The abundance of insects indicates that they are one of the most adaptable forms of life on earth. Genetic, physiological, and biochemical plasticity and the extensive reproductive capacity of insects are some of the main reasons for such domination. The endocrine system has been known to regulate different stages of physiological and developmental processes such as metabolism, metamorphosis, growth, molting, and reproduction. However, in this review, we focus on those aspects of the endocrine system that regulate female insect reproduction. The proper understanding of the endocrine system will help us to better understand the insect reproductive system as well as to develop new strategies to control the insect pest population. The juvenile hormone analogs and molting hormone analogs have been widely used to control the insect pests. Such insect growth regulators are usually more specific and cause little harm to the beneficial organisms. Therefore, a proper understanding of these signaling pathways as well as their interaction with each other and other signaling pathways is very crucial. Further, the interaction of microbiome with the endocrine system is also discussed.The proper synthesis and functioning of ecdysteroids and juvenile hormones (JHs) are very important for the regulation of vitellogenesis and oogenesis. However, their role and function contrast among different orders, and even in the same insect order. For example, the JH is the main hormone that regulates vitellogenesis in hemimetabolous insect orders, which include Orthoptera, Blattodea, and Hemiptera, while ecdysteroids regulate the vitellogenesis among the insect orders of Diptera, some Hymenoptera and Lepidoptera. These endocrine hormones also regulate each other. Even at some specific stage of insect life, they positively regulate each other, while at other stages of insect life, they negatively control each other. Such positive and negative interaction of 20-hydroxyecdysone (20E) and JH is also discussed in this review article to better understand the role of these hormones in regulating the reproduction. Therefore, the purpose of the present review is to deeply understand the complex interaction of endocrine hormones with each other and with the insulin signaling pathway. The role of microbiomes in the regulation of the insect endocrine system is also reviewed, as the endocrine hormones are significantly affected by the compounds produced by the microbiota. Reproductive physiology of insects includes all the physiological and behavioral processes from the development of the embryo to the production and oviposition of the fertile eggs . RegulatBombyx mori, the ecdysone oxidase was reported to be present in the cytoplasm throughout the yolk granules of the oocyte, and responsible for catalyzing 20E to 3-dehydroecdysone (3DE) through encoding an enzyme. Downregulation of BmEO by RNAi resulted in a significantly lower titer of 20E and hatching rate . Th. Th41].  chamber . Mutatio chamber . While ioduction . Furtherpendent) . Howeverectively  45]. Ho. HoDrosomericana . Meanwhiecretion .The role of juvenile hormones in controlling insect metamorphosis and reproduction is of great importance . JuvenilB. dorsalis, the treatment of dsMet significantly reduced the expressions of BdKr-h1, BdMet, BdVg1, and BdVg2. Over 50% reduction in expressions of BdKr-h1, BdVg1 and BdVg2 were observed after 72 h treatment of dsKr-h1 [T. castaneum [Locusta migratoria, H. armigera, and Nilaparvata lugens, suppression of Kr-h1 decreased the egg production by reducing the Vg expression, oocyte maturation, and ovarian development [Vg gene expressions in Pyrrhocoris apterus fat body [S. gregaria, using RNAi, it was then observed that this insect never enters the previtellogenic stage. The ovaries of such treated insects were found to be arrested. Further, the mRNA level of kr-h1 was decreased by 88% and 73% in CA and fat body, respectively. Therefore, the JHs’ receptor Met is found to be necessary for the vitellogenesis, ovary maturation, and accessory ecdysteroid biosynthesis. Delayed copulation behavior is also found to be associated with the knockdown of SgMet. Further, a notable decrease in insulin-related peptides was also observed against dsSgMet-treated female adults of S. gregaria [Vg genes. Met knockdown was responsible for the lower number of egg deposition. While in female adult mosquitoes, Met/Tai complex, a transcription factor, stimulates Kr-h1 expressions, which in turn promotes vitellogenesis [Diploptera punctata, ovarian growth was blocked owing to the silencing of Met, which in turn was responsible for a remarkable decrease in the size of developing eggs [Reticulitermes speratus, the Japanese termite, increase in the JH titers initiates Vg synthesis and results in the development of neotenic reproductivity [D. melanogaster, JH regulates female mating and pheromone production [A. aegypti adult female mosquitoes, the expression of large number of genes is regulated by JH [C. lectularius, common bed bug, silencing of Kr-h1 does not decrease the number of oviposited eggs; however, it significantly affected the hatching of eggs [H. armigera, knockdown of HaKr-h1 also decreased the transcription of vitellogenin and interrupted oocyte maturation [Grapholita molesta, silencing of GmKr-h1 increased preoviposition period and decreased fecundity [Vespula vulgaris releases honest signals to change the fertility status of subordinate workers, so their workers become reproductively repressed and help in colony. However, such signals are found to be controlled by endocrine hormones. To test if JH is the main hormone that maintains such fertility and fertility signaling, the workers were treated with JH analogue (methoprene) and JH inhibitor (precocene). The results showed that the oocyte size was increased after treatment with the methoprene; nevertheless, oocyte size was decreased against precocene. Thus, JH affects both fertility and fertility signaling in workers [After the identification of Met as a JHs’ receptor, a regulatory model was also developed that involved JH-Met-Kr-h1 . Kr-h1 k dsKr-h1 . The kr-astaneum . In Locuelopment ,55. RNAifat body . When thgregaria . In cockogenesis . In Dipling eggs ,60. In Ructivity . Meanwhioduction . In A. aed by JH , while A of eggs . In H. aturation . For Graecundity . The que workers .Streblognathus and Diacamma have queenless societies. Reduction of JH titer in gamergates corresponds with high individual ranks within the hierarchy. As the alpha is responsible for the reproduction of offspring, JH treatment of such individual results in the loss of its reproductive status [Dinoponera quadriceps, JH regulates the female reproduction by affecting the ovary development. The ants with increased JH levels develop reduced ovarioles, which in turn decreased their reproductive potential by reducing the number of vitellogenic eggs [Pteromalus puparum, endoparasitic wasp, promoted Vg gene expression, while JH application resulted in the reduction of Vg mRNA levels [C. lectularius [Meanwhile, JH also negatively affects the reproduction in some insects. Both e status . In Dinonic eggs . 20E treA levels . Howevertularius .Many aspects of reproduction, including vitellogenesis and oogenesis, are regulated by JH and 20E ,71. ProtDrosophila, an increase in JHs’ level was observed under the influence of stress conditions, which in turn resulted in the accumulation of eggs and oviposition seize. However, increase in 20E resulted in breakdown of oocytes, which ultimately reduced fecundity. Therefore, the proper balance between JHs and 20E is necessary for the normal development of oogenesis [In ogenesis . Howeverogenesis .T. castaneum, JH regulates the expression of Vg gene in fat body, while 20E controls the synthesis of Vg by its effect on ovarian development and oocyte maturation [T. castaneum. Both JHs and feeding are found to be required for the proper synthesis of Vg in the fat body, while the JHs’ signaling pathway acted via Met and increased the production of ILP2. Feeding triggered the production of ILP3, and later the insulin like peptides stimulated the phosphorylation of AKT resulted in the FOXO phosphorylation and ultimately its depletion from the nucleus. The depletion of FOXO later allowed the expression of the Vg gene. Therefore, these results suggested that the JHs modulated the Vg expressions through the insulin-like peptide signaling pathway, which ultimately affect FOXO localization in the fat body. The JHs also indirectly regulate the vitellogenesis by inducing the production of insulin-IGF, which in turn activates IIS [B. germanica and P. americana, JHs’ biosynthesis is promoted by insulin-IGF signaling (IIS) [In turation ,53. In aturation . The JHsates IIS . Meanwhing (IIS) ,77.Anopheles gambiae significantly reduced when the female was infected with Serratiochelin and Pyochelin [Candidatus Erwinia, increases the female reproductive output of Bactrocera oleae by increasing the production of essential amino acids [Ceratitis capitata, a group of nitrogen-fixing bacteria from Enterobacteriaceae family supports reproduction [Insects harbor different microbial communities that affect their biology. However, in this review, we specifically focused on the regulation of insect reproduction by the interaction of microbiomes and the endocrine system. Both endocrine hormones and microbiota influence each other. Endocrine hormones affect the metabolism of microbiota by a number of different channels . Reproduyochelin . A bacteno acids . Meanwhioduction .Beauveria bassiana is an entomopathogenic fungus, and it affected different life stages of Bemisia tabaci [Metarhizium anisopliae significantly influenced the reproductive system of Plutella xylostella, by decreasing the life span and egg laying ability. A significant difference in the fecundity of treated insects was observed, where the mean number of laid eggs was reduced to 101.55 eggs/female as compared with control of 192.55 eggs/female [Ostrinia nubilalis larvae concerning to the odors from plants of genus Chenopodium. Except for C. album and C. polyspermum, the neonate larvae showed repellent behavior from all the tested phytosteroid-positive species [T. castaneum [Mythimna separata was treated with the entomopoxovirus, it reduced the titer of ecdysone and resulted in the death of sixth instar larvae. Further, JHs’ titer was increased in such treated insects  is also transferred from males to females, which helps in the egg development. On the other hand, in the diamondback moth, exogenous application of 20E results in the reduction of female fecundity. Recent findings also explained the role of these endocrine hormones in controlling the fertility of insects. However, such a complex interaction of these endocrine hormones is also necessary for regulating the reproduction of such insects.The role of AA/TOR and insulin signaling pathways