Service sire has a considerable impact on reproductive success in dairy cattle. Most gene mapping studies for bull fertility have focused on additive effects, while non-additive effects have been largely ignored. The main goal of this study was to assess the relevance of non-additive effects on Sire Conception Rate (SCR) in Holstein dairy cattle. The analysis included 7.5 k Holstein bulls with both SCR records and 57.8 k single nucleotide polymorphism (SNP) markers spanning the entire genome.ADAM28, DNAJA1, TBC1D20, SPO11, PIWIL3 and TMEM119, that are directly implicated in testis development, male germ line maintenance, and sperm maturation.The importance of non-additive effects was evaluated using an efficient two-step mixed model-based approach. Four genomic regions located on chromosomes BTA8, BTA9, BTA13 and BTA17 showed marked dominance and/or recessive effects. Most of these regions harbor genes, such as This study provides further evidence for the relevance of non-additive effects in fitness-related traits, such as male fertility. In addition, these findings may point out new strategies for improving service sire fertility in dairy cattle via marker-assisted selection. Reproduction efficiency is arguably a very important economic trait in dairy cattle. Reproductive inefficiency results in increased calving intervals, increased involuntary culling rates, decreased milk production, and delayed genetic progress, among other problems. All this causes significant economic losses . It is cMAP1B and PPP1R11, were implicated in male fertility in Holsteins [FOXJ3 genes related to bull sperm function [There is growing evidence that genetic factors explain part of the differences in fertility among dairy sires. Indeed, some semen production and quality traits, such as sperm concentration, sperm motility, and percentage of abnormal sperm, have moderate to high heritability . In addiolsteins . A recenfunction . Our grofunction . These rfunction , 18. OveIt is important to remark that association studies primarily focus on the identification of genetic variants with additive effects. On the other hand, non-additive genetic effects receive in general much less attention. Indeed, it remains controversial the relative importance of non-additive effects on complex traits , 20. It Since August 2008, the US dairy industry has access to a national phenotypic evaluation of service sire fertility called Sire Conception Rate (SCR). This bull fertility evaluation is exclusively based on cow field data. The current model considers not only factors closely related to the bull under evaluation , but also different factors (nuisance variables) associated with the cow that receives the unit of semen, such as cow age, parity, and milk yield , 22. Thehttps://www.uscdcb.com/). For bulls with multiple fertility evaluations, the most reliable SCR record (i.e. the SCR record with most breedings) was used in the analyses.A total of 7458 Holstein bulls with SCR data were used in this study. Sire conception rate values ranged from − 16.6% to + 14.1%. These SCR records were obtained from 27 consecutive evaluations provided to the US dairy industry between August 2008 and August 2017. These bull fertility evaluations are available at the Council of Dairy Cattle Breeding (CDCB) website (Genome-wide single nucleotide polymorphism (SNP) data for the 7458 Holstein bulls were provided by the Cooperative Dairy DNA Repository (CDDR). SNP information  was based on the bovine genome reference UMD3.1. Markers that mapped to the sex chromosomes, or were monomorphic, or had minor allele frequency less than 1% were removed from the dataset. After data editing, a total of 57,838 SNP markers were retained for subsequent analysis.Figure y = Xβ + Zu + e, where y is the vector of SCR records, β is the vector of fixed effects included in the model, u is the vector of random animal effects, and e is the vector of random residual effects. The matrices X and Z are the incidence matrices relating phenotypic records to fixed and animal effects, respectively. The random effects are assumed multivariate normal with K is a kinship matrix that can be calculated using either pedigree or genotypic information, and R is typically an identity or a diagonal matrix.The association between phenotypes and SNP genotypes using related individuals can be implemented within the framework of the classical animal model, y = Xβ + XSNPβSNP + Zu + e, where XSNP is the design matrix for the SNP under study and βSNP is the regression coefficient or SNP effect. In this context, the significant effect of the SNP marker can be tested using a standard Wald statistic computed from the ratio of the estimate of βSNP and its standard error. However, in our case, the application of this test across the whole genome is computationally demanding, and hence, a two-step mixed model-based approach was implemented [For the whole-genome scan, the model can be rewritten as, lemented , 24.y = Xβ + Zu + e, without including SNP was fitted. The random effects were assumed multivariate normal with G is the genomic relationship matrix and I an identity matrix. Note that for computational reasons, the reliabilities of the SCR values were not considered in the analysis, and hence, the