Significantly, compared to non-diabetics, BVRA and PKCζ expression was low and haptoglobin was high in T2D patients leukocytes. Sustained TLR4 activation may deregulate homeostatic anti-inflammatory BVRA/mTORC2 signaling and thereby contribute to chronic inflammatory diseases.TLR4 is implicated in diseases associated with chronic low-grade inflammation, yet homeostatic signaling mechanisms that prevent and/or are affected by chronic TLR4 activation are largely uncharacterized. We recently reported that LPS/TLR4 activates in human leukocytes signaling intermediates (SI), abbreviated TLR4-SI, which include mTORC1-specific effectors and targets, and that leukocytes of patients with T2D or after cardiopulmonary bypass (CPB) expressed similar SI. Extending these previous findings, here we show that TLR4-SI expression post-CPB was associated with low serum bilirubin and reduced preoperative expression of biliverdin reductase A (BVRA), the enzyme that converts biliverdin to bilirubin, in patient’s leukocytes. Biliverdin inhibited TLR4 signaling in leukocytes and triggered phosphorylation of mTORC2-specific targets, including Akt, PKCζ, AMPKα-LKB1-TSC1/2, and their association with BVRA. Torin, PP242, and a PKCζ inhibitory peptide, but not rapamycin, prevented these biliverdin-induced responses and TLR4 inhibition. In contrast, LPS/TLR4 triggered decreases in BVRA, AMPKα and PKCζ expression, and an increase in haptoglobin, a heme binding protein, in leukocytes  Our understanding of homeostatic molecular mechanisms that limit TLR4 signaling, and whether/how chronic TLR4 activation might impact these homeostatic signaling processes remains limited. TLR4 is activated by lipopolysaccharide (LPS) derived from pathogenic and commensal gut-residing Gram-negative bacteria, and endogenous ligands that include free fatty acids4. LPS is a potent inducer of inflammatory responses in humans6. However, whereas LPS doses above 0.5 ng/kg elicited inflammatory responses that included IL-6 and TNF-α production and broad changes in leukocyte’s mRNA and signaling intermediates (SI) expression7, doses below 0.5 ng/kg triggered only changes in leukocyte’s SI expression8. These observations suggested the hypothesis that the pattern of SI detected in leukocytes challenged with low LPS doses may be clinically relevant and serve as a tool for interrogating whether and how chronic low-grade TLR4 activation may deregulate homeostatic signaling mechanisms that prevent unwarranted TLR4 activation.Toll-like receptor 4 (TLR4) is implicated in morbidities associated with chronic low-grade inflammation, including insulin resistance and type 2 diabetes (T2D)10, including AMP-activated protein kinase (AMPK) and hypoxia inducible factor subunit α (HIF-1α)8. In general, mTORC1 is activated when nutrients are abundant. Scarcity of energy and/or glucose triggers AMPK activation11. AMPK can prevent mTORC1 signaling via two mechanisms: (i) AMPK can phosphorylate Raptor on Ser792 thus preventing mTORC1 assembly12, and (ii) AMPK can phosphorylate and activate TSC1/213, which acts as a GAP for Rheb14. Activated TSC1/2 inhibits Rheb and consequently mTORC114. Raptor is phosphorylated at Ser792 in resting leukocytes10. LPS/TLR4 triggers Raptor dephosphorylation at Ser792 via a unique matrix metalloproteinase 9 (MMP9)-dependent molecular mechanism9. Though MMP9 is best known for its role in extracellular matrix degradation15, in LPS activated leukocytes intracellular MMP9 contributes to AMPKα cleavage10, mTORC1 activation, and the phosphorylation of S6K1 at Ser38917. mTORC1 also upregulates TLR4 and HIF-1α expression. HIF-1α and HIF-1β form the transcription factor HIF-1, an essential regulator of myeloid cells bioenergetics and antimicrobial functions18. LPS does not activate mTOR complex 2 (mTORC2)10. Hereafter, we refer to the panel of SI that include elevated MMP9, HIF-1α, TLR4, and proteolytically cleaved AMPKα, as ‘TLR4-SI’.Among the SI affected by TLR4 activation are several mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling components10. However, T2D patients treated with insulin, in general, did not express TLR4-SI10. Further investigation revealed that insulin counter-regulates TLR4 signaling in leukocytes, and vice-a-versa10. Building on this prior work, in this study we sought to determine why the majority, but not all CPB patients expressed TLR4-SI postoperatively, hypothesizing existence of TLR4 signaling regulator(s) in addition to insulin. Here, a prospective blinded study of CPB patients uncovered an inverse association between postoperative TLR4-SI expression and serum bilirubin levels. Bilirubin is the end product of heme catabolism. Heme is converted to biliverdin by heme oxygenases and then reduced to bilirubin by biliverdin reductase A (BVRA). We then discovered that TLR4-SI expression postoperatively was also inversely associated with preoperative BVRA expression, suggesting that TLR4- and BVRA-signaling in leukocytes are interconnected. We report that biliverdin/BVRA activates mTORC2 and an AMPK signaling axis that inhibits mTORC1, a key TLR4 signaling node. Furthermore, we identify biliverdin/BVRA SI that are altered by TLR4 causing mTORC2 inhibition. The data identify a novel counter-regulatory signaling loop involving BVRA and TLR4. We propose that in T2D patient’s leukocytes this loop is deregulated and that the signals are skewed in favor of TLR4.We recently reported that leukocytes of patients with T2D and a majority of patients after cardiopulmonary bypass (CPB) surgery expressed SI similar to TLR4-SIAll methods were performed in accordance with the relevant guidelines and regulations.Rutgers, Robert Wood Johnson Medical School Institutional Review Board approved the study. Patient participation was voluntary and without compensation. Informed written consent was obtained from all participants before enrollment in the study. Details concerning study protocol, inclusion exclusion criteria and surgical procedure are included in the Supplement.CPB patient’s blood was drawn pre-anesthesia (P), within 30 minutes of patient’s arrival in the recovery room (RR), and in the early hours of day 1 post-operatively (D1). Additional daily samples were collected from 4 randomly selected patients in the early hours of days 2, 3, 4 . IL-6 was measured using Quantikine ELISA kits, Cat# D6050 . Insulin was measured using an ELISA assay .Antibodies used: Actin  was from Sigma. Heme oxygenase 2 (HO-2)  was from Boster Biological Tech Co., Pleasanton CA. HIF-1α , AMPKα , MMP9 , TLR4 , BVRA , Akt1 , p-LKB1 Ser431 , LKB1 , p-CaMKKβ Thr286 , CaMKKβ , p-mTOR Ser2481 , mTOR , p-PKCζ Thr410 , PKCζ , heme oxygenase 1 (HO-1) , heme oxygenase 2 (HO-2) , tuberin (TSC1/2), haptoglobin β , p-NOS3 Ser1177  and NOS3  were all from Santa Cruz Biotechnology. p-Raptor Ser792 , Raptor , p-p70 S6 kinase Thr389 , p-AMPKα Thr172 , p-Akt Ser473 , p-Akt Thr308 , tuberin/TSC2  and p-tuberin/TSC2 Ser1387  were all from Cell Signaling Technology.Eschericnia coli 0111:B4; Sigma) . Biliverdin (50 μM) (Sigma) was freshly dissolved in 0.2 N NaOH, adjusted to a final pH of 7.4 with HCl and kept in the dark. Metformin (10 μM) was from Sigma. GSK2334470 (3 μM) was from Cayman. Rapamycin (100 nM), torin (50 nM), PP242 (100 nM) were from Tocris. The PKCζ inhibitory peptide (ζ-pseudosubstrate inhibitory peptide) (10 μM) was from Fisher Scientifics.Reagents, source and final concentrations (unless otherwise indicated): LPS  were isolated, treated or not, and lysed as described10. Raw 264.7 cells were obtained from ATCC and cultured for up to 5 passages. Cell lysates were normalized for protein content and analyzed by western blotting. In brief, samples were subjected to SDS-PAGE separation followed by blotting onto polyvinylidene difluoride membrane. Immunoreactive bands were detected using Super Signal Chemiluminescence (Thermo Scientific Pierce) and visualized by autoradiography. All figures shown are an accurate representation of the data and no image was manipulated.Blood drawn into heparin-containing tubes was separated into aliquots and treated with LPS (10 ng/ml) or biliverdin (50 μM), unless otherwise indicated. Leukocytes were then isolated as described4 sample buffer and analyzed by western blotting as described earlier.Pierce crosslink IP kit  was used to crosslink BVRA antibody to agarose. Leukocyte pellets were washed once with PBS and then lysed for 5 min on ice with 500 μl of RIPA buffer. Cellular debris was removed by centrifugation and supernatants were normalized for protein concentration. Lysates (500 μg protein) were pre-cleared for 2 h at 4 °C with 40 μl of protein A/G agarose beads and were then incubated overnight at 4 °C with 2 μg of BVRA-agarose with gentle rotation. Samples were then washed three times with washing buffer . Bound proteins were eluted with 5X NaDodSOt-test was used to analyze continuous variables and Mann-Whitney test for comparison of non-parametric data. Categorical variables were compared using Fisher exact test. Normality was determined using D’Agostino and Pearson test. Demographic and clinical variables recognized in unadjusted comparisons as statistically significant at p-value of less than 0.1 were selected for inclusion in a logistic regression model. Data are presented as mean and 95% confidence interval (95%CI) of mean if data were normally distributed, or median and interquartile  range if data were not normally distributed. Within group values were compared using ANOVA with repeated measure and Tukey’s multiple comparisons test, or Friedman test and Dunn’s multiple comparisons test if data were not normally distribution. Data and statistical analyses were performed using Prism 7.0 for Mac OSX  and STATISTICA 12.0 for Windows . p-values less than 0.05 were considered statistically significant.Clinical and laboratory data were analyzed with respect to the detection of TLR4-SI on day 1 post-CPB. 9. MMP9, HIF-1α and TLR4 expression was low and AMPKα was intact in leukocytes of patients prior to or immediately after CPB  body surface area (BSA)  bilirubin  than those without TLR4-SI. Those factors showing a bivariate association with the dependent variable, i.e., TLR4-SI at p < 0.1, were entered into a multivariate logistic regression model. Detection of TLR4-SI was independently associated with decreased levels of total bilirubin  and increased body surface area .Next, patient’s demographics, clinical parameters, and blood chemistries were all analyzed with respect to the expression of TLR4-SI postoperatively Table . PatientSA) Fig. , but notbin Fig.  and insuBiliverdin reductase A (BVRA), the enzyme that reduces biliverdin to bilirubin, is ubiquitously expressed. BVRA expression in CPB patients leukocytes obtained on the morning of the surgery varied among patients Fig. . Strikin21. Analyses of leukocytes and neutrophils from three randomly selected CPB patients showed that the transient decline in BVRA expression seen in leukocytes was reproduced in neutrophils 25 phosphorylate AMPKα at Thr172. Activated LKB1 is phosphorylated at Ser43126 whereas CaMKΚβ autophosphorylates at Thr28627. Though both LKB1 and CaMKΚβ where detected in leukocytes, biliverdin induced only LKB1 Ser431 phosphorylation  responses to biliverdin. Reproducing the pattern seen in leukocytes, Akt Ser473, AMPKα Thr172, and LKB1 Ser431 phosphorylation were all detected in neutrophils but not mononucleated cells  phosphorylation at Ser1387, but not at Thr1462  and AMPKα Thr172 phosphorylation37. Other showed that mTORC2-dependent phosphorylation at Thr410 activates PKCζ38. Here, both biliverdin and metformin induced PKCζ phosphorylation at Thr410 , an inhibitor of 3-phosphoinositde-dependent protein kinase (PDK1)41. In LPS treated leukocytes  (HO) convert heme to biliverdin, the substrate of BVRA. Two HO isoforms were described in leukocytes: HO-1 and HO-2. The level of HO-1 is resting leukocytes was below detection Fig. , suggestivo Fig. . Despite53. Neutrophils synthesize and store haptoglobin in their granules and release it following activation54. Leukocytes treated with LPS in