not only cleared the vitellogenin intake by the developing oocyte, but also explained its role in the biosynthesis of the JHs’ hormones and ecdysteroids. Little information is available regarding the proper understanding of these signaling pathways in less studied insects. Therefore, in the present review, we also focused on the role of endocrine hormones in such less studied insects. Further, the interaction of these endocrine hormones is also explained. However, the comparative contribution and the interaction of these signaling pathways with each other further require clarity.C. Erwinia. Such bacteria increased the female reproductive output by increasing the production of essential amino acids. Female insects also transfer microorganisms to their partner as well as to their offspring. Therefore, in this review, we not only focused on the mechanism of transcriptional activation of 20E and JHs, but also discussed the complex interaction of these signaling pathways together with each other and with other signaling pathways . In addition, the role of microbiomes in regulating the endocrine hormones has been discussed. However, further studies should focus on the entire microbiome and the type of molecules they create in order to better understand their interaction with the endocrine hormones. As endocrine analogs are environmentally safe, the proper understanding of female endocrine system and reproductive system together with the microbiota will help us to control the insect pest more effectively.When the insect comes into contact with the microorganism, a change in the titer of endocrine hormones is observed. Such changes in the endocrine hormone titer significantly affect their reproduction in several different ways. A significant increase in reproduction of the olive fruit fly is observed after treatment with


Microbes heavily rely on cellular signaling pathways for their effective replication; hence, JNKs may play important roles in infectious diseases. In this review, we describe the basic signaling properties of MAPKs and JNKs in apoptosis, autophagy, and inflammasome activation. Furthermore, we discuss the roles of JNKs in various infectious diseases induced by viruses, bacteria, fungi, and parasites, as well as their potential to serve as targets for the development of therapeutic agents for infectious diseases. We expect this review to expand our understanding of the JNK signaling pathway’s role in infectious diseases and provide important clues for the prevention and treatment of infectious diseases. Mitogen-activated protein kinases (MAPKs), a new serine/threonine kinase, comprise an intracellular signaling cascade network that activates when cells recognize and respond to various extracellular stimuli . MAPK sic-Jun N-terminal kinases (JNKs) represent the MAPK family and play critical roles in balancing cell survival and death in response to both extracellular and intracellular stresses . JNKs arComparable to other members of the MAPK family, JNKs are activated by upstream MAPKKs (MKK4 and MKK7) via phosphorylation on threonine and tyrosine residues within their conserved Thr-Pro-Tyr (TPY) motif . MAPKKs This review aims to summarize the roles of JNKs in cellular signaling transduction and infectious diseases caused by microbes. We hope this review elucidates the relationship between JNK signaling and various infectious diseases and emphasizes the potential of JNKs to serve as targets for the development of therapeutic agents for infectious diseases.−/− mice showing a reduction in hippocampal neuron apoptosis induced by glutamate [Many studies have indicated that all three JNKs are involved in stimulating apoptotic cell death. Initially, delayed and persistent JNK1 activation was highly correlated with apoptosis induced by exposure to gamma radiation . A studylutamate .Two distinct mechanisms are currently involved in JNK-mediated apoptosis. In one mechanism, phosphorylated JNK is translocated to the nucleus upon activation, where it phosphorylates c-Jun and other transcription factors. Then, JNK promotes apoptosis by increasing the transcription of known pro-apoptotic genes, such as Fas/FasL signaling pathway-related genes, via the transactivation of c-Jun/AP-1-dependent or p53-dependent mechanisms. Finally, the binding of FasL to Fas mediates the activation of caspase 8, which further activates the downstream effector caspase 3 to trigger apoptosis ,30. In aThe JNK signaling pathway has also been reported to participate in autophagy regulation in response to various stress signals. For instance, a previous study showed that JNK1, but not JNK2, is required for starvation-induced autophagy . By contViruses are a group of strictly intracellular parasites that invade the cells of virtually all known organisms; they use the host cell’s machinery to replicate effectively, produce new progeny, and generally damage or kill the host cell in the infection process. Viruses cause a wide variety of human, animal, and plant diseases that negatively affect human health, breeding, and planting industries . The lonData from previous research have shown that a variety of viruses can manipulate JNK signaling to regulate their replication, i.e., RNA viruses including human immunodeficiency virus type 1 (HIV-1) , echovirNumerous viruses have evolved distinct mechanisms to activate JNK signaling by exploiting their encoded proteins. For instance, the NS1 protein of most influenza A virus subtypes can activate JNK, and the amino acid positioned at 103 in NS1 plays a critical role in this signaling event . The EpsApoptosis and autophagy are two highly regulated forms of programmed cell death in mammalian cells that control homeostasis-related cell growth and the cellular immune response to invading pathogens ,45,55. CAlthough JNK activation enhances viral replication among a variety of viruses, JNKs plays an important role in inhibiting the replication of some viruses. For example, JNK controls oncolytic vaccinia virus replication through PKR pathway activation, and JNK deficiency can significantly enhance oncolytic vaccinia virus replication . SimilarIn addition to viruses, bacteria are the second most common cause of infectious diseases. Infection by bacterial pathogens and the host’s defense against infection is a lifetime battle between bacteria and the host, where a combination of host defense mechanisms, including MAPK pathways and some virulence factors of invaded bacterial pathogens, are involved .Vibrio alginolyticus and Staphylococcus haemolyticus, suggesting that ChJNK may be involved in the host’s defense against bacterial infection [JNK has been shown to activate at higher levels upon bacterial infection in multiple mammalian cell types ,71. Recenfection . A similnfection . These fStreptococcus pneumoniae infection by activating cytokines [Escherichia coli (E. coli), triggers a ribotoxic stress response depending on its enzymatic activity, leading to JNK activation and induction in apoptosis for intestinal epithelial cells [Pseudomonas aeruginosa-encoded ExoS, a virulence factor produced and secreted directly into the host cell by the type III secretion system (T3SS), also activates JNK phosphorylation and triggers cellular apoptosis depending on the subsequent JNK-mediated signaling [Bacteria have evolved various mechanisms to activate the JNK signaling pathway . For insytokines . Shiga tal cells . The oppignaling   to modify serine, threonine, and lysine residues in MKKs (MKK1 and MKK6); consequently, all three MAPK signaling cascades including the JNK pathway are abolished [E. coli transfer the effector protein NleD  into host cells via the T3SS; this directly cleaves JNK and p38 between the Gly/Pro and Tyr of the TPY motif, rendering them inactive [Bacillus anthracis, as a gram-positive bacterium, secretes a major virulence factor: anthrax toxin. Similar to NleD, the lethal factor subunit of anthrax toxin is a zinc metalloprotease that cleaves the N-terminus of several MKKs, including MKK1-4 and MKK6-7, resulting in the permanent inactivation of these kinases. The inactivation of these kinases also leads to abolishing the ERK1/2, p38, and JNK signaling pathways [Mycobacterium tuberculosis, the pathogen causing tuberculosis, secretes an enhanced intracellular survival protein (an efficient N-acetyltransferase) that activates the JNK-specific phosphatase DUSP16 through the acetylation of Lys55. Therefore, JNK phosphorylation is abolished by increased levels of dephosphorylation, facilitating pathogen survival inside cells by reducing cell death and inflammation [In addition, many bacteria have evolved strategies to manipulate and subvert host MAPK signaling through translocated effector proteins that directly catalyze the post-translational modification of proteins in the MAPK network  Figure . The Avrresponse . Similarbolished . By continactive . The caupathways ,86,87. Mammation  , have been developed; for example, JNK peptide inhibitor 1 and JIP1 ,124 haveThe recent emergence of the SARS-CoV-2 virus and the subsequent pandemic have caused unprecedented healthcare and economic problems worldwide. Meanwhile, the continued prevalence and threat of COVID-19 also exposes the limitations of vaccine efficacy and the lack of more effective antiviral therapies. Hence, developing new antiviral strategies is highly urgent and desirable. Targeting host cell pathways that support the virus and microbial replication can directly inhibit the infection of various microbes; therefore, this approach is a promising anti-infection therapy.The JNK signaling pathway regulates many cellular events, including the cell cycle, differentiation, survival, apoptosis, and inflammatory responses. Viruses, bacteria, fungi, and parasites exploit various cellular processes and events to ensure their survival and cause infectious diseases in humans, animals, and plants. Viruses and other pathogens have been shown to lead to JNK activation and maintain successful infections in a JNK-dependent manner. Therefore, JNK can be considered a potential therapeutic target for many infectious diseases. However, the understanding of JNK functions in various infectious diseases is still limited and complicated. JNK activation exhibits different effects in different pathogens and shows diametrically opposite effects on the same pathogen. Hence, when selecting JNK as a target for treating infectious diseases, its effect as a suppressor or promoter must be considered. In addition, widely used JNK inhibitors currently contain a certain degree of toxicity and lack the required specificity for each JNK isoform. Researchers must explore more specific and less toxic inhibitor molecules, and clinical trials at different stages must be advanced. Although challenging, there remains a bright future for JNK inhibitors and their application in therapies for infectious diseases, particularly in important viral diseases.