R matrix was reduced to an identity matrix. The variance-covariance matrix was then estimated as At the first step, the following model, y = Xβ + XSNPβSNP + ϵ, assuming R package MixABEL [At the second step, the following model was fitted for every SNP,  MixABEL .GenABEL [e−07) was used to control for multiple testing.The genomic control procedure was applied to correct for a possible inflation of the test statistics using the function VIFGC implemented in the R package GenABEL . BonferrImproving reproductive performance remains a major goal in dairy cattle. Service sire has been recognized as an important factor affecting herd fertility. Selective breeding is one strategy for improving bull fertility. Indeed, recent studies have identified and evaluated potential fertility-related biomarkers that could be used to select high fertility bulls , 17, 27.Pvalue < 0.01). Table Figure The distribution of SCR values for each of the four SNP loci with significant dominance effects is displayed in Fig. ADAM28 and DNAJA1, that might be directly related to service sire fertility. Gene ADAM28 encodes a member of the disintegrin and metalloproteinase family, a group of membrane proteins implicated in a variety of biological processes involving cell-cell and cell-matrix interactions, including gametogenesis and fertilization [ADAM28 is predominantly expressed in the epididymis, denoting a direct role in sperm maturation [DNAJA1 is a member of the DnaJ family, a group of molecular chaperons that assists other proteins in their folding, transport and assembly into complexes [DNAJA1 plays an important role in testis development and spermatogenesis through the regulation of androgen receptor signaling in Sertoli cells [DNAJA1 function in mice results in the accumulation of androgen receptor protein in Sertoli cells, which in turn hyperactivates the androgen receptor signaling pathway, leading to testicular hypogonadism and severe defects in spermatogenesis [The significant region in BTA8 located at 73–76 Mb harbors at least two candidate genes, namely turation . Moreoveomplexes . Interesli cells . Indeed,ogenesis .TBC1D20, SPO11, RAD21L1 and BCL2L1, are located in the significant region detected in BTA13 at 58–62 Mb. Gene TBC1D20 encodes a GTPase-activating protein that is directly implicated in sperm biology, especially in acrosome assembly [TBC1D20 gene in mice results in defects in the acrosomal formation, which in turn leads to male infertility [SPO11 and RAD21L1 are directly implicated in the meiosis, a key step during gametogenesis. Gene SPO11 is a topoisomerase-like protein responsible for the formation of DNA double-strand breaks that occur during meiotic recombination [SPO11 leads to severe gonadal abnormalities, including the apoptotic death of spermatocytes during early meiosis [SPO11 significantly associated with male infertility [RAD21L1 encodes a protein that is part of large multi-subunit complexes, known as cohesins, that play important roles in many aspects of meiotic chromosome dynamics [RAD21L1 is specially implicated in male meiosis; indeed, the disruption of RAD21L in mice precludes the initial association of homologous chromosome as well as the subsequent pairing in spermatocytes [BCL2L1 encodes a member of the BCL-2 protein family, a well-studied group of proteins that either promote or inhibit apoptosis [BCL2L1 is directly involved in the regulation of germ line apoptosis in the testis [At least four putative candidate genes for bull fertility, assembly . Indeed,ertility . Moreovebination . It has  meiosis . Interesertility . On the dynamics . Recent atocytes . Finallypoptosis . Notablye testis .PIWIL3 and TMEM119. Gene PIWIL3 encodes a member of the PIWI family, a group of proteins that appear to be restricted to the germ line and bind to a subclass of germ cell-specific small RNAs [PIWIL3 was significantly associated with an altered risk of oligozoospermia in humans [TMEM119 encodes a transmembrane protein actively involved in osteoblast differentiation. Recent studies have shown that TMEM119 also affects different male fertility traits. The gene TMEM119 is expressed in spermatocytes and spermatids in the developing testis, and TMEM119-deficient mice showed impaired testis development and spermatogenesis, resulting in a significant decrease in both testis weight and sperm number [TMEM119 perturbs late spermatogenesis, specifically at the round spermatid stage [The region in BTA17 located at 64–68 Mb harbors two putative genes for service sire fertility, all RNAs . These pall RNAs . Notablyn humans . Moreovem number . Currentid stage .In this study, we investigated the relevance of non-additive effects on bull fertility in Holstein cattle. Four genomic regions in BTA8, BTA9, BTA13, and BTA17 showed marked dominance and recessive effects. These regions harbors genes with known roles in male fertility, including testis development, spermatogenesis, and sperm maturation. Overall, our results provide more evidence for the relevance of non-additive effects in traits closely related to fitness such as male fertility. In addition, these findings may contribute to the development of new strategies for improving sire fertility via selective breeding and marker-assisted selection.