vitro  biliverdin induced Akt phosphorylation at Thr473, the mTORC2 phosphorylation site55, but failed to induce S6K1 phosphorylation at Ser389, the mTORC1 phosphorylation site16, and (ii) torin and PP242, which act on mTORC1 and mTORC2, suppressed responses to biliverdin, whereas rapamycin, which under acute conditions inhibits only mTORC1, did not. These findings are significant since they demonstrate that bilirubin/BVRA inhibits TLR4/mTORC1 signaling in leukocytes via mTORC2. A similar mechanism may prevent TLR4 activation at steady state.Leukocytes form the first line of defense against invading microorganisms. In humans, minute LPS concentrations and multiple endogenous ligands that are produced by damaged tissues activate leukocyte’s TLR4. Since activated leukocytes may cause sever damage to hosts, we hypothesized existence of physiological mechanisms that prevent unwarranted TLR4 activation, and furthermore, that failure of these mechanisms may contribute to chronic, low-grade inflammatory diseases. We recently reported that insulin is a TLR4-signaling regulator/inhibitor in leukocytes10. Consequently, whereas PKCζ-LKB1-AMPK-TSC1/2 inhibited mTORC1 in the context of biliverdin/BVRA signaling in leukocytes, insulin suppressed TLR4 signaling via mTORC2-Akt Ser473 and Foxo1/3 without LKB1-AMPK-TSC1/2 involvement. These data demonstrate that mTORC2 can engage distinct SI subsets to inhibit TLR4. Whereas the insulin and biliverdin/BVRA signaling pathways that converge on mTORC1 differ, we noted striking mechanistic similarities between biliverdin/BVRA and metformin signaling in leukocytes. As one of the oldest and most commonly used drugs for treating type 2 diabetics, metformin’s mechanism of action has been studied extensively. In general, metformin activates LKB1-AMPK-TSC1/2 signaling33. Our data now demonstrate that both metformin and biliverdin use mTORC2 to inhibit mTORC1. How biliverdin/BVRA activates mTORC2 remains unanswered. Maines and colleagues suggested that BVRA acts as a scaffold that supports oligomers formation56. Our data support this possibility and suggest that in leukocytes, BVRA facilitates formation of signaling complexes that include mTORC2 and it’s signaling targets.It is interesting to compare the mechanisms by which insulin and biliverdin/BVRA inhibit TLR4 signaling in leukocytes. Contrasting biliverdin/BVRA signaling dependence on mTORC2, insulin signaling in leukocytes, and in general, involves both mTORC1 and mTORC2 activation58. Consistent with this possibility, a recent study demonstrated an increase in oxidative stress indicators in BVRA KO mice59. Others reported that bilirubin is a ligand of PPARα, a regulator of lipids metabolism in mice livers60. On the other hand, BVRA was implicated in insulin, IGF-1 and TNF-α signaling47. In IGF-1 treated HEK293 cells, recombinant BVRA co-immunoprecipitated with Akt1, Akt2 and PDK1 and facilitated Akt phosphorylation at Thr308, the PDK1 phosphorylation site45. BVRA also interacted with PKCδ in this model cell system45. However, in TNFα treated HEK293 cells, BVRA regulated PKCζ45. Of these, only PKCζ activation was reproduced in biliverdin-treated leukocytes. Consistent with our findings, Wegiel and colleagues reported that biliverdin triggered TLR4 signaling inhibition in Raw 264.7 cells28. However, the TLR4 inhibitory mechanism in Raw 264.7 cells involved endothelial nitric oxide synthase (eNOS) activation by CaMKΚβ and increased nitric oxide generation. Although we observed similar biliverdin-induced responses in Raw264.7 cells, i.e., eNOS and CaMKΚβ activation, biliverdin did not induce CaMKΚβ/eNOS signaling in leukocytes, neutrophils or mononucleated cells. The data establish existence of cell-type specific biliverdin/BVRA signaling mechanism(s), and identify a novel biliverdin/BVRA signaling pathway in leukocytes.Earlier studies have focused on the possibility that bilirubin and biliverdin act primarily as antioxidants and may reduce oxidative stress damage by suppressing intracellular oxides levels65. Low serum bilirubin were also shown to correlate with higher C reactive protein levels in patients with T2D and impaired glucose tolerance66. It is also known that patients with Gilbert’s syndrome, a syndrome associated with higher than normal bilirubin, have in general a lower incidence of coronary heart disease and arteriosclerosis67. Together, these clinical studies suggested that whereas high bilirubin levels are health beneficial, low bilirubin levels could be detrimental to health. We hypothesize that higher bilirubin levels are associated with decreased risk for human diseases, and vice-a-versa, since these levels reflect, at least in part, how well the biliverdin/ BVRA signaling network operates. When fully functional, biliverdin//BVRA may contribute to higher serum bilirubin levels and at the same time suppress TLR4 signaling in leukocytes.Prior clinical and epidemiologic studies found that low serum bilirubin levels were associated with increased risk of pre-diabetes, T2D, metabolic syndrome, as well as strokeNotably, our data are first to show that activated TLR4 can counter-regulate BVRA signaling. One of the key biliverdin/BVRA signaling components targeted by TLR4 is BVRA itself. TLR4 may also disrupt biliverdin/BVRA signaling by reducing AMPKα/AMPK, PKCζ, and heme availability. Significantly, leukocytes of patients with T2D expressed low BVRA and PKCζ levels and elevated haptoglobin, reproducing the expression pattern seen in LPS treated leukocytes. Compared to untreated leukocytes, LPS-treated leukocytes with reduced BVRA expression were less responsive to biliverdin. Higher BVRA expression may enhance leukocyte’s resistance to stressors that act on TLR4, or in other words, that biliverdin/BVRA impact leukocyte’s activation threshold. Our data suggest that activated TLR4 deregulates this safety mechanism.In summary, we discovered a novel TLR4 and biliverdin/BVRA counter regulatory mechanism that controls TLR4 activation in human leukocytes. Furthermore, our data suggest that the balance between TLR4 and biliverdin/BVRA signaling in leukocytes of patients with T2D is deregulated and chronically shifted in favor of TLR4. A fully functional biliverdin/ BVRA signaling network may, in general, suppress unwarranted TLR4 signaling, whereas suboptimal network activity may fail to prevent TLR4 activation thus contributing to non-resolving chronic low-grade inflammation.Supplement


The effort to characterize intrinsically disordered regions of signaling proteins is rapidly expanding. An important class of disordered interaction modules are ubiquitous and functionally diverse elements known as short linear motifs (SLiMs).S. cerevisiae. Using a single cell, reporter-based flow cytometry assay in conjunction with a fluorescent reporter driven by a pathway-specific promoter, we quantitatively assessed pathway output via systematic deletions of individual motifs. We found that, when deleted, 34% (10/29) of predicted SLiMs displayed a significant decrease in pathway output, providing evidence that these motifs play a role in signal transduction. Assuming that mutations in SLiMs have quantitative effects on mechanisms of signaling, we show that perturbations of parameters in a previously published stochastic model of HOG signaling could reproduce the quantitative effects of 4 out of 7 mutations in previously unknown SLiMs.To further examine the role of SLiMs in signal transduction, we used a previously devised bioinformatics method to predict evolutionarily conserved SLiMs within a well-characterized pathway in Our study suggests that, even in well-characterized pathways, large numbers of functional elements remain undiscovered, and that challenges remain for application of systems biology models to interpret the effects of mutations in signaling pathways.The online version of this article (10.1186/s12918-018-0597-3) contains supplementary material, which is available to authorized users. There is an increasing proportion of known signaling interactions mediated by regions of proteins lacking a defined tertiary structure under native conditions, known as intrinsically disordered regions (IDRs). Comparative proteomics studies have used evolutionary conservation to identify short functional elements in these intrinsically disordered regions  and suggOne of the best-understood functions of SLiMs is to mediate signaling interactions, both through post-translational modifications and peptide-domain interactions –9. RecenBecause each SLiM is thought to mediate a single, usually transient interaction, we expect mutations in them to have quantitative effects on protein function. We therefore decided to employ a quantitative, single cell reporter assay , 17 to dOverall, we identified 7 previously uncharacterized short protein sequences (between 3 and 17 amino acids in length) that significantly impact pathway output when deleted. Among these and 3 previously known motifs, we observed a wide quantitative range of effects on pathway output and tested whether these effects could be explained by perturbations of parameters in a previously proposed quantitative model of the HOG pathway . We founWe searched for short motifs in disordered regions in model signaling pathways in yeast using previously described bioinformatics methods . We consAnalysis of the Sho1-dependant branch of the HOG pathway yielded 29 predicted short linear motifs (pSLiMs) within 14 disordered regions  has been the subject of extensive molecular characterization , 27 and We sought to test for functional importance of the short, conserved elements (pSLiMs) in the HOG signaling pathway. We first confirmed that previously characterized pSLiMs in the HOG pathway affected signaling function in a reporter-based flow-cytometry assay that can quantitatively measure pathway activity using a fluorescent reporter downstream of Hog1- specific transcriptional output see . We madeWe found that when deleted, three of these four known motifs showed reduction in reporter activity Fig. . This con = 38). To assess whether a mutant affected HOG signaling, we compared the divergence (in replicate experiments) of the mutant distribution to that of the wild-type. If the divergence of the mutant to the wild-type was significantly greater than the wild-type compared with itself, we consider that mutant to impact signaling , Unlike the binding site in Opy2, deletion of the known Bem1 binding site in Ste20  showed nn = 11 vs. 38, P < 0.00001, corrected, Additional file Perturbation of Hot1 activity is known to lead to stochastic responses in the HOG pathway . InteresP < 0.05).Using the same assay as above, we tested a cohort of previously uncharacterized pSLiMs in the disordered regions of the HOG pathway Fig. . Of the These varied effects include nearly complete abolishment of the pathway activity for deletion of a previously uncharacterized proline-rich sequence in Pbs2, while most mutants showed subtle quantitative effects. The large effect of the mutation in the Pbs2 pSLiM is consistent with Pbs2’s role in localizing the signaling complex , 27, 31,In principle, quantitative models of signaling can be used to connect genotype to phenotype, and could therefore be used to predict the impact of mutations in disordered regions. Considering that signaling proteins are often mutated in human disease, this represents a potentially important area of application for quantitative models.The yeast MAPK signaling pathways have served as a foundation for the understanding of signaling in more complex organisms , and then = 38), which is larger, but on the same order as the divergence between replicates (0.016 +/− 0.014). This indicates that the previously developed model  can explain the distribution of reporter expression in our experimental setup.We therefore first tested whether a previously published model of HOG signaling  could exTo predict the effect of mutations in the pathway, we assumed that each mutation would affect only one of the 5 parameters associated with signaling components in the model . Because the simplified