Chloridea virescens life history traits and flight performance. Sublethal concentrations of chlorantraniliprole and flubendiamide increased larval and pre-pupal development times and decreased larval weight; flubendiamide increased pupal development times. Chlorantraniliprole increased adult male longevity and reduced female fertility, while flubendiamide reduced fecundity. Overall life table parameters were negatively impacted by both treatments. Males exposed to either insecticide showed significant reductions in flight duration and distance for unsustained flights (<30 min). The duration and distance of the first flights were reduced when exposed to chlorantraniliprole. Sustained flights (>30 min) were generally unaffected by insecticide exposure and both sexes flew >6400 m in a single flight. The sublethal effects of flubendiamide and chlorantraniliprole on C. virescens’ population dynamics could lead to generation asynchrony and provide insufficient susceptible moths when sprayed on refuge crops. However, the distance and duration of flight may still be sufficient to ensure mixing of potentially resistant and susceptible populations from refuge plots.High-dose and refuge are the most important strategies for delaying resistance evolution in Bt crops. Insecticide sprays in refuge areas could be necessary and may limit refuge effectiveness. Here, we evaluated the sublethal effects of two diamide insecticides (chlorantraniliprole and flubendiamide) on  Chloridea (=Heliothis) virescens Fabricius [Lepidoptera: Noctuidae]) is an important economic pest of several crops in southern Canada, the United States, and throughout South America, except for Chile and southern Argentina [®) were commercially introduced in Brazil. This soybean event expresses genes encoding the insecticidal protein Cry1Ac of Bacillus thuringiensis Berliner (Bt) and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) protein of Agrobacterium sp. that confers tolerance to the herbicide glyphosate (RR) [The tobacco budworm, rgentina ,2,3. In rgentina . In the C. virescens [B. thuringiensis insecticidal proteins to ensure mortality of more than 95% of heterozygous insects [Previous work revealed that soybean MON 87701 × MON 89788 provides a “high dose” against major lepidopteran pests such as irescens ,7. The h insects ,11.Bt-susceptible pests, which can mate with rare survivors from the Bt crop field, decreasing the abundance of the resistant insects [C. virescens, supplemental foliar insecticide applications to control and reduce pest populations could be necessary in non-Bt soybean refuge areas [To manage evolution of resistance, the main insecticide resistant management (IRM) strategies are “high dose” and planting of “structured refuge” (non-Bt soybean). The structured refuge areas provide sources of  insects ,9. Recen insects . However insects ,13,14. Tge areas . Thus, tge areas .C. virescens [2+ from intracellular stocks, a process necessary for muscle contraction. In intoxicated insects, symptoms begin with cessation of feeding and uncoordinated muscle contraction, eventually causing mortality [Diamides (IRAC Group 28) are the most recent chemical group introduced to the insecticide market for use in soybean refuge areas to manage irescens ,17,18. Tirescens . Diamideortality ,22,23,24C. virescens in Bt soybeans as a model system to study potential effects of sublethal insecticide exposure on aspects of a resistance management system. Our objectives were to evaluate the sublethal effects of flubendiamide and chlorantraniliprole on life history traits of C. virescens, life table parameters, and flight performance in order to better understand the implications for soybean refuge area management, and by inference, refuge management strategies in other Bt crops.In the field, it is likely that in addition to direct mortality , some target pests may be exposed to sublethal concentrations, where they survive but suffer negative biological effects ,26. ManaC. virescens were obtained from Benzon Research . The larvae were reared on tobacco budworm artificial diet purchased from Southland Products . This diet was a dry premix and was prepared following the manufacturer’s suggested protocol. The C. virescens adults were fed a 10% honey solution and water via cotton wicks in Petri dishes placed on the bottom of adult cages. All insect stages were maintained in chambers at constant environmental conditions .Susceptible strains of ® 5 SC  supplied by DuPont and Belt®SC  by Bayer CropScience.The commercial formulations of diamide insecticides used in the bioassays were Prevathon−1) and nine concentrations of flubendiamide (1.8 to 180 ng mL−1) on a logarithmic scale were tested on newly molted third instar larvae of C. virescens using a diet-incorporated bioassay [C. virescens larvae were placed in each cup after the diet cooled. Ninety third instar larvae were tested per treatment. Insect mortality was evaluated daily for seven days following exposure to treated diets. Larvae were considered dead if they did not show head movement or peristaltic contractions when touched with a paintbrush. Moribund larvae were scored as alive [®, Berkeley, CA) to analyze the concentration-mortality relationship [50, LC40, LC30 and the corresponding confidence intervals (95% CI) for chlorantraniliprole and flubendiamide were estimated and the values were considered different when there was no overlap of the 95% CI .For determination of sublethal concentrations, eight concentrations of chlorantraniliprole  and examined daily for adult emergence. A representative number of newly emerged adults (<24 h) were weighed before they fed. The numbers of normal and deformed adults were recorded. Adults were considered deformed if they were unable to shed the pupal exuvium or had wing deformities. A pair of nondeformed, newly emerged moths (<24 h) was introduced into a rearing cage. The rearing cage was 12-cm-high with top and bottom diameters of 16 and 14 cm, respectively, and contained an inner sheet of paper and a transparent fabric top, both of which served as oviposition substrates. A Petri dish (6 cm diameter) was placed on the cage bottom with a wick of cotton soaked in a 10% honey solution for adult food. Oviposition substrates and food supplies were replaced as needed. In all treatments, at least 24 pairs of moths were used for studies. Eggs laid by each pair were counted daily until female death.r), the finite rate of increase (λ), net reproductive rate (oR), and mean generation time (T).Male and female adult longevity was recorded. Males were not replaced if they died before the female. To evaluate fertility (percentage of eggs that hatched), a minimum of 800 eggs from five random pairs per treatment were collected on the third day of oviposition. Immature development and survival, female oviposition period, fecundity, fertility, and female longevity were used to construct life tables for each treatment. Parameters measured included the intrinsic rate of increase  was used to attach the tether to the moth’s prothorax after clearing the scales with a small paintbrush. To minimize wing movement and stress before the flight tests, the moth was placed in a Styrofoam box and positioned, so its legs were in contact with the substrate. Moths were refrigerated (4 °C) for 10 min before the start of the flight assay. Due to variable effects of the insecticides on insect development and the large number of insects that needed to be reared, treatments could not be blocked over time in the flight chamber. To the degree possible, the insects flown on any given day were all either male or female from one insecticide treatment and the control.Adults were fed with a 10% honey solution for 4 h before tethering while being maintained in environmental chambers (25 ± 2 °C and 60 ± 10% RH). Moths were anesthetized in a freezer (−15 °C) for 5–7 min. Stainless-steel entomological pins (number 00) with the nylon head removed were used to tether the moths. The cut end was inserted into one end of a computer connector pin that could then be connected to the flight mill arm. A small cork (ca. 2 mm square) attached to the pointed end of the pin served as the tethering point. Gel super glue with bond activator  and assays were conducted from 7 p.m. to 7 a.m. during the dark phase of the daily cycle. Each moth was flown a single night. A custom LabView  computer program automatically recorded data for each of 24 stations including the clock time of the beginning and end of each flight, and the number of revolutions of each flight. These data were then used to calculate flight duration (s), flight distance , and flight speed (m/s). The time between flight bouts also was calculated (arrest time in s). Additional calculations were made to estimate total flight time, total distance, and total arrest time over the 12-h assay.C. virescens’ individual flight durations were categorized as sustained (>30 min) or unsustained (≤30 min). This delineation was consistent with a gap in the distribution of flight durations and has been used by many other researchers to delineate sustained flights in various insect species [For analyses,  species ,44,45. A2; α = 0.05) [The data were subjected to exploratory analyses to assess the assumptions of normality of residuals , homogen = 0.05) .C. virescens were estimated using a matrix model approach in Pop Tools [r, intrinsic rate of increase; Ro, net reproductive rate; and T, generation time (days) for each treatment. Confidence intervals were estimated by bootstrap resampling with 5000 iterations. Permutation testing was used to compare life table parameters between treatments with a Bonferroni correction for multiple comparisons. The test statistic was simply the difference between treatment parameters and the p-value was estimated as the number of times the resampled test statistic exceeded the original test statistic out of 5000 iterations.Life table statistics for op Tools . Matriceop Tools  to estimChloridea virescens’ third instar larvae were more susceptible to the lethal concentrations of chlorantraniliprole than flubendiamide. The LC50 of chlorantraniliprole was 4.819 ng mL−1 and corresponded to about a 7-fold lower concentration than the flubendiamide LC50 (27.972 ng mL−1). Likewise, chlorantraniliprole LC40 and LC30 concentrations (4.007 and 3.289 ng mL−1) were approximately 4.5- and 4-fold lower than flubendiamide LC40 and LC30 concentrations (18.583 and 11.997 ng mL−1) (ng mL−1) .p < 0.0001; C. virescens’ survival was lowest in the chlorantraniliprole treatment during this period, in comparison with flubendiamide and the control. After placing exposed larvae on untreated diets, significant differences also were observed among treatments, but here insecticide exposure reduced survival compared with the control.   = 2, 2547;  0.0001) .Both male and female larval development times were longer with exposure to sublethal insecticide doses and the effect was more pronounced with chlorantraniliprole . LikewisFollowing 7 days of exposure to insecticides, larval weight was much lower compared with the control . For expr), net reproductive rate (oR), and the resultant generation time (T)  . Net repp = 0.05), but not females (p = 0.62)  A. Males  = 0.62) B–D, or t = 0.62) B,D. In cp = 0.0337)  A. Controp = 0.002; p = 0.0006; p = 0.0003; For first flights greater than 30 min in duration, no differences were observed in either sex for duration, distance, speed, or rest periods A–D. Howe2 = 12.15; df = 2, 280; p = 0.0023) and males   A. AveragC. virescens [C. virescens larvae. In addition, sublethal concentrations of these insecticides at a dose represented by the LC30, were associated with changes in life history traits that ultimately affected population growth characteristics and phenology. Additional effects were observed in the flight behavior of male moths.In Brazil, Bt soybean has been highly effective in controlling irescens ,7. Neverirescens ,15,27. Sirescens ,26. Modeirescens ,51,52. Wirescens ,54. Hereirescens ,56, are These life history trait effects included reductions in larval, pupal, and adult weight; prolongation of larval and pupal development times; reductions in adult male longevity; and reductions in female fertility and fecundity. There also were additional reductions in larval survival, which is usual in studies of this nature, but suggests that our sublethal doses may have been slightly too high. Overall, our results are consistent with other studies on diamide sublethal effects ,31,57,58C. virescens’ larvae from first through fifth instar are highly susceptible to Cry 1Ac, leading to 100% mortality [Several additional factors need to be considered in determining whether and to what degree these sublethal effects will disrupt resistance management. For example, diamides insecticides have been shown to have low toxicity to natural enemies ,61. Thusortality . Thus, iortality ,65. OverC. virescens females may have invested in flight energy at the expense of reproduction, but additional work will be needed to test this hypothesis.Given the need for both sufficient numbers of susceptible moths and populations synchronized to produce adults during the same time period in Bt and non-Bt fields, the dispersal ability of susceptible moths should be sufficient to ensure cross mating of susceptible and resistant moths ,51,52,66C. virescens’ thoracic muscles was four times higher than cyantraniliprole, another anthranilic similar to chlorantraniliprole [30 on C. virescens fecundity might be associated with differential ryanodine binding on the RyR complex [Sublethal concentrations of flubendiamide reduced pupal and adult weight, in addition to fecundity. Studies have shown that the binding of flubendiamide on iliprole . Therefo complex ,73. This complex ,74, corr complex . Thus, t complex . Future C. virescens males, both sexes flew approximately 1.7 to 2.7 times the maximum distance (800 m) required between Bt soybean fields and refuge areas when engaged in unsustained flight in a single 12-h assay period. This figure jumped to 10- to 14-fold for moths engaged in sustained flight, although only about a third of moths do so. Thus, both sexes have the potential to more than adequately move the required 800 m. Still, fewer moths exposed to flubendiamide would be expected to engage in unsustained flight, while those exposed to chlorantraniliprole seemed to be more active than the control. The overall impact of these differences is difficult to judge in the field, but flubendiamide use could reduce the number of moths traveling the required 800 m. Previous work has demonstrated that C. virescens adults can fly more than 7.5 km in their lifetimes [Flight mills have been widely used to measure flight behavior in a wide diversity of insects and to examine the effects of a wide array of biological and ecological factors ,43,44,45ifetimes ,82, althifetimes ,77,78. OC. virescens to several common diamide insecticides can reduce population growth and alter phenological timing. This could potentially disrupt random mating between susceptible moths from the refuge and resistant moths from Bt fields and interfere with resistance mitigation. The extent of the impact would likely depend on the actual sublethal dose the insects would encounter and this would certainly change over time [C. virescens is an economically important pest, such as Bt cotton in Mexico, Puerto Rico, Colombia, and Brazil [Overall, we found that sublethal exposure of ver time ,60,83,84d Brazil , and to


Follicle-stimulating hormone receptor (FSHR) is a G protein-coupled receptor (GPCR) with pivotal roles in reproduction. One key mechanism dictating the signal activity of GPCRs is membrane trafficking. After binding its hormone FSH, FSHR undergoes internalization to very early endosomes (VEEs) for its acute signaling and sorting to a rapid recycling pathway. The VEE is a heterogeneous compartment containing the Adaptor Protein Phosphotyrosine Interacting with Pleckstrin homology Domain and Leucine Zipper 1 (APPL1) with distinct functions in regulating endosomal Gαs/cAMP signaling and rapid recycling. Low molecular weight (LMW) allosteric FSHR ligands were developed for use in assisted reproductive technology yet could also provide novel pharmacological tools to study FSHR. Given the critical nature of receptor internalization and endosomal signaling for FSHR activity, we assessed whether these compounds exhibit differential abilities to alter receptor endosomal trafficking and signaling within the VEE. Two chemically distinct LMW agonists  were employed. T1 was able to induce a greater level of cAMP than FSH and B3. As cAMP signaling drives gonadotrophin hormone receptor recycling, rapid exocytic events were evaluated at single event resolution. Strikingly, T1 was able to induce a 3-fold increase in recycling events compared to FSH and two-fold more compared to B3. As T1-induced internalization was only marginally greater, the dramatic increase in recycling and cAMP signaling may be due to additional mechanisms. All compounds exhibited a similar requirement for receptor internalization to increase cAMP and proportion of FSHR endosomes with active Gαs, suggesting regulation of cAMP signaling induced by T1 may be altered. APPL1 plays a central role for GPCRs targeted to the VEE, and indeed, loss of APPL1 inhibited FSH-induced recycling and increased endosomal cAMP signaling. While T1-induced FSHR recycling was APPL1-dependent, its elevated cAMP signaling was only partially increased following APPL1 knockdown. Unexpectedly, B3 altered the dependence of FSHR to APPL1 in an opposing manner, whereby its endosomal signaling was negatively regulated by APPL1, while B3-induced FSHR recycling was APPL1-independent. Overall, FSHR allosteric compounds have the potential to re-program FSHR activity via altering engagement with VEE machinery and also suggests that these two distinct functions of APPL1 can potentially be selected pharmacologically. Membrane trafficking of G protein-coupled receptors (GPCRs) tightly orchestrates the signaling from this superfamily of signaling receptors. In turn, receptor signaling is known to drive or regulate GPCR trafficking. This inter-relationship is further highlighted through the ability of a growing number of GPCRs to not only activate heterotrimeric G protein signaling from the plasma membrane, but also internal membrane compartments including the endosomal membrane . Thus, tEndosomal signaling of GPCRs can alter the temporal profile of G protein signaling, such that the ligand-induced internalization of receptors to endosomes generates a “second wave” or persistent second messenger signaling profile. This has been demonstrated for distinct Gαs-coupled GPCRs such as thyrotropin-stimulated hormone receptor, parathyroid-stimulating hormone receptor and the β2-adrenergic receptor , but alsFSHR plays pivotal roles in the regulation of male and female reproduction. Although a Class A/rhodopsin GPCR, FSHR has a very large extracellular, horseshoe-shaped N-terminus through which it binds their orthosteric ligands . This deAmong glycoprotein receptors, most of the LMW allosteric compounds developed so far target FSHR; these include positive allosteric modulators , negativIn this study, we took advantage of LMW FSHR ligands from two distinct chemical classes, to assess whether these compounds could alter receptor endosomal signaling via impacting its trafficking compared to FSH. We demonstrate that LMW agonistic compounds exhibit enhanced endosomal signaling and recycling due to a differential sensitivity to APPL1 regulation. Such ligands offer novel potent tools to pharmacologically program FSHR to specific sorting and/or signaling outputs by their differential ability to engage the VEE machinery.The FSHR low molecular weight ligands  were provided by TocopheRx . Purity of compounds ranged from 95–97%. Chemical structures are shown in FLAG-hFSHR has been previously described . Nb37-GFsiRNA-mediated knockdown of APPL1 was achieved by transfection of duplex RNA oligos (Life Technologies) corresponding to GACAAGGTCTTTACTAGGTGTATTT. Control cells were transfected with non-sense duplex RNA oligos (AATTCTCCGAACGTGTCACG).2. Both transient and stable transfections of DNA were performed with Lipofectamine 2000 (Life Technologies). Transfection of siRNA was performed using RNAiMAX (Life Technologies). For transient expression, cells were assayed 48- or 96-h post-DNA and siRNA transfection, respectively.HEK 293 cells (ATCC) were maintained in DMEM containing 10% FBS and penicillin/streptomycin (100 U/mL) at 37°C in 5% COReceptor imaging in live or fixed cells was monitored by treating cells with mouse anti-FLAG antibody  in phenol-red-free DMEM prior to agonist treatment. Cells were washed three times in PBS/0.04% EDTA to remove FLAG antibody bound to the remaining surface receptors and then fixed . Cells were permeabilized and treated with goat anti-mouse secondary antibodies conjugated to Alexa Fluor 488 or 555 and imaged using a TCS-SP5 confocal microscope (Leica) with a 63 × 1.4 numerical aperture (NA) objective. Leica LAS AF image acquisition software was utilized. All subsequent raw-image files were analyzed using ImageJ or LAS AF Lite (Leica) to measure levels of co-localization. Briefly, co-localization was assessed by a manual object-based analysis where first FSHR endosomes (objects) were identified and defined as ROIs, then presence of either APPL1, EEA1 or Nb37 was tested in those ROIs. For 3-colour TIRF this was confirmed by line-scan analysis.Measurement of whole-cell cAMP was carried out with the HTRF-based cAMP Dynamic 2 kit (Cisbio Bioassays) as per manufacturer’s instructions. Cells were ligand treated in