It has been shown that saturated fatty acids (FAs) have a detrimental effect on pancreatic β-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in β-cells remain unclear; however, mitogen-activated protein (MAP) kinase and endoplasmic reticulum (ER) stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA) affects the effect of saturated stearic acid (SA) on the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic β-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.  Increased levels of fatty acids (FAs) in blood are considered to be one of the main factors responsible for pancreatic β-cell death in type 2 diabetes ,2,3,4,5.The p38 MAPK pathway becomes mainly activated by the dual-specific MKK3/6 (mitogen-activated protein kinase kinase 3 and 6) kinase due to various extracellular stimuli such as chemical stresses (reviewed in ). p38 MAThe ERK pathway is mostly activated by growth factors. Growth factors, acting through receptor kinases and adaptor protein son of sevenless (SOS), activate Ras GTPase, which is responsible for c-Raf phosphorylation . This kiER stress signaling is represented by three known pathways. Their activation seems to begin on the ER membrane by the activation of three proteins: (1) IRE1α (inositol-requiring protein 1α); (2) PERK (protein kinase RNA (PKR)-like ER kinase); and (3) ATF6 (activating transcription factor 6). Activated IRE1α causes unconventional splicing of mRNA for XBP1 (X-box binding protein 1), which leads to the translation of active transcription factor (XBP1s). It also leads to JNK  activation by phosphorylation, which further phosphorylates c-Jun. Activated PERK results in the inhibition of protein translation via eIF2α (eukaryotic initiation factor 2α) phosphorylation. ATF6 translocation to the nucleus, where ATF6 functions as a transcription factor, represents the activation of the ATF6 pathway. The main purpose of these signaling patterns is to restore ER homeostasis, e.g., by decreasing protein translation and increasing the expression of chaperones, such as the ER chaperone BiP (immunoglobulin heavy chain-binding protein) . HoweverIn the present study, we tested how unsaturated OA affects the effect of saturated SA on the p38 MAPK and ERK pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic β-cells NES2Y. We demonstrate that OA is able to inhibit all effects of SA. OA alone had minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.We showed that OA (0.2 mM) inhibited SA-induced (1 mM) cell death of NES2Y cells. OA applied alone has no or only minimal effects on NES2Y cells A. Cell dWe previously showed the activation of the p38 MAPK signaling pathway and the inhibition of the ERK signaling pathway within 24 h of the application of apoptosis-inducing SA (1 mM) in NES2Y β-cells . In ordeThe effect of SA on the activation of the p38 pathway members, i.e., an increase in the level of phosphorylated MKK3/6, p38 MAPK, and MAPKAPK-2, was inhibited by co-incubation with OA as early as 12 h after the treatment in all of the tested proteins. OA applied alone resulted in low or nearly no changes in the level of phosphorylated p38 MAPK pathway members. No change was detected in the level of total p38 MAPK within 24 h of FA treatment A.The effect of SA on the inhibition of the ERK pathway members, i.e., a decrease in the level of phosphorylated c-Raf, MEK1/2, and ERK1/2, was also inhibited by OA co-incubation within 24 h of the treatment. Separate application of OA resulted in nearly no effect on the level of phosphorylated ERK pathway members. We did not detect any change in the level of total ERK1/2 within 24 h of FA treatment B.We previously documented the activation of ER stress signaling pathways (IRE1α and PERK pathways) within 24 h of apoptosis-inducing SA (1 mM) treatment in NES2Y β-cells . In ordeThe effect of SA on the activation of ER stress pathways, i.e., an increase in the levels of phospho-IRE1α (and IRE1α), phospho-JNK (JNK level was not changed), phospho-c-Jun (and c-Jun), phospho-eIF2α (eIF2α level