model does not explicitly include signaling components (or their protein interactions) we do not know in advance which parameter each mutation would affect, nor do we know the magnitude of the effect the mutation in a SLiM would have on the parameter. However, through simulations of where the different parameters were reduced individually (to model the effects of mutations), we noticed that the effects on the parameters fell into two groups  which is expected in part because the mutant model has added flexibility. Interestingly, we could not achieve a significantly better fit for the models when the remodeling parameter was perturbed  suggesting that the mutation in the Opy2 binding site affects the signal activation upstream of Hog1, which is consistent with the known position of Opy2 in the pathway  pSLiMs had significant effects on pathway output. We predict a large fraction of pSLiMs found in other signaling pathways would also be functionally significant. As an example, we applied the same phyloHMM software   [SSK2 Δ::HisMX3 SSK22 Δ::0; HO Δ::STL1pr-GFP, LEU2). All subsequent deletions/mutations of the genomic sequence were performed using the Delitto Perfetto method for seamless mutagenesis of the yeast genome [PBS2 Δ180–188; PBS2 Δ269–274; STE50 Δ341–346) and created C-terminal fusions with a fluorescent tag (mCherry) of each deletion (and the corresponding wild-type counterpart) for subsequent confocal analysis before and after induction of the HOG pathway. The mCherry tag was amplified in conjunction with the Ura3 auxotrophic marker and integrated as a fusion into each designated strain. Confocal analysis of fixed (see below) uninduced and induced cells revealed no difference in either expression or localization as compared to wild-type  lacking the Sho1-independent branch of the osmoresponse pathway ( ssk22Δ) . The GFP ssk22Δ) , integrat genome , and wert genome  . A secot genome  essentiaHOG pathway induction experiments were performed as follows: Replicate overnight cultures inoculated from single colonies were diluted 10-fold into the appropriate selective media and grown to mid-log phase (4 h @ 30 °C). Un-induced samples were taken at this point, and cells were pelleted and re-suspended in an equivalent volume of fixative . Cultures were then induced by addition of NaCl to a final concentration of 0.4 M and incubated @ 30 °C for an additional 60 min. Induced samples were then taken and treated as above. All fixed samples were stored @ 4 °C pending flow cytometry analysis (within 48 h).Reporter activation in fixed cells from all induction experiments was subsequently measured by flow cytometry. Flow cytometry analysis was performed on a BD FACSAria IIU High Speed Cell Sorter, incorporating 3 air-cooled lasers at 488, 633, and 407 nm wavelengths, and equipped with BD FACSDiva™ software (v. 6.1.3). The instrument was calibrated prior to each experiment using a blend of size-calibrated fluorescent beads. The GFP fluorescence was excited at 488 nm and collected through 530/30 nm bandpass filter. All samples were further diluted by a factor of 10, and a total of 50,000 events (cells) were counted for every sample within gated SSC and FSC populations that excluded doublets and small debris. Typical acquisition rates were 700–900 events/second. Initial flow cytometry data was analyzed using FLowJo software .Data analysis of flow cytometry data was performed using in-house Matlab scripts. We used KL divergence to compare distributions. Because KL divergence is based on continuous probability distributions, to implement it on discrete data set, we used an approximation to estimate the KL divergence based on the empirical distribution .We pooled WT data from the same experiment day (3 to 4 replicates) which we refer to as the WT pool. We then used this WT pool as the Q distribution to calculate KL divergence. Used in this way, the KL divergence can be thought of as the error one would make if the wild type distribution were used in place of a given mutant distribution. The KL divergences of each WT replicate from the WT pool of the same experiment day is calculated and grouped to estimate the variance in WT reporters. We then calculated the KL divergence of a mutant replicate from the WT pool of the same experiment day. The divergences of the same mutant from different experiment days are then grouped together. We tested if the mutants’ divergences from WT pools are different from WT’s divergences from WT pools with the Wilcoxon Rank Sum test (WRS) and Bonferroni correction for multiple tests  were performed with 50,000 runs to represent 50,000 cells of each.The value of the stress parameter was set to 18 at The distribution obtained for each perturbation was analyzed using the same method that we used to compare the mutant distributions to the wildtype distribution (described above) with the following modifications: first, a small lognormally distributed constant  was added to the results of each run to represent basal gene expression . SecondAdditional file 1:Table S1. Predicted short linear motifs (pSLiMs) in the hypotonic stress and canonical Wnt signaling pathways. A. Signaling components of the hypotonic stress pathway in S.cerevisiae identified from KEGG, with 36 SLiMs predicted in 11 hypotonic stress signaling proteins using PhyloHMM. B. Signaling components of the human canonical Wnt signaling pathway identified from KEGG, with 24 SLiMs predicted in 16 Wnt signaling proteins using SLiMPrints. Periods indicate positions where any amino acid can match. (PDF 32 kb)Additional file 2:Table S2. P-values from Wilcoxon Rank Sum Tests comparing the distribution of KL divergence from mutants to wild-type pools. (PDF 37 kb)Additional file 3Figure S1. Effects of perturbations in model parameters fall into two groups. The activity parameter that affects the pathway activation steps upstream of the MAPK Hog1  affect Additional file 4Figure S2. Controls for mutant protein expression. Confocal images of dual fluorescent reporter strains with a pSTL1::GFP HOG pathway reporter combined with C-terminal mCherry fusions of Pbs2 Δ180–188, Pbs2 Δ269–274, Ste50 Δ341–346, wild-type Pbs2 and wild-type Ste50 before and after a 60 min induction with 0.4 M NaCl. The top row shows the pSTL1::GFP wild-type strain (no mCherry) for comparison. The left two columns show the mCherry channel, and we find no difference of mCherry fusion protein expression levels or localization in SLiM deletion strains as compared to their wild-type counterparts. The right two columns show the GFP channel and confirm that the HOG pathway has been induced as expected (based on our flow cytometry results) in the mCherry tagged strains. (TIF 1835 kb):


Toxoplasma gondii (T. gondii) that induces toxoplasmosis, particularly in humans. The anti-parasitic effects of ursolic acid against T. gondii-infected cells and T. gondii were evaluated through different specific assays, including immunofluorescence staining and animal testing. Ursolic acid effectively inhibited the proliferation of T. gondii when compared with sulfadiazine, and consistently induced anti-T. gondii activity/effect. In particular, the formation of parasitophorous vacuole membrane (PVM) in host cells was markedly decreased after treating ursolic acid, which was effectively suppressed. Moreover, the survival rate of T. gondii was strongly inhibited in T. gondii group treated with ursolic acid, and then 50% inhibitory concentration (IC50) against T. gondii was measured as 94.62 μg/mL. The T. gondii-infected mice treated with ursolic acid indicated the same survival rates and activity as the normal group. These results demonstrate that ursolic acid causes anti-T. gondii action and effect by strongly blocking the proliferation of T. gondii through the direct and the selective T. gondii-inhibitory ability as well as increases the survival of T. gondii-infected mice. This study shows that ursolic acid has the potential to be used as a promising anti-T. gondii candidate substance for developing effective anti-parasitic drugs.This study was carried out to evaluate the anti-parasitic effect of ursolic acid against  Toxoplasma gondii (T. gondii). T. gondii not only induces chronic infection in various sites of human body, but it also causes brain infection through the central nervous system as zoonotic parasitosis. In addition, T. gondii has unique infectious subcellular organelles, including apicoplast, conoid, rhoptries, dense granules, and micronemes [T. gondii forms a unique proliferative membrane, such as a parasitophorous vacuole membrane (PVM) including reticular network (RN) during its proliferation after invasion into host cells, and grows in it [T. gondii, and they are being used to treat toxoplasmosis’ patients in the clinic. However, their side effects are still clear in the clinic, and it is also being exposed to drug-resistance gradually [T. gondii, which inhibits consistently synthesis of the DNA and/or RNA in the proliferation of protozoa species, including malaria. Toxoplasmosis is caused in all age groups, including young children or adults globally, which is one of parasitic diseases as zoonosis infected through cronemes . In genews in it . Until rradually ,4,5. In T. gondii activities/effects [T. gondii has not yet been developed through the clinical study. Furthermore, despite various efforts for developing anti-parasitic drugs, zoonotic parasitosis derived from parasites has been consistently worsening a crisis of the public health worldwide.Until recently, it was reported that various extracts and/or natural products derived from medicinal plants had the possibility of useful medical resources for treating infectious virus diseases, including middle east respiratory syndrome (MERS), dengue fever or Zika fever, as well as showed in vitro inhibitory effects against chronic diseases, such as hepatitis, cancer, and tuberculosis ,9,10,11./effects ,14,15,16T. gondii activity of ursolic acid has not been reported yet. In addition, a novel and/or effective drug for the treatment of toxoplasmosis has not been developed yet, even though significant results regarding anti-T. gondii activity have been reported through various studies globally [T. gondii, causing toxoplasmosis in human. This study was carried out to evaluate the anti-T. gondii effect of ursolic acid which is known as a bioactive substance, and to determine its potential as a promising candidate substance for developing novel anti-toxoplasmosis drugs. In this aspect, various researches for discovering effective drugs against toxoplasmosis, and studies on new substances of relatively low toxicity with safety are urgently required to inhibit zoonosis. Ursolic acid is a bioactive substance contained in various medicinal plants that are used as natural resources in oriental medicine and in folk medicine, and is also known to have a variety of effects and bioactivity, such as anxiolytic activity , anti-anglobally ,31. For T. gondii causes parasitic disease on both animals and human, as well as having unique micro network systems and a specific structure, including various micro-organelles, such as mitochondria, rhoptries, and micronemes. We evaluated the anti-parasitic activity and the effects of UA on the proliferation and growth of T. gondii using an MTT assay and UA showed the anti-T. gondii activity in the range of 25–200 μg/mL. T. gondii-infected cells were incubated with different concentrations (25–200 μg/mL) of UA for 24 h, and their viability was markedly decreased in a dose dependent manner  showed a significant decrease of T. gondii including T. gondii fragmentation as well as morphological changes such as cell shrinkage and cell fragmentation when compared with the untreated T. gondii-infected cells (Data not shown), which suggests that UA has anti-parasitic activity and the inhibitory effect against T. gondi in infected cells  value of UA against T. gondii and T. gondii-infected cells was measured as 94.62 μg/mL and 162.25 μg/mL, respectively. These results demonstrate that UA strongly induced anti-proliferation activity of