triplicate, and experiments were repeated at least three times. All cAMP concentrations were corrected for protein levels.In order to measure cAMP real-time response in living cells, HEK293 cells were transiently transfected with two plasmids coding for FSHR and the BRET-based cAMP sensor CAMYEL . Forty hCells were imaged using an Elyra PS.1 AxioObserver Z1 motorized inverted microscope with a scientific complementary metal-oxide-semiconductor (sCMOS) or electron-multiplying charge-coupled device (EMCCD) camera and an alpha Plan-Apochromat 100×/1.46 Oil DIC M27 Elyra objective (Zeiss), with solid-state lasers of 488, 561, and/or 642 nm as light sources. For live-cell imaging, cells were imaged for 1 min at a frame rate of 10 frames per second (fps) at 37°C in phenol-red-free Opti-MEM supplemented with HEPES (Life Technologies). ZEN Lite image acquisition software was utilized to collect time-lapse movies and analyzed as tiff stacks using the ImageJ plugin Time Series Analyzer. The number of exocytic events were counted manually by visual inspection and was normalized by cell area. Maximum intensity projections, kymographs and fluorescence intensities were generated using ImageJ. For fixed-cell imaging, cells were prepared as described for confocal imaging. Counting of FSHR-, APPL1-, EEA1-, Nb37- single, double or triple-positive endosomes was conducted by manually selecting ROIs using ImageJ; line scan analyses and fluorescence intensity profiles were also obtained using ImageJ.p < 0.05.Data are presented as mean ± SEM. Statistical significance was determined using GraphPad Prism v8 . Normality tests were performed with D’Agostino-Pearson omnibus tests. For non-normally distributed data, Kruskal-Wallis tests were used. Normally distributed data sets were analyzed using unpaired Student’s t test, one-way ANOVA followed by Dunnett post-test, or two-way ANOVA followed by Bonferroni post-test when comparing two groups, more than two groups, or at least two groups under multiple conditions, respectively. Differences were considered significant at 50 values, and only T1 exhibited a higher efficacy, expressed as Emax compared to FSH  which recognizes the active, nucleotide free conformation of Gαs, and was previously employed by us and others to directly visualize G protein signaling from endosomes . TIR-FM We have previously demonstrated that APPL1 plays two key and distinct functions in regulating FSHR activity: it is required for receptor recycling and negative regulation of endosomal cAMP when stimulated with its natural ligand . One posde novo receptor biogenesis, as pre-treatment with cycloheximide had no effect on plasma membrane insertion events of FSHR when stimulated with either B3- or FSH  and a benzamide compound (B3); chemical classes that have been reported to be allosteric activators of FSHR . T1 has in vivo . The benin vivo . The curin vivo , and is in vivo , althougWhile T1 exhibited enhanced efficacy in cAMP signaling of FSHR, both B3 and T1 could induce a greater rate of exocytosis, with an increased brightness and size for each individual fusion event, or “puff”. The latter suggests that a greater number of receptors are recycled within each vesicle. Again, this was more pronounced for T1, exhibiting nearly a three-fold increase in exocytosis rate. While the increased number of T1-induced FSHR endosomes may in part explain the enhanced recycling, it is also possible that the increased maximal cAMP responses induced by this compound play a role, in view of our findings that LHR recycling is driven by its cAMP signaling . HoweverAs described above, APPL1 plays two key functions in the VEE; driving plasma membrane recycling and negative regulation of G protein signaling . These tThese ligands have provided novel tools to further probe these regulatory features of the VEE and potentially toward understanding its downstream impact. At present how the VEE impacts FSHR, or LHR, signaling in the ovary or testes is unknown. While T1 could induce follicular development in rats and estradiol production in primary human granulosa cells, interestingly with a greater Emax than FSH , it is iCompartment or location bias in signaling, not only refers to signaling between plasma membrane and endomembrane, but spatial control across distinct intracellular compartments, including between distinct endosomal compartments . Our stuThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SS, FDP, RR, and NS performed experiments under supervision of AH, ER, and DP. HY, SP, and SN provided the FSHR LMW compounds. AH conceived the study and with SS, FDP, and ER designed research. SS, FDP, and RR analyzed data. SS, FD, and AH wrote the paper. All authors read and approved final manuscript.This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) project grant awarded to AH (BB/S001565/1) and a Society for Endocrinology Early Career Grant to SS. The Facility for Imaging by Light Microscopy (FILM) at Imperial College London is part-supported by funding from BBSRC grant BB/L015129/1. ER is supported by the French National Research Agency under the program “Investissements d'avenir” Grant Agreement LabEx MabImprove: ANR-10-LABX-53; “ARD2020 Biomédicaments” and “APR-IR MODUPHAC” grants from Région Centre Val de Loire. FDP was a recipient of a doctoral fellowship from INRA and Région Centre.HY, SP, and SN were employees of TocopheRx when reagents were shared. Author SN is employed by the company Mitobridge Inc. Author HY is employed by the company Canwell Pharma.The remaining 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.


Cancer stem cells (CSCs) have both the capacity for self-renewal and the potential to differentiate and contribute to multiple tumor properties. The function of CSCs can be regulated by well-balanced process of ubiquitination and deubiquitination of proteins related to the specific stemness of the cells executing various stem cell fate choices. Growing evidence suggests that the involvement of deubiquitinating enzymes (DUBs) in altering several signaling pathways leading to survival of CSCs. In this review, we have compiled all the evidences of DUBs and summarized its role in regulating several signaling network in cancer stem cells.Cancer stem cells (CSCs) have both the capacity for self-renewal and the potential to differentiate and contribute to multiple tumor properties, such as recurrence, metastasis, heterogeneity, multidrug resistance, and radiation resistance. Thus, CSCs are considered to be promising therapeutic targets for cancer therapy. The function of CSCs can be regulated by ubiquitination and deubiquitination of proteins related to the specific stemness of the cells executing various stem cell fate choices. To regulate the balance between ubiquitination and deubiquitination processes, the disassembly of ubiquitin chains from specific substrates by deubiquitinating enzymes (DUBs) is crucial. Several key developmental and signaling pathways have been shown to play essential roles in this regulation. Growing evidence suggests that overactive or abnormal signaling within and among these pathways may contribute to the survival of CSCs. These signaling pathways have been experimentally shown to mediate various stem cell properties, such as self-renewal, cell fate decisions, survival, proliferation, and differentiation. In this review, we focus on the DUBs involved in CSCs signaling pathways, which are vital in regulating their stem-cell fate determination. Signal transduction is the cellular response to a physical or chemical signal and is mediated by a cascade of molecular events initiated when a particular ligand binds to its corresponding receptor, known as activation of a signaling pathway. Various cellular signaling pathways interact with one another and form networks that coordinate cellular responses through combinatorial signaling events. These responses include variations in transcriptional, translational, and post-translational activities at the molecular level, which can either change the conformation of proteins or their subcellular locations and ultimately influence cellular mechanisms.Stem cells in multicellular organisms are defined as cells having the ability to self-renew or differentiate into various types of cells and proliferate to generate more of the same stem cells . Stem ceMostly, stem cells use their capacity for unlimited proliferation to produce differentiated cells. However, recent evidence indicates that differentiated cells can de-differentiate into stem cells with phenotypic plasticity . GeneralTumors can originate from normal stem cells and acquire the hierarchical characteristic of normal tissue. Therefore, tumors are assumed to be maintained by so-called cancer stem cells (CSCs), which are similar to ASCs in their ability to self-renew and generate tumor cells indefinitely. CSCs can self-renew through the asymmetric division of cells to differentiate into a non-tumorigenic bulk tumor mass . In otheSignaling pathways, such as Notch, Wnt, and Sonic hedgehog (Shh), are associated with normal stem cell development and regulation of CSCs, and their dysregulation can cause cancer. The growth of CSCs can be controlled by the phosphatase and tensin homolog (PTEN) and signaling molecules from the polycomb family. The precise regulation of those pathways through ubiquitination/deubiquitination activities of regulatory proteins is crucial for the proper execution of developmental programs, whose manipulation can alter stem cell properties and lead to cancer . MultiplUbiquitination is a post-translational modification (PTM) process by which the highly conserved 76-amino acid ubiquitin protein is covalently conjugated to a lysine residue on a substrate protein as the result of a cascade of enzymatic reactions . The firMonoubiquitination, the attachment of short Lysine (K)63 -linked ubiquitin chains to a protein, has primarily been linked to chromatin regulation, protein sorting, and trafficking. In contrast, polyubiquitination has chains of more than three ubiquitins, with the C-terminus of one ubiquitin connected to the K48 of the following ubiquitin, and it has been associated with protein signaling and clearance through proteasomal degradation ,18. The Ubiquitin has seven Lys residues that can be ubiquitinated to form isopeptide-linked ubiquitin chains. Proteomics studies have revealed the existence of all possible linkage types in cells . VariousThe ubiquitination process is reversed by specialized enzymes—i.e., DUBs, that oppose the action of the E3 ligases by cleaving the iso-peptide linkage between the N-terminal lysine and the C-terminal glycine ubiquitin residues. Analyses of the human genome have identified about 100 functional DUBs that have been divided into seven broad classes based on active site homology: ubiquitin-specific proteases (USPs), ubiquitin carboxy-terminal