Myocardial fibrosis after myocardial infarction (MI) is one of the leading causes of cardiovascular diseases. Cardiac fibroblasts (CFs) are activated and promoted by MI to undergo myofibroblast transformation (CMT). Urolithin A (UA) is an active and effective gut metabolite derived from polyphenolics of berries and pomegranate fruits, which has been reported to have anti-inflammatory and anti-oxidant functions. However, whether UA affects the CMT process during myocardial fibrosis remains unclear.TGF-β1-treated primary rat cardiac fibroblasts were used for in vitro study. Cell proliferation ability was evaluated by MTT assay. Cell migration and invasion abilities were tested by wound healing and Transwell assays. The expression of CMT process-related markers were measured by qRT-PCR and western blot. The rat MI model was established by left anterior descending coronary artery (LAD) ligation and evaluated by H&E and Masson staining.Our data demonstrated that UA treatment could inhibit the CMT process in TGF-β1-induced CFs, including cell proliferation, migration and invasion abilities. Knocking down of Nrf2, which was activated by UA treatment, could mitigate the effects of UA treatment on CMT process. Moreover, in vivo administration of UA in rat MI model successfully up-regulated Nrf2 expression and improved the myocardial damage and fibrosis.The study discovered the function and mechanism of UA on myocardial fibrosis and demonstrated the protective effects of UA administration through activation of Nrf2 pathway. Myocardial fibrosis is one of the most important pathological changes after myocardial infarction (MI), which is the main cause of sudden death around the world  is a kind of leucine zipper transcription factor that is mainly expressed in the cytoplasm  were isolated from Sprague–Dawley rats of 1–3 days old using the protocol as previously described  and Urolithin A  with indicated concentrations were used to treat cardiac fibroblasts. Negative control siRNA (si-NC) and Nrf2 specific siRNA (si-Nrf2) were designed and purchased from GenePharma . Cell transfection was performed by Lipofectamine 3000  for 48 h.−ΔΔCt method.Cells and cardiac tissues from rats were dissolved in TRIzol reagent . Total RNA was obtained according to the manufacturer’s protocol. The RNA was then tested for quality and synthesized into cDNA using an iScript cDNA Synthesis Kit . qRT-PCR was performed using iQ™ SYBR Green Supermix . GAPDH and U6 were used as endogenous controls for normalization. Relative expression levels were normalized and analyzed using the 2Cells were washed with cold PBS and incubated with lysis buffer on ice for 30 min. Then, the cells were scraped, and after centrifugation, the supernatant containing the lysate was collected and stored at − 80 °C. Cardiac tissues from rats were homogenized and then incubated with lysis buffer on ice for 30 min. A BCA assay Kit  was used to determine protein concentration. Protein samples were denatured and then separated by SDS-PAGE and transferred to PVDF membranes . After blocking with non-fat milk for 1 h, membranes were incubated overnight at 4 °C with the following primary antibodies from Cell Signaling Technology : α-SMA (#19245), vimentin (#5741), DDR2 (#12133), tensin (#11990), Nrf2 (#12721), SOD1 (#37385), HO-1 (#82206), NQO-1 (#62262) and β-actin (#3700), and all were used at a 1:1000 dilution. After washing 3 times, the membranes were incubated with goat anti-mouse (#7076) or anti-rabbit (#7077) HRP-conjugated secondary antibodies . The signals were analyzed using an ECL detection Kit .4 cells were collected and seeded into 96-well plates and 20 µL of MTT solution was added to each well. After 2 h cultures, the MTT solution was replaced with 200 μL of DMSO. The absorbance at 490 nm was measured using a spectrophotometer.The 3--2,5-diphenyl tetrazolium bromide (MTT) assay  was used to detect cell viability according to the manufacture’s protocol. Briefly, after indicated treatments, 1 × 10The protocol was carried out as previously described  with matrigel pre-treatment. The lower chamber was filled with medium with serum. The plate was incubated in the incubator for 8 h. Cells remaining in the upper insert were removed using cotton swabs, and the invasive cells were fixed with 4% paraformaldehyde for 10 min. After washing with PBS for 3 times, the cells were stained with a crystal violet solution and imaged using brightfield microscopy  and quantified.A Transwell assay was performed according to a reported protocol  using left anterior descending coronary artery (LAD) ligation method as described previously , the sections were dewaxed in xylene and rehydrated in a descending series of alcohol. Slides were then stained with hematoxylin and eosin (H&E) or Masson’s trichrome  according to the manufacture’s protocol.For H&E staining, the myocardial injury was scored according to the following standard: Score 0: no myocardial structure injury; Score 1: slight myocardial mesenchymal edema, gap increase, and local necrosis; Score 2: extensive myocardial swell and mesenchymal edema, and medial local necrosis; Score 3: severe small vessel damage and myocardial necrosis, massive inflammatory cellular infiltration, and formation of contraction bands; Score 4: severe diffusive myocardial necrosis and hemorrhage, accompanied by massive small vessel damage and formation of contraction bands.For Masson’s trichrome staining, the extent of fibrosis was expressed as the relative fibrosis area, which equals the ratio of Masson’s trichrome-stained area to the whole of myocardial area in view.Statistical analysis was performed using GraphPad Prism 5. All experiments were conducted at least three times. All data are presented as the mean ± standard deviation  followed by Tukey post hoc test for multiple comparison. P < 0.05 was considered statistically significant.To test the effects of UA on myocardial fibrosis, primary rat cardiac fibroblasts were used as the model for in vitro study. Dose evaluation of UA was firstly determined and the UA (≤ 20 μM) exhibited no effect on cell viability of CFs Fig. A, thus 1In order to determine the mechanism of UA’s function in myofibroblast transformation, several markers were tested. The mRNA and protein levels of fibrosis markers including vimentin, DDR2, tensin and α-SMA were significantly increased upon TGF-β1 treatment in CFs compared to control group  is an important event in the initiation of myocardial fibrosis (Zhou et al. It has been shown that gut microbiota plays important roles in the pathogenesis of cardiovascular diseases (Miele et al. As a critical anti-oxidative gene, studies about the functions of Nrf2 on cardiovascular and metabolic diseases are increasing recently (Duan et al. Moreover, our study also tested the UA’s function in vivo via rat MI model. We found that UA treatment could significantly alleviate the levels of tissue damage and fibrosis caused by MI surgery in rats’ hearts. However, the conclusion was limited due to the lack of zoomed out images showing the full LV section. Comprehensive comparison will be included in the future study. Consistent with the in vitro results, MI-induced myocardial fibrosis represented by the alteration of markers was significantly inhibited by UA administration. Meanwhile, the MI surgery inhibited Nrf2 pathway in cardiac tissues, which was then rescued by UA treatment. Overall, these results provided the evidences to demonstrate UA’s function in vivo.In summary, the present work demonstrated that UA could inhibit TGF-β1-induced CMT process in cardiac fibroblasts via upregulating Nrf2 expression. In vivo administration of UA could significantly improve myocardial fibrosis from myocardial infarction surgery in rat model. Regarding the current clinical exploration of UA on skeletal muscle function, iron metabolism and endurance performance, our study further explored the function and mechanism of UA in cardiac fibrosis and highlighted the application of UA in the clinical treatment of cardiovascular diseases in the future.