T. gondii in the infected host cells by effectively blocking T. gondii as well as the direct inhibitory action against T. gondii.t manner . In addied cells . Further00 μg/mL . The 50%T. gondii induces anti-apoptotic steps and features through the inactivation of apoptotic proteins during its proliferative phase in host cells, which shows the unique parasitic life-cycle of T. gondii. In particular, the formation of PVM is accelerated in a time-dependent manner after cell invasion of T. gondii. For this reason, we investigated the inhibition of PVM that is caused by the interaction between T. gondii and UA during the proliferation stage of T. gondii in host cells. As shown in T. gondii were markedly decreased in T. gondii-infected host cells when it was treated with 100 μg/mL of UA and SF, respectively, and their changes were clearly observed under a UV fluorescence. The results indicate the inhibitory effect of UA against PVM formation and the viability of T. gondii. Therefore, these results show substantial evidence that UA effectively inhibited or blocked the PVM formed by T. gondii during the proliferative stage as well as the proliferation of T. gondii in the infected host cells after cell invasion. T. gondii induces infectious symptoms and diseases, such as lymphadenopathy and brain injury, particularly in human as a zoonotic parasite that is infected in both human and animals. In this aspect, we evaluated the anti-proliferative effect and the parasitic inhibitory activity of UA against the viability of T. gondii in T. gondii-infected mice. The mice were carefully observed during the experimental periods after the injection of T. gondii treated with 100, 200, and 400 μg/mL of UA for 24 h. As mentioned above, T. gondii-infected mice treated with different concentrations (200 and 400 μg/mL) of UA showed higher activity and viability than T. gondii-infected mice, and the mice indicated the dynamic and energetic activity as the normal group. In particular, it clearly confirmed a significant difference of the survival rate between T. gondii-infected mice and the infected mice treated with UA. However, T. gondii-infected mice treated with 100 μg/mL of UA showed that the effect of UA did not consistently maintain the survival of the infected mice by decreasing after 11.8 days. This result suggests that the death of mice may be owing to tissue damage and cytolysis by invasion of T. gondii than the loss of body weight of mice. Furthermore, symptoms and side effects, such as the loss of body weight, were not observed or induced in all of the groups treated with 200 and 400 μg/mL of UA for the experimental periods , the mice showed the vitality and the viability such as the uninfected control group without the loss of body weight and the cytotoxicity during the experiment. In particular, it was recently reported that UA has the anti-protozoa effects against Leishmania amazonesis and Leishmania infantums, causing leishmaniasis among the neglected tropical diseases, and its mechanism of action is associated with programmed cell death and nitric oxide (NO) production [T. gondii effect of UA suggests that UA may induce the anti-proliferation and the growth inhibitory action of T. gondii through the mechanism of action that is caused by nitric oxide (NO) production or programmed cell death in T. gondii and the infected host cells.In this study, we evaluated the anti-parasitic activity/effect of UA which inhibits the proliferation of oduction ,36. In tT. gondii-infected cells was selectively inhibited or blocked in the host cells treated with UA, as well as significantly increased in infected cells. Furthermore, the T. gondii-infected mice treated with UA showed a stable life-cycle and survival rates when compared with infected positive group during the experimental period. The results clearly provide the inhibitory effect and activity of the compound against T. gondii, and indicate whether UA induces the direct inhibition of T. gondii or increase the survival of mice by effectively blocking and inhibiting the viability of T. gondii. Taken together, these results indicate that the expression of PVM in ®, Waltham, MA, USA). MTT -2,5-diohenyl-2H-tetrazolium bromide; Thiazolyl blue), RPMI medium 1640, sulfadiazine, dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), 0.4% trypan blue solution, and Hoechst 33342 were purchased from Sigma-Aldrich Chemical Co., Ltd. . All the other chemicals and reagents were purchased from Merck Chemical Co., Ltd.  and Sigma-Aldrich Chemical Co., Ltd. . Fetal bovine serum (FBS), antibiotics, and trypsin-EDTA were purchased from Invitrogen Corporation (Gibcon = 25) were purchased from DaeHan Bio-Link Co., Ltd. , and all animals were kept at 23 ± 0.5 °C and 12 h-light/dark cycle in a controlled environment of a central animal care facility. Food and water were provided ad libitum to all animals. The facility was strictly maintained in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. BALB-c/mice , was dissolved in DMSO and ursolic acid (UA) was also dissolved in DMSO to a concentration of 50 mg/mL, according to the manufacturer’s instruction. Sulfadiazine was used as a standard drug to evaluate whether or not ursolic acid has an anti-parasitic effect and activity against T. gondii. All of the compounds were filtered using 0.2 µm membrane syringe filters  before use, and were stored at −80 °C deep-freezer until use. The anti-l-glutamine, supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin  in a humidified atmosphere containing 5% CO2 in air at 37 °C. The RH strain of T. gondii was suspended with 1X PBS, which was injected in the abdominal cavity of each BALB-c/mouse. Five days after the injection, T. gondii was collected from the peritoneal fluids of each mouse kept in the abdominal cavities of the mice before use in the studies. In the in vitro study, cells were infected with T. gondii (cells:T. gondii = 1:5). Glioma cells (C6 cells) were purchased from Korean Cell Line Bank at Seoul National University. C6 cells were cultured in RPMI medium 1640 containing 2 mM T. gondii, we investigated the viability of T. gondii exposed to UA and SF. Briefly, after T. gondii was seeded in a 24 well plate (1 × 107/well), T. gondii was incubated with different concentrations (50–200 μg/mL) of UA and SF for 24 h, respectively, and their viabilities were determined by MTT assay. It is to determine whether or not UA has direct anti-parasitic activity and/or effect against T. gondii. The survival rate (SR) of T. gondii was calculated, as follows: % of SR = (ODdrug-tested wells − ODblank)/(ODcontrol − ODblank) × 100. The optical density (OD) was measured at a wavelength of 570 nm using an ELISA leader. To evaluate the inhibitory effects of UA against the viability of 5/well), which were incubated at 37 °C for 24 h. The cells were infected with T. gondii (5 × 105 tachyzoites/well), and then T. gondii-infected cells were treated with 100 μg/mL of UA and SF for 24 h, respectively. The morphological changes of T. gondii-infected host cells were observed under a light microscope . The cells were seeded in a 24 well plate , which were infected with T. gondii (5 × 105 tachyzoites/well) after 24 h. The T. gondii-infected cells were incubated with 100 μg/mL of UA and SF for 24 h respectively. After washing with 1X PBS, the T. gondii-infected cells were fixed in 1X PBS containing 5% formaldehyde for 30 min, and then washed with 1X PBS and stained with a final concentration of 20 μM  for 30 min in the dark. After nuclear staining, the cells were washed with 1X PBS three times, and their nuclear changes were observed under a UV fluorescent microscope . The anti-eviously . The cel5 cells/well), which were infected with T. gondii (5 × 105 tachyzoites/well). T. gondii-infected cells were incubated with 100 μg/mL of UA and SF for 24 h, respectively. After washing with 1X PBS, they were fixed with 5% formaldehyde for 10 min and 0.05% (v/v) Triton X-100 for 5 min. The cells were blocked with 1X PBS containing 1% BSA for 1 h at room temperature after washing. A mouse monoclonal anti-PVM antibody was diluted with 1:100 (v/v) using 1% BSA/PBS, and then the cells were incubated with anti-PVM antibody solution at room temperature for 1 h. After washing, goat anti-mouse IgG-FITC-conjugated secondary antibody was diluted with 1:100 (v/v) using 1X PBS, which was added to each well. The cells were incubated at room temperature for 1 h, and were washed with 1X PBS every 15 min, four times. Their fluorescence was observed under a UV fluorescent microscope . The cells were seeded onto cover slips in a 24 well plate (1 × 10n = 25) were divided into normal (n = 5) and experimental groups . T. gondii was seeded in a 12 well plate (4 × 106/well), which was incubated with different concentrations  of UA for 24 h, respectively. T. gondii treated with the compounds was harvested before the injection in the abdominal cavity of each mouse in the experiment group, which was washed with 1X PBS three times. The pellets were suspended with 1X PBS, which were injected in the abdominal cavity of each BALB-c/mouse in the experimental groups. T. gondii-infected mice untreated with UA were used as an infected positive group, and the animals were kept in a central animal care facility during the experiment. Twenty-five animals . * p < 0.05 was considered to be statistically significant. All of the results were expressed as mean ± S.D. Statistical analysis of the data was performed using Student’s T. gondii activity causing the direct inhibition of T. gondii, but also causes anti-parasitic effect against T. gondii by strongly inhibiting the proliferation and growth of T. gondii in infected host cells. In addition, our results show clearly for the first time that UA has anti-T. gondii effect which consistently induces the survival of T. gondii-infected mice. Therefore, this study provides substantial results and the potential that UA can be utilized as a promising candidate substance for anti-T. gondii drug development of next-generation through T. gondii-infected cells and infected-mice model. In addition, UA suggests novel perspectives of the approach for anti-T. gondii drug development, which shows the need of further study regarding the safety and/or efficacy against toxoplasmosis through preclinical study in the near future.The results of this study demonstrate that UA not only has anti-


