EphrinB2–EphB4 signaling is critical during embryogenesis for cardiovascular formation and neuronal guidance. Intriguingly, critical expression patterns have been discovered in cancer pathologies over the last two decades. Multiple connections to tumor migration, growth, angiogenesis, apoptosis, and metastasis have been identified in vitro and in vivo. However, the molecular signaling pathways are manifold and signaling of the EphB4 receptor or the ephrinB2 ligand is cancer type specific. Here we explore the impact of these signaling pathways in neurooncological disease, including glioma, brain metastasis, and spinal bone metastasis. We identify potential downstream pathways that mediate cancer suppression or progression and seek to understand it´s role in antiangiogenic therapy resistance in glioma. Despite the Janus-faced functions of ephrinB2–EphB4 signaling in cancer Eph signaling remains a promising clinical target. The receptor tyrosine kinase (RTK) EphB4 and its ligand ephrinB2 are part of a larger group of signaling receptors, the Ephs, which have been shown to steer a multitude of physiological processes during adulthood and embryogenesis . Eph recThe molecular signaling between EphB4 and ephrinB2 is a crucial regulator of cellular interactions in a multitude of developmental and physiological processes. The underlying principal for the majority of these ligand–receptor contacts lies within the regulation of cellular repulsion and adhesion . Most coDuring development of the nervous system, Eph–ephrin signaling is crucial for cellular migration in neurons by guiding axons and mediating their formation and plasticity as well as apoptosis of neural progenitors ,10,11,12During carcinogenesis, cancer cells can hijack these physiologic functions and subsequently increase their migratory and invasive properties . A multiIt is important to bear in mind, that under pathologic conditions an increase in receptor expression does not necessarily have to correlate with increased downstream signaling, as demonstrated in a study by Noren et al . DespiteMultiple targeted drugs against ephrinB2 and EphB4 have been developed to pharmacologically manipulate this interaction in different ways . Some haSeemingly opposing effects of ephrinB2–EphB4 signaling on cancer progression are found throughout the literature . The oveSoluble ephrin-Fc ligand has repeatedly been shown to reduce malignancy in different tumor cells in vitro and in vivo ,59,63,64EphrinB2 and EphB4 are essential proteins for the development of the cardiovascular system ,65. GeneCurrently only Bevacizumab, a monoclonal antibody targeting VEGF, is used as a therapeutic agent for recurrent glioblastoma in the US only, based on two phase-II studies . In factMalignant brain tumors, derived from glial cells, such as astrocytoma or glioblastoma, represent the largest entity of non-metastatic, primary lesions of the central nervous system (CNS) . These tInteractions of EphB4 with its ligand ephrinB2 are important for vessel network formation. EphB4 is primarily expressed on venous endothelial cells (EC), while ephrinB2 is predominantly expressed by arterial ECs . InteresEphrinB2–EphB4 interaction as well as their bidirectional signaling seem to exert pivotal and, at times, converse effects on progress and invasiveness of tumor cells in different tumor entities. Concerning glioma, receptor as well as ligand primarily seem to carry out tumor promoting effects by means of their respective signaling. Analysis of human glioma specimens, has shown increased mRNA expression levels of EphB4 and ephrinB2 in ECs as well as in xenograft tumor cells . Also, gIn summary, it appears that the balance of EphB4 and ephrinB2 expression, their respective signaling pathway activation, and special distribution influence glioma growth, development, and invasion essentially. The bidirectional signaling and receptor–ligand expression is challenging and, despite intensive research, not sufficiently understood to generalize its effects on biological behavior of malignant glioma.Regarding specific angiogenetic effects of EphB4 and subsequent antiangiogenic resistance, endothelial overexpression of EphB4 during glioma growth in vivo, effectuates significantly enlarged tumor vessels, which seemingly organized themselves to a more structured network . FurtherLive imaging of the tumor vessels, in two modalities , revealed large, well perfused EphB4 tumor vessels . EphB4 eA possible molecular pathway, that involves ephrinB2–EphB4 mediated therapeutic resistance, is the Notch signaling pathway, which is equally relevant for viability of embryonic mice . ActivatDLL4-overexpressing human glioblastoma cell lines, treated with Bevacizumab, displayed a clear antiangiogenic effect of DLL4 towards the VEGF receptor inhibitor . Inhibitγ1 phosphorylation, a critical downstream effector mediating tumor invasiveness and resistance towards antiangiogenic treatments such as Sunitinib  receptor were discovered . EGF sigunitinib  105]. A. Aγ1 phoβ- signaling pathway, following activation by the hypoxia-inducible factor (HIF)-1α , de, de7], dThe widespread expression of Eph–ephrin on distinct cancer types as well as its major functions in crucial steps of carcinogenesis, suggest significant therapeutic opportunities. Our group recently evaluated the effects of pharmacological EphB4 stimulation and inhibition in our spinal metastatic melanoma model ,134. BotWe start to understand how tissue morphogenesis is regulated in a cell–cell contact-dependent manner. The ephrins and their receptors (Ephs) are major players involved in this signaling. Tumors are hijacking these systems to gain access to new territory and form new blood vessels. Here we described the importance of ephrinB2 and EphB4 for neurooncological diseases in particular. They are involved in antiangiogenic resistance in glioma and are critical for extravasation of metastatic melanoma cells into the spinal bone. However, clinical translation is currently still difficult, and it is important to point out, that a lot of the studies reviewed here included experiments that were performed in different settings (in vivo vs. in vitro), as well as in different species. Moreover, detailed research of the bidirectional signaling pathways through individual and simultaneous receptor/ligand stimulations in combination with bilateral cell observations remain mostly unaccounted for. Still a positive perspective for the future resembles that multiple multi kinase inhibitors  are now in clinical phase-IV studies (approved), although how much of their effectiveness in patients is caused by the inhibition of ephrinB2–EphB4 is unclear. Moreover, most of these inhibitors target the EphB4 kinase domain even though non-kinase dependent signaling and reverse signaling are induced after ephrinB2 binding. This problem is solved by a second generation of peptide inhibitors based on TNYL-RAW, targeted substitution of amino acids resulted in the BIDEN-AP peptide that was found to inhibit both forward and reverse signaling alike . From th


Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole—enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery.The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs.  Candida albicans is the main causative agent of such infections, since the early 2000s there has been a marked increase in the prevalence of non-albicans-related invasive candidiasis worldwide n/([azole]n + dnK), using GraphPad Prism 9 , where ΔAmax is the maximum change in the absorbance, [azole] is the azole concentration and n is the Hill coefficient. The UV-visible absolute absorbance spectrum of the purified protein was obtained using an Ultrospec protein . The spe protein . The spiin state . Type IIin state ,44 usinggorithm)  by fittiv/v in 0.1 M glycine-NaOH buffer in the pH range of 9.3 to 9.5. The drop volume was 1 μL or 2 μL in a 1:1 ratio of reservoir solution and 20 to 30 mg/mL of protein in SEC buffer. Crystals were picked using an appropriately sized nylon loop  and flash-cooled in liquid nitrogen. Data sets were collected on the MX2 beamline at the Australian Synchrotron using a Dectris EIGER 16M detector. The data were indexed and integrated using XDS [Ni-NTA affinity and SEC-purified ScCYP51-6×His was co-crystallised with VT-1129 (40 µM) using a hanging-drop vapor diffusion method at 18 °C [sing XDS  and scalsing XDS . Moleculsing XDS  from Phesing XDS  using thsing XDS  and Cootsing XDS , respectsing XDS  and was A homology model of CpCYP51 was generated using Modeller 9.22  using thS. cerevisiae strains generated in the present study are described in S. cerevisiae strains were constructed expressing recombinant CpCYP51-6×His and CpCYP51-6×His Y132F, with or without a recombinant version of their cognate NADPH cytochrome P450 reductase (CpCPR-6×His) co-expressed from the PDR15 locus and deleted of endogenous CYP51. In some strains, expression of the native CYP51 was regulated using the GAL1 promoter, and in other strains, the native CYP51 was deleted. The viability of the resultant strains when grown in medium containing glucose as an energy source demonstrated that CpCYP51-6×His was functional. S. cerevisiae strains expressing recombinant CYP51s and CPR are presented in PDR5 locus by strains Y2719, Y2721, Y2711 and Y2712  was attained . The seqGAL1 promoter, were examined. The strains tested are described in The susceptibilities of the recombinant strains towards azole drugs were initially screened using agarose diffusion assays. Susceptibility to the short-tailed triazoles FLC and VCZ, the long-tailed triazoles itraconazole (ITC) and posaconazole (PCZ), medium-tailed tetrazoles VT-1161 and VT-1129 and the control drugs micafungin (MCF) and amphotericin B (AmpB) was assessed. The antifungal susceptibilities of the strains expressing wild type CpCYP51-6×His or CpCYP51-6×His Y132F, with or without co-expression of the cognate NADPH-cytochrome P450 reductase (CpCPR) and with native CYP51 either expressed or deleted/repressed by the GAL1 promoter, i.e., similar sized zones of inhibition were obtained for Y2718 and Y2719, Y2720 and Y2721, Y2713 and Y2714, and for Y2715 and Y2716. This indicated that expression of the native ScCYP51 was repressed effectively in glucose media and that the azole resistance phenotypes of these strains were due to the functional expression of wild type CpCYP51 and CpCYP51 Y132F. The viability and drug susceptibilities of these strains confirm that the recombinant CpCYP51 is a functional drug target. Co-expression of CpCPR did not cause any change in susceptibility towards the azoles. However, a slight reduction in susceptibility to AmpB was observed, e.g., strains Y2720 and Y2721 compared to Y2718 and Y2719, respectively. The same effect was seen among the Y132F expressing strains, i.e., between Y2713 and Y2715 or Y2714 and Y2716. The zones of inhibition produced by the recombinant strains and the reference strain Y2411 expressing the native ScCYP51 are shown in Comparison of strains Y2718–Y2721 to Y2713–Y2716 showed that the CpCYP51 Y132F protein conferred significant resistance to FLC and VCZ. With 12.5 nmole of FLC or 0.3 nmole VCZ, no inhibition zone observed with strains Y2713–Y2716 compared to the wild type strains a. Y2713–80 values of the drugs for the recombinant strains and strain Y2411 were determined  or CpCYP51 Y132F (Y2713 versus Y2714), or those pairs which co-expressed from the PDR15 locus the cognate reductase CpCPR (Y2720 versus Y2721 and Y2715 versus Y2716), gave comparable susceptibility to each drug tested. None of the recombinant strains expressing CpCYP51 or CpCYP51 Y132F showed increased MIC80 values for FLC and VCZ when the cognate reductase was co-expressed  confirmed that the enzyme was of high purity. The ratio ΔA393–470 nm/ΔA416.5–470 nm equal to 0.34 implied that the enzyme was completely in the low spin state [The absolute absorbance spectrum of Ni-NTA and SEC-purified CpCYP51-6×His a exhibitin state . The simin state . Amax) was almost two-fold less on binding to the tetrazoles VT-1161 and VT-1129 compared to that of the triazoles VCZ and PCZ, with peaks shifted from 429–427 nm for triazole binding to 423–425 nm for tetrazole binding and the troughs shifted from 411–407 nm for triazole binding to 411–405 nm for tetrazole binding (dK and Hill coefficient) are tabulated in dK) values significantly lower than the enzyme concentration showed that all the drugs bound tightly to the enzyme [Typical type II binding spectra were obtained on titration of Ni-NTA-purified CpCYP51-6×His (1 μM) with VCZ, PCZ, VT-1161 and VT-1129. The difference in absorbance , VCZ (PDB ID: 5HS1), ITC (PDB ID: 5EQB) and VT-1161 (PDB ID: 5UL0), but there was no crystal structure for a CYP51 in complex with VT-1129. The X-ray crystal structure of full-length ScCYP51-6×His in complex with VT-1129 (PDB ID: 7RYX) a was obtStrong electron density within the active site allowed modelling of VT-1129 b. The liUnexpectedly, the water mediating the hydrogen bond network previously seen between the heme ring C propionate carboxyl (RCC), the Y140 residue and the tertiary hydroxyl group in VT-1161  was not To better understand the interaction of azoles with CpCYP51, a homology model of CpCYP51 was obtained using ScCYP51 (PDB ID: 4K0F) as a template. The interactions between CpCYP51 and ligands can be gleaned by the superimposition of the known liganded structures with the model . LookingC. parapsilosis CYP51s, followed by co-expression with their redox partner CPR, was successful in our S. cerevisiae expression system as were the previously published C. albicans and C. glabrata CYP51s [PDR5 locus of a S. cerevisiae host strain deleted of seven ABC transporters has provided a stable platform to characterise these enzymes functionally and biochemically. Due to the deletion of the Pdr3 transcriptional regulator and seven ABC transporters in our host strain, which prevents drug transport from the cells by efflux pumps, the susceptibility to azole drugs of the resultant recombinant yeast strains was primarily due to the constitutive overexpression of CYP51s from the PDR5 locus. As constitutive overexpression was controlled by the mutant gain of function pdr1-3 transcriptional regulator, azole drug concentrations had no effect on the expression of the efflux pumps or CYP51 in our host system. In addition, the recombinant wild type CpCYP51 or mutant CpCYP51 Y132F enzymes were shown to sustain growth when the native CYP51 was deleted or their GAL promoter-dependent expression was blocked in presence of glucose. Heterologous overexpression of functional, full-length hexa-histidine tagged a CYP51s . The conKd) indicative of tight 1-1 binding and Hill coefficients in the same range as obtained previously with S. cerevisiae, C. albicans and C. glabrata CYP51-6×His constructs expressed in the same S. cerevisiae host system [Functional CpCYP51-6×His was demonstrated by showing type II binding of VCZ and PCZ. Both drugs caused a red shift in the Soret peak of CpCYP51 from 417 nm to 420–422 nm. Type II binding of these drugs gave dissociation constants  of fungal CYP51s.S. cerevisiae [C. albicans and C. glabrata [C. albicans and A. fumigatus CYP51s [Crystal structures of full-length CYP51 of revisiae ,50,51,67glabrata , as wells CYP51s  in compls CYP51s ,50,51,67s CYP51s . The crys CYP51s . CompariThe resistance mechanism can be interpreted by comparing the crystal structures of ScCYP51 in complex with FLC and VCZ with the structures of the Y140F mutant with FLC (PDB ID: 4ZDZ) and VCZ (PDB ID: 4ZE0), respectively . The watEach recombinant strain expressing CpCYP51 showed equivalent susceptibility to VT-1161 and VT-1129 but with strains expressing recombinant CpCYP51 being four to six-fold more susceptible than strains expressing recombinant CpCYP51 Y132F.A. fumigatus CYP51B was suggested to contribute significantly to binding the experimental tetrazole VT-1598 [Hargrove et al. previously highlighted the significance of a H-Bond (2.8 Å) between the trifluoroethoxyphenyl oxygen of VT-1161 and the imidazole side chain of H377 residue in the structure of the CaCYP51 catalytic domain that would strengthen the inhibitory activity of VT-1161 . Further VT-1598 . However VT-1598 ,41 nor VC. parapsilosis CYP51 and CYP51 Y132F in S. cerevisiae is expected to enable phenotypic and biochemical screens that can identify antifungals targeting these CYP51s from an increasingly important fungal pathogen. This will help the discovery of novel antifungals in compound libraries and assist the further development of drug scaffolds used to design broad-spectrum antifungals. The yeast strains developed here have allowed for study of the differential behaviour of the CYP51s to both established and novel antifungals, indicating the potential of these strains as screening tools. Co-expression of CpCYP51 with its cognate CPR also provides opportunity for in vitro biochemical assays using fluorescent substrates such as BOMCC [S. cerevisiae host has broadened our understanding of structurally related mutations. The 10- to 12-fold increase in resistance to the short-tailed azoles conferred by CpCYP51 Y132F mutation was reduced to six-fold for VT-1161 and its congener VT-1129, indicating that the impact of water-mediated hydrogen bond network involving the tertiary alcohol in FLC, VCZ and VT-1161 was partially reduced by the medium-length tails in VT-1161 and VT-1129. The expression of functional, full-length recombinant as BOMCC . The pre


P < 0.001). Furthermore, a non-linear relationship was detected between UA and SBP. In multivariate piecewise linear regression, the inflection point of UA was 4.13 mg/dl , the levels of SBP increased with the increase in UA when the UA level was >4.13 mg/dl . However, we did not observe a significant relationship between UA and SBP when the UA level was <4.13 mg/dl . Our study found a nonlinear relationship between UA and SBP in Chinese children and adolescents with ISS and showed that SBP levels were associated positively with the rise of UA levels when the UA levels reached the inflection point.The purpose of this study was to investigate the relationship between systolic blood pressure (SBP) and uric acid (UA) in patients with idiopathic short stature (ISS). The present study was a cross-sectional study. A total of 210 Chinese children and adolescents with ISS were included, and their anthropometrics and biochemical parameters were measured. Growth hormone peak levels were assessed after provocation tests with L-dopa and insulin. The univariate analysis results showed a significant positive association between UA and SBP levels ( Short stature refers to individuals in a similar living environment and of the same race, same sex and age who are two standard deviations lower than the average height of the normal population. Despite the short stature, for many affected children and adolescents, there are no clear reasons for the identified growth problems. This heterogeneous group of children and adolescents is often described as having ISS. ISS is one of the major causes of short stature , and ourShort stature is considered a risk factor for adult cardiovascular diseases (CVDs), such as coronary heart disease and hypertension , 4. The UA is the final product of purine metabolism. Dietary intake affects the production and metabolism of UA to a large extent. Picky eating, partial eating and the excessive intake of high-sugar drinks are poor eating habits that are common in short stature children and adolescents and that may lead to an increase in uric acid levels . An elevTherefore, it is necessary to evaluate the relationship between UA and SBP in ISS children and adolescents. However, to the best of our knowledge, few studies have focused on this issue. The aim of this study was to examine the relationship between SBP and UA in Chinese children and adolescents with ISS.enrolled, as described in the flow chart . The detailed procedure for systolic and diastolic blood pressure (DBP) measurements is described as follows: The BP of the participants was measured using a standard sphygmomanometer after 5 min of seated rest on days 1 in the morning. The trained nurse then measured BP twice while the participant was seated and then calculated and recorded the mean of the two measurements of SBP and DBP, if the difference between two measurements was >5 mmHg, another measurement was performed and and then calculated and recorded the mean of the three measurements of SBP and DBP. According to Tanner stage [Heights were measured by a specially designated individual using the same measuring instrument . The height of each participant was measured in the morning with the participants without shoes, and the allowable error range was 0.1 cm. Height SDS was calculated based on the normal range of Chinese children . The weier stage , the staer stage , 19.Blood sample was collected on days 2 in the morning after overnight fasting (at least 8 h). To assess GH secretion peaks, L-dopa  was administered orally or insulin  was administered subcutaneously after overnight fasting. Blood samples were collected after 0, 30, 60, 90 and 120 min to obtain serum GH concentrations at each time point. GH was measured by the chemiluminescence method  with an analytical sensitivity of 0.010 μg/L. Serum IGF-1 levels were measured by the chemiluminescence immunometric method  with intra- and inter-assay CVs for IGF-1 of 3.0% and 6.2%, respectively. Liver function index measurements including alanine aminotransferase and aspartate aminotransferase, renal function indicators including creatinine and uric acid (UA), and lipid profiles including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) and fasting blood glucose (FBG) were tested by a biochemical automatic analyser . Thyroid function, including free T3, free T4, thyroid-stimulating hormone, gonadotropin, cortisol rhythm and adrenocortical hormone, and urinary free cortisol (24UFC) were assessed using a luminescence immunoassay system .http://www.R-project.org, The R Foundation) and EmpowerStats . We express continuous variables with a normal distribution as the mean (standard deviation) and continuous variables with a non-normal distribution as the median (quartile). Categorical variables are expressed in frequency or as a percentage. A univariate analysis model was used to determine the significance of the association between SBP and UA as well as the other independent variables. We then investigated the relationship between SBP and UA using smooth curve fitting after adjusting for potential confounders. Finally, we further applied a multivariate piecewise linear regression model to examine the threshold association between SBP and UA. P values < 0.05 (two-sided) were considered statistically significant.All analyses were performed with the statistical software packages R . Other variables that remained significantly positively associated with SBP were age, BMI, IGF-1, 24UFC and pubertal stage (P < 0.05), whereas a significant negative association was found between SBP and sex (P < 0.05). No significant association was observed between SBP and TG, TC, HDL-C, LDL-C, or FBG (P > 0.05).Univariate linear regression analysis was performed to determine the relationships between clinical parameters and SBP. As shown in Table P = 0.03). Specifically, SBP levels