JAK/STAT signaling pathway is one of the important regulatory signaling cascades for the myriad of cellular processes initiated by various types of ligands such as growth factors, hormones, and cytokines. The physiological processes regulated by JAK/STAT signaling are immune regulation, cell proliferation, cell survival, apoptosis and hematopoiesis of myeloid and non-myeloid cells. Dysregulation of JAK/STAT signaling is reported in various immunological disorders, hematological and other solid malignancies through various oncogenic activation mutations in receptors, downstream mediators, and associated transcriptional factors such as STATs. STATs typically have a dual role when explored in the context of cancer. While several members of the STAT family are involved in malignancies, however, a few members which include STAT3 and STAT5 are linked to tumor initiation and progression. Other STAT members such as STAT1 and STAT2 are pivotal for antitumor defense and maintenance of an effective and long-term immune response through evolutionarily conserved programs. The effects of JAK/STAT signaling and the persistent activation of STATs in tumor cell survival; proliferation and invasion have made the JAK/STAT pathway an ideal target for drug development and cancer therapy. Therefore, understanding the intricate JAK/STAT signaling in the pathogenesis of solid malignancies needs extensive research. A better understanding of the functionally redundant roles of JAKs and STATs may provide a rationale for improving existing cancer therapies which have deleterious effects on normal cells and to identifying novel targets for therapeutic intervention in solid malignancies. The majr pylori . Besidesr pylori . Recent is of GC .Although, several drugs have been developed for the treatment of GC, the overall prognosis for GC patients is still dismal . Current757FF GC established mouse models. STAT3 is known to control the transcriptional epithelial-to-mesenchymal (EMT) regulators through G1 and G2/M cell-cycle progression, leading to the progression of metastasis in gastric malignancies , MMP7, MMP9, and survivin which regulates cell proliferation and survival  is among the top 10 ranked malignancies in terms of both incidence and mortality. Histopathologically two major types of EC are esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC). ESCC, on the other hand, accounts for almost 90% of all EC cases worldwide . Regardlp-JAK1 and p-STAT3 in primary ESCC tumors predicted the poor prognosis of ESCC patients  was employed in vitro and in vivo as a potent compound for the treatment of ESCC , purified root extract from medicinal herb  of ESCC   . HCC hasA number of pharmacological inhibitors can be used for the inhibition of the JAK/STAT signaling pathway in controlling HCC . SSI-1 . Ruxolitin vitro and in vivo settings (Head and neck squamous cell carcinoma (HNSCC) is mostly derived from mucosal epithelial of the buccal cavity . The consettings . Increassettings . Furthersettings . Moreovesettings .p-JAK tyr1007 as well as mRNA expression of respective genes in HNSCC cells. Leeman-Neill et al. also demonstrated growth inhibitory effects of guggulsterone in HNSCC preclinical models mediated by modulation of STAT3 signaling  is the most common heterogeneous malignancy and the second leading cause of cancer-associated mortalities in females. Thanks to worldwide screening for early diagnosis, BC has a better survival time compared with other gynecological malignancies. Apart from the genetic predisposition of BRCA1/2, PTEN and TP53 mutations in high-risk groups, the other genetic expression profile of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 (HER-2) contributes to the pathogenesis and stratification of BC . Besides+ BC, but STAT1 actively promotes tumor growth in ER− tumors or ER+ tumors in premenopausal BC  is the most common cause of cancer-related deaths from gynecologic malignancies accounting for 4.3% of female deaths worldwide . The higogenesis . Recent ogenesis . Despiteogenesis . To dateogenesis . In precogenesis , 2020. Rogenesis . Also, rogenesis . Curcumiogenesis . Curcumiogenesis . Similartivation . The raptivation . Curcumitivation . In a sitivation . A studytivation . Also, Htivation . Besidestivation . Huang etivation . Severaltivation . Varioustivation . The dectivation . Src, a tivation . Howevertivation . The IL-tivation . Anothertivation   have been found to modulate the expression of molecules involved in the JAK/STAT signaling network. Preclinical studies in cancer models have shown the effect of various inhibitors of JAK/STAT signaling which resulted in inhibition of cellular proliferation and tumor progression.Thus, JAK/STAT signaling appears to be an important target with the potential for a high therapeutic index. Although it takes more than two decades to get approval from FDA to target JAK/STAT signaling. In the coming future, we expect more specific compounds with the least deleterious effects on normal cells which target particularly the kinase activity of JAK/STAT mediators to attenuate its amplification in malignancies. However, the big question is whether next-generation compounds/inhibitors will be able to attenuate the functional part of mutated JAKs and leave wild-type JAKs unaffected. This approach of inhibition could decrease the deleterious effects of compounds/inhibitors on normal cells and will improve the drug efficacy. Besides various preclinical and experimental settings, effective STAT inhibitors in clinical settings are still a dream to come true. Although targeting transcriptional factors is not common, however, this approach will lead to more specific inhibition and in the coming future could be a promising therapeutic approach against JAK/STAT signaling in solid malignancies. However, it is important to validate the mechanism of action of small molecule inhibitors (SMIs) of the JAK-STAT pathway before conclusions can be drawn about their clinical use. The hope is to administer SMIs of JAK-STAT pathway with minimum risk to human subjects and that these inhibitors will produce a significant therapeutic effect against the solid malignancies.


Much of online conversation today consists of signaling one’s political identity. Although many signals are obvious to everyone, others are covert, recognizable to one’s ingroup while obscured from the outgroup. This type of covert identity signaling is critical for collaborations in a diverse society, but measuring covert signals has been difficult, slowing down theoretical development. We develop a method to detect covert and overt signals in tweets posted before the 2020 US presidential election and use a behavioral experiment to test predictions of a mathematical theory of covert signaling. Our results show that covert political signaling is more common when the perceived audience is politically diverse and open doors to a better understanding of communication in politically polarized societies. Individuals often signal identity information to facilitate assortment with partners who are likely to share norms, values, and goals. However, individuals may also be incentivized to encrypt their identity signals to avoid detection by dissimilar receivers, particularly when such detection is costly. Using mathematical modeling, this idea has previously been formalized into a theory of covert signaling. In this paper, we provide an empirical test of the theory of covert signaling in the context of political identity signaling surrounding the 2020 US presidential elections. To identify likely covert and overt signals on Twitter, we use methods relying on differences in detection between ingroup and outgroup receivers. We strengthen our experimental predictions with additional mathematical modeling and examine the usage of selected covert and overt tweets in a behavioral experiment. We find that participants strategically adjust their signaling behavior in response to the political constitution of their audiences. These results support our predictions and point to opportunities for further theoretical development. Our findings have implications for our understanding of political communication, social identity, pragmatics, hate speech, and the maintenance of cooperation in diverse populations. Individuals constantly emit signals of their identity, consciously and unconsciously, informing others about the sort of person they are. Identity signals are any components of communication that inform receivers of the signaler’s membership (or nonmembership) in a subset of individuals –3. Such 413Identity signaling is especially important in vast and diverse social communities, in which little can be assumed about strangers in the absence of identity information. This type of scenario is made all the more common in the digital age . In receGiven the social importance of political identity in the United States and other countries , 20, we 21Overt, unambiguous signals of identity are useful when individuals can sufficiently benefit from their role in supporting positive assortment—preferentially interacting with similar others. A wide literature on social tags and ethnic markers has documented and modeled the utility and likely emergence of such signals for this purpose , 28–31. 28We refer to identity signals that are accurately received by their intended audience but obscured when received by others as covert signals , 32, 33.3437Recently, Smaldino and colleagues have developed a theory of covert signaling, using formal mathematical and agent-based models of cultural evolution to examine the circumstances under which overt or covert identity-signaling strategies should be favored , 33. The4851It is likely that a great deal of online speech is covert, especially on social media platforms on which users can be personally identified, such as Twitter. Although other social media sites have more users than Twitter , TwitterOur data were collected during an especially salient period for political identity signaling on Twitter: the 2020 COVID-19 pandemic, in which millions of people were restricted from gathering and communicating in person, making online engagement one of the principal ways to interact with other humans. Moreover, our tweets were collected and assessed in the wake of many high-profile sociopolitical events in the United States, including the Black Lives Matter protests following the murder of George Floyd, the confirmation of Supreme Court Justice Amy Coney Barrett, and the 2020 presidential election. Opinions on these events were often polarized, making expressions of those opinions into signals of political identity.While they are likely to be common, covert signals are also inherently challenging to study empirically because, by definition, they require insider knowledge to be detected. In this paper, we introduce a theoretically motivated measurement of covertness, focusing on identity signals in the context of political speech online. Essentially, covertness was measured in terms of how people from ingroup and outgroup political groups perceived different