was not changed), CHOP and BiP, as well as the induction of XBP1 splicing, was significantly inhibited by OA co-incubation within 24 h of the treatment. Separate application of OA resulted in nearly no changes in the level of tested proteins and XBP1 splicing .We, as well as other authors, have shown that unsaturated FAs have nearly no detrimental effect on pancreatic β-cells, but that they are in fact capable of inhibiting the pro-apoptotic effect of saturated FAs ,5,6,7,8.Our data showed that the activation of the p38 MAPK pathway, inhibition of the ERK pathway, and activation of the IRE1α and PERK ER stress pathways by SA were inhibited by OA see . The inhWe also provided a pilot experiment , in which we tested the effect of OA on the SA-induced activation of the ATF6 pathway of ER stress signaling. We found (data not shown) that SA-induced ATF6 activation and translocation into the nucleus did not seem to be inhibited by OA co-incubation. Separate administration of OA had no effect on ATF6 translocation. However, this finding was not confirmed by an independent method.Our findings concerning both MAP kinase signaling pathways as well as ER stress signaling pathways support the fact that the point of OA inhibitory intervention in SA-induced apoptotic signaling is located upstream of this signaling. Our preliminary data, as well as some previous studies ,30, indiIn conclusion, we have shown, employing human pancreatic β-cells NES2Y, that unsaturated OA is able to inhibit the effects of saturated SA on the activation of the p38 MAPK signaling pathway and inhibition of the ERK signaling pathway, as well as activation of the IRE1α and PERK ER stress signaling pathways. OA alone had minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.Chemicals were from Sigma-Aldrich , unless otherwise stated. The following primary and secondary antibodies for Western blot analysis were used: anti-phospho-MKK3/6 (#9236), anti-p38 MAPK (#8690), anti-phospho-p38 MAPK (#4511), anti-phospho-MAPKAPK-2 (#3007), anti-phospho-c-Raf (#9427), anti-phospho-MEK1/2 (#9154), anti-ERK1/2 (#5013), anti-phospho-ERK1/2 (#4370), anti-BiP (#3177), anti-CHOP (#2895), anti-eIF2α (#9722), anti-phospho-eIF2α (#9261), anti-IRE1α (#3294), anti-SAPK/JNK (#9258), anti-phospho-SAPK/JNK (#4668), anti-c-Jun (#9165) and anti-phospho-c-Jun (#9261), anti-PARP (#9542), anti-cleaved caspase-7 (#9491), anti-cleaved caspase-8 (#9496), anti-cleaved caspase-9 (#9505) from Cell Signaling Technology , anti-phospho-IRE1α (ab 48187) and anti-GAPDH (ab9485) from Abcam , and anti-actin (clone AC-40).The human pancreatic β-cell line NES2Y employed in the experiments was kindly provided by Roger F. James  ,32. NES2Our previous studies showed that SA, at a concentration of 1 mM, leads to the activation of the p38 MAPK and ER stress signaling pathways and to the inhibition of the ERK signaling pathway within 24 h of the treatment . Therefo3 cells/100 μL of culture media. After a 24-h pre-incubation period  the culture medium was substituted with a serum-free medium containing 2% BSA with or without fatty acid(s). The control medium contained 2% BSA only. After 96 h of incubation, the number of living cells was assessed using a hemocytometer counting system, after staining with trypan blue.Cells were seeded into the wells of a 96-well plate at a concentration of 5 × 106 cells per sample) were seeded and, after a 24-h pre-incubation period , the culture medium was substituted with a serum-free medium containing 2% BSA with or without FA(s)  at required concentrations. The control medium contained 2% BSA only. After the required incubation period, cells were harvested and Western blot analysis was performed as described previously [Cells (approximately 1 × 10eviously . All pri6 cells per sample) were seeded and, after 24 h pre-incubation, FAs and thapsigargin were applied as described above (see Cells (approximately 1 × 10bove see . After 3The statistical significance of observed differences was determined using the Tukey test.