Giardia duodenalis in Xinjiang, northwest China, we collected 801 fecal specimens from seven large-scale pig farms and screened them using PCR targeting the SSU rRNA gene. Twenty-one (2.6%) of the specimens from five farms were G. duodenalis-positive, with a significant difference in prevalence among different farms (0–8.7%) (p < 0.01). Giardia duodenalis prevalence was highest in fattening pigs , followed by sows , post-weaning piglets , and pre-weaning piglets , but there was no significant difference in prevalence among the age groups (p > 0.05). Sequence analysis of the SSU rRNA gene revealed that the 21 G. duodenalis strains belonged to three assemblages: A (n = 2), B (n = 16), and E (n = 3). Assemblage B was the predominant assemblage and was widely distributed in all G. duodenalis-positive farms and age groups. All G. duodenalis-positive specimens were further assayed at the β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes, and two tpi, four gdh, and two bg sequences were identified. These data indicate that pigs may be a zoonotic risk and can potentially spread G. duodenalis infection from animals to humans.To study the presence of  Giardia duodenalis is a common protozoan parasite that can infect humans, livestock, companion animals, and wildlife [G. duodenalis is considered a complex species and has been grouped into eight distinct assemblages or genotypes (A–H) based on genetic analysis. Among them, assemblages A and B have a wide host range and are responsible for the majority of known human disease cases [wildlife . G. duodse cases , while ase cases . In pigsse cases , 20, 21.G. duodenalis infection has been frequently reported in a wide range of animals in China, including cattle (1.7–74.2%), sheep (0–13.1%), goats (0–27.78%), rabbits (3.9–8.3%) and other mammals  [G. duodenalis infection in pigs in China are available. In the published studies, assemblages A and E were identified in pigs, with assemblage E being the predominant assemblage [G. duodenalis in pigs in the Xinjiang Uygur Autonomous Region (hereafter referred to as Xinjiang), northwest China, to assess zoonotic transmission risk and elucidate the public health significance of this protozoan parasite.To date,  horses) . Althougsemblage , 20, 21.A total of 801 fresh fecal specimens were collected from 169 pre-weaning piglets (<20 days old), 281 post-weaning piglets (21–70 days old), 129 fattening pigs (71–180 days old), and 222 sows (>181 days old) from seven large-scale pigs farms in Marabishi, Alaer, Yarkant, Baicheng, Shaya, Changji, and Ruoqiang in Xinjiang between September 2017 and June 2018. Each of the sampled farms ranged from 10,000 to 80,000 pigs. These farms were visited on a single occasion and specimens were randomly collected from the animals by a veterinarian. At the time of collection, no diarrhea was apparent in the herds. Using sterile gloves, specimens were collected directly from the rectum or immediately from fresh feces deposited on the ground after animal defecation. The fresh feces were placed into clean plastic bags marked with the date, age, and farm, and immediately placed onto ice packs in an insulated container. Specimens were transported to the laboratory, stored at 4 °C, and processed no later than a week after collection.® Stool DNA Kit , according to the manufacturer’s instructions. Extracted DNA specimens were used as a template for polymerase chain reaction (PCR)-based analyses. Positive (dairy cattle-derived assemblage E DNA) and negative controls (distilled water) were included in each PCR assay.Genomic DNA was extracted from approximately 200 mg of each fecal specimen using the E.Z.N.A.RG. duodenalis was identified using the SSU rRNA gene, as described previously [eviously  (Table 1bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes, as described previously [bg, gdh and tpi genes.DNA from all SSU rRNA-positive specimens was further tested using PCRs targeting the β-giardin  using the ClustalX 2.1 program to determine the assemblages of G. duodenalis in each specimen.PCR amplicons of the correct size were DNA sequenced by GENEWIZ . Sequence accuracy was confirmed by bidirectional sequencing. The resulting sequences were aligned against reference sequences downloaded from the National Center for Biotechnology Information GenBank database . Differences of p < 0.05 were considered significant.Differences in prevalence between ages and farms were compared with the G. duodenalis based on the SSU rRNA gene. The prevalence in pigs in this study is within the range reported in previous studies. The highest prevalence was observed in a farm from Ruoqiang , followed by Baicheng , Shaya , Yarkant , and Changji . G. duodenalis was not detected in specimens from farms in Alaer and Marabishi. The prevalence of G. duodenalis in pigs was significantly different among different farms  , higher than sows , post-weaning piglets , and pre-weaning piglets , but the differences between age groups were not significant   from five farms tested positive for  < 0.01) . The pre > 0.05) .n = 2), B (n = 16), and E (n = 3), were detected among 21 G. duodenalis-positive specimens . Of two tpi sequences, one was identified as assemblage B and the other was identified as assemblage E (Table S1). The assemblage B sequence was identical to the sub-assemblage BII sequence (GenBank accession no. KX468987) from humans in Spain, while assemblage E was identified as a novel sequence and showed 99% similarity to the assemblage E sequence (KJ668134) from pigs in China. The four gdh sequences obtained in this study were identified as assemblages A (n = 1), B (n = 1), E1 (n = 1) and E2 (n = 1) (Table S1). Assemblage A and B sequences were identical to the sub-assemblage AII sequence (EF507661) from humans in Brazil and the assemblage B sequence from chinchillas in China, respectively. Assemblage E1 and E2 sequences were identical to the assemblage E sequence (KJ668145) from a pig in China and assemblage E sequence (MG820464) from cattle in USA, respectively. Of two bg sequences, one was identified as a novel sequence of assemblage B, showing 99% similarity to the assemblage B sequence (LC436571) from humans in Japan, while the other was identified as assemblage E and was identical to the assemblage E sequence (KU668892) from wild boars in China.Based on the molecular analysis of the SSU rRNA gene, three assemblages, A .G. duodenalis has been reported in pigs worldwide, ranging from 0% in pigs from Preah Vihear, Cambodia (0/74), to 66.4% (81/122) in pigs from Ontario, Canada [G. duodenalis in domestic pigs was 1.7% (15/897) from Henan Province, 8.0% (45/560) from Shaanxi Province in domestic pigs, and 3.1% (11/357) from Sichuan Province in captive Eurasian wild boars [G. duodenalis. The discrepancy between previous studies was potentially due to farm hygiene management, including differences in animals stocking density, hygiene regimes, or water supply [G. duodenalis cysts is by vectors such as flies and rodents, which could be explored in future studies.Varying prevalence of , Canada . In Chin66.4% 81/2 in pigsld boars , 21. In r supply . To prevG. duodenalis prevalence was in post-weaning pigs (20–30 kg) , and the lowest was in the piglets (<7 weeks)  [G. duodenalis prevalence in post-weaning pigs (4 weeks to 6 months) , and the lowest in pre-piglets (11 days to 3 weeks)  [G. duodenalis prevalence in sows , and the lowest in pre-piglets (2–5 weeks)  [G. duodenalis prevalence was in fattening pigs (70–180 days) , and the lowest was in pre-weaning piglets (<20 days) . Because of the absence of uniform age divisions and the different sample sizes across studies, it is difficult to evaluate the association between pig age and G. duodenalis infection; more studies should be undertaken to illustrate this association.A previous study in Denmark found that the highest , 3/152) . Similar%, 2/32) , and a sing pigs –30 kg  have been reported in pigs, with assemblage E being the predominant assemblage , which were genetically different from previous samples from northwestern China [G. duodenalis in pigs (such as geographic or host segregation), thus further epidemiological surveys should be undertaken to analyze the genetic differences.To further clarify the genetic diversity of rn China . BecauseG. duodenalis infection  was identified in this study, the identification of zoonotic assemblages A, B, and E, and the predominance of assemblage B, suggest that pigs pose a potential risk for the zoonotic transfer of G. duodenalis in the studied region.Although a low prevalence of


Drosophila, there are fewer pathway components, which has enabled significant discoveries regarding well-conserved regulatory mechanisms. Work across species illustrates the relevance of these regulators in humans. In this review, we showcase fundamental Jak/STAT regulation mechanisms in blood cells, stem cells, and cell motility. We examine the functional relevance of key conserved regulators from Drosophila to human cancer stem cells and metastasis. Finally, we spotlight less characterized regulators of Drosophila Jak/STAT signaling, which stand as promising candidates to be investigated in cancer biology. These comparisons illustrate the value of using Drosophila as a model for uncovering the roles of Jak/STAT signaling and the molecular means by which the pathway is controlled.Over the past three-decades, Janus kinase (Jak) and signal transducer and activator of transcription (STAT) signaling has emerged as a paradigm to understand the involvement of signal transduction in development and disease pathology. At the molecular level, cytokines and interleukins steer Jak/STAT signaling to transcriptional regulation of target genes, which are involved in cell differentiation, migration, and proliferation. Jak/STAT signaling is involved in various types of blood cell disorders and cancers in humans, and its activation is associated with carcinomas that are more invasive or likely to become metastatic. Despite immense information regarding Jak/STAT regulation, the signaling network has numerous missing links, which is slowing the progress towards developing drug therapies. In mammals, many components act in this cascade, with substantial cross-talk with other signaling pathways. In  Drosophila [Drosophila has been very informative about its functional mechanisms in humans.The Janus kinase (Jak) and Signal transducer and activator of transcription (STAT) signaling pathway is crucial in the regulation of the immune response, in stem cell regulation, and in determining cell identities in diverse organisms. In the late 1980s and early 1990s, this signaling cascade was shown to be central to the interferon response in humans (reviewed in ), and itosophila ,4,5,6,7.osophila ,11, illuosophila ,13,14. GDrosophila. We review studies linking key Jak/STAT activity regulators with human disease, especially blood cell cancer, cancer stem cells, and metastatic cancers derived from breast and prostate. We also describe well-characterized cell types and phenotypes affected by loss and gain of function of Jak/STAT pathway components in Drosophila, and discuss how flies can be useful for the identification of new pathway regulators. Finally, we explore connections between genes determined to be Jak/STAT regulators in Drosophila and their human homologs that are linked to disease, and highlight candidates for further study based on their involvement in both contexts.Here, we broadly compare and contrast the Jak/STAT signaling cascade in mammals and Drosophila genes, the gene encoding Jak is named after its loss of function phenotype; due to defective segmentation and skipped segments in the cuticular patterns of late embryos and early larvae, the mutant was named hopscotch  [Extracellular cues trigger Jak/STAT signaling, which ultimately leads to transcriptional activation of target genes . The basmutants) ,28. Janumutants) . Activatmutants) ,7,30. Comutants) . Non-phomutants) ,32,33. Lmutants) ,34,35. Hmutants) . Thus, tDrosophila is important to provide a tractable, but still very relevant, system for characterization of this signaling cascade and its essential regulators.As the various STAT proteins in humans can homo- or hetero-dimerize, and can be activated by numerous permutations of the ligands, receptors, and Jaks, the combinatorial outcomes are very complex. Thus, the stripped-down pathway that exists in In both flies and mammals, normal early development requires correct Jak/STAT signaling, and spatiotemporal pathway misregulation later in life is detrimental. Humans with inborn errors in Jak/STAT genes that are important in blood cell lineages are immunocompromised . AdditioJak1 and Jak3 mice have severe combined immunodeficiency (SCID), and Jak1 mutants also have neurological defects and poor survival past birth; knock out mutations in Jak2 are embryonic lethal, and mutations in the Jak family member Tyk2 result in poor response to pathogens [Stat1 mutant mice have abnormal immune responses and are more susceptible to infections than wild type [Stat3 mutant mice die in early embryogenesis and tissue-specific mutations result in changes in the proliferation–apoptosis balance in blood cells, poorer cell motility, and inflammation [Stat5a and b have overlapping, required roles in mammary gland and ovary development, as well as being important in blood cell proliferation and cytotoxic activity [Stat5a/b double knock out mice are sterile.While some null Jak/STAT pathway mutations cause tissue-specific defects, presumably others would not allow human development to term, as evidenced by mouse genetic studies. Mutations in genes encoding positive signaling components result in early lethality in mice or cell type-specific effects ,40. For athogens . Similarild type . Stat3 