increased as UA increased when the UA level was >4.13 mg/dl . However, the SBP levels displayed a decreasing trend as UA increased when the UA level was <4.13 mg/dl, but the difference was not statistically significant .As shown in Fig. P = 0.03). The positive relationship between UA and SBP was significant only when the UA levels reached the inflection point.In this cross-sectional study, we observed a nonlinear relationship between UA and SBP in children and adolescents with ISS, and the UA turning point was 4.13 mg/dl (95% CI 3.28, 6.65; Hyperuricaemia has been reported to have increased in children and adolescents in recent years. According to the known standards reported in the literature, a UA level > 5.5 mg/dl is considered abnormal . AccordiMost previous literature has reported a linear positive association between UA and SBP in healthy adults and adolescents: The Framingham study found a significant positive association between UA levels and changes in SBP and DBP after 4 years in adults . In chilThe present study has several limitations. First, our research was conducted in a homogeneous population of children and adolescents with ISS and the results cannot be extrapolated to other populations. Second, we did not collect data regarding other potential confounders of SBP, such as diet and family history. We intend to conduct a large-sample prospective study to specifically address factors affecting BP changes in children with ISS, including questionnaires about the dietary habits of the participants and the effects of a family history of hypertension. Third, the cross-sectional design of this study does not allow us to determine causality.In conclusion, we described a nonlinear relationship between UA and SBP levels in children and adolescents with ISS after adjusting for potential confounders and SBP levels were associated positively with the rise of UA levels when the UA levels reached the inflection point, however, it is uncertain whether the association is causal, and some relative future research are required to further confirm its potential mechanism and provide clinical evidence for controlling uric acid levels in children and adolescents with ISS.Short stature is considered a risk factor for adult hypertension, adult primary hypertension is likely derived from risk factors already present in childhood.Hyperuricaemia is a recognised risk factor for hypertension in adults and children.The univariate analysis results showed a significant positive association between uric acid and systolic blood pressure levels in children and adolescents with idiopathic short stature.There was a nonlinear relationship between uric acid and systolic blood pressure, and the uric acid turning point was 4.13 mg/dl, a multivariate piecewise linear regression model displayed a significant positive association between uric acid and systolic blood pressure when the uric acid levels more than 4.13 mg/dl.


Self- and non-self ligand discrimination is a core principle underlying T cell-mediated immunity. Mature αβ T cells can respond to a foreign peptide ligand presented by major histocompatibility complex molecules (pMHCs) on antigen presenting cells, on a background of continuously sensed self–pMHCs. How αβ T cells can properly balance high sensitivity and high specificity to foreign pMHCs, while surrounded by a sea of self-peptide ligands is not well understood. Such discrimination cannot be explained solely by the affinity parameters of T cell antigen receptor (TCR) and pMHC interaction. In this review, we will discuss how T cell ligand discrimination may be molecularly defined by events downstream of the TCR–pMHC interaction. We will discuss new evidence in support of the kinetic proofreading model of TCR ligand discrimination, and in particular how the kinetics of specific phosphorylation sites within the adaptor protein linker for activation of T cells (LAT) determine the outcome of TCR signaling. In addition, we will discuss emerging data regarding how some kinases, including ZAP-70 and LCK, may possess scaffolding functions to more efficiently direct their kinase activities. The immune system maintains a homeostatic state within an organism while remaining poised to vigorously respond to life-threatening challenges. T cells, as a major component of the adaptive immune system, contribute to this difficult task by maintaining a level of tonic signaling which is required for their survival under homeostatic conditions. However, these same T cells are capable of rapidly responding to challenges by pathogens or cancerous cells to induce remarkably precise immune responses through the generation of signals that lead to clonal expansion and acquisition of effector functions only by appropriate antigen-specific T cells.The T cell antigen receptor (TCR) is the major surface receptor used by every T cell to survey host cells expressing short self-peptides derived from host self-proteins bound to self-MHC molecules (self–pMHC). At the same time, peptides derived from pathogen proteins that are likewise bound to MHC molecules can also be recognized by the TCR and serve as agonists to initiate T cell responses in a highly sensitive and specific manner. One to ten foreign agonist–pMHCs are sufficient to activate an antigen-specific T cell ; each TCR clonotype also reacts with one foreign agonist–pMHC  , 2. Howevia interactions with self–pMHCs which generates survival signals, so called tonic signaling -induced signaling requires more ligand-binding events and is less sensitive than natural/conventional TCR signaling –23. Suche.g., cell-cell interactions) and intrinsic ones  contribute to T cell ligand discriminatory capability. A TCR typically binds to an agonist foreign–pMHC with a longer half-life than it does to a self–pMHC  versus slip bonds  (D in the range of ~1000 µM) at 37°C, a recent study showed that the level of TCR discrimination is lower than the level that was estimated by earlier work  , 39–41. lf–pMHC) . Interesier work . RegardlThus, it is unclear how TCR ligand discrimination ability can be chemically encoded to control the T cell fate decision process and transform the TCR:pMHC signal to a digital activation outcome , 43, suckinetic proofreading mechanism that functions as a molecular timing device to set an activation threshold for T cells  LCK and ζ chain-associated protein kinase 70 (ZAP-70), as two proximal and essential T cell specific kinases in TCR signal initiation, have attracted great interest regarding their supportive roles in kinetic proofreading.CK to subsequently phosphorylate and activate the ITAM-bound ZAP-70 (γ1 (PLCγ1) which is required for second messenger generation leading to calcium increases as well as Ras and protein kinase C (PKC) activation. Signaling beyond this point might be considered an irreversible (or a highly energy-consuming if it was to be reversed) signaling step since at least some T cell responses can be rapidly initiated subsequent to these events.LCK initiates the immediate signaling step after TCR:pMHC engagement , 54, 55.d ZAP-70 , 59. AftLike other SFKs, LCK has an SH3 domain, an SH2 domain, a tyrosine kinase domain and a C-terminal inhibitory tyrosine . HoweverIndeed, each of the LCK structural domains are also suggested to play important roles in regulating TCR signaling. The LCK SH2 domain binds to phospho-Y319 of ZAP-70, promoting the open, active conformations of both LCK and ZAP-70 and sustaining coreceptor localization to the stimulated TCR , 67, 68.). In examining the sequences of LAT among 41 mammalian species, we found that there are sixteen proline residues within the membrane-proximal region of LAT (from P33 to P95 in human LAT). The positions and frequencies of proline residues within this region are highly conserved in all mammalian LAT sequences that were examined. Yet the functionality of this region had not been appreciated. We found one particular proline motif in this region, PIPRSP (P80−P82−P85), interacted with the LCK SH3 domain and that this interaction was functionally important  in a coreceptor-bound form, LCK bridges these signal initiating proteins, ZAP-70 and LAT, to the engaged TCR:pMHC complexes.Using a series of cellular experiments as well as molecular and biochemical approaches, we concluded that LCK, This trimolecular protein interaction may facilitate weak TCR:pMHC interactions, especially those at the borderline of T cell activation thresholds, and contribute to the reversible kinetic proofreading signaling steps. In addition, this ZAP-70–LCK–LAT intramolecular coordination also supports TCR signal transduction in the proximity of engaged TCR:pMHC complexes. From the perspective of kinetic proofreading, this may be a key event to allow the internal signaling network to properly discriminate TCR interaction with an agonist or a self–pMHC. Thus, only the TCR:foreign–pMHC interaction would successfully propagate downstream signaling steps. Indeed, these data, combined with computational modeling, further revealed that this higher order of coreceptor-coupled protein-protein interaction is particularly important to endow TCRs with the sensitivity to detect and respond to weak ligands. Elimination of the proline-rich motif in LAT compromised TCR signaling and T cell development .The kinetic proofreading model assumes that the signaling intermediates among the proofreading signaling steps can instantly reflect the status of TCR:pMHC interaction. Put differently, once the TCR is disengaged from pMHC, all the proofreading steps are quickly reversed  –79, whicγ1. Replacement of this key tyrosine with phenylalanine disrupted thymic development of T cells and TCR induced T cell activation  is especially important for the recruitment and activation of PLCtivation –95. Impofreading , 52. Theγ1 pathway, has some unique features. Biochemical and structural analysis of ZAP-70 revealed that the substrate binding site in its kinase domain is unusually enriched in basic residues  increased the phosphorylation rates of LAT Y132, the recruitment, phosphorylation and activation of PLCγ1, and the magnitude and the rate of calcium elevation  can only associate with the LAT signalosome  T cells, have successfully leveraged our knowledge of T cell signal transduction and T cell activation to treat cancers and/or autoimmune diseases. Although CARs can elicit T cell effector functions, these artificial receptors appear to be much less efficient at detecting foreign antigens than genuine TCRs. Moreover, most CARs lack the ability to fully propagate signals through the canonical TCR signaling pathway and appear to bypass some key signaling steps , 103–105W-LL and AW wrote the manuscript and approved the submitted version. All authors contributed to the article and approved the submitted version.AW was supported by NIH P01 AI091580 and 1R37AI114575.