hydrolases, ovarian-tumor proteases, Machado–Joseph disease protein domain proteases, JAB1/ Mpr1, Pad1 N-termina (MPN)/Mov34 metalloenzyme  ,29. MoreThe metastatic potential of tumors is determined by the prevailing lineage-specific cell fate determinants, which can include transcriptional regulators expressed in niche cells. Any modification or loss of controlled differentiation can result in the development of metastatic characteristics, such as de-differentiation, procurement of stem cell-like activities, or cellular plasticity . MultiplSomatic cells can be reprogrammed to become pluripotent stem cells through transient ectopic overexpression of transcription factors that play an essential role in regulating the growth of CSCs, such as Oct3/4, Sox2, Nanog, KLF4, c-Myc and other reprogramming factors . These fOctamer-binding transcription factor 4 (Oct4), a homeodomain transcription factor of the Pit-Oct-Unc family, is recognized as one of the most important transcription factors . Oct4 isSRY (sex determining region Y)-box 2 (Sox2) is a crucial transcription factor for CSCs and regulates their differentiation and stemness. Sox2 belongs to a family of high-mobility group transcription factors and plays a significant role in early development and maintenance of undifferentiated ESCs . PhosphoKrüppel-like factor 4 (KLF4) is expressed in many tissues, plays an essential role in many physiological processes, and acts as a bifunctional transcription factor (activator or repressor depending on the function of the target gene). Ubiquitination of KLF4 is a vital PTM that can control its turnover within cells. In response to TGF-β signaling, KLF4 expression is downregulated and inhibited by the ubiquitin–proteasome pathway . Kim et Myc is a family of proto-oncogenes that encode transcription factors critical to DNA binding proteins of the basic helix–loop–helix (bHLH) superfamily . Myc conNanog, a differentiated homeobox (HOX) domain protein, has multipotent transcriptional regulatory function and self-renewal capability . Nanog iInhibitors of DNA binding (ID) proteins are transcriptional regulators that control cell fate determination and the timing of differentiation in progenitor stem cells during normal development . ID protThis section will cover signaling pathways, such as Sonic hedgehog, Wnt, Notch, TGF-β and Hippo, and others that regulate CSC properties. The Hedgehog (Hh) pathway plays a significant role in the development and patterning of numerous organs during embryogenesis by influencing cellular differentiation, proliferation, and migration. The Hedgehog pathway includes three secreted Hedgehog ligands—Sonic, Desert, and Indian—their cognate receptor Patched, the transmembrane protein Smoothened, and three Gli translation components that balance the activation and suppression of the pathway. Hh activity regulates normal stem cell (NSC) populations, and aberrant Hh signaling is associated with many cancers, including colon cancer, basal cell carcinoma, glioblastoma, multiple myeloma (MM), adenocarcinoma, bladder cancer, esophageal squamous cell carcinoma, gastric cancer, lung squamous cell carcinoma, and chronic myeloma leukemia (CML) . The Hh Gli proteins are transcriptional effectors of Hh signaling; several DUBs are reported to interact and stabilize Gli proteins. For instance, USP7 acts as a positive regulator of Hh signaling by deubiquitinating and stabilizing Ci and Gli proteins, indicating that Gli is a significant potential therapeutic target for Hh-related cancers . AnotherThe expression of USP37 is critical in the maintenance of key Hh components. Genetic depletion of USP37 leads to a reduction in the protein level of key Hh components and stem cell markers. In contrast, the activation of Hh signaling by purmorphamine results in the up-regulation of USP37, which, in turn, leads to the stabilization of Gli1 and impacts on Epithelial–mesenchymal transition (EMT) in breast cancer stem cells .Hh signaling is critical to osteoblast differentiation during embryogenesis. OTUB2 deubiquitinates and stabilizes the GLI2 protein by preventing its proteasomal degradation. Moreover, depletion of OTUB2 reduces the expression level of Hh target genes, such as N-myc, Patch1, and Gli1, leading to the suppression of Hh-induced osteoblast differentiation in mesenchymal stem cells (MSCs) . USP8 promotes Hh signaling by preventing its ubiquitination and changing its subcellular localization, where it removes modified ubiquitin from Smo that enhances its signaling activity . Usp14 pThe Wnt/β-catenin signaling pathway plays an essential role in tissue self-renewal and cell fate determination and is involved in tumorigenesis . The WntRecent research has focused on the role of Wnt activation in regulating CSCs in cancers of the mammary glands, skin, nervous system, intestines, lungs, urinary tract, and blood . Loss ofUSP4 positively regulates Wnt/β-catenin signaling in colon cancer. The C-terminal catalytic domain of USP4 is responsible for β-catenin binding and nuclear transport. Knockdown of USP4 in a colon cancer cell line decreased invasion and migration activity, indicating that USP4 is a positive regulator of β-catenin and is a potential target for anti-cancer therapeutics .USP6 activates Wnt signaling by deubiquitinating FZD and increasing its cell surface abundance, which is an important mechanism in regulating sensitivity to Wnt ligands. Chromosomal translocations in nodular fasciitis promote USP6 overexpression, which results in transcriptional activation of the Wnt/β-catenin pathway. During tumorigenesis, Wnt signaling is the main target of USP6 because inhibition of Wnt signaling decreases USP6-driven xenograft tumors . UbiquitUSP9X stabilizes and deubiquitinates β-catenin by removing the Lys 48-linked polyubiquitin chains that mark it for proteasomal degradation, thereby promoting glioma cell proliferation and survival and indicating it as a therapeutic target in high-grade gliomas . AdditioUsp14 deubiquitinates Dvl, a key regulator of Wnt signaling. Suppression of Usp14 activity leads to increased Dvl polyubiquitination and impairs downstream factors of Wnt signaling, suggesting that Usp14 is a positive regulator of the Wnt signaling pathway . USP14 iUSP25 acts as a positive regulator of Wnt/β-catenin signaling. USP25 interacts with and deubiquitinates tankyrase, which destabilizes Axin and positively regulates Wnt signaling . USP47 aIn contrast, several DUBs act as negative regulators of Wnt/β-catenin signaling. For instance, USP7 inhibits Wnt/β-catenin signaling by promoting stabilization of Axin by direct interaction through its N-terminal TRAF domain and stabilizes and deubiquitinates the Axin protein. Depletion of USP7 promotes osteoblast and adipocyte differentiation by activating Wnt signaling. Thus, USP7 is a specific target for sensitizing cells in response to Wnt/β-catenin signaling . USP34 sThe Notch pathway consists of a cascade of signaling molecules involved in development of cells and maintenance of stemness . Notch sNotch signaling denotes a primordial, cell-fate-determining, and evolutionarily conserved pathway with strong relevance to CSCs. Various studies have revealed that the Notch pathway is involved in regulation of CSCs in pancreatic cancer, esophageal adenocarcinoma, breast carcinomas, and many other cancers ,113,114.Notch signaling plays a role in regulating neural stem cell expansion in vivo and in vitro . In vivoUSP7 acts as a regulator of UbE2E1, an E2 ubiquitin conjugation enzyme with a unique N-terminal extension. USP7 attenuates UbE2E1-mediated ubiquitination, an effect that requires the N-terminal ASTS sequence of UbE2E1 as well as the catalytic activity of USP7 . AnotherUSP9X interacts with and stabilizes ITCH, an E3 ligase that ubiquitinates Notch. USP9X interacts with and protects Itch auto-ubiquitination through its deubiquitinating activity . USP12 aCYLD showed that the JAG2 protein levels and Notch target genes were upregulated [USP9X deubiquitinates Mind Bomb (MIB) 1, an E3 ligase required for Jagged1 ubiquitination-mediated endocytosis and Notch activation . Tribbleegulated .USP10 stimulates Notch signaling in the endothelium by binding with the Notch1 intracellular domain (NICD1). USP10 stabilizes NICD1 by extending its half-life, which is necessary for prolonged cellular Notch responses. Inactivation of USP10 reduces NICD1 and subsequently downregulates Notch-induced target gene expression in endothelial cells, indicating its importance in endothelial Notch responses during angiogenic sprouting . USP11 deubiquitinates and stabilizes promyelocytic leukaemia (PML) to control Notch-induced malignancy in brain tumors. The Notch effector Hey1 is recruited to the USP11 promoter to repress expression of USP11. Thus, Notch-induced downregulation of USP11 and PML promotes multiple malignant features of glioblastoma multiforme (GBM) and glioma-initiating cells (GICs), indicating the importance of this pathway in GBM malignancy . The transforming growth factor-β/bone morphogenic proteins (TGFβ/BMP) signaling pathway controls several processes, such as cell differentiation, proliferation, survival, and motility of cells . The TGFUSP2a positively regulates the TGF-β signaling pathway by removing the K33-linked ubiquitin chain. USP2a binds with heterodimeric type II and type I TGF-β serine/threonine kinase receptors (TGFBR1 and TGFBR2) upon TGF-β stimulation and promotes the recruitment of SMAD2/3. The phosphate groups on the Ser207/Ser225 of USP2a are phosphorylated by TGFBR2, which disassociates SMAD2/3 from TGFBR1. The phosphorylation of USP2a and SMAD2 is positively linked in tumor tissues, with USP2a hyper-phosphorylated in lung cancers. The inhibition of USP2a impairs TGF-β-induced EMT and metastasis, suggesting USP2a as a potential target for the treatment of metastatic cancers .USP4 directly interacts with and deubiquitinates type 1 TGF-β receptors that control TβRI level at the plasma membrane. Akt, a serine/threonine-specific protein kinase, phosphorylates USP4, which promotes its re-localization from the nucleus to the membrane, where it binds with TβRI. The depletion of USP4 restricts TGF-β-induced EMT and metastasis .USP9X enhances TGF-β signaling by countering SMAD4 monoubiquitylation. USP9X binds and deubiquitinates monoubiquitinated Smad4, opposing the activity of Ectodermin, which reversibly represses Smad4 activity . The depRNAi inhibited TGF-β-induced phosphorylation of SMAD and TGF-β-mediated transcriptional responses [USP11 binds and deubiquitinates Alk5, a type I TGF-β receptor, which results in enhanced TGF-β-induced gene transcription. USP11 knockdown mediated by esponses . USP11 tesponses ,135.USP15 enhances TGF-β signal by interacting with the SMAD7-SMAD E3 ligase (SMURF2) complex that is responsible for deubiquitination and stabilization of the type I TGF-β