mammation ,40. Statactivity . Female Drosophila result in early lethality; however, this can be overcome experimentally using sophisticated genetic tools that allow fly researchers to test mutations in individual cell types or at certain times in development. These types of experiments can be performed by using temperature sensitive mutations, by tissue-specific expression control through the Gal4/UAS system, or by using clonal mosaic analysis, in which most cells of the organism are heterozygous and small clones of cells are homozygous mutant. . Theols, see ,46,47,48ols, see ,50, wingols, see ,52, eye ols, see , gut steols, see ,55,56, aols, see ,58,59, aols, see ,24,60,61xts, see ,63,64,65xts, see  (tumors)xts, see ,50,67 . These proteins bind to activated STATs to block DNA binding and transcriptional activity, and have roles in the sumoylation and downregulation and degradation of signaling components ,75,76,77 the eye . Members the eye ,79. Thes the eye ,81. Eigh the eye ,83,84 an the eye ,86. When the eye . Fly Soc the eye , similar pathway ,90,91,92 pathway ,93,94, r pathway . So far,osophila ,73,96. Aosophila ,26,27. Fosophila ,101,102.Drosophila Jak/STAT regulators in metastasis.More recently, flies have been used to identify novel regulators of Jak/STAT signaling. This has been accomplished by identifying mutants with similar phenotypes, by leveraging unbiased genetic enhancement and suppression screening, and by assaying pathway activity in cell culture. Many of the proteins identified in this way have human homologs that may play analogous roles. Given that Jak/STAT activity has important functions in immune response, cell motility, and stem cell maintenance in both humans and flies, genetic analysis in the fly provides a valuable strategy to elucidate critical regulators in these processes. Thus, studying different cellular contexts and using multiple approaches will be useful for determining how these components are controlled and how they normally fine-tune the pathway to prevent pathological states. We will describe Jak/STAT regulation in three contexts: Mammalian blood cell differentiation and proliferation, stem cell signaling, and cell motility and metastasis. Next, we will draw parallels with regulation of these processes in flies via Jak/STAT signaling. Finally, we will explore the potential contributions for homologs of In humans, blood cells are produced from hematopoietic stem cells (HSC), which reside in a stem cell niche in bone marrow. HSCs divide asymmetrically to renew and make multipotent daughter cells; depending on local factors, these will produce multipotent stem cells restricted either to lymphoid or myeloid lineages. Jak/STAT signaling is important in both stem cell regulation and differentiation. STAT5 is expressed in HSCs and is necessary for their ability to self-renew ,103, andMultiple different mutations in the human Jak/STAT signaling pathway or its positive regulators result in a defective immune response or proliferative disorders in blood cells ,21,37,39Since activating mutations lead to disease, several drugs are currently used therapeutically to suppress Jak/STAT signaling in patients. AG490 was the first Jak inhibitor characterized, and it has been used in the treatment of acute lymphoblastic leukemia; other drug analogs have been shown to be effective in blocking acute myeloid leukemia progression and survival in cell culture ,107,108.Drosophila hemocytes show many similarities to human white blood cells and play critical roles in the immune response. The fly innate immune system acts through the production of antimicrobial peptides and the phagocytosis of pathogens. Drosophila have an open circulatory system and do not require oxygenated red blood cells. However, three types of blood cells differentiate from hemocytes: Plasmatocytes, which phagocytose bacteria and are most similar to human macrophages; crystal cells, which are responsible for melanization of pathogens; and lamellocytes, which encapsulate large foreign invaders like wasp eggs [asp eggs ,70,110. asp eggs ,111. An asp eggs . HemocytDrosophila, the pathway is necessary for an effective immune response in larva and adults [Like in mammals, Jak/STAT interacts with multiple signaling pathways in the hematopoietic compartment to control hemocyte differentiation and proliferation. Interactors include the combined homolog of platelet-derived growth factor and vascular endothelial growth factor, PVF2 ,114; thed adults ,123,124.Drosophila Jak, HopTumorous-lethal (HopTum), has provided an invaluable tool in the study of Jak/STAT signaling in flies [T42, creates an activating amino acid substitution in the kinase-like domain, which results in phenotypes similar to those due to HopTum expression [Drosophila hemocytes provide a reasonable model for leukemia mechanisms.A dominant, activating mutation in in flies . This poin flies ,9,10,11.in flies . A diffepression , supportDrosophila embryonic hemocytes [Drosophila.A number of drug screens for Jak/STAT inhibitors have been performed using a cultured cell line derived from emocytes ,129,130.emocytes , which cemocytes . AlthougDrosophila has provided strong insight into the molecular regulation of stem cells and the niches that support them. Besides its involvement in blood cell cancers, Jak/STAT signaling activation is common in carcinomas. Activated STAT3 and STAT5, in particular, promote tumorigenesis and cancer stem cells, and this has been extensively reviewed ,135,136.Drosophila testis is an outstanding model for examining stem cell regulation, as it is well-characterized, clearly organized, and genetically accessible. Adult testes have two stem cell populations, the germline cells and the somatic cyst stem cells [Socs36E led to excessive signaling, overproliferation of stem cells, and changes in stem cell adherence to the niche [The em cells ,137. Disem cells ,138,139.em cells ,140. Theem cells . Higher em cells . Loss ofhe niche . While the niche .Socs36E expression and downregulate STAT activity. These function to counteract Jak/STAT signaling and promote somatic cell differentiation instead of stem cell maintenance. Similarly, the transcription factor, Apontic, suppresses Jak/STAT signaling to limit the number of somatic stem cells and thus limit the size of the stem cell niche, also by promoting expression of Socs36E and probably a STAT-directed microRNA [Key transcription factors and epigenetic regulators have been found to impact the output of Jak/STAT activity in testes, and provide feedback on signaling to prevent it from getting too high or too low. The B-cell lymphoma Bcl6 homolog, Ken and Barbie (Ken), is needed in the somatic stem cells to repress the Jak/STAT inhibitor Ptp61F; thus, Ken indirectly maintains high enough levels of Jak/STAT activity for stem cell self-renewal . Ken oppmicroRNA ,147. TheDrosophila—the border cells —and we outline how border cell behaviors capture aspects of metastatic cell migration.Jak/STAT signaling activation has been linked to more metastatic cancers for a number of tumor types . A numbeAbout 80% of diagnosed breast cancers are invasive, which threatens advancement to metastatic disease . MetastaLayered over the core signaling, conserved regulators of Jak/STAT signaling also impact human breast cancer metastasis, although not always as expected. Given the key role of Jak2, it is clear that regulators mediating its degradation have a critical function in preventing overactivation. SOCS protein family members are known for their negative regulation of Jak/STAT signaling ,155. SOCE-cadherin in breast cancer cells. In breast metastatic lesions, reduction of activated nuclear STAT5a levels correlated to increased BCL-6 cellular expression [Additional conserved regulators are relevant to breast cancer, although direct modulation of Jak/STAT activity has not been defined in all cases. The transcriptional repressor, BCL6, appears to be important to promote mesenchymal properties of breast cancer cells . E-cadhepression . PTP1B ipression . Breast pression . Lastly,pression . FurtherAs with other carcinomas, Jak/STAT core components, IL-6, Jak2, and STAT5, and other key regulators of this pathway are implicated in promotion of prostate tumor growth and metastasis, and there is no highly effective treatment to cure metastatic disease. Activated STAT5a/b indicates a poor prognosis . AbnormaFurther evidence for activated Jak2-STAT5a/b signaling leading to metastasis comes from the findings that this pathway regulates EMT markers . The actDisruptions of key, conserved regulators of Jak/STAT signaling are also implicated in human prostate cancer. The SOCS family appears to be involved. SOCS1 caused significant reductions in wound closure and invasive behavior when it is stably expressed in prostate cancer cell lines . In mousOther typical Jak/STAT regulators may function differently than expected in prostate cancer. Although PTP1B inhibits STAT activity, other PTPs seem to promote tumorigenesis or metastasis. PTP1B is overexpressed at the protein level in clinical samples of prostate tumors . The copDrosophila border cells, a subset of somatic follicular cells in the adult ovary [The first evidence for Jak/STAT signaling promoting cell motility came from lt ovary ,24,60. Elt ovary ,174). Atlt ovary ,175. Earlt ovary . Secretelt ovary ,178. In lt ovary . In addilt ovary . A key elt ovary ,181,182.Border cell motility has been likened to an EMT event; however, border cells move as a collective that retain some epithelial character. In particular, apical–basal polarity components are required ,184,185.Socs36E, a Socs5 homolog, or protein tyrosine phosphatase, Ptp61F, a homolog of the human gene encoding PTP1B, both disrupt border cell migration by allowing too many cells to become motile, due to excessive Jak/STAT signaling [Socs36E transcription is activated in response to STAT activity, functioning as an autoregulatory break on the signaling system [Since high levels of Jak/STAT signaling results in additional motile border cells, loss of function mutations in negative regulators also yield this phenotype. Mutations in ignaling ,96. Integ system . Ptp61F g system ,96.Stat92E-targeted microRNA, mir-279 [mir-279 and Socs36E to keep Jak/STAT signaling from getting too high [Brahma, genetically interacts with Stat92E, indicating that epigenetic regulation plays a key role in modulating the transcriptional output of the pathway in motile cells [Drosophila testes stem cells [A number of additional Jak/STAT regulators involved in border cell migration have been identified through unbiased genetic screens and further characterized. In particular, numerous transcriptional regulators influence pathway output. The Bcl6 homolog Ken functions to promote sufficiently high Jak/STAT activity levels by transcriptionally suppressing a  mir-279 . While a mir-279 . An addi mir-279 . Apontictoo high ,193. Recle cells , as is tem cells ,145,194.In border cells, Jak/STAT regulation is also controlled by cellular trafficking mechanisms. Exocytic regulation of Upd release from polar cells requires Snap Receptor (SNARE) components, N-ethylmalemide-Sensitive Factor (NSF) and alpha-Soluble NSF attachment protein (αSNAP), which reset the SNARE complexes to permit vesicle trafficking . EndocytDrosophila genetics lies in the ability to identify new components of a process in an unbiased way. This strategy has been fruitful in determining many Jak/STAT signaling regulators in the cell types discussed above, as well as in other contexts that are beyond the scope of this review. As is clear, Jak/STAT signaling is functionally and mechanistically conserved between flies and humans, and many regulators are shared between these pathways. However, more regulatory players have been characterized to act on Drosophila Jak/STAT signaling but are not yet connected to this pathway in humans. In the following section, we discuss the human homologs of positive and negative Drosophila Jak/STAT regulators in cancer progression and metastasis  is predictive of distant metastasis and reduced