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The modulation of vascular tone and the process of cerebral ischemia are mediated via the interactions between G protein-coupled receptors (GPCRs) and their ligands, including 5-HT, angiotensin II (Ang II), and urotensin II (UII). Thus, the potential synergistic effects of the different components of XXMD on the regulation of vasoconstriction and vasodilation were further investigated by molecular docking based on network pharmacology. We constructed and analyzed a database comprising 963 compounds of XXMD and studied the interactions between five vascular GPCRs (5-HT1A receptor (5-HT1AR), 5-HT1B receptor (5-HT1BR), Ang II type 1 receptor (AT1R), beta 2-adrenergic receptor (β2-AR), and UII receptor (UTR)) and the various herbal constituents of XXMD using molecular docking. By constructing and analyzing the compound-target networks of XXMD, we found that Glycyrrhizae Radix et Rhizoma, Ginseng Radix et Rhizoma, and Paeoniae Radix Alba were the three major herbs that contained a large number of compounds with high docking scores. We additionally observed that several constituents of XXMD, including gallotannin, liquiritin apioside, nariutin, 1,2,3,4,6-pentagalloylglucose, folic acid, and ginsenoside Rb1, targeted multiple vascular GPCRs. Moreover, the interactions between the components of XXMD and the targets related to vascular tone constituted the comprehensive cerebrovascular regulatory function of XXMD and provided a material basis of the vasoregulatory function of XXMD. The study reports the contributions of various components of XXMD to the regulatory effects on vascular tone and provides scientific evidence for the multicomponent and multitargeting characteristics of XXMD.Xiaoxuming decoction (XXMD) is a traditional Chinese herbal medicine (CHM) that is used for the treatment of stroke in China. Stroke injury damages the cerebral vasculature and disrupts the autoregulation of vasoconstriction and vasodilatation, which is crucial for maintaining constant cerebral blood flow (CBF). It has been reported that XXMD exerts a positive effect on cerebral circulation in animal models of stroke. However, the mechanisms underlying the regulatory effect of XXMD on vascular tone, and the interactions among the multiple components of XXMD, remain unclear. In this study, XXMD was found to induce relaxation of the basilar artery rings of rats precontracted by 5-hydroxytryptamine (5-HT)  Scutellariae Radix, Paeoniae Radix Alba, Glycyrrhizae Radix et Rhizoma, Stephaniae Tetrandrae Radix, Ginseng Radix et Rhizoma, Cinnamomi Ramulus, Armeniacae Semen Amarum, Ephedrae Herba, Chuanxiong Rhizoma, Aconiti Lateralis Radix Praeparata, Saposhnikoviae Radix, and Zingiberis Rhizoma Recens at a 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1.5 : 5 ratio. Preclinical studies from our laboratory and other groups have demonstrated that XXMD has neuroprotective effects in rats with acute or chronic cerebral ischemia methyl}-3,4-dihydro-1,6-naphthyridin-2(1H)-one, respectively, were defined as the active ligand-binding pocket. For β2-AR .The LibDock module of Dock Ligands in Discovery Studio 4.1 was used to perform molecular docking. The conformation method was set to fast to ensure docking accuracy and velocity, and the other parameters were set to default. In this semiflexible docking approach, 100 or less conformations of each ligand were generated to ensure computational speed and comprehensiveness. The output index and LibDock scores were subsequently obtained. The scores indicate the binding affinity between the receptors and the ligand conformations. The higher the score of a small molecule, the stronger the protein binding affinity. For each target, the 50 top-scoring compounds were selected, and their scores were considered to be the highest among all the conformations for each ligand. The LibDock scores of the agonists and antagonists, retrieved from the IUPHAR database, were also determined for each target, for assessing the potential activity of the herbal compounds in XXMD. The following agonists and antagonists were used for molecular docking: 5-HT1AR -UH 301 and WAY-100635); 5-HT1BR ; AT1R , The information pertaining to the chemical composition and related compound-target network interactions were analyzed using Cytoscape version 3.7.1. The compound-target network was established using Cytoscape, and the interactions were subsequently visualized.XXMD was found to induce relaxation of the rat basilar artery rings . The maxIn order to identify the potential material basis of the modulatory effects of XXMD on the vascular tone, 963 compounds from the twelve herbs of XXMD were retrieved from China Natural Product Database and from the literature. The number of compounds in each herb was determined, and the results are depicted in In order to identify the potentially effective constituents of XXMD, the compounds derived from the different herbs of XXMD were retrieved and analyzed. The names, plant sources, and chemical categories of forty compounds are enlisted in β2-AR, and UTR) using the LibDock program in Discovery Studio 4.1. The ligands dihydroergotamine, 5,7-diethyl-1-{[2′-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl}-3,4-dihydro-1,6-naphthyridin-2(1H)-one, and BI167107 were extracted from the crystal structures of 5-HT1BR, AT1R, and β2-AR, respectively, and redocked into the active site of the corresponding receptors. The root mean square deviation (RMSD) values between the docked poses and the ligand conformation in the crystallographic complexes were 0.3074, 0.3168, and 0.4324 Å, which indicated that the docking protocol was highly accurate and reliable.The cocrystallized ligands were redocked to the five vascular GPCR targets  are depicted in The compound-target network interactions were subsequently analyzed. Several of the 50 top-scoring compounds interacted with multiple targets, including 5-HT1AR, 5-HT1BR, AT1R, To date, there are no definitive treatment strategies for ischemic stroke. With the development of recent research on multi-component-multi-target drugs, the functions of TCM in the treatment of complex diseases have become increasingly important. Owing to the potent compound group formulation used in TCM prescriptions, the efficacy of TCM is attributed to the interactions between multiple active ingredients and multiple disease targets. Several animal experiments and clinical studies have demonstrated that XXMD, a CHM with relaxant effects on cerebral blood vessels, can improve the treatment outcomes in both acute and chronic ischemic brain injury , 5, 10. β2-AR, and UTR. The 50 top-scoring compounds with high docking scores to each of the GPCR targets were selected and sorted. The herbal sources of these compounds were determined for identifying the roles of the twelve herbs of XXMD in regulating the vascular tone via modulation of GPCR activity.In this study, the vasorelaxant effects of XXMD on rat basilar arteries contracted by 5-HT were determined. The modulation of vascular tone and the process of cerebral ischemia are mediated via the interactions between GPCRs and their ligands, including 5-HT, Ang II, and UII. Therefore, molecular docking was performed to determine the potential synergistic effects of the components of XXMD on the regulation of vascular tone via modulation of the activity of vascular GPCRs. First, a database of 963 compounds of XXMD, with different chemical structures, was constructed and analyzed. Molecular docking was used to investigate the interactions between the chemical constituents of XXMD and the five vascular GPCRs, namely, 5-HT1AR, 5-HT1BR, AT1R, The results of our study suggested that several overlapping compounds and their analogues are widely distributed in the different medicinal herbs of XXMD. The contents of these ingredients are relatively high in the XXMD prescription, compared to those of the other compounds. Furthermore, previous studies have demonstrated that most of these compounds have similar physiological activities, including antitumor, anti-inflammatory, antioxidative, neuroprotective, blood lipid-regulating, and vasorelaxant properties. These compounds, derived from a variety of different plants in XXMD, have different chemical structures and can produce synergistic therapeutic effects under the same pathological conditions. Therefore, they are likely to be the key components that play a crucial role in the therapeutic effect of XXMD.Liang [Apart from their pharmacological activities, some of the chemical ingredients, including essential oils, promote the absorption of other drugs. It is speculated that ginger is indispensable in the treatment of cerebral apoplexy by XXMD, and the maximum dosage should be five Liang . FurtherLiang . It can Glycyrrhizae Radix et Rhizoma, Ginseng Radix et Rhizoma, and Paeoniae Radix Alba. The therapeutic potential of these herbs and their constituents in cerebral ischemia has been extensively studied. It has been demonstrated that the aqueous extract of licorice (Glycyrrhizae Radix et Rhizoma) reduces infarct volumes and exerts a neuroprotective effect by regulating the expression of apoptosis-related proteins in mice with middle cerebral artery occlusion (MCAO) [β-glycyrrhetinic acid (GA) [Ginseng Radix et Rhizoma compounds in herbal medicine, has been shown to attenuate damage due to ischemic stroke via suppression of oxidative stress and inflammation [Paeoniae Radix Alba and Rhizoma Ligustici Chuanxiong was reported to ameliorate focal cerebral ischemia in rats with MCAO by attenuating the endoplasmic reticulum stress-dependent apoptotic signaling pathway [Glycyrrhizae Radix et Rhizoma, Ginseng Radix et Rhizoma, and Paeoniae Radix Alba play a key role in the modulatory effect of XXMD on vascular tone and its effects on suppressing ischemic stroke via regulation of the compound-target interaction network in XXMD and necessitate further investigation.Classification of the herbal sources of the 50 top-scoring compounds revealed that the three major herbs were n (MCAO) . Glycyrrn (MCAO)  and 18β-cid (GA) , isolatecid (GA) . Ginsenoammation . The actammation . A combi pathway . TherefoGlycyrrhizae Radix et Rhizoma, Aconiti Lateralis Radix Praeparata, and Zingiberis Rhizoma Recens. SNT improves cardiac function by inhibiting the excessive activation of the renin-angiotensin-aldosterone system, which involves a reduction in the levels of plasma angiotensin II and downregulation of the protein and gene levels of AT1R in heart failure after myocardial infarction in rats [in vitro 5-HT2B receptor antagonistic activity [2A receptors [It has been reported that TCM influences disease progression by modulating GPCR activity. The Chinese herbal formula, Sini Tang (SNT), comprises  in rats . ISL hasactivity . Ginsenoeceptors . The resPaeoniae Radix Alba, binds to 4 vascular GPCR targets and has been reported to induce vasorelaxation and suppress the vascular inflammatory process by activating the NO/cGMP signaling pathway [Ginseng Radix et Rhizoma and Chuanxiong Rhizoma, was found to bind to 4 GPCR targets. Interestingly, folic acid has been reported to improve endothelial function [Ginseng Radix et Rhizoma, was found to bind to three GPCR targets and has been reported to promote poststroke axonal regeneration by activating the cAMP/PKA/CREB signaling pathway [By reviewing the relevant literature, we found that most of the components of XXMD that target multiple vascular GPCRs possess vasorelaxant effects and suppress ischemic stroke. For instance, 1,2,3,4,6-pentagalloylglucose, a major component of  pathway . Folic afunction  and allefunction . Ginseno pathway . Few stuIn conclusion, the current study clearly demonstrated that XXMD was able to relax the cerebral artery rings of rats. XXMD comprises numerous compounds with different chemical structures and is derived from multiple plant sources. Therefore, XXMD might regulate cerebrovascular function via the network of interactions between the constituents of XXMD and the GPCRs involved in vasoconstriction. Our results demonstrated that the various herbal constituents of XXMD contribute to the regulation of vascular tone via the modulation of the target GPCRs. By investigating and analyzing the monomeric component of XXMD from the perspective of chemical composition and the relationships between the chemical constituents and targets, we were able to provide evidence for understanding the mechanisms underlying the effect of XXMD in the treatment of stroke.


Mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved regulators of eukaryotic cell function. These enzymes regulate many biological processes, including the cell cycle, apoptosis, differentiation, protein biosynthesis, and oncogenesis; therefore, tight control of the activity of MAPK is critical. Kinases and phosphatases are well established as MAPK activators and inhibitors, respectively. Kinases phosphorylate MAPKs, initiating and controlling the amplitude of the activation. In contrast, MAPK phosphatases (MKPs) dephosphorylate MAPKs, downregulating and controlling the duration of the signal. In addition, within the past decade, pseudoenzymes of these two families, pseudokinases and pseudophosphatases, have emerged as bona fide signaling regulators. This review discusses the role of pseudophosphatases in MAPK signaling, highlighting the function of phosphoserine/threonine/tyrosine-interacting protein (STYX) and TAK1-binding protein (TAB 1) in regulating MAPKs. Finally, a new paradigm is considered for this well-studied cellular pathway, and signal transduction pathways in general. Signal transduction is an intricate dance of cells sensing and responding to their environment, as well as influencing the behavior of neighboring cells or an entire organism . These c42−) to a substrate, while phosphatases remove the attached phosphate. The most common phosphorylation events occur on serine, threonine, and tyrosine residues /rhodanese homology) non-catalytic domain and a C-terminal catalytic phosphatase domain , which is essential for phosphatase activity  of wild-type (WT) MK-STYX is larger than the mutant (black arrow), which substituted the arginine and restored the KIM motif, 48ITALRRKKKN57 (WT residue shown in yellow and mutated residue shown in red)  C. This cThe prototypical pseudophosphatase STYX ,24, whic2+-dependent protein phosphatase) , which has a fold similar to MPP (Mgphatase) , also haphatase) . There at manner . TAK1 ist manner ,77,79. St manner ,80. TAB1t manner  and has t manner  and pro-t manner ,79. TAB1t manner  techniqut manner ,25.Mitogen-Activated Kinases: New Insights for Old Cell Signaling Pathways. Once perceived to be “dead” and molecular “duds” [C. elegans pseudophosphatases EGG4 and EGG5 trap mini brain kinase 2 (MBK-2) to regulate the oocyte-to-zygote transition though DYRK [Reviewing the contribution of pseudophosphatases in MAPK signaling is ideal for this special edition, r “duds” ,31,82, tr “duds” ,32. For ugh DYRK ,36. In tugh DYRK ,32,35,36Continued study of pseudophosphatases has provided new insights into cell signaling pathways, while shifting the paradigm for better understanding of the complexity of signaling cascades. The roles of STYX and TAB1 in MAPK signaling demonstrate that pseudophosphatases must be considered when investigating the tight control of MAPK activation and downstream regulation. Pseudophosphatases are not limited to the PTP family but are found among the serine/threonine phosphatases as well ,83,84. LUnderstanding the structure–function relationship of pseudophosphatases is important for understanding their function ,25. MutaThe combination of proteomics, biochemistry, structural biology, and transgenic animal models has been instrumental in understanding the molecular mechanisms of pseudophosphatases ,23,67. P


HTLV-1 infects approximately 5–10 million people worldwide and is the infectious cause of adult T-cell leukemia/lymphoma (ATL) and several chronic inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), dermatitis, and uveitis. Unlike other oncogenic retroviruses, HTLV-1 does not capture a cellular proto-oncogene or induce proviral insertional mutagenesis. HTLV-1 is a trans-activating retrovirus and encodes accessory proteins that induce cellular transformation over an extended period of time, upwards of several years to decades. Inarguably the most important viral accessory protein involved in transformation is Tax. Tax is a multifunctional protein that regulates several different pathways and cellular processes. This single viral protein is able to modulate viral gene expression, activate NF-κB signaling pathways, deregulate the cell cycle, disrupt apoptosis, and induce genomic instability. The summation of these processes results in cellular transformation and virus-mediated oncogenesis. Interestingly, HTLV-1 also encodes a protein called Hbz from the antisense strand of the proviral genome that counters many Tax functions in the infected cell, such as Tax-mediated viral transcription and NF-κB activation. However, Hbz also promotes cellular proliferation, inhibits apoptosis, and disrupts genomic integrity. In addition to viral proteins, there are other cellular factors such as MEF-2, superoxide-generating NAPDH oxidase 5-α (Nox5α), and PDLIM2 which have been shown to be critical for HTLV-1-mediated T-cell transformation. This review will highlight the important viral and cellular factors involved in HTLV-1 transformation and the available  Orthoretrovirinae subfamily. Isolated over 40 years ago from a patient with cutaneous T-cell lymphoma, HTLV-1 was the first discovered human retrovirus [+ T-cell malignancy [Human T-cell leukemia virus type 1 (HTLV-1) is a complex δ retrovirus within the trovirus . Large plignancy , HTLV-1-lignancy , and sevlignancy . Approxiin vitro study has suggested that transmission of HTLV-1 through a route such as breastfeeding requires transcytosis of free infectious HTLV-1 virions through the epithelial barrier [+ T cells are the primary and preferential target cell of HTLV-1 infection, with CD8+ T cells constituting roughly 5% of the total infected cells [HTLV-1 has several routes of transmission, including breastfeeding, sexual intercourse, and exposure to infected blood products such as through blood transfusions or sharing of needles. Mother-to-child transmission through breastfeeding is typically the most common transmission route within endemic regions . While m barrier . These v barrier . Among ted cells ,13. Duried cells .gag, pro, pol, and env) essential for viral replication (Hbz), all the HTLV-1 genes are encoded from the sense strand of the proviral genome. One of the most critical accessory genes for HTLV-1 is Tax. Tax protein is required for transformation and functions as a transcription factor to enhance viral transcription from the promoter in the 5′ LTR [The HTLV-1 viral genome consists of a relatively small positive-sense RNA genome of approximately 9 kb in size. Two copies are packaged in the virus particle. Upon entry into a host cell, the ssRNA genome is reverse transcribed into dsDNA and integrated into the host genome. The integrated dsDNA form of the retroviral genome is termed the provirus. The viral genome is flanked by long terminal repeats (LTRs) at both the 5′ and 3′ ends. These direct repeats consist of three regions: the unique 3′ (U3), the repeated (R), and the unique 5′ (U5) regions. The LTRs also contain important elements necessary for viral transcription, polyadenylation, and integration. Like all retroviruses, HTLV-1 contains the standard structural and enzymatic genes  mouse models . As a viOne of the key roles of Tax is transactivation of the promoter located in the 5′ LTR. Tax-mediated viral transcription drives synthesis of all plus strand gene products including the structural and enzymatic viral proteins, in addition to regulating its own expression. Tax mediates viral transcription through three discontinuous G/C-rich 21-base pair repeats, known as Tax responsive elements (TREs), located within the U3 region of the 5′ LTR ,27. ThesIn addition to regulating viral gene expression, Tax also has a strong influence over several cell signaling pathways such as NF-κB . This ac+ T-cell line Jurkat leads to enhanced expression of the pro-apoptotic ligands CD95L, TNF-α, and TNF-related apoptosis inducing ligand (TRAIL), ultimately leading to widespread cell death [Persistent activation of NF-κB results in stimulation of growth factors, cytokines, and proto-oncogenes—all of which influence the host cell cycle. NF-κB signaling also has the ability to activate expression of anti-apoptotic proteins. Not surprisingly, Tax-mediated activation of NF-κB has been reported to enhance expression of the anti-apoptotic proteins X-linked inhibitor of apoptosis (XIAP), survivin, and Bcl-xL . Tax canll death . In addill death . In addill death ,57. Tax ll death ,59. By e+ T cells transduced with various Tax-expressing retroviral vectors produced ROS as a direct consequence of Tax expression, subsequently causing DNA damage and the initiation of cell senescence [Through modulation of several proteins critical for proper cell cycle regulation, Tax can also promote the transformation of HTLV-1-infected T cells by preventing the recognition and repair of DNA damage. The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases exert cell cycle delay in response to compromised genomic integrity such as DNA damage or stalled replication forks . Tax prenescence . The intnescence . The abiin vivo, Tax by itself is not able to efficiently transform primary human cells in vitro [+ T cells is reliant on the Hbz-mediated down-regulation of Tax and NF-κB expression. Given the pathogenic and transformative properties of Tax, Hbz-mediated inhibition of this viral transcriptional activator additionally functions as a means to regulate the transformation process. However, loss of Hbz does not affect HTLV-1-mediated T-cell immortalization in cell culture [Although the expression of Tax alone is sufficient to promote tumorigenesis in Tg mouse models in vitro ,67,68. Tin vitro . Hbz intin vitro . In addiin vitro . Decreas culture . Interes culture . These shbz mRNA is also able to support cell proliferation, although the precise mechanism(s) remain unclear [in vitro studies with murine cell lines have demonstrated that the independent expression of Hbz significantly promotes soft agar cell proliferation and colony formation [Hbz can also interact with the AP1 superfamily proteins ATF3 and JunD . ATF3 is unclear . Like Ta unclear . Additioormation . Altogetormation . Furthertax/rex mRNA transcripts and prevents their export from the nucleus [in vitro studies using immortalized human fibroblasts demonstrated that p30 can enhance viral transformation when coexpressed with Myc [+ T-cell proliferation