receptor. Moreover, high expression of USP15, which is predominantly expressed in glioblastoma and breast and ovarian cancer, correlates with high TGF-β activity . UCH37 iOTUB1 is critical for cellular migration and TGF-β-mediated gene transcription. Additionally, OTUB1 stabilizes the active SMAD2/3 complex by preventing the ubiquitination of phospho-SMAD2/3 through the inhibition of the E2 ubiquitin-conjugating enzymes, independent of its catalytic activity . USP4 can suppress the TGF-β signaling pathway through a non-SMAD pathway. TNFα induces binding between USP4 and TAK1, resulting in deubiquitination of TAK1 and suppression of TAK1-mediated NF-κB activation . CYLD acThe Hippo pathway is linked to regeneration and tumorigenesis . TranscrUSP9X influences the Hippo pathway by negatively regulating YAP/TAZ activity. The Angiomotin (AMOT) protein, which is involved in the repression of YAP/TAZ function, was deubiquitinated by USP9X at lysine 496 to stabilize AMOT and lower YAP/TAZ activity . AdditioUSP11 deubiquitinates and stabilizes the vestigial-like family member 4 (VGLL4) protein, which directly competes with YAP by forming a complex with the TEA domain proteins (TEADs), thereby acting as a tumor suppressor. VGLL4 blocks the interaction of YAP and TEAD, reducing cell progression and migration. USP11 increases VGLL4 expression and modulates YAP-TEAD complexes by removing ubiquitin moieties from VGLL4 ,153. USPYOD1, a member of the ovarian tumor protease (OTU) subfamily of DUB enzymes, interacts with and regulates ITCH E3 ligases, resulting in the degradation of the LATS kinase that enhances the transcriptional activity of YAP and TAZ . YOD1 isOTUB2 stimulates CSC traits through angiogenesis. It interacts with and activates YAP/TAZ directly through sumoylation, which allows YAP/TAZ to interact with TEAD transcription factors and thus drives oncogenic properties both in vitro and in vivo . OTUD1 rAltogether, these findings demonstrate how the properties of the NSC signaling pathways, such as self-renewal, proliferation, differentiation, and survival, are unbalanced in tumorigenesis and CSCs, which are regulated by multiple exogenous/endogenous genes or microRNAs. These complex pathways are initiated by downstream signals, such as cytokines and developmental, proliferation, expression, apoptosis, anti-apoptotic, and metastatic genes in CSCs. The main DUBs which are discussed above, that play a critical role in regulating signaling pathways, such as Sonic hedgehog, Wnt, Notch, TGF-β and Hippo, are represented in CSCs have much greater resistance to cytotoxic treatment than non-tumorigenic cells, indicating the importance of targeting CSCs to prevent tumor relapse. In CSCs, apoptotic proteins (Bim) are downregulated, and anti-apoptotic factors , DNA repair capacity, and caspase inhibitors are upregulated, contributing to therapeutic resistance . AldehydThe inhibitors of apoptosis (IAPs) are critical regulators of apoptosis and other fundamental cellular processes. The USP11-dependent mechanism selectively and specifically regulates cIAP2 stability independent of cIAP1 . USP19 eClaspin is a checkpoint kinase 1 (Chk1) interacting protein that is essential for Chk1 phosphorylation and activation by the ATR kinase, which can also promote DNA replication fork progression and stability. Claspin and USP7 interact in vivo to maintain a steady-state level of Claspin . HERC2/UThe anti-apoptotic protein FLIPS (caspase eight family inhibitors) is a crucial suppressor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)–induced apoptosis. USP8, a downstream target of Akt, regulates the ability of Akt–atrophin-interacting protein 4 (AIP4) to control FLIPS stability and TRAIL sensitivity . Chk1 isSeveral ligands on one cell can activate corresponding receptors on neighboring cells, and those interactions can involve several cell types, as well as the extracellular matrix (ECM) and vasculature, in a dynamic microenvironment . Such inAs noted above, the microenvironment of CSCs plays a significant role in maintaining stemness. To date, several signaling pathways in the CSC microenvironment—i.e., NF-κB, Hypoxia-inducible factors (HIFs), and Pattern recognition receptors (PRRs)—have been found to be associated with hypoxia, low ROS, hyper-angiogenesis, and immunosuppression ,189,190.Crosstalk between signaling pathways is a crucial activity for maintaining the robustness of biological systems and can be initiated by a specific stimulus—i.e., an extracellular cue or individual gene suppressor or protein inhibitor. Several pathways act not just as a particular unit, but also as part of a biological network during cellular processes. Crosstalk among the Hh, Wnt, TGF, and other pathways has been reported in cancer and CSCs . For insThe interplay among complex signaling pathways is vital for maintaining embryogenesis, stem cell diversity, and tissue homeostasis. Dysregulation in these signaling networks leads to several pathological conditions, including cancer and other anomalies. TGF-β plays a significant role in EMT by activating the Smad pathway, in which lymphoid enhancer factor 1 (LEF1) is activated by phosphorylated Smad2 and binds with the promoter region of the E-cadherin gene to repress its transcription in response to the TGFβ3 signaling pathway . Both LEIn cancer treatment, targeting tumorigenic CSCs directly is considered a more advanced technique than attempting to treat the tumor as a whole. Tumor recurrence after years of disease-free survival has incited interest in stem cells and intense investigation into the role of CSC self-renewal capacity and multi-lineage differentiation. DUBs protect the stemness of CSCs and regulate cell processes, such as cell proliferation, signal transduction, and apoptosis. DUB inhibitors target the most truculent DUBs, regulating CSC-related proteins and eliminating some of the challenges of cancer treatment, such as drug resistance and tumor recurrence. DUB inhibitors are superior to proteasomal inhibitors in curing refractory tumors—e.g., b-AP15, a DUB inhibitor targeting USP14 and UCHL5, can overcome resistance to bortezomib, a 20S proteasome inhibitor, in MM ,206,207.DUB inhibitors counteract the activity of DUBs associated with cancer stemness and EMT, including pimozide, which inhibits USP1 in osteosarcoma and glioblastoma ,208. ML3HBX19818 is a potent inhibitor of USP7 that signals p53-mediated apoptosis in HCT116 colon cancer cells by blocking the deubiquitinating activity of USP7 ,211. P50P22077 and HBX19818, which was originally reported as an inhibitor of USP7, inhibit USP10 deubiquitinating activity, which produces anti-cancer activity . SpautinWP1130 inhibits USP9X, USP5, UCHL1, USP14, and UCH37 in liver and breast cancer ,218,219,A proteasome inhibitor, b-AP15, that targets 19S regulatory particle associated DUBs, including USP14 and UCHL5, showed anti-tumor activity. b-AP15 effectively inhibited the dissemination of an acute mouse model of C1498 leukemia, inhibiting tumorigenesis in multiple solid tumor mouse models including lung, colon, and breast carcinoma xenografts ,213,222.PX−478 is a potential CSC-targeted therapeutic drug molecule because of its inhibitory effect on HIF−1α signaling, which is hyperactivated in the hypoxic niches of CSCs . C527 isAlthough DUBs are attractive targets, clinical development of small-molecule DUB inhibitors has been limited by several obstacles. Most DUBs have a catalytic cysteine active site that is optimal for inhibitor development, and those catalytic pockets are conserved. Screening for potent small-molecules that show selectivity among related DUBs with conserved catalytic pockets is a big challenge in developing DUB inhibitors for clinical use. Another big obstacle for DUB inhibitor screening is oxidative hydrolysis of the active-site cysteine. Because most DUBs transfer ubiquitin molecules through a reactive thiol group, the assays used to screen for inhibitors are prone to non-selective redox or alkylating false-positives . AdditioThus, despite the growing attractiveness of DUBs as cancer targets, only a few DUB inhibitors have advanced through clinical trials for cancer therapy. VLX1570, an inhibitor of USP14, was advanced to a phase I/II clinical trial in combination with dexamethasone (NCT02372240) by the US Food and Drug Administration (FDA) in 2017 . AnotherTaken together, our findings indicate that CSCs are cancer cells that underlie tumor relapse, heterogeneity, and metastasis. Recent evidence identifies CSCs as the possible cells of origin in numerous cancers. CSCs can be regulated by stemness markers , signaling pathways , extracellular factors , and other factors. To maintain their pluripotency and differentiation, CSCs have to maintain a balance between ubiquitination and deubiquitination. DUBs maintain CSC activity and stemness, implying their role in development of CSC-specific treatments. This article summarized the role of DUBs in regulating stem cell pluripotency, CSC-associated transcription factors, proteins, and signaling pathways and their prospective uses in cancer research. However, additional research is required to identify effective ways to eliminate CSCs. First, neither the precise role of DUBs in CSCs nor which particular cancers are primarily driven by CSCs have been elucidated with certainty. Second, only a small amount is known about the interaction between DUBs and cell-fate determinants, such as Oct4 and Nanog. Third, information is lacking about the roles of DUBs in regulating malignant transformation of healthy stem cells and the signaling pathways that regulate CSC stemness and malignant potential. Fourth, little is known about the role of DUBs and the crucial JAK/STAT, Notch, and PI3K signaling pathways in CSCs. Fifth, there is inadequate evidence supporting CSCs as mediators of tumor development, that therapeutic resistance is driven by the tumor microenvironment, and that DUBs are involved in those processes. Answering those questions will be crucial to development of therapeutic DUB inhibitors to limit tumor progression and relapse. The use of genome editing, epigenetics, and cellular metabolism studies should be considered in cancer therapy because DUB-mediated signaling and regulatory functions also contribute to CSC stemness. DUB inhibitors, natural products, and agents that specifically target CSCs can also be included in future studies. Although we have come a long way in our understanding of the signals that drive cancer growth and how those signals can be targeted, effective control of cancers remains a key scientific and medical challenge. Using conventional treatments to target CSCs is difficult because of epigenetics and aberrant signaling. Thus, an improved understanding of CSCs and their signaling pathways could improve modern therapeutic approaches for diverse cancers in the clinical setting.


