripts showed significantly higher expression levels in hot appendices by both qRT-PCR and cycleave PCR (AmAOX transcripts were ∼7–8-times more abundant in hot appendices than in pre-hot samples. The expression of transcripts encoding the QNT type of AmAOX (AmAOX1e) was significantly higher in hot appendices, supporting the notion raised in our previous report that AmAOX1e is the major AOX molecule expressed in thermogenic appendices of A. maculatum [AmAOX1e, in the appendices. To determine whether gene transcripts other than AmAOX1e are induced in the thermogenic appendices of A. maculatum, expression analysis for voltage-dependent anion channel (VDAC) and ubiquitin genes was conducted using the same RNA samples from pre-hot, non-hot and hot appendices that were used in the AmAOX1e expression analysis. VDAC has a role in regulating metabolic and energetic flux across the outer mitochondrial membrane and its gene expression is reflective of mitochondrial metabolism [VDAC transcript levels between pre-hot and hot appendices were almost the same, whereas those in non-thermogenic appendices were significantly lower than in the other samples  that degrade AmAOX proteins, antibodies to Hsp60 and VDAC were also used in the western analysis (To next characterize the protease(s) that degrade AmAOX proteins, we tested four types of protease inhibitor via an analysis . We founanalysis . EDTA ananalysis . This suanalysis .A. maculatum and that AmAOX proteins are their natural target substrates for degradation in a temperature-dependent manner. Moreover, the E-64-sensitive degradation of AmAOX proteins proceeded faster in the mitochondria from non-hot and hot appendices at either 15°C or 30°C  and matrix (SUP) fractions . PurifieWe further characterized the E-64-sensitive protease(s) via activity profiling with the E-64 analogue DCG-04 containing a biotin tag . RepeatePhoenix dactylifera (NCBI Reference Sequence: XP_008775532.1) was found to be present in the mitochondria from A. maculatum . The maximum temperature of the appendix reaches 32°C at the δ-stage during floral development in A. maculatum [P. dactylifera  appendices, their protein translation levels remain unchanged across different developmental stages . These data are consistent with our previous paper which demonstrated that AmAOX proteins constitutively express at all stages during floral development in A. maculatum [AmAOX transcript levels were significantly higher in the hot appendices , whereas the QDT type of pyruvate-insensitive AOX (AmAOX1e) is principally expressed in mitochondria from non-hot and hot appendices [AmAOXs gene expression from pre-hot to hot appendices is a ‘switching out’ of the pyruvate-sensitive AOX form for a pyruvate-insensitive AOX form. In this case, activities of pyruvate insensitive AmAOX1e might then no longer be dependent upon the threshold level of pyruvate concentration within the matrix of mitochondria in thermogenic appendices of A. maculatum.Ubiquinol is a common substrate for both AOX- and COX-mediated respiration pathways, and previous study showed that the ubiquinone pool in ogenesis . Becauseogenesis , it is cpendices . It is tA. maculatum is a fundamental question to arise from our current data. AOX proteins in plant mitochondria generally exist as either a non-covalently associated reduced dimer (active form) or an oxidized dimer (inactive form) through the formation of a disulfide bridge between the conserved Cys I residue [A. maculatum.The significance of increased AmAOX protein turnover in  residue ,69. The  residue ,70, an iA. maculatum. The temperature-inducible degradation of AOX proteins and the subsequent stimulation of their gene transcription may contribute to the endogenous heat-production in this plant.In conclusion, AOX proteins are a potential substrate of an E-64-sensitive cysteine protease of the thermogenic appendices of