tweets. On Twitter, cross-partisan followers are rare , and thuMaterials and Methods.Our paper constitutes a direct empirical test of the theory of covert signaling. Based on this theory, we predict that covert signaling will be more prevalent among 1) individuals in more heterogeneous communities or individuals with minority status and 2) individuals who face higher costs from being recognized as dissimilar. We derive a simple mathematical model of our experimental design, which yields more precise predictions concerning the relationships between covert signaling, the frequency of outgroup members in the audience, and the cost of being disliked. We test these predictions in a behavioral experiment in which participants select from a set of tweets that contain either overt or covert political signals to communicate with and be evaluated by an audience of varying partisanship C. We comAudience members in the behavioral experiment liked and disliked covert and overt tweets in line with our definitions of covert and overt signaling. At the same time, as might be expected from a comparison between theoretical modeling and empirical reality, participant behavior was more nuanced than our original theoretical assumptions. Overall, as shown in In order to better interpret the results of our experiment, we developed a simple mathematical model in which we consider the relative expected payoffs to pure strategies of covert or overt signaling. The model allows us to make predictions under the assumption of rational behavior, though the source of that rationality could stem from either individual strategizing or population-level selection on optimal signaling strategies .n, a proportion, d, will be members of the outgroup, and the rest will be members of the ingroup. We assume that overt signals are received  by all audience members with probability, R, and that covert signals are received by ingroup members with a probability Or < Ir. Audience members who perceive similarity will like the signal, conferring a benefit, b, on the signaler, while audience members who perceive dissimilarity will dislike the signal, imposing a cost, c, on the signaler. These costs and benefits are added to the baseline benefit for participation, w0.In an audience of size The expected payoff to an overt signaler is therefore the following:Similarly, the expected payoff to a covert signaler is the following:Covert signalers avoid some of the cost of being disliked but also receive fewer liking benefits. The relative advantage of covert signaling depends heavily on the proportion of outgroup members in the audience, as well as the signal efficiency of both overt and covert signals.n = 10, R = 0.8, Or = 0, indicating the importance of testing these models empirically.We calibrated the model to our experiment by setting artisans . Note thartisans , 33 thatd. We therefore expect a smaller effect of cost compared to outgroup size in our experiment. It is also worth noting that this simplified model focuses on pure strategies, predicting a total switch from overt to covert signaling as the audience reaches a critical threshold of outgroup members. In reality, experimental subjects often played mixed strategies, indicating that real world signaling is much more fraught with uncertainty than the model indicates.The theory of covert signaling , 33 predSI Appendix, Fig.S9). In these models, we controlled for the participants’ age, gender, race, education, and political group . SI Appendix, section 4.3) across the distribution of gender, age, race, and education in our sample. Our estimates and CIs, therefore, represent the effects for a population similar to our sample: politically engaged and social media–literate Americans across the political spectrum . The resulting average marginal effect (AME) of outgroup size on the use of covert tweets represents the difference between the average predictive margins between two specific values of outgroup size. We used the Spost13 package  and cost of dislikes (0.5 or one cent) within individuals and between individuals, we estimated a mixed-effects Poisson model on the count of tweets shared per round, with each outgroup experimental condition nested within individuals and random intercepts for each individual. We used a Poisson distribution to approximate the distribution of the number of covert tweets shared and total number of tweets shared in our sample .In line with our first theoretical prediction, we found that participants used more covert signaling as the number of outgroup individuals in the audience increased, as long as the outgroup was conceptualized as cross-partisans B. As theThere was no effect of outgroup size on the use of covert tweets when the outgroup consisted entirely of members of the same political party, copartisans holding either more moderate or more radical views , with no consistent differences observed for purely copartisan audiences in which the outgroup was defined only by the far-left/right mainstream split (Left).The main results hold descriptively across all four political groups : As the am split , Left.There were small variations across the four political groups. In the copartisan condition, participants on the right side of the political spectrum used slightly more covert tweets compared to participants on the left. In the cross-partisan condition, the patterns also varied between moderates and radicals. On the one hand, mainstream right and left individuals had quite an increase in their covert signaling when the number of outgroup audience members changed from four to six. On the other hand, radical right and left participants exhibited a more linear increase in their proportion of shared covert tweets by outgroup size. Radical right and left participants were also more bimodal, with one subgroup sharing almost only covert tweets when they were in the minority and another subgroup sharing a relatively lower proportion of covert tweets. The difference between moderates and more extreme participants was greater on the right compared to the left. Far-right participants had fewer changes in their signaling strategy over varying outgroup sizes compared to moderate right participants. Though we could not determine an explanation for these differences with such small groups, our results point to some variation in the perceived ability to assort with similar others and the perceived cost of signaling.We have shown how individuals may be strategically altering their online communication based on their likely audiences and that they do so in ways mostly consistent with the theory of covert signaling. This work connects to older theories of both identity signaling , 61, 63 The theory predicts that individuals should use more covert signaling in more heterogeneous groups or when they are in the minority. We found support for this prediction in the ways people shared political speech in a behavioral experiment. We observed the highest levels of covert signaling when audiences consisted almost entirely of cross-partisans, supporting the notion that covert signaling is a strategy for avoiding detection by hostile outgroup members. Of note, we selected tweets for our study at a time of heightened partisan divisions: the four weeks preceding the 2020 US presidential election. Consequently, these tweets mostly discussed the opposing political party. This focus was reflected in our behavioral experiment, in which we did not observe an effect of audience composition when all members were (more or less extreme) copartisans. In that societal context, participants might have perceived the cost of dislikes to be minimal and have likely focused on partisan disputes in their real-life conversations happening around that time. Future work testing the theory of covert signaling should also examine signaling strategies in copartisan conversations during times of salient intragroup political divisions.Results, helps to explain why the effect of audience composition  should be so much stronger than the effect of cost. However, the effect of cost is even smaller than we expected. Future work should take into account stronger differences in potential losses to investigate differences in the cost of dislikes.We found no significant effect of cost in our experiment. In previously published, formal models , 33, cosWe found small differences in the use of covert tweets between far-left/right participants and moderates. The theoretical models that inspired this study , 33 makeOur study reflects the complexity of how strategies of identity signaling can vary across communities and time. In the real world, including Twitter, communities are highly assortative, and individuals can often restrict interaction partners to members of their ingroups –68. TwitAnother direction for future empirical work on covert signaling in more natural settings is the analysis of the dynamic relationship between covert signaling and social networks. Specifically, individuals who are more likely to use overt signaling might end up in more homogeneous networks, either by choice or because they are shunned by disagreeing audiences. In our experimental design, the audience composition was fixed, but in reality, networks change after individuals express their opinions and identities over time. Signal type and utility can also be affected by changes in the network. For example, as familiarity with initially covert signals grows beyond insider networks, those signals can become overt [as with the recent case of the anti-Biden catchphrase “Let’s go Brandon.” ]. In ourOur empirical study could not discern intent on the part of either signalers or receivers. Signalers did change their signaling strategy by using proportionally more covert tweets as the audience contained more outgroup members. We tried to construct a situation that would resemble identity signaling, in which each participant could only share tweets from their own political side and were rewarded for being only correctly perceived by outgroup members. However, the participants may have been trying to maximize payoffs irrespective of sharing their identity. To further test the theory of covert signaling, we should study situations in which we know individuals want to share their identity. Furthermore, we should differentiate outgroup receivers who are “churlish,” disliking any signal that they do not see as ingroup , from thThis work provides three specific pathways to develop and refine the theory of covert signaling. First, our findings shed light on the complexities of measuring the covertness of signals in a real-world setting. Our study design assumed a relatively clear dichotomy between overt and covert signaling, in line with prior modeling work , 33. HowSecond, our findings highlight other strategies that individuals might use when signaling in heterogeneous networks beyond switching from overt to covert signals. We find here that participants were overall more cautious with what they shared in heterogeneous environments or when in the minority, sharing fewer tweets overall. In case of a potential