This review will summarize the current evidence for biased signaling through cannabinoid receptor subtypes CB1 and CB2.An agonist that acts through a single receptor can activate numerous signaling pathways. Recent studies have suggested that different ligands can differentially activate these pathways by stabilizing a limited range of receptor conformations, which in turn preferentially drive different downstream signaling cascades. This concept, termed “biased signaling” represents an exciting therapeutic opportunity to target specific pathways that elicit only desired effects, while avoiding undesired effects mediated by different signaling cascades. The cannabinoid receptors CB Biased signaling is the concept that different ligands acting on the same G protein-coupled receptor (GPCR), in the same tissue, can give rise to markedly different cellular responses . Quantifying biased signaling involves determining the effects of two or more agonists on two or more cellular responses, and comparing the agonist profiles for each pathway. More recently, the operational model of bias has been utilized5 Hence, the development of an agonist that preserves the analgesic (G protein-mediated) properties of activated μ-opioid receptors without any respiratory side effects would be beneficial to avoid adverse effects, and such compounds have recently been developed in light of this hypothesis.7Therapeutically, it is hoped that the study of biased signaling of GPCR-directed therapeutics will enable engagement of desired pathways over those not involved in the therapeutic effect. Ideally, this would eliminate on-target but unwanted or adverse effects; agonists or allosteric modulators that induce signaling in a biased manner could potentially revolutionize future therapeutic drugs. The μ-opioid receptor presents a prime example where biased signaling might be exploited at the translational level. Although opioid receptors are useful targets of analgesics, μ-opioid receptor activation also causes respiratory depression, which is suggested to be a product of β-arrestin2 recruitment.1) and cannabinoid receptor 2 (CB2), and they will be the focus of this review. CB1 plays a role in regulating neurotransmission in many brain regions. When activated, CB2 regulates immune responses and inflammatory pathways. Mice lacking CB2 often demonstrate an exacerbated inflammatory phenotype8 and besides roles in the periphery expression in brain microglia suggest a role for CB2 in neuroinflammation.10 Recently, CB2 has been suggested to contribute to neuronal plasticity in mouse hippocampal neurons11 potentially expanding the role of this receptor in the brain. The endogenous lipids anandamide and 2-arachidonoylglycerol (2-AG) are the physiological cannabinoid receptor agonists and are hence known as endocannabinoids.12–14 A great deal of interest has centered on the potential role of CB1 in targeting a range of central nervous system (CNS) disorders such as pain,15 anxiety,16 multiple sclerosis,17 obesity,18 nicotine addiction,19 Huntington disease,20 and Parkinson's disease.21 In more recent times CB2 has also become a focus in peripheral inflammatory disorders such as nephrotoxicity.22The cannabinoid receptor family consists of two GPCRs, cannabinoid receptor 1  channels26 and glycine receptors,27 respectively. The potential contribution of these receptors to the therapeutic effects of cannabinoids or the physiological effects of endocannabinoids are only beginning to be explored, and much less is known about how ligands regulate their signaling.In addition to the two well-established cannabinoid receptors, several other GPCRs have been reported to be activated by cannabinoid drugs or endocannabinoids and related molecules, including GPR55,1 in the CNS provides a strong rationale for developing ligands with biased profiles to potentially avoid the consequences of activating multiple signaling pathways in many different brain regions. Biased ligands could also potentially have context-dependent effects, providing effective modulation of pathways dysregulated by disease in restricted subsets of neurons. Modulating selected downstream pathways would result in a more targeted pharmacological response, but considerable research is still required to understand which of the characterized pathways are therapeutically desirable, and under which disease condition.The widespread distribution of CB29 both CB1 and CB2 were initially described as exhibiting pertussis toxin-sensitive signaling through Gαi/o-type G proteins,30 however, differences in signaling were reported from the outset. When expressed in AtT-20 cells, CB1 mediated an inhibition of P/Q-type calcium channels and an activation of G protein-gated inwardly rectifying potassium channels (GIRKs) in addition to inhibition of adenylyl cyclase.30 By contrast, CB2 was not reported to modulate the activity of either