survival in human melanoma . Silenciosophila , Jak/STAMicroRNA 140-5p targets IGF2BP1 in this tissue. In vitro assays showed that IGF2BP1 downregulation by another microRNA (miR-150) reduced migration and invasion of an osteosarcoma cell line [miR-150 suppressed tumor growth in an osteosarcoma xenograft mouse model via repression of IGF2BP1. In both hepatocellular carcinoma and glioblastoma cell lines, downregulating IGF2BP1 via different microRNAs reduces tumor proliferation and invasion potential [IGF2BP1 is an mRNA binding factor implicated in multiple cancers . Silenciell line . Moreoveotential ,204. In otential . Thus, Iotential .PRDX4 is linked to cancer progression in multiple cell types. It is overexpressed in non-small cell lung cancer and prostate cancer ,208. MolThe transcription factor CEBPD acts as a pro-metastatic factor in several cancers. CEBPD overexpression increased urothelial cell migration and showed an increase in invasion potential in vitro . InhibitDrosophila, these are worthy of attention. The human proteins BPTF, UBAP2, REST, and EYA2 have Drosophila counterparts that act to repress Jak/STAT signaling in various cell types ) in Jak/STAT signaling, we hypothesize that BPTF could be involved in human cancer metastasis, in part, by suppressing Jak/STAT signaling. However, BPTF can be linked with pro- or anti-metastatic roles, which may mean changes in its expression are indirect, that it has cell type specific effects, or that it may act in human cancer pathogenesis via multiple mechanisms.The nucleosome-remodeling factor BPTF has been shown to be present in various cancers with different metastatic potentials. Examination of patient tumor samples indicated that BPTF is a suppressor of lung cancer metastasis to the brain . In a cuUBAP2 was found to be overexpressed at the gene level in samples collected from castration-resistant prostate cancer patients [UBAP2 copy number significantly reduced cell growth. On the other hand, UBAP2 was lowly expressed in hepatocellular carcinoma (HCC) patient samples [Drosophila homolog Lig mutates to embryonic lethality, but has mainly been shown to affect Jak/STAT signaling in eye progenitor cell growth [UBAP2 contains a ubiquitin-associated domain, so is presumed to function in protein turnover. patients . Additio samples . In HCC l growth , suggestReduction of another putative Jak/STAT regulator, the transcription factor REST, increased LIN28A expression and tumor growth both in cell culture and in models in vivo . The tumDrosophila, the corresponding protein Eya activates Apontic, which negatively regulates Jak/STAT signaling in ovarian follicle cells [Lastly, the transcription factor EYA2 was shown to be misregulated in a number of cancers. In lung adenocarcinoma patient samples, it was upregulated, and upon its knockdown in cell culture, tumor growth and invasion potential were suppressed . EGFR hale cells , but EyaDrosophila positive Jak/STAT regulators are shown to be linked to human cancer progression and/or metastasis. Understanding their mode of action in cancer progression via, possibly, Jak/STAT signaling could provide us a new direction towards therapeutic interventions.In summary, many of the human homologs of Drosophila is warranted and useful for uncovering new genes with roles in human disease. Interestingly, new levels of pathway regulation are becoming apparent in multiple Drosophila cell types. These findings suggest that future studies are needed to evaluate further vesicle trafficking regulation and epigenetic control of Jak/STAT activity and target gene expression. Additional drug screening in Drosophila is also likely to be very informative. Further studies of the candidates described here and new ones will likely shed light on the regulation of cancer metastasis by Jak/STAT signaling or additional linked pathways.The portrait of Jak/STAT signaling in human cancer metastasis is far from complete, and is obviously very complex. The description we provide here fortifies the idea that further explanation of Jak/STAT regulation in


G protein coupled receptors (GPCRs) were first characterized as signal transducers that elicit downstream effects through modulation of guanine (G) nucleotide-binding proteins. The pharmacotherapeutic exploitation of this signaling paradigm has created a drug-based field covering nearly 50% of the current pharmacopeia. Since the groundbreaking discoveries of the late 1990s to the present day, it is now clear however that GPCRs can also generate productive signaling cascades through the modulation of β-arrestin functionality. β-Arrestins were first thought to only regulate receptor desensitization and internalization – exemplified by the action of visual arrestin with respect to rhodopsin desensitization. Nearly 20 years ago, it was found that rather than controlling GPCR signal termination, productive β-arrestin dependent GPCR signaling paradigms were highly dependent on multi-protein complex formation and generated long-lasting cellular effects, in contrast to G protein signaling which is transient and functions through soluble second messenger systems. β-Arrestin signaling was then first shown to activate mitogen activated protein kinase signaling in a G protein-independent manner and eventually initiate protein transcription – thus controlling expression patterns of downstream proteins. While the possibility of developing β-arrestin biased or functionally selective ligands is now being investigated, no additional research has been performed on its possible contextual specificity in treating age-related disorders. The ability of β-arrestin-dependent signaling to control complex and multidimensional protein expression patterns makes this therapeutic strategy feasible, as treating complex age-related disorders will likely require therapeutics that can exert network-level efficacy profiles. It is our understanding that therapeutically targeting G protein-independent effectors such as β-arrestin will aid in the development of precision medicines with tailored efficacy profiles for disease/age-specific contextualities. The β-arrestin family comprises four members: visual arrestins (arrestin1 and arrestin4) and the non-visual arrestins . Furthermore, an α-arrestin family, structurally related to the β-arrestins has been identified . To provreceptorsome’) which functions both to target the receptor-kinase complex to clathrin-coated pits and additionally to recruit activated non-receptor tyrosine kinase c-Src to the plasma membrane. This ‘receptorsome’ formation process engenders a receptor-based capacity to induce a novel signaling cascade distinct from the G protein-dependent paradigm. Thus, it was elegantly demonstrated that β-arrestins did not only ‘arrest’ G protein signaling, but transformed the signaling activity and initiated supplementary signaling cascades where the ‘desensitized’ receptor functioned as a part of a mitogenic signaling complex  receptor signaling pluridimensionality  and (2) ffectors . The conffectors . This fuWhile signaling bias using functionally selective xenobiotic ligands has been a topic of ongoing research for nearly two decades, it has become clear that natural ligand bias also occurs for GPCRs and likely represents another physiological level of receptor signaling complexity. In discussing the natural biased agonism of chemokine receptors (CCRs), i and Ang II/Gαq pathways, it however selectively promotes β-arrestin activation , which is physiologically implicated in the development of hypertension and the natural aging process . Furthertivation . These r1, HPRD (Human Protein Reference Database2), IntAct3, MINT (The Molecular INTeraction Database), STRING4, DIP (Database of Interacting Proteins5) and CORUM6. The cumulated known interaction partners for β-arrestin1 or β-arrestin2 are detailed in Supplementary Tables 7 enables the identification of biomedical text items, i.e., Gene Symbol identifiers of proteins, that possess latent semantic associations with the input interrogator concept term . Latent semantic analysis is a natural language processing data extraction mechanism (etc….) and specific text entities, in this case Gene Symbol targets identified using the database of gene-word documents assembled in the GeneIndexer database  MAP kinase cascades . ThroughThe MAP kinases are a family of serine/threonine kinases involved in the transmission of external signals regulating cell division, growth, differentiation, and apoptosis. Mammalian cells contain three major categories of MAP kinases: ERKs, JNKs, and p38/HOG1 MAP kinases . In 2000While ubiquitination was long thought to control only protein degradation, it has now become clear it also plays a role in productive signal transduction cascades. β-Arrestins can act as adapters for E3 ubiquitin ligases such as MDM2, which mediates ubiquitination of p53 and as such plays a role in DDR  by contrβ-arrestin2 has been shown to possess the ability to inhibit nuclear factor κB (NF-κB) signaling through the physical stabilization of IKBα  . Next toβ-Arrestin can also control NF-κB-mediated signaling through AT1aR activation, which recruits this scaffold protein. This was analyzed in rat vascular smooth muscle cells. Genetic knockdown of β-arrestin1 and 2 inhibited Ang II-induced p64 NF-κB nuclear localization. The internalization of the activated AT1AR mediated by β-arrestin, stimulated the NF-κB signaling pathway, engendered a nuclear localization of the transcription factor and resulted in the initiation of COX-2 (Cytochrome c oxidase subunit 2) protein synthesis. As such, this research line linked receptor internalization with the NF-κB pathway .In addition, β-arrestin2 exerts anti-inflammatory action through the prevention of NF-κB activation. This action is mediated through the direct inhibition of p38 MAPK. On the other hand, a simultaneous anti-inflammatory effect can also be initiated through cAMP and PKA activation via G protein signaling which exerts an inhibitory effect on NF-κB . Such ouThe epidermal growth factor receptor (EGFRs) can be functionally transactivated by GPCRs as well as being functionally regulated by β-arrestins, through the stimulation of a transmembrane matrix metalloprotease which causes cleaving of membrane bound EGF ligand . In addiThe β2AR has been proposed to maintain an active conformation, even in endosomes distant from the plasma membrane, which has the ability to signal productively through G proteins to generate cAMP . This su‘tailored efficacy’ therapies that possess a more focused efficacy with fewer off-target side effects. Perhaps one of the best characterized arrestin pathway-selective biased agonist is that mediated through the type 1 human parathyroid hormone receptor (PTH1R). The PTH1 receptor regulates calcium homeostasis and bone metabolism. 