The molecular signaling pathways that lead to cell survival/death after exposure to organophosphate compounds (OPCs) are not yet fully understood. Mitogen-activated protein kinases (MAPKs) including the extracellular signal-regulated protein kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and the p38-MAPK play the leading roles in the transmission of extracellular signals into the cell nucleus, leading to cell differentiation, cell growth, and apoptosis. Moreover, exposure to OPCs induces ERK, JNK, and p38-MAPK activation, which leads to oxidative stress and apoptosis in various tissues. However, the activation of MAPK signaling pathways may differ depending on the type of OPCs and the type of cell exposed. Finally, different cell responses can be induced by different types of MAPK signaling pathways after exposure to OPCs. There is evidence that organophosphate pesticides (OPs) contribute to the etiology of many diseases including neurodegenerative, cardiovascular, respiratory, gastrointestinal, diabetic, dyslipidemic, and kidney diseases . In addiOxidative stress-induced by OP exposure can stimulate the mitogen-activated protein kinase (MAPK) signaling pathway in the tissue . MAPK moThe activation of phospholipase C, gamma (PLC-gamma), and the proto-oncogenic tyrosine-protein kinase c-Src (c-Src) phosphorylate Ras and Raf by ROS both directly and indirectly activates ERK signaling .ERK, one of the main signaling cassettes of the MAPK signaling pathway, contributes to the modulation of meiosis and mitosis functions in differentiated cells. In mammals, ERK consists of ERK 1/2, ERK3/4, ERK5, ERK7, and ERK8 classes. The ERK family plays the main role in the regulation of cell growth cycles, survival, and differentiation under normal and pathological conditions . In addiThis review was intended to evaluate and show the influence of OPs on the activation of the MAPK signaling pathway by oxidative stress and the association of the activated pathway with various diseases.50 for humans is between 0.5 and 5 g/kg, and oral or respiratory exposure to 10 to 200 mg parathion is fatal for adults [It has been shown that organophosphorus compounds (OPCs) are organic compounds consisting of a phosphoryl (P=O) bond or a thiophosphoryl (P=S) bond, and several OPCs with different chemical, physical, and biological properties are commercially used. Furthermore, most of them are dissolved in oils and only a little in water, and the classification of OPCs is quite complex. There is therefore no uniform classification system for them. Indeed, OPCs derived from phosphoric acid and phosphonic acid derivatives usually have anti-cholinesterase activity, whereas OPCs derived from phosphonic acid do not have this property . Exampler adults . HoweverThe estimated lethal doses of DZN, malathion, and parathion in humans are 25 g, 60 g, and 10 to 200 mg respectively . InteresMost symptoms of OP intoxication are caused by the stimulation of several muscarinic receptors, and tachycardia and hypertension are sometimes observed in acute intoxications associated with the cholinergic effects of OPs on the CNS, sympathetic ganglion synapses, or the adrenal medulla . A few nMany of the OPs have no tumorigenic effect in animals, but some of them could induce cancer in experimental models ,20. TeraPON1 and Nrf2 gene expression. The study showed that DDVP increased MDA levels and decreased glutathione (GSH), SOD, and CAT activities in the liver [Drospphila melanogaster, CPF increased the phosphorylation of p38-MAPK and JNK [Oxidative stress is caused by a disturbance of the balance between oxidative and antioxidative systems, which leads to an impairment of the antioxidative defense . It has he liver . Furtherhe liver  pointed he liver . Further and JNK . Shou an and JNK  investig and JNK . In fact and JNK .Apoptosis or programmed cell death is regulated also by the MAPK signaling pathways under stress conditions. Three conventional MAPK signaling pathways  play an important role in modulating the apoptotic pathway. Indeed, it was shown that activated JNKs and p38-MAPKs stimulate apoptosis cascades, while ERK1/2 plays an important protective role against apoptosis . It has Several members of the MAPK family, such as JNK, p38-MAPK, and ERK, are implicated in many neurodegenerative processes. The effects of OPCs on the MAPK signaling pathway-mediated neurodegenerative diseases have been reported in several scientific works of literature. OPCs disturb the balance between JNK, p38-MAPK, and ERK inducing apoptotic, oxidative, and inflammatory damages in neurons. Because exposure to CPF is more common among people, the majority of studies focused on the CPF neurotoxicity to study the impact of insecticides in the MAPK signaling pathways. In addition, it has been reported that CPF induces neuronal damage by activating three main pathways of MAPK including p38-MAPK, JNK, and ERK signaling. The effects of CPF on the MAPK pathways indicated that CPF has a major role in the induction of apoptosis, inflammation, and oxidative stress. The investigation on neurotoxic effects of CPF in PC12 cells indicated that treatment with 0, 25, 50, 100, and 200 μM CPF induced dose-dependently apoptosis via activating the p38-MAPK, JNK, and ERK, suggesting that activated p38-MAPK, JNK, and ERK signaling pathways acted as death signals and also activated caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage . CPF alsIt is known that Drp1 is necessary for the translocation of p53 into the mitochondria after CPF-increased ROS production. Blocking the Drp1 function-inhibited CPF caused mitochondrial-dependent apoptosis by interaction with p53 . Ki et aTreatment with two activators of ERK1/2, such as nerve growth factor (NGF) and carbachol, inhibited the delayed neurotoxicity induced by CPF, which shows the influence of the inhibition of ERK1/2 phosphorylation on the delayed neurotoxicity caused by CPF . MoreoveA study by Lazar et al.  showed aIt has been reported that cardiomyocyte survival and death are regulated by extracellular ligands, cytokines, and growth factors that bind to cell surface receptors and induce intracellular specific signal transduction pathways . CardiacChronic obstructive pulmonary disease (COPD) is one of the most important respiratory diseases in which an increase in activated MAPK signaling is found in bronchial epithelial cells, alveolar macrophages, and pulmonary lymphocytes. Further, activated p38-MAPK is involved in local and systemic inflammation in COPD . It has -)-ICP induces a change in the Bax/Bcl-2 ratio and hepatotoxicity by the sustained activation of JNK [The activated MAPKs signaling contributes to hepatic metabolic processes. The liver modulates the metabolism of glucose and lipids. Pathophysiological conditions such as diabetes, obesity, and fatty liver disease associated with disturbed liver functions. In addition, stress stimulates the activation of hepatic MAPKs, which leads to a disorder of insulin action and lipid metabolism. In this context, OPCs can influence the balance of MAPK signaling pathways in liver metabolism and can contribute to metabolic disorder . Furthern of JNK . The addn of JNK .−1 significantly increased the phosphorylation of JNK and reduced the expression of Bcl-2 and ClAP-2. In fact, TCEP also increased the expression of caspase-3 and caspase-9 in primary cultured proximal renal tubule cells [Recent studies have shown that the MAPK/ERK signaling pathways play a major role in nephrogenesis and lead to differentiation of the nephrogenic mesenchyme. Moreover, an imbalance in MAPK signaling pathways causes several illnesses, such as cancers . Interesle cells .A large body of evidence has demonstrated a link between the dysfunction of the MAPK signaling pathways and the dysregulation of reproductive function . In addi−4 to 10−5 M) for 24 h caused time-dependent cell death and reduced phosphorylated ERK2 in a dose-dependent manner, but phosphorylation of Raf-1, an upstream ERK molecule in the Ras-ERK cascade, could not affect cell death time [It has been reported that OPCs can affect the immune system, which in turn can alter the differentiation and subversion of abnormal cells, thereby assessing the risk of cancer. Natural killer (NK) cells are an important component of the immune system and can interfere with tumor- and virus-infected cells, and the risk of cancer and infection can increase if the function of NK cells is impaired . In thisath time . Indeed,ath time .It is known that environmental pollutants are the main factors involved in diabetes and its complications. Studies have shown a link between exposure to OPCs and the occurrence of diabetes . FurtherIt has been reported that the effect of OPCs on lipid metabolism is controversial. In this context, exposure to DZN disturbed lipid metabolism by altered MAPK signaling pathways. In addition, subacute DZN  on the regulation of lipid metabolism, ERK, and expression of the low-density lipoprotein receptor (LDLr) in rat liver was evaluated. DZN significantly increased cholesterol, triglyceride, and LDL levels. Additionally, DZN reduced the activated ERK1/2 and LDLr transcripts, suggesting that DZN caused dyslipidemia and increased the expression of LDLr by inhibiting the ERK signaling pathway .It is known that exposure to OPCs causes apoptosis through activation of ERK, JNK, and p38-MAPK signaling pathways. The members of the MAPK family appear to be affected differently by each OPC, depending on the cell type. In addition, MAPKs protect cell survival or induce equilibrium and can distinguish cell death between antiapoptotic and proapoptotic signals transmitted by ERK, JNK, and p38-MAPK signaling pathways .The present review suggests that OPCs may induce apoptosis by interrupting the modulation of the MAPK signaling pathway. The activation of MAPKs in the target organs has a significant effect on the toxicity of OPCs. These compounds induce apoptosis and oxidative stress by disrupting the downregulation of Nrf2 and the equilibrium of phosphorylation of Erk, JNK, and p38-MAPK in tissues from animal and in vitro models. In addition, Erk, JNK, and p38-MAPK have pro- and antiapoptotic effects triggered by OPC-induced tissue damage. Moreover, MAPK signal transduction has both inhibitory and stimulatory effects on apoptosis, depending on cell type, stimulating factor, and latency of MAPK signaling activation. In particular, MAPK has a modulating effect on numerous downstream molecules, including transcriptional and translational components, cell cycle molecules, kinases leading to cell proliferation, cell cycle arrest, migration, differentiation, and apoptosis. These multiple downstream targets, cell type, and cell cycle-specific molecular cross-talk or kinetics and time of activation can cause the complexity of MAPK effects on apoptosis. Moreover, their balance ultimately determines the fate of the cells. However, the new findings could also clarify the controversial data on the various effects of OPCs, including their effect on blood glucose and lipid profile obtained in different cell types and animal models exposed to different OPCs.It was found that some antioxidants with beneficial effects against toxic damage induced by OPCs could regulate the MAPK signaling pathways. Chemical antioxidants such as vitamin C and E, melatonin, N-acetylcysteine, etc. as well as flavonoids including curcumin, quercetin, berberine, etc. may be able to combat against OPC toxicity by modulating MAPK signaling.In summary, the effect of OPCs in the initiation and progression of various diseases may be related to their effect on the regulation of MAPK signaling pathways. Studies need to be performed to determine the effect of the antidote of OPCs on MAPK signaling to find more suitable therapeutic approaches.