channel in AtT-20 cells but did inhibit adenylyl cyclase.30 This poor coupling may be an example of functional selectivity as it was recently found that mouse CB2 (mCB2) can inhibit voltage-gated calcium channels, in a manner strongly dependent on the CB2 ligand used, with CP55,940, but not WIN55,212-2, able to inhibit the channels via a Gβγ pathway.31 It is unclear how far this apparent biased signaling extends, however, as both WIN55,212-2 and CP55,940 hyperpolarize AtT-20 cells expressing human CB2 (hCB2), and this response almost certainly reflects Gβγ subunit-mediated activation of GIRKs.32First cloned in the early 1990s,33 demonstrated that while CB1 and CB2 had similar affinity for Gαi-type G proteins, CB2 had significantly lower affinity for Gαo than Gαi. CB1 is promiscuous in its G protein coupling, with activation of Gαs- and Gαq-dependent signaling under some conditions, or in some cells. Gαs-like coupling has been suggested under circumstances where Gαi activation is limited, such as following pertussis toxin treatment, or simultaneous activation of CB1 with other Gαi linked receptors such as D234–36; indeed it appears that in some cell types the Gαs linkage is dominant.38 Studies have suggested that the second intracellular loop of CB1 mediates both Gαs and Gαi coupling specificity,39 but a detailed understanding of what regulates the specific G protein coupling is still lacking. Gαq coupling of CB1 has been suggested for only a few ligands40 and will be discussed in more detail below. Finally, in vivo studies have also suggested coupling of CB1 to the Gαi/o-related G protein Gαz.41 The ability to activate such a diverse range of G proteins strongly suggests that biased signaling between different G protein pathways could be achieved, and examples of this are emerging and described below. Apart from the interactions with Gαi and Gαo less is known about additional G proteins interacting with CB2.In studies using purified G proteins, Glass and Northupi, Gαo, Gαs, and Gαq heterotrimers have been described for many GPCRs, and this signaling seems similar for the most part for CB1 and CB2. Gαi/o subunits inhibit adenylyl cyclase or couple to the mitogen-activated protein kinase (MAPK) pathway. Gαs stimulates adenylyl cyclase (and subsequent phosphorylation of cAMP response element-binding protein [CREB]). Gαq couples to phospholipase C and promotes release of intracellular calcium ([Ca]i). Gβγ subunits derived from Gαi/o activate GIRKs (comprising KIR 3.X heteromers), activate phosphatidylinositide-3-kinase, and inhibit voltage-dependent calcium channels (ICa).42–44The consequences of activating Gα45 Arrestin signaling has been generally thought to occur in the absence of bound G protein, and although this has been recently challenged,46 arrestin-mediated signaling is likely to be mediated by a different ligand/receptor conformation from those involved in Gα interactions.Originally described as “G protein-coupled” proteins based on their modulation of classical signaling pathways via heterotrimeric G proteins, GPCRs also recruit other proteins for signaling, most prominently β-arrestin-1 and -2. Arrestins serve multiple functions as regulators of G protein coupling and receptor localization, and are essential elements of multiple GPCR signaling cascades involving kinases, phosphatases, and ubiquitin ligases.47MAPK family members have been found to regulate diverse biological functions by phosphorylation of specific target molecules (such as transcription factors) and thereby participate in the regulation of a variety of cellular processes, including cell proliferation, differentiation, and apoptosis.1 activation can lead to phosphorylation of extracellular signal-regulated kinase 1 or 2 (ERK1/2). In Chinese hamster ovary (CHO), U373 MG, and PC-3 cells, phosphatidylinositide 3-kinase activation is required for CB1 activation of ERK1/2, and the Gβγ subunit, rather than Gα, may transduce the CB1 signal.49 In contrast to this, ERK1/2 activation in N1E-115 mouse neuroblastoma cells, or mouse hippocampal slices, is reported to be downstream of inhibition of cyclic adenosine monophosphate (cAMP)/protein kinase A.51As for most GPCRs, there are multiple pathways by which CB1 may also transactivate members of the receptor tyrosine kinase family leading to subsequent activation of ERK1/2. Several tyrosine kinase receptors have been implicated in this pathway, including the vascular endothelial growth factor receptor52 and epidermal growth factor receptor.53 The Src-family kinase, Fyn, is also likely to be involved in the activation of ERK1/2 by the CB1-transactivated tyrosine kinase receptors, as Fyn knockout (KO) mice show no elevation of phosphorylated ERK1/2 (pERK1/2) in response to cannabinoid administration.51CB1 activation, all of the above studies found the activation to be downstream of Gαi/o protein signaling (and therefore