12,Tyr34]bovine PTH(7-34) showed β-arrestin bias, acting as an inverse agonist for PTH1R-Gαs coupling  [hPTH(1-34)], which is the current conventional therapy for osteoporosis  can be activated by both endogenous enkaphalin peptides and xenobiotic opiates such as morphine or fentanyl . G proteapeutics .q-mediated activation of phospholipase C (PLC) and induces receptor phosphorylation by GRKs, thus recruiting β-arrestin to the receptor. This β-arrestin recruitment desensitizes Gαq signaling, causes receptor internalization and induces non-G protein-dependent β-arrestin signaling , which competitively antagonizes G protein signaling through the AT1R, yet stimulates β-arrestin recruitment, activating several kinase pathways in a β-arrestin-dependent manner. This biased ligand increased cardiomyocyte contractility in vitro. In vivo characterization of the ligand, in rats, showed a reduction in mean arterial pressure, and most importantly (and unlike other unbiased antagonists) TRV120027 increased cardiac performance and preserved cardiac stroke volume  has been demonstrated to possess unique survival benefits in congestive heart failure, through a β-arrestin-selective pathway in absence of the Gαs-dependent activation of adenylate cyclase (AC)  . This biWhile for some cardiovascular diseases most so-called ‘β-blockers’ (βAR antagonists) are effective, in others only a subset of such agents exert favorable actions. The βAR receptor has also been targeted to treat asthma, a chronic inflammation of the airways. The use of glucocorticosteroids and β2AR agonists represent the cornerstones of asthma therapy . Recentltip of the spear’ of aging? Alterations in complexity and diversity of GPCR systems will likely impact nearly all physiological systems implicated in aging, thus an enhanced appreciation of how altered GPCR signaling diversity controls the aging process is vital for the future development of pharmacotherapeutics with augmented efficacy profiles in the specific contexts of stress and aging. As a caveat to this posit however, it is likely that alterations in GPCR β-arrestin bias during age-related pathology are not simply unilateral or unidirectional, as signaling diversity from GPCRs may be vital for both allostatic and homeostatic mechanisms. Thus, in addition to increased expression levels, decreases  in β-arrestin expression/functionality may also be important  . This fimportant . To diss2O2) treatment leads to the activation of multiple stress kinase cascades, such as the apoptosis signal-regulating kinase 1 (ASK1) signaling pathways, and then induce cell apoptosis , which has been the focus for both new anti-diabetic and anti-neurodegenerative therapies . Sonoda In addition to alterations of metabolism mediated by glycoregulatory disruption, global metabolic functions in aging are affected by changes in thyroid hormone activity. Thyroid hormones serve to control the generic metabolic rate in the body through the control of glucose/lipid/protein catabolism. With advancing age however there is an increased prevalence of hypothyroidism, which is furthermore associated with coronary heart disease, heart failure and cardiovascular mortality . An animportmanteau description of this process as ‘inflammaging’ was warranted. β-Arrestins have been shown to act as scaffold proteins or signal transducers for key inflammatory signaling molecules in receptor tyrosine kinases (RTKs) signal transduction pathways, such as the NF-κB pathway , where they found that this antidepressant increased β-arrestin2 expression and enhanced the association of β-arrestin2 with transforming growth factor beta-activated kinase 1 (TAK1) binding protein 1 (TAB1) and disrupted the TAK1-TAB1 interaction, which attenuates the IκB (inhibitor of κB) degradation and NF-κB nuclear translocation , as well as the presence of a secretory dysfunction of the excess plasma-borne glucose  . Recent in vivo, as such aiding in the development of insulin resistance and thus the progression of T2DM  has previously shown to cause strongly neurotrophic pharmacological activities, and is thus a possible treatment of neurodegenerative disorders such as AD. Acute cellular treatment of human neurons showed alteration in the physical interaction between the neurotrophic tyrosine kinase receptor 2 (NTRK2), and APP, and APH1A and β-arrestin2, suggesting that changes in this multi-protein complex may be associated with the beneficial therapeutic actions of amitriptyline  signaling as being beneficial in cognitive disorders  can stimulate the contractility of isolated cardiac myocytes via the AT1R and β-arrestin2  receptor ubiquitination and degradation, and (ii) a decrease in cell viability and ERK signaling activation through β-arrestin1. As such, this signaling bias, i.e., β-arrestin1 regulation, suggests a possible therapeutic strategy to enhance response to anti-IGF-1R therapies .In addition to the demonstration that the EGFR receptor forms functional complexes with GPCR systems , further‘biased ligands’ can selectively activate G protein and/or β-arrestin functions, thus eliciting novel biological effects for even the best studied GPCRs . While they have only been recently identified, biased ligands have actually been in pharmaceutical drug development for a long time, but they were labeled as classical ‘antagonists’  , Multi-PJvG, JOH, HL, PS-O, LL, BM, and SM contributed to the writing and editing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Uterine adenosarcomas (UAs) account for 5–8% of cases of uterine sarcomas. Treatment includes total abdominal hysterectomy (TAH) and bilateral salpingo-oophorectomy (BSO).Fertility preservation is an emerging concept in gynaecology oncology and is particularly relevant in UA, where cases are diagnosed as young as 15-year-old. This manuscript demonstrates a case of UA which was treated conservatively, achieved successful livebirths and underwent completion hysterectomy after two decades of follow-up.This was a retrospective case note review.An 18-year-old nulliparous woman presented with abnormal vaginal bleeding. Ultrasound identified an endometrial polyp, which was histologically diagnosed as low-grade adenosarcoma. She was advised to undergo TAH and BSO, but instead decided to preserve her fertility and opted for conservative management. She was monitored with pelvic ultrasound, hysteroscopy and endometrial biopsy bi-annually, with annual pelvic magnetic resonance imaging for 10 years which was uneventful. 11 years post-operatively she conceived following in-vitro fertilization (IVF) but suffered a miscarriage at 16 weeks likely due to cervical incompetence. She subsequently conceived with twins. She delivered spontaneously preterm at 28 weeks. Both children are alive and well. After 20 years of follow-up, she underwent a laparoscopic hysterectomy with no evidence of recurrence. She remains disease free.Whilst radical completion surgery should be advised in UA, this case, in addition to all published conservatively managed cases of UA, demonstrates that conservative management is possible in appropriately selected women. Intensive monitoring post-operatively is essential owing to the risk of recurrence; however, this may pose deleterious side effects which require consideration. Uterine adenosarcoma (UA) accounts for 5–8% of cases of uterine sarcomas, a rare form of neoplasm comprising less than 10% of uterine malignancies . The staAdenosarcomas are typically defined by the presence of a benign appearing epithelial component in combination with a low-grade sarcomatous component often resembling endometrial stromal sarcoma . CarcinoUA most commonly presents with vaginal bleeding, but may cause pelvic pain, vaginal discharge, and symptoms related to uterine enlargement , 8, 12. Fertility preservation is an emerging concept within gynaecological oncology. This is secondary to the fact that up to 10% of invasive cancers occur in women aged under 45 . FertiliWith the rising age of motherhood globally , the demWe present a case of a young woman with UA who was treated conservatively, subsequently achieved livebirth and finally underwent completion hysterectomy after two decades of follow-up.An 18-year-old nulliparous woman presented with abnormal vaginal bleeding. Ultrasound detected the presence of an endometrial polyp, which was confirmed on hysteroscopy and removed using a resectoscope. The polyp was approximately 2 cm in length. Histological analysis revealed benign endometrial glands with moderately cellular stromatolites showing some degree of periglandular condensation and mild pleomorphism. No SO or MI was noted. The histological diagnosis of low-grade adenosarcoma was made. Subsequently, the patient was advised to undergo hysterectomy, BSO and lymphadenectomy, but she declined to preserve her fertility. She was referred for a second opinion and following extensive counselling, opted for conservative management.Over the following 10 years, she was monitored with pelvic ultrasound, hysteroscopy and endometrial biopsy every 6 months, and with annual pelvic magnetic resonance imaging. Follow-up was largely uneventful, with normal imaging and histology. Nine years post-operatively an abnormal appearing area on the posterior aspect of the lower uterine segment was identified on hysteroscopy and subsequently resected. Histology was benign with no features of recurrence.Eleven years post-operatively, she underwent in vitro fertilization (IVF) and successfully conceived following the first cycle. Unfortunately, she suffered a second trimester miscarriage at 16 weeks’ gestation, with a history suggestive of cervical incompetence after admission with a dilated cervix and bulging membranes. The following year she conceived twins following another IVF cycle. She underwent an uncomplicated elective trans-vaginal cervical suture. She delivered male twins at 28 weeks’ gestation following pre-term labour, which was attributed to placental abruption. Both children are alive and well. She was offered hysterectomy after delivery, and at each subsequent follow-up appointment, but declined as she was uncertain as to whether or not her family was complete. Following pregnancy, her follow-up intensity was reduced to annually with continued ultrasound scans along with hysteroscopy and endometrial biopsy. She continued to defer definitive treatment until, after a total of 20 years of follow-up, she finally underwent an uncomplicated laparoscopic hysterectomy with ovarian conservation. There was no histological evidence of disease recurrence.n = 4; 40%) underwent dilatation and curettage (D&C) ± polypectomy. Two (20%) women received adjuvant chemotherapy: one had nine cycles of vincristine, dactinomycin and cyclophosphamide following polypectomy with no residual disease at a subsequent D&C [This case demonstrates the feasibility of FSS in appropriately selected women with UA. Conservative management of adenosarcoma is rare, with only ten previously published cases, as summarised in Table uent D&C , whilst uent D&C . Follow-uent D&C . Three . One patient had stage IA disease with SO whilst the second had stage IB disease [Whilst there are limited data on recurrence following conservative treatment in UA, previous studies have demonstrated that women can remain disease free for beyond 10 years following FFS . Convers disease . The othIn conservatively managed cases, intensive monitoring and follow-up post-operatively are essential , 4. LimiAn important consideration is the potentially deleterious physical and psychological effects of increased surveillance. The management described herein utilised multiple hysteroscopies and endometrial biopsies, initially every 6 months, then annually from 10 years onwards. This resulted in over 20 hysteroscopies throughout an 11-year period between index operation and first pregnancy, which resulted in a late miscarriage at 16 weeks’ gestation. Whilst no known association between multiple hysteroscopies and cervical incompetence can be found, it is highly likely that this contributed. A previous systematic review on miscarriage did not identify hysteroscopy as a risk factor , multiplThe final consideration is the need for completion surgery once the woman’s family is complete. Along with a hysterectomy, traditional management of UA includes BSO to exclude and treat ovarian involvement , 24. FurIn the context of rising maternal age and a preponderance to impact women of reproductive age, FSS may become increasingly requested in UA. We present herein a case which was treated with local excision. The patient subsequently went on to have a successful pregnancy and remained disease free for 20 years before undergoing completion surgery with ovarian preservation. Whilst we would advocate that all cases of UA should be advised to undergo hysterectomy ± BSO, conservative management can be considered in women of childbearing age who have not yet completed their families. Criteria for management would be a low-grade tumour and stage IA disease without SO or MI. A proposed management algorithm for UA is demonstrated in Fig. Post-operatively, expert ultrasonographic surveillance should be undertaken 3 monthly for the first 2 years, 6 monthly until 5 years post-operatively, and annually thereafter, in a similar fashion to other moderate to high-risk gynaecological cancers . A low t