Therefore, HTLV-1 infection may be inversely associated with hypertension by activating endothelial maintenance, including atherosclerosis. To clarify these associations, a cross-sectional study was conducted using 2989 Japanese individuals aged 60–99 years participating in a general health check-up.Human T-cell leukemia virus type 1 (HTLV-1) activates inflammatory cascades by activating the NF-κB pathway. The minor allele of single nucleotide polymorphism (SNP) in breast cancer suppressor BRCA1Logistic regression models were used to clarify the association between HTLV-1 and hypertension. Platelet levels stratified analyses were also performed since platelet production, which plays a crucial role in endothelium maintenance, can be stimulated by activating the NF-κB pathway.HTLV-1 infection was found to be significantly inversely associated with hypertension, particularly in subjects with high platelet levels (≥ second tertiles of platelet levels); the fully adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were 0.75  for total and 0.64  for high platelet levels, respectively. Further analysis of the non-hypertensive subjects demonstrated that HTLV-1 infection was significantly positively associated with atherosclerosis in subjects with the highest tertile of platelet levels  but not in subjects with low platelet levels (first and second tertiles of platelet level) .Asymptomatic HTLV-1 infection is inversely associated with hypertension, possibly by activating endothelial maintenance, including atherosclerosis progression. Although human T-cell leukemia virus type 1 (HTLV-1), which is the earliest recognized human retrovirus, has been shown to induce adult T-cell leukemia/lymphoma , the maj-associated protein (BRAP) activate an inflammatory cascade via the activation of the NF-κB pathway , followed by logarithmic transformation. A trend test was performed using a regression model for the mean values.Logistic regression models were used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) to determine associations between HTLV-1 and hypertension, the HTLV-1 infection status specific association between platelet and hypertension, and the category of platelet levels specific to the association between HTLV-1 and hypertension.To emphasize the beneficial influence on the prevention of hypertension, further analysis was performed using subjects without hypertension.Using logistic regression models, we analyzed the participants without hypertension and evaluated the specific association of HTLV-1 infection status between platelet count and atherosclerosis as well as the specific association for the category of platelet levels between HTLV-1 and atherosclerosis.For all of the subjects, we evaluated the effect of HTLV-1 infection on the association between platelet and hypertension as well as the effect of platelet categories on the association between HTLV-1 infection and hypertension by logistic regression analysis. For subjects without hypertension, we evaluated the effect of HTLV-1 infection on the association between platelet count and atherosclerosis as well as the effect of platelet categories on the association between HTLV-1 infection and atherosclerosis by logistic regression analysis.2), HDL-cholesterol (mg/dL), triglycerides (mg/dL), HbA1c (%), white blood cell (cells/μL), and γ-GTP (U/L). Smoking status and drinking status are known cardiovascular risk factors. However, these factors were not taken into account as they did not influence the endothelium directory; those factors induced physical stress on the endothelium by activating inflammation  and with [22.2 ± 0.3 (× 104/μL)] HTLV-1 infection was observed (p = 0.091).Conversely, this stimulated platelet production due to HTLV-1 infection can be masked through hypertension-related endothelial injury-induced platelet production. Thereafter, among the hypertensive subjects, no significant difference between without [22.7 ± 0.1 (× 10In terms of the strengths of our study, all of our results can be explained by simple mechanisms. Multi-faceted analyses allowed us to determine the possible mechanisms underlying the present results. Furthermore, this study also indicates the mechanism for the activation of atherosclerosis progression, which could have a beneficial influence on preventing hypertension. This is partly compatible with a previous study, which reported no association between CIMT progression and the risk of a cardiovascular event , whereasThe potential limitations of this study warrant consideration. First, the activation of the NF-κB protein may play an important role in our results; however, no data concerning NF-κB protein were available. Future studies considering the activity of NF-κB protein will be necessary. The factor that directly inhibits platelet activity and angiogenesis could act as a strong confounding factor in the present analysis as under the influence of these factors; high platelet levels no longer indicate enhanced activity of endothelial repair. However, we found significant associations even without information on these factors. Furthermore, since this was a cross-sectional study, causal relationships could not be established. However, multi-faceted analyses enabled us to determine the possible mechanism underlying our results.HTLV-1 infection is inversely associated with hypertension, particularly in subjects with high platelet levels. Among subjects with non-hypertension, HTLV-1 infection is positively associated with atherosclerosis, limited to subjects with the highest platelet level. Endothelial repair, including the development of atherosclerosis, may have an inverse association with hypertension.


Small molecule inhibitors and peptide inhibitors are identified for therapeutic intervention of JNK signaling pathway. SP-600125, an anthrapyrazole small molecule inhibitor for JNK with high potency and selectivity has been widely used for dissecting JNK signaling pathway. CC-401 is the first JNK inhibitor that went into clinical trial for inflammation and leukemia. Inhibitor for mixed lineage kinase (MLK), CEP-1347 also negatively regulates JNK signaling, and tried for potential use in Parkinson’s disease. Cell-permeable peptide inhibitor D-JNKI-1 is being developed for the treatment of hearing loss. The current status of these JNK inhibitors and safety issue is discussed in the minireview.Targeting protein kinases has been active area in drug discovery. The c-Jun N-terminal kinases (JNKs) have also been target for development of novel therapy in various diseases, since the roles of JNK signaling in pathological conditions were revealed in studies using


Avian color patterns function in varied behavioral contexts, most being produced by only a handful of mechanisms including feather nanostructures and pigments. Within a clade, colors may not occupy the entire available space, and incorporating complementary colors may increase the contrast and efficacy of visual signals. Here, we describe plumage patterns in four ecologically and phylogenetically diverse bird families to test whether they possess complementary colors. We present evidence that plumage colors in each clade cluster along a line in tetrachromatic color space. Additionally, we present evidence that in three of these clades, this line contains colors on opposite sides of a line passing through the achromatic point . Finally, interspecific color variation over at least some regions of the body is not constrained by phylogenetic relatedness. By describing plumage patterns in four diverse lineages, we add to the growing body of literature suggesting that the diversity of bird visual signals is constrained. Further, we tentatively hypothesize that in at least some clades possessing bright colors, species-specific plumage patterns may evolve by swapping the distributions of a complementary color pair. Further research on other bird clades may help confirm whether these patterns are general across bird families. Summary: In four diverse bird radiations, we reveal the presence of complementary colors, putatively for higher contrast. Color patterns may have diversified by redistributing a complementary color pair over the body. Avian color patterns represent important visual signals that serve diverse functions , and theMany studies aimed at quantifying color use color space models, which typically involve mapping measured colors onto axes centered around an ‘achromatic point’, with more saturated (or vivid) colors being further away from this point . Colors Relatively few bird clades have had their overall color space quantified across species, and much remains to be understood about patterns of color space occupancy. It is thus important to describe the color space of additional bird clades to examine which regions of tetrachromatic color space they occupy . This fuPsittacula parakeets (Psittacidae), fruit and seed-eaters inhabiting deciduous forests and woodland  -visible light reflectance spectrometry . They arwoodland , and (4)woodland   . We firsHydrornis occupies 36.03% of the volume of total avian color space, Erythropitta 29.92%, and Pitta 26.93%). Barbets largely distribute between the green (MWS) and red (LWS) vertices, with a few blue-violet patches . Plumage colors of Psittacula parakeets lie between the middle of red-green space and the middle of blue-UV space . An XYZ color space using a noise-limited model of color space  recovers a linear axis that explains the bulk of color variation, suggesting that plumage colors are largely distributed along an ‘axis’ in tetrachromatic color space. The results of principal components analysis to quantify the proportion of variation explained by this line are summarized below for each avian clade, and also in Table S1 (see Supplementary Data).Across the four avian clades, we find that plumage colors distribute between two points in tetrahedral color space. The color signals of pittas lie between red (LWS) and violet (VS) color vertices (indicating highly-saturated colors) A  of the XYZ coordinates in color space explains 85% of chromatic variation B. PC1 loPC1 explains almost 74% of chromatic variation D, loadinPC1 explains 75% of variation in color F, loadinPC1 explains about 70% of color variation, loading weakly negatively on X (−0.32) and strongly positively on Y and Z (0.64 and 0.7).Across all four clades, the Z coordinate loads most strongly on PC1, thus suggesting that most variation in perceptual coordinate space occurs along the elevational rather than azimuthal direction along the PC1 line. Therefore, in subsequent analyses, we transformed this XYZ space into a spherical coordinate system, and used the elevational coordinate Φ and the sign of this coordinate as an indicator of where different colors lie along this line. Although this does not take variation in the azimuthal plane into account, the results of our analysis suggest that this variation is relatively low compared to variation along the elevational axis in all four families. Thus, colors with opposite signs of Φ in this dataset should lie on opposite sides of the achromatic point  species within each clade exhibit colors lying on both sides of the achromatic point, and (2) whether the distribution of colors along a major axis is constrained by phylogenetic relationships . In summary, we uncover evidence that most species in three of the four avian clades (with