higher cost of being disliked by the outgroup, many might believe that the benefits of identity signaling to the ingroup might not be strong enough to compensate for the potential cost of being disliked by the outgroup and thus decide to reduce the amount of signaling altogether. Future iterations of the theory of covert signaling could model not only the benefits of covert versus overt signals but also consider the decision to signal in the first place.Finally, in large, multicultural societies, identity is both critical and complicated. It is multidimensional and contextual , 70, andOur analysis suggests that people adjust their political speech according to strategic incentives, in ways predicted by our formal mathematical and computational models. Our work also points to specific areas in which the theory of covert signaling could be improved. We believe this work provides support for calls to increase the integration of formal modeling with mainstream social science research –76. FormOur work highlights that even deciding whether to classify communication as “political” is inherently context dependent. The significance of a statement depends on the circumstances in which it is communicated and on the background knowledge of the receiver. This indicates that automated methods to detect political speech online  selecting Twitter users who might be more likely to use covert or overt political speech and downloading their tweets, 2) rating a selection of tweets on different dimensions and marking a subset as more likely to be covert or overt, and 3) conducting a behavioral experiment with the selected covert and over tweets to test whether people indeed use them to communicate their political beliefs in theoretically predicted conditions.Our study took place between September 15 and November 1, 2020. Our study was deemed exempt by University of California Merced’s Institutional Review Board office (UCM2020-65) on May 5, 2020. The anonymized data and code are available on the Open Science Framework (OSF): DOI 10.17605/OSF.IO/XMZRA .We developed a process for selecting candidate tweets that would maximize the proportion of overt and covert political signals on both sides of the political spectrum A. To dowSI Appendix, Table S1). These news accounts generally corresponded to the progressive left wing, more closely affiliated with the Democratic party, and the Trumpist or Tea Party right wing, more closely affiliated with the Republican Party. As Twitter’s application programming interface (API) limits the download of lists of followers, we randomly sampled 10,000 followers of each of the eight news accounts. Any account that had not tweeted 4 wk prior to sampling was rejected and replaced through continued sampling. We were able to download the list of followers for 73,869 of these accounts .First, we selected seed Twitter accounts from a list of followers of eight news accounts representing far-left and far-right factions of the US political landscape on Twitter . While these sets of accounts might not represent a coherent political identity, they can still be combined to approximate the orientation and degree of political engagement of a single Twitter user. We downloaded the follower list for each of the news accounts and ranked far-left/right seed accounts by the proportion of far-left/right news accounts they followed from our list. We selected the top 20% of these seed accounts on the left and right, representing the most “engaged” accounts and thus more likely to use political identity signaling for a total of 16,398 engaged users.Second, we developed a schema for classifying the political orientation of each seed account based on their media diet as represented by the proportion of far-left or -right political news sources they followed. We identified highly “polarized” US-based news outlets using data from mediabiasfactcheck.com and selected those whose Twitter accounts had at least 10,000 followers. This produced a set of 43 far-left news accounts and 50 far-right news accounts  were rare, and embeddedness in a cross-partisan follower network was practically nonexistent. Instead, we operationalize homogeneity and heterogeneity in terms of the extent of engagement with copartisan far-left/right news accounts. Homogeneous follower networks are those that are about as engaged or committed to strong partisanship as their seed accounts, while heterogeneous follower networks are much less engaged than their seed accounts. Drawing on the theory of covert signaling, we expect seed accounts with heterogeneous networks to be less likely to overtly share their political identity than seed accounts with homogeneous networks.Followers of our seed accounts were labeled as engaged if they followed at least as many news accounts as the 50th percentile of our initial pool of seed accounts (three for the left and four for the right) and as disengaged if they followed at most one far-left/right news site. In order to have a pool of tweets with both many overt and covert tweets, we selected seed accounts that had the most homogeneous and heterogeneous networks. Specifically, we selected the top 20% of engaged seed accounts, ranked by the proportion of engaged followers in their networks, and labeled them as having “homogeneous networks” . Then, we selected the top 20% of engaged seed accounts, ranked by the proportion of disengaged followers in their networks, and labeled them as having “heterogeneous networks” . We ended up with 1,834 far-left accounts and 1,446 far-right engaged accounts with either homogeneous or heterogeneous follower networks.Rating and Classification of Tweets), removing tweets that were posted more than 6 wk prior, were replies to other users, contained images or news article links, were not in English, or were too short . To further improve legibility, we deleted all other links from tweets. Finally, we removed retweets to avoid duplicates. This procedure produced between 1,303 and 2,100 tweets for each of the four groups of seed accounts (far-left/right with homogeneous/heterogeneous follower networks) for a total of 6,594 tweets. Of those, we randomly selected 1,303 or 1,304 tweets for each group for a total of 5,215 tweets  to be rated and classified as covert or overt, as described in the following sections.Fourth, we downloaded tweets from the resulting four groups of engaged seed accounts (far left/right with homogeneous/heterogeneous follower networks). Using the Twitter API, we downloaded up to the maximum number of available tweets from each user  in the 4 wk leading up to the behavioral experiment. We filtered out tweets that would be difficult to understand for independent raters .To determine whether tweets downloaded were overt or covert, we recruited human raters B. To estSI Appendix, section 3). For the political orientation dimension, raters were asked to estimate the perceived political orientation of the tweets’ authors on a seven-point scale from extreme left to extreme right or to answer “not political.” For the affective response dimension, raters were asked two questions: to estimate how negatively they felt toward a tweet and how offensive the tweet would be to people similar to themselves (from very offensive to not offensive). The two ratings were highly correlated (r =.70) and were averaged in one summative scale. We pretested a third question on the offensiveness for other groups but excluded it, given that it did not clearly reflect how the ingroup or outgroup reacted to the political signal.We asked raters several questions about each tweet to determine the tweet’s position on two dimensions: political orientation and affective response . To investigate interrater reliability, we fitted a random effects linear model to probe how much variation in the political leaning ratings could be attributed to individual raters. The multilevel regression model estimates random effects for tweet, rater, and partisanship of rater (our four groups). We used Shapley regression to decompose the explained variance . The multilevel regression accounts for 50.0% of the total variance. Of this, 89.3% of the variance can be attributed to the tweets themselves. Raters account for 10.3% of the explained variance, which is just 5.2% of the total variance. Thus, we conclude that while each rater saw a different subset of tweets and rating is somewhat noisy, interrater reliability was high.Each rater received 50 randomly selected tweets, a number that, according to our pretests, one person could evaluate in 20 min, approximating the feasible duration for an online study . Of the SI Appendix, Table S6) drawing on the theoretical framework of Smaldino and colleagues : large copartisan difference in tweets’ perceived political orientation and affective scores, neutral ratings of political orientation given by moderates, neutral or positive affective score by moderates, and political content recognized by far copartisan raters. We marked tweets as potentially overt if they satisfied the following four theory-based criteria : small cross-partisan difference in tweets’ political orientation score, large difference in cross-partisan and small difference in copartisan affective score, and political content recognized by all raters. We set up percentile cutoffs to obtain around 30 tweets that had the highest probability to represent each of the four categories—covert left  . Together, these show our ratings and criteria carry useful information related to political signaling and suggest that covert and overt tweets should be perceived differently by participants in our behavioral experiment. While our methods were designed to select the most likely covert and overt tweets in our sample, we refer to these tweets as “covert” and “overt” in our results for simplicity.In We used the tweets selected as overt and covert political signals in the previous section to conduct an experimental study testing our theoretical predictions in a controlled setting C. We desSI Appendix, section 2 who were screened as being far-left/right in their political views and 240 who were more moderate, with an equal number of participants with left and right political identities in far and moderate groups. In the first part of the experiment, participants were asked if they liked, disliked, or were neutral toward each of the 80 left- and right-leaning tweets selected for this stage. In the second part of the experiment, participants were asked to select and share tweets to an audience of 10 other participants. Each participant saw a randomly sorted list of 40 only left- or right-leaning tweets, matching their own political orientation, split between covert and overt messages. Participants were paid a baseline of $3.40 and were given a bonus for each audience member who liked the majority of the tweets they shared and charged a cost for each audience member who disliked the majority of their tweets. The instructions to the participants are shown in SI Appendix, Figs. S6 and S7.We selected a subset of 240 participants from the pool of raters described in Participants were randomly split in two experimental groups: one having a strictly copartisan audience (in which audience members were identified as either extreme or moderate copartisans) and one having a partly cross-partisan audience (in which audience members were identified simply as either co- or cross-partisans). Each group participated in eight successive rounds, randomly ordered to vary two within-subject factors: 1) the number of people in the audience with political leanings different from their own  and 2) the cost of dislikes, either a low-cost condition in which the cost of dislikes (0.5 cents) was half of the bonus for likes (1 cent) or a high-cost condition in which the cost of dislikes was the same as the bonus for likes (both 1 cent). Bonuses were calculated based on the actual likes and dislikes recorded from participants in the first part of the experiment.Supplementary File