through activation of nuclear factor of activated T cells (NFAT) and signal transducers and activators of transcription-5 (STAT-5) signaling pathways [Similar to Hbz, many of the remaining viral accessory proteins of HTLV-1 also play a key role in regulating Tax activity and the transformation potential of virally infected T cells . The vir nucleus . Further nucleus . Previou nucleus . Despitewith Myc . An addiwith Myc . p13 haswith Myc ,88. In cpathways . Severalpathways .Tax is the major driver of 5′ viral transcription and thus has tight control over its own expression. In addition, Tax regulates cellular epigenetic proteins and miRNA machinery which influences the immortalization process. Several viral accessory proteins, including Hbz, function to regulate Tax activity, viral gene expression, and T-cell transformation through a variety of different mechanisms.Transformation of HTLV-1-infected T cells is mediated by a wide variety of both viral and cellular proteins. Although viral proteins such as Tax and Hbz play an important role in T-cell transformation, there are several additional cellular factors that contribute to this process . These c. revealed that superoxide-generating NAPDH oxidase 5-α (Nox5α) is a cellular protein that is required for the maintenance of HTLV-1-transformed T cells [in vivo down-regulation of Nox5α led to an observable decrease in the tumor growth rate of HTLV-1 tumor transplant mice. In summation, expression of Nox5α is critical for maintaining the survival, proliferation, migration, and oncogenic potential of HTLV-1 transformed cells, and therefore serves as a key cellular factor in promoting the transformation phenotype of HTLV-1-infected T cells [Notably, a study by Shigemura et al T cells . This st T cells .. demonstrated that myocyte enhancer factor-2 (MEF-2), a transcription factor for Interleukin 2 (IL-2) produced during peripheral T-cell activation, is substantially up-regulated within HTLV-1-infected cells and ATL patient samples [in vitro co-culture system, MEF-2 was found to be essential for the transformation of CD4+ T cells by HTLV-1. Furthermore, direct inhibition of MEF-2 activity and expression resulted in decreased viral replication and transactivation of the 5′ LTR promoter, which was attributed to the propensity of MEF-2 to colocalize with Tax and stabilize its interaction with CREB. Therefore, MEF-2 is not only required for viral transformation but is also critical for the ability of Tax to efficiently promote viral gene expression from the 5′ LTR promoter [., which characterized PDLIM2 mRNA and protein to be highly expressed in HTLV-1-negative Jurkat cells and significantly down-regulated in HTLV-1-transformed T-cell lines such as C8166, MT4, Hut-102, and SLB-1. Co-expression of PDLIM2 and Tax in HTLV-1-transformed cell lines led to a dose-dependent decrease in Tax-mediated NF-κB activation and viral transcription, while PDLIM2 knockout reversed this effect. In addition, artificial up-regulation of PDLIM2 in Tax-expressing Rat-1 fibroblasts suppressed the growth of these cells in soft agar, suggesting that PDLIM2 plays an important role in reducing the capacity of HTLV-1-infected cells to transform and proliferate in vitro [in vivo since severe combined immunodeficiency (SCID) mice transplanted with C8166 and MT4 cells exhibited no tumorigenesis when co-expressed with both PDLIM2 and Tax [Several cellular proteins have been shown to be involved in HTLV-1-mediated T-cell transformation by interfering with the activity and expression of Tax. A study by Jain et al samples ,99. Thropromoter . This roin vitro . Express and Tax . Regulat and Tax . Altogetin vitro [Another notable transforming factor for HTLV-1 is parathyroid hormone-related protein (PTHrP), which is constitutively expressed in HTLV-1 carriers and highly up-regulated during HTLV-1-mediated transformation of T cells ,103. Comin vitro . This prin vitro . Althoug. outlines a large array of different miRNAs that have been reported to be up-regulated in HTLV-1-transformed T-cell lines and ATL-derived patient cell lines [+ T cells [miRNAs have also been shown to play many diverse and notable roles in the process of HTLV-1-mediated T-cell transformation. A review by Ruggero et alll lines . As descll lines ,79. Howell lines . miR-93  T cells . In addi T cells . However T cells . Cellula+ T cells in vitro using a co-culture immortalization assay [+ T cells from the PBMCs mixed cell population. This type of assay allows for not only the study of early viral infection events, but also the ability of HTLV-1 to immortalize a target cell. Cell viability, cell number, and the number of transformed wells can be measured at weekly time points to assess the immortalization process. This type of assay has been useful for studying the function and biochemistry of individual viral proteins and protein modifications and their role in transformation [in vivo, such as Hbz, are not required for transformation in vitro [HTLV-1 is able to transform primary CD4on assay . The useormation ,111–113.in vitro ,111.+ T cells were developed [+ Foxp3+ T(reg) cells and effector/memory CD4+ T cells. Tg mice have been especially useful for establishing the role of Tax and Hbz in transformation and oncogenesis. These mice also provide a valuable tool to test various future treatments, particularly those targeted at Tax or Hbz, and to investigate additional cellular factors involved in transformation and disease development.Mouse models have provided a useful tool for studying cellular and viral factors required for development and maintenance of HTLV-1-mediated transformation. Some of the earliest mouse studies within the HTLV-1 field involved the use of Tax Tg mice . These ieveloped . Hbz Tg in vitro co-culture immortalization assays [in vivo. NOD/SCID γ c (null), or NOG mice, have immunological dysfunctions of T, B, and NK cells. Successful transplantation of various HTLV-1-transformed cell lines  into these mice has been beneficial for examining viral-induced cell proliferation and tumorigenesis [in vivo [in vivo model for studying HTLV-1-mediated transformation and the development of novel drugs and targets to reduce disease burden.In the early nineties, SCID mice were used to successfully model the proliferative and tumorigenic potential of ATL cells . Of noten assays . Taken tigenesis . For exa[in vivo . Like TgscidIl2rgtm1WjlPrkdc /SzJ) mice, lack mature T, B, and NK cells, are deficient in multiple cytokine signaling pathways, and have many defects in innate immunity. HIS mice are created by injecting human umbilical-cord stem cells (CD34+) into the livers of immunodeficient neonatal NSG mice, resulting in the development of human lymphocytes that appear phenotypically normal but cannot mount an adaptive immune response [+ T cells, and (3) development of lymphoproliferative disease. A recent study using these mice found that the envelope protein of HTLV-1 is important to induce CD4+ T-cell proliferation (vs. CD8+ T-cell proliferation), suggesting that T-cell transformation in vivo may also be influenced by the Env protein of the virus [+ stem cells into NOG mice (IBMI-huNOG mice) [+ T cells was observed in the periphery and atypical lymphocytes which represent ATL-specific flower cells were observed after 4–5 months. Proliferation and proviral load were also detected in CD4+ T cells. Importantly, the authors also report the presence of an HTLV-1-specific adaptive immune response in these animals. While technically challenging to create, this model will undoubtedly be useful for investigating in vivo mechanisms of transformation and disease development, as well as drug and vaccine candidates.In efforts to develop an improved model of HTLV-1 infection and disease progression, ‘humanized immune system’ (HIS) mouse models were developed. NSG mice, or NOD-SCID/γ(null)  . After HDrosophila melanogaster model expressing Tax in the compound eye and plasmatocytes (a leukocyte-like cell) has been described [Drosophila homolog of IKKγ/NEMO. Conversely, an Hbz transgenic Drosophila model neither induces transformation nor NF-κB activation in vivo [Drosophila models possess several advantages including fast generation time, high numbers of progeny, and several genetic screens (near complete collection of mutants and RNAi lines). In addition, several key signaling pathways are highly conserved between mammals and flies. However, while initial findings support this model system, any results from this model still require further validation in mammalian systems.Recently, a Tg escribed . Tax exp in vivo . InsteadA key to the HTLV-1 transformation process is establishment of viral infection and viral persistence. HTLV-1 infection of rabbits mimics early infection in humans. New Zealand white rabbits inoculated with HTLV-1 become persistently infected ,113,130.+ T-cell malignancy that is often fatal, there is an urgent need to understand the viral and cellular mechanisms that regulate or influence HTLV-1-mediated T-cell transformation. Serving as the primary driving force for viral transformation, the HTLV-1-encoded Tax oncoprotein is critical for viral gene expression, immortalization, and pathogenesis. Through its ability to directly interact with a multitude of different cellular proteins and pathways, Tax can transform infected T cells by modulating viral gene expression at the 5′ LTR promoter, deregulating the cell cycle, disrupting the apoptotic response, and decreasing genomic stability. Many of these processes are heavily dependent on Tax-mediated activation of NF-κB. Antagonization of Tax-driven NF-κB induction and viral transcription is mediated by the viral protein Hbz. Derived from the anti-sense strand of the proviral genome, Hbz is required for the controlled proliferation and clonal expansion of HTLV-1-infected CD4+ T cells. Hbz can also promote the transformation of infected T cells by preventing apoptosis and reducing genomic stability. Tax and Hbz are not the only viral accessory proteins that influence the transformation of HTLV-1, as p12, p13, and p30 have individually been shown to play unique roles that contribute to this dynamic process. In addition to viral proteins encoded by HTLV-1, a multitude of cellular factors have also been illustrated to either promote (Nox5α and MEF-2) or repress (PDLIM2) the transformation of HTLV-1-infected cells.HTLV-1 is a transactivating retrovirus that has the potential to induce ATL and/or various inflammatory diseases following an extensive clinical latency period of up to several decades. Given the nature of ATL to be an extremely aggressive and chemotherapy resistant CD4in vivo models including rabbits, mice, and Drosophila have been critical in our understanding of this oncogenic virus. The role of Tax and Hbz in the transformation process is undisputed, however several unknowns remain: (1) How important are other viral proteins in the transformation process? (2) What is the timing and expression level of Tax and Hbz required for efficient transformation? (3) Which cellular factors are critical for regulating Tax/Hbz? and (4) Which cellular factors are required to push an immortalized cell to oncogenesis/a diseased state? Since the discovery of HTLV-1 over 40 years ago, there has been considerable progress made towards understanding the viral transformation process. As the molecular tools and models improve, we will undoubtedly continue to refine our understanding of the dynamic HTLV-1-mediated cellular transformation process.Molecular tools such as co-culture immortalization assays and