the exception of the sandgrouse) possess color scores lying on either side of the achromatic point, indicating complementary colors within each species, putatively for higher contrast. Further, phylogenetic comparative analyses suggest that phylogeny does not appear to explain significant variation in color and luminance scores. Regions with high color variation exhibited low or non-significant phylogenetic signal throughout. We summarize the results of these analyses below.Hydrornis baudii has, on average, a color score of −1.15, and the deep-red crown of the sympatric . Color scores are consistent with a Brownian motion model of evolution on the cheek, wing and tail, and exhibit weak and non-significant phylogenetic signal across other body regions (Supplementary Data). In addition, the regions with non-significant phylogenetic signal all possess relatively high coefficients of variation in color scores  crown of the male ympatric  Erythropic point A . Color and luminance scores (Supplementary Data), corroborating the results from phylogenetic signal. In addition, all head regions possess relatively high coefficients of variation (CVs) for color scores, but not body regions . Taken together, these results are also consistent with body colors being a constrained feature within this clade, but colors diversifying on the head regions.Color scores span between −1.4 and +1.57, and thus both sides of the achromatic point. For example, the red throat of ympatric  P. rafflribution B demonste scores  exhibit Psittacula longicauda nicobarica (−1.38) exhibits the opposite sign to the red shoulder patch of male P. cyanocephala (+1.32). Color histograms again indicate that all species possess colors lying on opposite sides of the achromatic point . Neither color nor luminance scores exhibit significant phylogenetic signal across any body regions (Supplementary Data). Color scores exhibit higher CVs than luminance scores . However, aside from luminance scores on the wing (Supplementary Data), interspecific color and luminance distances are not significantly correlated with phylogenetic distance. Coefficients of variation of color scores  and +1.33. Sandgrouse are clustered in chromatic space to one side of the achromatic point, further supported by color histograms D, althouthe wing , neitherr scores  are geneientalis  represenAcross diverse bird families, we thus uncover consistent evidence that plumage colors within a clade do not occupy the entire available color space, in keeping with previous studies. In three clades, each species possesses complementary colors occurring on opposite sides of the achromatic point, and phylogenetic comparative analyses indicate generally low or non-significant phylogenetic signal . Taken together with the distribution of color scores along a single line in color PC space, we suggest that colors on opposite sides of the achromatic point represent complementary colors. Their presence across most species within at least three clades, together with the lack of phylogenetic signal, suggests tentatively that evolutionary diversification of color patterns in at least some bird clades occurs by redistributing a complementary color pair across body regions. We discuss this further below.To summarize, we find that plumage colors in each of the four bird clades distribute along an axis between two colors (or regions of the avian-visible spectrum) that are complementary (spanning either side of the achromatic point), except the arid-country sandgrouse whose colors are found to only one side of the achromatic point (we discuss this further below). Additionally, our phylogenetic comparative analyses uncover heterogeneous phylogenetic signal across body regions, suggesting that colors on at least some regions of the body are not phylogenetically constrained. This suggests that the restricted color space occupied by each clade is not the result of related species closely resembling each other. Our study includes only four clades, and not a broader sample of other bird groups. Further studies are required to test these hypotheses across a broader selection of avian clades. However, we do identify our analyses of four diverse, representative clades as a launching point for further broad studies. Although not directly confirmed in our study systems, it is important to note here that tetrachromatic visual systems (avian and reptilian) possess a number of opponent color processes to compare cone outputs . In humaHydrornis baudii possesses a blue crown and underparts, and a reddish-brown back, whereas the sympatric Erythropitta granatina possesses a deep blue-violet back and a bright red crown patch and belly. For Asian barbets, this redistribution of colors appears to occur primarily on the head, and an examination of their color patterns supports this. Most members of the family possess largely green bodies, and bright colors are confined to the head regions. It is noteworthy here that relatively few species in these clades are sexually dimorphic . Psittacula parakeets and sandgrouse are sexually dimorphic, but this has little broad effect on the plumage patterns we observed  may additionally constrain plumage diversity or the position of complementary colors on the body. All four families studied here experience predation, and possess both cryptic colors, and colors that offer maximal contrast in their preferred habitats. For example, blue-violet and saturated reds are very conspicuous against a forest understory background , and alsColor vision is challenging to study comparatively in speciose bird lineages containing rare or range-restricted species, and many of the species we examine are poorly known. Thus, although our study is primarily descriptive of color patterns in four diverse bird clades, and our design does not permit us to conclusively identify the ecological driver of these patterns, we do find that at least three clades possess apparently complementary colors. Based on this, we tentatively hypothesize that color patterns in some bird clades may have diversified by redistribution or replacement of these complementary colors between species. Further research will focus on testing this hypothesis. Additionally, our study did not quantify evolutionary rates or shifts in color scores. With recent analytical frameworks , this prSupplementary Data). For the pittas, we measured 80 specimens of 27 species [out of 34 according to the previous taxonomy; more recent taxonomic sources  of Asian barbets, 55 specimens of 12 species of Psittacula (one species was not sampled), and 57 specimens of 16 species of sandgrouse. Although our dataset did not include some species, we sampled the majority of recognized species in each family. Missing species are mostly qualitatively similar in plumage to the other species sampled, and we consider their addition unlikely to alter the broad patterns we observe (Supplementary Data). Where possible, we measured specimens collected relatively recently (Supplementary Data) to obtain a comprehensive estimate of the color space occupied by each clade. These clades were selected partly because they are all monophyletic, but also because they are ecologically diverse, representing environments from forest understory all the way to arid environments. This phylogenetic and ecological disparity allows us to potentially make broader inferences about the evolution of bird color.We measured museum specimens of four avian clades, held in the collections of the Division of Birds, Smithsonian National Museum of Natural History (USNM), Washington, D.C., USA  selecting specimens with no qualitative evidence of fading and (2) where possible, measuring specimens of diverse collection ages , includiPsittacula parakeets, we did not treat the throat as a separate region from the cheek, owing to the structure of plumage patterns across their heads . Secondly, owing to the long tails of these parakeets, we could obtain reliable measurements of undertail colors in all species, and these are therefore included in our analyses. For reasons of accessibility due to the methods of skin preparation, we did not measure the underwing and undertail colors of any other species except parakeets, and we could not reliably access the white wing patches or tails of certain Pittidae, which have also therefore been excluded from our analyses   in our tetrahedral plots of color space, but not in subsequent analyses .We measured plumage reflectance of museum skins, using an S2000 UV-visible fiber-optic reflectance spectrophotometer  with a DT1000 deuterium-tungsten halogen light source. Measurements were referenced to a CIE D65 (white under average daylight illumination) white standard , and dark referenced to a black surface. We first moved the probe over each region of the body, looking at the computer display to ensure that (to the best of our ability) we did not miss any patches that are not visible to the human eye . We then measured one reflectance spectrum for each color patch on each specimen using the Overture  software. Although our dataset did not take within-patch variation into account as a result, we generally observed that intraspecific variation  for the same patch was lower than interspecific variation, and is thus unlikely to alter the patterns we observe. Using photographs of each specimen , we divided the body of the bird into seven regions commonly used by ornithologists to describe plumage patterns: the crown , the cheek , the throat , the upperparts (the back and rump), wing, tail , and underparts  . For Psied bird) . FinallyCyanistes caeruleus)  program TetraColorSpace  and the eruleus) . Again, eruleus) . We alsoeruleus)  as a refLeiothrix lutea . First, we performed one-sample t-tests on the maximum and minimum values to test whether they differed from a mean of zero, and then a paired two-sample t-test to test whether they differed from each other. These two tests together served to test whether each clade possessed colors lying on opposite sides of the achromatic point (zero).First, we estimated the proportion of variation in coordinate space explained by the first major axis using principal components analysis (PCA) on the XYZ coordinates obtained above, following published studies . We usedSupplementary Data). This essentially calculated the pairwise difference in average color score between each pair of species, for each body region. Thus, we obtained a matrix of ‘color distances’ that could be correlated to the phylogenetic distance matrix. This test provided additional quantification on the effects of phylogenetic relatedness on interspecific color variation, and we predicted that a lack of signal, together with the presence of complementary colors, would be consistent with pattern diversification by redistributing a complementary color pair across body regions.Finally, we used phylogenetic comparative analyses to investigate whether color and luminance scores across each body region exhibited phylogenetic signal. We first sorted all the patches measured in each of the four avian clades into crown, cheek, throat, back, wing, tail and underpart patches  for both males and females. Next, we calculated the average color score and luminance index (double cone photon catch) for each region of the body for the male plumage of each species . Using published phylogenetic information for each family  and the