Drosophila, where they initiate a signaling pathway that regulate growth by influencing Hippo signaling and morphogenesis by regulating Planar Cell Polarity (PCP). In this review, we discuss recent advances in our understanding of the mechanisms by which Ds-Fat signaling pathway regulates these critical developmental processes. Further, we discuss the progress in our understanding about how they function in mammals.Dachsous (Ds) and Fat are evolutionarily conserved cell adhesion molecules that play a critical role in development of multiple organ systems, where they coordinate tissue growth and morphogenesis. Much of our understanding of Ds-Fat signaling pathway comes from studies in  Drosophila, based on lethality in the mutants and were subsequently shown to cause the dramatic overgrowth of the imaginal discs, the larval precursor for adult organs . These cell adhesion molecules interact in a heterophilic manner to initiate bidirectional signaling , mediated by their cytoplasmic domains to regulate a number of critical developmental processes including growth, tissue patterning, convergent extension and directed cell migration. Mutations in FAT4 and DCHS1 are associated with many cancers and multi system developmental defects such as Van Maldergem syndrome and Hennekam syndrome, characterized by craniofacial anomalies, intellectual dysfunction, digital contractures, hypoplastic kidneys, sternal and auditory defects  and a relatively small intracellular domain (ICD) . Unlike  defects .Drosophila Ds-Fat mediated cell-cell adhesion is a key upstream regulator of Hippo signaling. However, in mammals Dchs1/Fat4 influences this pathway only in specific tissues. Further, the mechanism by which they influence Hippo signaling seems to have evolutionarily diverged , Warts (Wts) and their cofactors Salvador (Sav) and MOB as tumor suppressor (Mats)  respectidiverged .Drosophila adult wings and abdomen, and the hair follicles in mammalian skin uniformly point to one direction. Similarly, the stereocilia in the cochlear hair cells in the mammalian inner ear are also uniformly organized, and disorganization of this leads to deafness. PCP is primarily regulated by a conserved signaling network mediated by the core pathway of PCP proteins, which consists of the transmembrane proteins Starry night (Stan) , Frizzled and Vangogh (Vang) , and the cytosolic proteins Prickle (Pk), Disheveled (Dsh) and Diego (Dgo)  . These pgo (Dgo) . Fat sigDrosophila, where the pathway is most extensively studied. Then we will summarize our understandings of how these protocadherins function in mammals.Here we will first describe recent advances in our understanding of Fat signaling in Drosophila Fat is a very large (560 KD) transmembrane protein with 34 cadherin repeats, 4 EGF like repeats and 2 lamin-G domains in the ECD. This is followed by a transmembrane domain and relatively small ICD, which does not have any identifiable domains. However, specific regions in the ICD have been identified that play important role in growth or PCP regulation  transcription factor, Ds is expressed in a steep decreasing gradient from the periphery to the center of the wing pouch. On the other hand, Vg promotes Fj expression in an opposite decreasing gradient from the center to the periphery of the presumptive wing primordium . Computadachs mutants suppressed the lethality and overgrowth phenotypes of fat mutants  and FBXL7 respectively  was isolated based on the reduced crossvein spacing phenotypes in the adult wings of the mutant animals . App encof Dachs . Howeverof Dachs  and juxtof Dachs .fat mutants can significantly destabilize Ex levels.Vamana (Vam) [also known as Dachs ligand with SH3 domains (Dlish)] was isolated by two groups independently . Misra aex transcription by activated Yki and this is thought to be the key mechanism by which loss of Fat induces overgrowth  is a FERM domain protein that functions as a crucial negative feedback regulator in the Hippo signaling pathway. Ex is transcriptionally induced by activated Yki and it localizes to the apical plasma membrane by interacting with the cytoplasmic domain of the cell adhesion protein Crumbs . It direergrowth . Howeverergrowth . ConcomiDco3. Dco3 mutants also display higher levels of Dachs. Dco encodes Casein Kinase-1ε and phosphorylates Fat-ICD in the D region , as the name suggests, was isolated based on the overgrowth of the wing discs in animals carrying a neomorphic gain-of-function mutant allele, D region , which tD region . Thus, ilft mutants exhibit reduced crossvein spacing  was reported as an interactor of Fat and Ds ICDs in a yeast two-hybrid screen. Subsequent genetic analysis revealed that the  spacing . At the Atrophin (Atro)  is a downstream effector of Fat in PCP regulation in the eye . Atro isfat mutants as well as cells expressing Dachs fused to Zyxin, Wts is destabilized  provides a connecting link between core PCP and Fat signaling. pk encodes Prickle and Spiny-legs (Sple) proteins and the relative ratio of the two isoforms regulate core PCP polarization relative to Ds-Fat (Drosophila abdomen.Fat and Ds regulate tissue patterning by influencing the core PCP signaling. Both Ds, Fat and the core PCP proteins localize in an asymmetric manner creating cellular anisotropies that regulates PCP . Ds and o Ds-Fat . Tissueso Ds-Fat . Ds and o Ds-Fat  (Ayukawao Ds-Fat . HoweverFat and Ds also regulate PCP in a Dachs independent manner. The transcriptional corepressor Atrophin binds to the cytoplasmic domain of Fat and regulates PCP in the equatorial region of the eye . Howeverfat mutant clones exhibit higher tension at the clone border abutting the wild type tissue but show less tension within the clones (Fat, Ds and Dachs also play an important role in polarization of adherens junction tension . Laser ae clones . Dachs ie clones .Drosophila, Fat signaling in vertebrates has diverged through evolution. The key downstream components such as Dachs, Vam/Dlish and Ex are not conserved in mammals (In contrast to Fat signaling in  mammals . Althoug mammals . Mutatio mammals . Mouse k mammals . In most mammals  (Zhang e mammals . In mous mammals . In spec mammals  (Ragni e mammals . Fat4 muDrosophila and mammals in the last few years, our knowledge of this pathway still remains rudimentary. It is not completely understood how Fat regulates Hippo pathway. Further, loss of Fat/Ds in Drosophila and Fat4/Dchs1 in mice results in altered aspect ratio of the organs. It remains a challenge to identify the mechanisms by which these protocadherins regulate organ shape. Further, it will be important to understand the molecular basis underlying the differences in signaling output in different tissues. Future studies in Drosophila and mammals will provide a unified mechanism by which Ds and Fat coordinate growth and morphogenesis in multiple organs.Precise coordination of growth and morphogenesis during development is critical to formation of optimally functioning organs, and Fat signaling plays a central role in coordinating these processes. Although there has been significant progress in our understanding of this pathway both in


Thymic stromal lymphopoietin (TSLP) is a Th2-like cytokine involved in asthma pathogenesis. Excessive reactive oxygen species (ROS) production can lead to airway inflammation, hyperresponsiveness and remodeling. Mitophagy, followed by ROS production, is the selective degradation of mitochondria by autophagy and often occurs in defective mitochondria. In the present study, we aimed to examine the effects of TSLP on ROS production and mitophagy in human monocytes and to investigate the underlying mechanisms, including epigenetic regulation.SHDA gene. TSLP-induced SHDA expression was inhibited by the histone acetyltransferase inhibitor anacardic acid (AA) and the histone methyltransferase inhibitor methylthioadenosine (MTA), and chromatin immunoprecipitation assays revealed that TSLP enhanced H3 acetylation, H4 acetylation, and H3K4 and H3K36 trimethylation in the SHDA promoter. Confocal laser microscopy showed that TSLP treatment increased the signals of the mitophagy-related proteins PINK1, LC3, phospho-parkin and phospho-ubiquitin, and pretreatment with AA and MTA reduced TSLP-induced PINK1 and LC3 accumulation in mitochondria. Western blot analysis showed that TSLP significantly increased phosphor-AMPK signal intensity, and the effects were inhibited by the antioxidant NAC. The increased signal intensities of the mitophagy-related proteins PINK1, Parkin and LC3 I/II were decreased by dorsomorphin, an AMPK inhibitor. TSLP decreased M1-related cytokine CXCL-10 production and increased M2-related cytokine CCL-1 and CCL-22 production, which was suppressed by the mitophagy inhibitor Mdivi-1 and PINK1 gene knockdown.TSLP induced ROS generation, and the effects were reversed by the antioxidant N-acetylcysteine (NAC) in THP-1 cells. Transmission electron microscopy images showed donut-shaped mitochondria that lost the cristae ultrastructure after TSLP stimulation. A decrease in mitochondrial membrane potential, decreased MTCO2 expression, and increased mitochondrial DNA release after TSLP stimulation were found. TSLP enhanced mitochondrial complex I and complex II/III activity and increased mitochondrial copy numbers and the expression of the complex II Epithelial-derived TSLP regulates ROS production and mitophagy through AMPK activation and histone modification and alters M1/M2 chemokine expression in human monocytes.The online version contains supplementary material available at 10.1186/s13578-022-00767-w. Asthma is one of the most common chronic diseases in childhood, and its prevalence has been increasing in industrializing countries. Thymic stromal lymphopoietin (TSLP), interleukin (IL)-25 and IL-33 are upstream epithelial cytokines and play important roles in allergic diseases , 2. TSLPOxidative stress, which is physiological damage due to reactive oxygen species (ROS), has been demonstrated to induce smooth muscle contraction, airway hyperresponsiveness, mucus secretion, and epithelial shedding , 9. ExceMitophagy, which is mitochondrial autophagy, is important for mitochondrial quality control and essential