EMBO Molecular Medicine by Kaur et al (2021) reveals that treatment of Wnt‐addicted cancer cells with inhibitors of Wnt signaling induces a state of BRCAness leading to hypersensitivity to PARP inhibitors. This is a new example of induced synthetic lethality that could pave the way for new indications for PARP inhibitors or may contribute to the long‐awaited clinical introduction of therapeutic agents targeting the Wnt pathway.Despite considerable efforts, therapeutic strategies targeting the Wnt pathway are still not clinically available. The pervasive role of Wnt‐βcatenin signaling for the control of stem cells during normal tissue homeostasis makes the on‐target toxicity of available therapeutic grade molecules an important limitation preventing their clinical introduction. The article in this issue of  et al (this issue of EMBO Mol Med) that shows that treatment of Wnt‐addicted cancer cells with inhibitors of Wnt signaling induces a state of BRCAness leading to hypersensitivity to PARP inhibitors.S. Angers discusses a new example of induced‐synthetic lethality reported by Kaur  This state of impaired HRR activity, which predicts the efficacy of PARP inhibitors, is commonly referred to as “BRCAness” as it phenocopies to some extent the germ line inactivation of the BRCA1/2 genes  induces a vulnerable state in tumor cells that can be exploited by a second treatment (in this case a PARP inhibitor) Fig . While PRNF43 mutations but that are resistant to Wnt pathway inhibition, possibly the result of additional mutations evading a requirement for βcatenin signaling for the proliferation of these cells. This result suggests that harnessing this induced synthetic lethal interaction could be effective in the context of tumors resistant to Wnt pathway inhibitors or alternatively slow down the emergence of therapeutic resistance.Finally, the functional interaction between Wnt‐βcatenin signaling and the DNA damage pathways is distinct from the well‐established role of the Wnt pathway in progenitor cell self‐renewal. Consistent with this, synergy between Porcupine and PARP inhibitors was also observed in pancreatic cancer cell lines that harbor


The new structures were elucidated on the basis of extensive 1D and 2D NMR, HR ESIMS spectroscopic analyses, and electronic circular dichroism (ECD) calculations. The absolute configuration of 2 was established by application of Mosher’s method. Compounds 4–8 exhibited moderate cytotoxicities against A549 and HeLa cell lines with the IC50 values less than 20 μM.Four new cytochalasans, phychaetoglobins A–D ( Chaetomium is the rich source of cytochalasans, among which Chaetomium globosum can produce plenty of such secondary metabolites +.1H NMR data of 2d (600 MHz in DMSO-d6): δH 10.91 , 8.20 , 7.46, 7.27 , 7.06 , 7.03 , 6.97 , 6.95 , 6.36 , 6.13 , 5.24 , 5.12 , 4.07 , 3.83 (m H-3), 2.85 , 2.71 , 2.67 , 2.60 , 2.49 , 2.18 , 2.16 , 2.01 , 1.98 , 1.64 , 1.27 , 1.21 , 1.18 , 1.03 , 0.89 , 0.82 , 0.80 . HRESI MS: m/z 703.1738 [M + H]+.A conformational search was carried out in the MMFF94 molecular mechanics force field using the MOE software package , and all1–16 against human carcinoma cells HeLa and A549 were determined using the 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The cells were maintained in RPMI-1640 containing 10% (v/v) fetal bovine serum (FBS) and 0.4% (v/v) penicillin–streptomycin solution  at 37 °C under 5% CO2. The cells were digested by trypsinization and then diluted to a concentration of 1 × 104 cells/mL. The diluted cell suspensions were then placed into 96-well microtiter plates and incubated with the test samples for 72 h. The control contained 2 μL MeOH. After incubation, the MTT solution was added and plates were incubated for 4 h. The supernatant liquid was removed, and the cells were disrupted with 150 μL DMSO for 10 min. The absorption was measured at 570 nm.The cytotoxic activities of 1–4), together with twelve known cytochalasans (5–16), were identified from the culture of C. globosum kz-19. All cytochalasans were tested for their cytotoxicities against HeLa and A549 tumor cell lines. Among compounds 4–8, those that contained a C-5/C-6 double bond and a C-7 hydroxyl group or a C-6/C-7 epoxide ring exhibited strong cytotoxic activities with the IC50 values of 3.7–13.7 μM. Compound 1 represents the first example of a cytochalasan with a five-membered lactone fused in the macrocyclic ring. Natural cytochalasans containing a cyclopentenone or a pyrrole moiety in the macrocyclic ring are rare. To our knowledge, only four natural cytochalasans containing a cyclopentenone ring have been reported, including cytoglobosin A ,,1–4), toobosin A . Furtherobosin A  and armoobosin A . Therefo


Public concern regarding environmental contaminants (ECs)-related reproductive disorders has increased due to increasing global rates of infertility. All kinds of ECs are on rise rapidly in developing and industrializing low- and middle-income countries. The aquatic environments throughout the world are repositories for enormous amounts of ECs. As the biology of the reproductive system is highly conserved in vertebrates, wildlife or laboratory studies on fish provide significant information to establish a detailed risk assessment, and to identify novel or more sensitive endpoints for ECs-related reproductive disorders. The adverse effects of ECs on endocrine regulation of reproduction in male fishes have been extensively studied and reviewed; however, our knowledge on the effects and mechanisms of action of ECs on determinants of male fertility is limited. The present study is a state-of-the-art comprehensive review on the ECs-related fertility threat in male fishes with emphasis on the ECs effects on sperm production, morphology, genome, and motility kinetics. After a brief introduction to reproductive biology, fertility indicators, and determinants of fertility in male fishes, wildlife evidences for reproductive disorders were reviewed in fishes from the polluted aquatic environment. The laboratory studies show that ECs detected in aquatic environment are capable of causing fertility threat at environmentally relevant concentrations associated with a decrease in fertility determinant(s). This study suggests an urgent need to better elucidate mechanisms through which ECs affect sperm functions to cause fertility threat.Increasing global rates of diminished fertility in males has been suggested to be associated with exposure to environmental contaminants (ECs). The aquatic environments are the final repository of ECs. As the reproductive system is conserved in vertebrates, studies on the effects of ECs on fertility endpoints in fishes provide us with valuable information to establish biomarkers in risk assessment of ECs, and to understand the ECs-related fertility threat. The aim of the present review was to evaluate associations between ECs and fertility determinants to better understand ECs-related male fertility threat in male fishes. Wildlife studies show that the reproductive system has been affected in fishes sampled from the polluted aquatic environment. The laboratory studies show the potency of ECs including natural and synthetic hormones, alkylphenols, bisphenols, plasticizers, pesticides, pharmaceutical, alkylating, and organotin agents to affect fertility determinants, resulting in diminished fertility at environmentally relevant concentrations. Both wildlife and laboratory studies reveal that ECs adverse effects on male fertility are associated with a decrease in sperm production, damage to sperm morphology, alternations in sperm genome, and decrease in sperm motility kinetics. The efficiency of ECs to affect sperm quality and male fertility highly depends on the concentration of the contaminants and the duration of exposure. Our review highlights that the number of contaminants examined over fertility tests are much lower than the number of contaminants detected in our environment. The ECs effects on fertility are largely unknown when fishes are exposed to the contaminants at early developmental stages. The review suggests the urgent need to examine ECs effects on male fertility when a fish is exposed at different developmental stages in a single or combination protocol. The ECs effects on the sperm genome are largely unknown to understand ECs-related inheritance of reproductive disorders transmitted to the progeny. To elucidate modes of action of ECs on sperm motility, it is needed to study functional morphology of the motility apparatus and to investigate ECs-disrupted motility signaling. Global rates of environmental contaminants (ECs)-related reproductive disorders have been increasing over the past 50 years. In human beings, the incidences of testicular dysgenesis syndrome, including hypospadias (urethra opens on the underside of the penis instead of the tip), cryptorchidism (one or both testes not descended into the scrotum), testicular cancer, low semen quality, and infertile men, show global increases associated with ECs ,7,8,9. LThe aquatic environment is at greatest risk from pollutants since all chemicals will eventually find