for providing cellular energy, calcium homeostasis, redox signaling and apoptotic signaling , 13. MitHistone acetylation and methylation controlled by histone acetyltransferase (HAT), histone deacetylase (HDAC), and histone methyltransferase epigenetically regulate gene expression. Acetylation of histone 3 (H3) and H4 and trimethylation of H3K4 and H3K36 are associated with gene activation . Asthma Both TSLP and ROS production are important for the pathogenesis of asthma and other allergic diseases. However, the effects of TSLP on ROS production remain unclear. In the present study, we investigated the effects of TSLP on ROS production and mitophagy and the detailed mechanisms, including epigenetic regulation.We first investigated whether TSLP could induce ROS production in monocytes. THP-1 cells were pretreated with different concentrations of TSLP. Pretreatment with TSLP  significantly increased ROS production in THP-1 cells at the 2-h time point  analysis to observe whether TSLP alters mitochondrial integrity and induces mitophagy. We found that mitochondria decreased in size, cristae were lost, and autophagic vacuoles contained damaged mitochondria after TSLP treatment Fig. A. We alsND1 (respiratory chain complex I), SDHA (respiratory chain complex II), CYTB (respiratory chain complex III), COX1 (respiratory chain complex IV) and ATP6 (respiratory chain complex V) was screened using qRT–PCR, and we found that TSLP significantly increased the expression of the complex II gene SDHA but not the others  and the histone methyltransferase inhibitor methylthioadenosine (MTA)  Fig. A, B. TheMitophagy often occurs in defective mitochondria following damage or stress. Owing to the finding of TSLP-induced ROS production and mitochondrial damage, we further examined whether TSLP could induce mitophagy-related protein  signals in human monocytes. Western blot analysis showed that TSLP  significantly increased the signal intensities of PINK1, phospho-Parkin and LC3 after TSLP treatment Fig. A–C. ConfAMPK is a critical sensor of cellular energy status and has been reported to mediate mitophagy and promote mitochondrial fission . Some stTo investigate the functional effect of TSLP-induced mitophagy on human monocytes, we examined the effects of TSLP on chemokine production. TSLP significantly decreased the production of the M1-related chemokine CXCL-10 Fig. A and cytTSLP, IL-33 and IL-25 are epithelial cell-derived cytokines that stimulate group 2 innate lymphoid cells (ILC2s) to release type 2 cytokines such as IL-5 and IL-13 and play an important role in asthma pathogenesis . It has SDHA gene expression were significantly increased by TSLP. These data indicated that TSLP regulated mitochondrial redox in human monocytes.Inflammatory cells that are recruited to the asthmatic airway have an exceptional capacity to produce a variety of ROS, contributing to tissue damage and airway inflammation . EnvironSHDA gene promoter. Confocal laser microscopy showed that pretreatment with AA and MTA also reduced TSLP-induced PINK1 and LC3 expression in mitochondria. These findings suggest that epigenetic regulation may be an important mechanism by which TSLP induces ROS production and mitophagy.Another novel finding of the present study is the epigenetic regulation of TSLP-induced ROS production. Histone acetylation and trimethylation, which are usually carried out by a variety of HATs or methyltransferases, are associated with gene activation . In thisTSLP shifts macrophage polarization to an M2-like phenotype . AccordiMitochondria not only play a vital metabolic role but also cause cell death via toxic products associated with oxidative phosphorylation and mitochondrial quality control at the protein, organelle and suborganelle levels . MitophaTSLP, a Th2-like cytokine, plays an important role in the pathogenesis of asthma, allergies, and other airway diseases. Understanding the possible mechanisms of the effect of TSLP on ROS production can help us identify targets for therapy or find novel pharmaceutical strategies to reduce asthma morbidity. All the findings in the present study suggest that epithelial-derived TSLP may induce not only Th2-related cytokine expression but also ROS production, subsequently influencing cellular oxidative stress and mitochondrial quantity, regulating mitophagy and shifting M1/M2 chemokine expression through the AMPK signaling pathway and histone modification in monocytes Fig. . These f5/mL and pretreated with or without TSLP  for 2 h. ROS production was measured with a dichlorodihydrofluorescein diacetate  assay by flow cytometry according to the methods described in previous studies [THP-1 cells  were cultured in RPMI-1640 medium . The cells were resuspended in fresh media at a concentration of 1 × 10 studies , 40. FolThe mitochondrial fraction was isolated from THP-1 cells using a mitochondrial isolation kit, and the activities of complex I and complex II/III in whole cell lysates or isolated mitochondria were measured using a microplate assay kit  according to the manufacturer’s instructions.THP-1 cells were treated with TSLP for 8 h, and JC-1 dye  was used to measure the mitochondrial membrane potential by LSR II flow cytometry . JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (525 nm) to red (590 nm). Mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. After TSLP treatment, the cells were prepared for JEM-2000EXII transmission electron microscopy  to observe the morphology of mitochondria and mitophagy.The total mtDNA of THP-1 cells were isolated after 8-h TSLP stimulation by a mitochondrial DNA isolation kit  following the manufacturer’s instructions. Two primer sets specific for the mitochondrial ribosomal 16SRNA, mtDNA-79 and mtDNA-230, were designed as described in a previous study , 42. TheTo detect mitophagy, we used the Mitophagy Detection Kit  according to the manufacturer’s instructions. Briefly, THP-1 cells were incubated with serum-free RPMI containing 100 nM mitophagy dye at 37 °C for 30 min. After washing and discarding the supernatant, the cells were stimulated with 40 ng/mL TSLP in complete medium for 12 h. Mitophagy dye fluorescence was detected by an LSR II flow cytometer  with a PerCP Cy5.5 channel. The data were analyzed using FCS Express 4 Flow Research .MT-ND1, MT complex I), succinate dehydrogenase complex, subunit A, flavoprotein , mitochondrial cytochrome b , cytochrome c oxidase I , ATP synthase, H + transporting, mitochondrial Fo complex, subunit F6 , and mitochondrial proton carrier: uncoupling protein 2 (UCP2) were detected to investigate mitochondrial involvement. After treatment with TSLP for different durations, total RNA (1 μg) was reverse transcribed by SuperScript II using an anchored oligo-dT primer as instructed by the manufacturer , and the resulting DNA was used as template for qRT–PCR. The primer sequences and qRT–PCR were performed as described in our previous work [The gene expression of mitochondrial copy numbers, respiratory chain complexes I–V, including NADH dehydrogenase, subunit 1  according to MatInspector . The primer sequences were as follows: forward: 5′-CTGACCACACTACCTCAGCA and reverse: 5′-CAGTGCTTGCTTCTTGGTGA. All TaqMan reagents were purchased from Applied Biosystems. The relative intensities of the amplified products were normalized to the input DNA levels.ChIP was performed as described previously with minor modifications . PrimersTHP-1 cells were treated with TSLP for 2, 4, 6 or 8 h and analyzed by western blotting with anti-MTCO2 antibody (Abcam), anti-5′-AMP-activated protein kinase , anti-phospho-AMPK , anti-PINK1 , anti-LC3 , anti- phospho-Parkin (Ser65) Antibody #36,866  and anti-GAPDH antibodies . Western blotting analysis was performed as described in our previous work .Cultured THP-1 cells were treated with or without TSLP and fixed on glass slides. The cell slides were washed twice with PBS and permeabilized with 0.5% Triton for 5 min. These slides were then incubated in blocking solution  for 30 min. After being blocked, the slides were incubated with PINK1, LC3, phospho-Parkin (Ser65) Antibody #36,866  or phospho-Ubiquitin (Ser65), Cat. No. ABS1513-I  antibodies, followed by Alexa Fluor 488-labeled and Alexa Fluor 568-labeled MitoTracker mitochondrial tracking dye for 1 h. After washing, 4′,6′-diamidino-2-phenylindole (DAPI)  dissolved in PBS was used as a nuclear staining dye, and the slides were mounted on Vectashield (Vector Laboratories Inc.), covered with glass coverslips, and imaged using an LSM 700 microscope .After 2 h of TSLP pretreatment, THP-1 cells were exposed to phorbol 12-myristate 13-acetate , LPS  and IFN-γ  to induce M1 polarization or exposed to PMA and IL-4  to induce M2 polarization. After being treated with or without PMA/LPS/IFN-γ or PMA/IL-4, the cell-free media were collected at 48 h, and CXCL10, TNF-α, IL-6, IL-1β, CCL-1, and CCL-22 were measured by ELISA . CCL-22 concentrations in THP-1 cells and PMA/IL-4-primed THP-1 cells with or without the mitophagy inhibitor Mdivi-1 were also analyzed by ELISA.SHDA  was used to investigate whether SHDA was involved in TSLP-induced mitophagy. PINK1-knockdown cells were pretreated with TSLP for 2 h and then polarized to form M2 macrophages with PMA (20 ng/mL) and IL-4 (20 ng/mL). The supernatants were collected, and the CCL-22 concentration was measured after 48 h by ELISA.THP-1 cells were transduced using shRNA lentiviral particles at a multiplicity of infection (MOI) of 1. A MOI is the number of transducing lentiviral particles per cell. All shRNA lentiviral particles were purchased from the RNA technology platform and gene manipulation core of Academia Sinica. The lentivirus encoded either a nontargeted (nt) shRNA (TRCN0000208001) or a shRNA directed against PINK1 . A nontargeting (nt) shRNA (#TRCN0000208001) or shRNA targeting p value < 0.05.GraphPad Prism  and IBM SPSS statistical software  were used for statistical analysis. The Mann–Whitney U test was used for pairwise comparisons between samples with and without TSLP treatment. The immunoreactivities of mitophagy-related proteins in western blot analysis were normalized to those of the total proteins. The western blot results were analyzed by the Wilcoxon signed-rank test. Multiple nonparametric comparisons to assess the effects of TSLP on chemokines were analyzed by the Kruskal–Wallis test with post hoc analysis. Statistically significant differences were considered if a Additional file 1: Figure S1. Respiratory chain complex II SHDA is involved in TSLP-induced mitophagy. A The knockdown efficiency of SHDA was determined by western blot. B Mitophagy detection dye to quantify THP-1 cell mitophagy showed significantly increased fluorescence upon treatment with 40 ng/mL TSLP in the nontargeting control. When SHDA was knocked down, the increased fluorescence intensity of TSLP-induced mitophagy was inhibited. *p < 0.05, **p < 0.01 and ***p < 0.001 between THP-1 cells with and without TSLP treatment.Additional file 2: Figure S2. The effects of TSLP on hypoxia-inducible factor (HIF)-1-BCL2-interacting protein 3 (BINP3) signaling.