themselves in the rivers, lakes, and oceans as the final repository . As biolWe have recently reviewed the toxicity of ECs on sperm morphology and motility in fishes, in vitro . The revThe present study is a state-of-the-art comprehensive review on the ECs-related fertility threat in male fishes with emphasis on the adverse effects of ECs on determinants of fertility, including sperm production, morphology, genome, and motility kinetics. After a brief introduction to reproductive biology, fertility indicators, and determinants of fertility in male fishes, wildlife evidences for reproductive disorders are reviewed in fishes from the polluted aquatic environment. To understand whether reproductive disorders in wildlife is associated with ECs, we review laboratory studies in which the adverse effects of particular ECs are studied on determinants of fertility, in vivo. Finally, we discuss urgent needs to better elucidate mechanisms through which ECs affect sperm functions to cause fertility threat. The present review provides us with valuable information to understand ecotoxicological impacts of ECs on fish fertility, which can be useful to establish biomarkers in ECs risk assessment.It is essential to review reproductive biology, fertility indicators, and determinants of fertility in male fishes before delving into the ECs-related fertility threat. These provide the basic information to better understand multiplicity of sites through which ECs interfere with fertility. To clarify the terminology, “semen” refers to seminal plasma and sperm and “sperm” refers to sperm cells in the present review.In general, the male reproductive organ consists of a paired testes, the testicular duct, and the sperm duct in fishes ,29 Figu. In someThe testes are divided into the “tubular type” and the “lobular type” according to the distributions of spermatogonia in the seminiferous region ,31,32,33Danio rerio) and guppy (Poecilia reticulate).In the tubular type, as spermatogonia divide and enter in meiosis, the cysts migrate towards the region of the spermatic ducts located in the central region of the testis, where the cysts open to release sperm E. This tOryzias latipes), common carp (Cyprinus carpio), goldfish (Carassius auratus), and rainbow trout (Oncorhynchus mykiss).In the lobular type, the testis is composed of numerous lobules that are separated from each other by a thin layer of fibrous connective tissue, and spermatogonia are spread along the germinal compartment throughout the testis. The cysts do not migrate or become displaced during their development, and sperm is released into the lobular lumen F. This tThe testicular compartment contains Sertoli cells, Leydig cells, blood/lymphatic vessels, macrophages and mast cells, and neural and connective tissue cells G. The LeThe testicular ducts are located adjacent to the testes, which continue into the sperm ducts on the ventral sides. Testicular and sperm ducts possess very similar structural and enzyme-histochemical characteristics, and play key roles in nutrition of sperm, storage of sperm, synthesis of steroids, secretion of proteins and enzymes, and formation of the seminal plasma ,35. MatuSperm is produced from spermatogonia following divisions ,38,39. DSperm is differentiated into a head, midpiece, and flagellum in fishes ,41,42 F. The heaThe seminal plasma is a product of Sertoli cells, testicular ducts, and sperm ducts, and its composition is different among fishes that may reflect species variations. The main role of seminal plasma is to create an optimal environment for the storage of sperm during maturation in the sperm ducts. Seminal plasma maintains sperm viability, motility, and fertilizing ability, and protects sperm against damage caused by proteolytic or oxidative attacks ,51.2+) ions in both freshwater and marine fishes, while cyclic adenosine monophosphate (cAMP) remains unchanged. However, studies show that demembranated sperm of salmonid and sturgeon fishes require cAMP for the axonemal beating 2 [194], 2 ,164.2 or BPA  to ECs causes damage to sperm and decreases sperm motility and velocity to decline sperm fertilizing ability; however, the effective concentrations are far exceeding the WHO recommended limits . The pre2, EE2, 17α-MT, 4-NP, BPA, BPS, and DEHP and its metabolite (MEHP) cause diminished fertility at environmentally relevant concentration (The present review shows that: (a) Unlike the source of contaminants from industrial, municipal, treatment plant, or agricultural activities, abnormalities in sperm morphology ,139, damntration . (c) Thentration . It is wntration . HoweverThe present review highlights the following aspects that should be taken into account in further studies to better elucidate the ECs-related fertility threat in male fishes.(a) One can easily distinguish that the number of contaminants that have been used in fertility tests are comparable to the number of contaminants in our environment. Future studies should examine the effects of other chemicals with major public concerns. Among them, there is an immediate need to examine the effects of pharmaceuticals  that are highly consumed in global medicine use, agriculture, and animal husbandry ,241. Fer(b) Examination of the adverse effects of ECs at environmentally relevant concentrations that cover the lowest and highest detected concentrations is highly needed. Most studies have failed to include the lowest environmental concentration among treatments .(c) The ontogeny-dependent effects of the ECs on male fertility are largely unknown. Basically, developmental stages are the main parameters to be carefully defined based on the objective. For instance, if the study is aiming at investigating the adverse effect of ECs on sperm production, it is a prerequisite to expose the fish from the early developmental stage of the testis. However, the present review shows that a number of studies that exposed fishes to ECs at early life stages is considerably lower than those that examined the adverse effects of ECs in adult-exposed individuals . When em(d) Assessment various types of determinants of fertility to better elucidate site of action of an EC that cause diminished fertility. There are a few studies that have evaluated the ECs effects on sperm production and motility .(e) The present study shows that ECs may cause phenotypic changes in the reproductive system and affect determinants of fertility . It has To better elucidate ECs effects on sperm morphology, future studies should investigate functional structure of sperm including nucleus, mitochondria, centrioles, and axoneme, with particular consideration to the organization of the microtubules and motor protein called “dynein” in the latter case ,246,247.Regarding the ECs effects on sperm genome, it is needed to examine effects of ECs on incidence of aneuploidy , to stud2+, and cAMP) that regulate sperm motility signaling are fully unknown and should be considered in future studies [2+ through activation of a Ca2+ channel to interfere sperm motility in human. The laboratory studies suggest that ECs may affect sperm swimming pattern as sperm velocity parameters are decreased  was exposed to 0.1 µg/L EE2, which was associated with decrease in VSL. This suggests that the adverse effects of EE2 on sperm velocity could be related to amount of ATP available to generate flagellar beating force. Further studies are needed to investigate sperm energetics along with sperm motility kinetics to better understand the mechanisms through which ECs cause fertility threat.(f) Sperm requires energy for motility and flagellar beating force ,68,106. However, assessment of the adenylate energy charge (AEC = ATP + 0.5 ADP/ATP + ADP + AMP) is suggested to better understand the adverse effects of ECs on sperm energetics and metabolism of energy ,66,249. (g) In an aquatic environment, there are mixtures of ECs that may exhibit pharmacodynamic interactions. These interactions can be antagonistic or synergistic, which may cause an increase in the toxicity of a particular chemical through the cocktail effect ,142. For(h) Our current knowledge on the ECs-related male fertility threat is mainly from freshwater fishes inhabiting lakes or rivers . Future (i) Recently, epigenetic inheritance of the adverse effects of ECs received high scientific and public considerations. As ECs have been shown to affect the sperm genome, further studies need to characterize transgenerational inheritance of the adverse effects of ECs to the offspring. There are studies that show the adverse effects of bisphenols and 4-NP that were inherited to the next generations ,167; how(j) The threat of ECs to fish populations is still an open question. There are historical reports that show populations of fishes have declined ,251,252;We also would like to offer the following suggestions to normalize the studies for better understanding of the ECs-related fertility threat in fishes.(a) The large variations among results of studies that have examined a particular contaminant suggest the need to optimize a protocol for harmonization of studies, which should encompass both biological and technical issues. These may include a protocol for optimizing the design of exposure tests, methods for evaluating male fertility, and laboratory tools to assess determinants of fertility. For example, (a-i) CASA has become very popular for the analyses of sperm motility kinetics. Technical conditions for recording of sperm motility and setting of CASA affect the results , thus ne(b) The present study suggests species-specific adverse effects of an EC on male fertility and its determinants. To better understand sensitivity of a species to an EC, the effects of ECs could be examined on small laboratory fishes including zebrafish, guppy and medaka, which serve as aquatic model organisms. Application of species with different biology of reproduction will provide us with valuable information to assess ecological risk assessment of ECs. Among species, zebrafish and Medaka, in contrast to guppy, are external fertilizers, and the testes are of the tubular type in zebrafish and guppy compared to those of medaka that are of the lobular type.(c) Although all studies have considered this note, it is worth reminding that one should take into account renewal of exposure medium based on the half-life of the EC.