pertussis toxin sensitive), however, pertussis toxin-insensitive ERK1/2 activation has also been demonstrated in rat CB (rCB1)-transfected human embryonic kidney 293 (HEK293) cells.54 The Gαi/o protein-independent pERK1/2 activation might arise from the recruitment of β-arrestin-1 as mutation of phosphorylation sites in the CB1 C-terminal (S426A/S430A) has been suggested to result in β-arrestin-1-mediated pERK activation,55 which in general occurs later than the G protein-mediated activation.56 Similarly, as discussed below, ORG27569, an allosteric modulator of CB1, has been suggested by some studies58 but not others60 to activate β-arrestin-1-mediated pERK1/2 signaling through CB1.Regardless of the precise pathway mediating pERK1/2 downstream of CB1 has been reported, although there is considerable divergence between tissues and ligands used among the studies, which makes reconciling contrasting results difficult. In rat hippocampal slices, anandamide, CP55,940, WIN55,212-2, and Δ9-tetrahydrocannabinol (THC) activate p38 MAPK, but not c-Jun N-terminal kinase (JNK).61 In cultured cortical neurons, JNK was activated when stimulating with THC in a pertussis toxin-sensitive manner,62 however, HU-210 failed to stimulate ERK1/2, p38 MAPK, or JNK in a subsequent study on cultured hippocampal neurons.63 In Neuro-2a cells endogenously expressing mCB1, HU-210 activated only ERK1/2, but not JNK or p38 MAPK pathways,64 although a different study found that HU-210 stimulated JNK activation in these cells.65 Rueda et al.66 showed that THC-mediated activation of JNK was dependent on Gαi/o proteins, phosphatidylinositide 3-kinase, and Ras, and involved platelet-derived growth factor (PDGF) receptor transactivation in CHO cells. In contrast, in the same cells, THC activation of p38 MAPK was not dependent on PDGF receptor activation,66 suggesting the possibility of multiple CB1-stimulated pathways for kinase activation in the same cells.Activation of other MAPKs downstream of CB2 activation of MAPK pathways. While several studies have investigated the ability of cannabinoid agonists to modulate ERK1/2 activation in response to inflammatory stimuli,68 only one study has examined the pathway by which CB2 may link to ERK activation. In this study, hCB2 coupling to ERK1/2 was shown to be pertussis toxin sensitive but independent of cAMP.69 Some of the variability observed in the pathways activating kinases downstream of both CB1 and CB2 may reflect different levels of receptor expression between cells as previous studies have demonstrated that the ability to activate some kinases (most notably pAkt) required high receptor expression, whereas ERK1/2 activation occurred at all levels of expression.70There are relatively few studies on CB71 In this sequence of events, G protein receptor kinases (GRKs) have a prominent role phosphorylating the GPCR after agonist binding, and this phosphorylation increases the affinity of arrestin for the receptor. Arrestin binds to domains of the receptor thought to interact with G proteins, preventing receptor activation of these effectors. It is also possible that phosphorylation of a receptor before arrestin binding disrupts receptor/G protein interactions. The ultimate consequences of arrestin binding are complex; for many receptors, arrestin binding is a step in a pathway that leads to removal of the receptor from the plasma membrane, however, arrestin also acts as a scaffolding molecule for non-G protein-mediated signaling pathways such as activation of ERK.A common pathway for uncoupling GPCRs from G protein-dependent signaling is receptor phosphorylation followed by binding of an arrestin.1-mediated inhibition of agonist-stimulated adenylyl cyclase activity in N18TG2 cells.72 This work included showing that desacetyllevonantradol  inhibition of adenylyl cyclase maximally desensitized within 30 min of drug exposure.72 The desensitization of CB1 responses was homologous, as it did not affect the muscarinic receptor-mediated inhibition of adenylyl cyclase and did not affect the maximum accumulation of cAMP produced by the stimulatory agonist, secretin.73 THC also produced desensitization in this assay, but it was slower and less complete than that produced by CP54,939. Similar effects of CP55,940 on hCB1 expressed in CHO cells indicate that rapid desensitization of CB1 coupling to adenylyl cyclase is common.74 While these assays provide a good indication that receptor desensitization is rapid, interpretation of the quantitative elements of the studies is difficult as the assays utilized 20 min or more of agonist exposure to define control responses, and this period almost certainly encompasses many significant regulatory events.In a classic series of studies, Howlett et al. delineated CBICa or activation of GIRK is measured.75 Unfortunately, there are very few cell lines where native CB1 receptors coexist with voltage-gated ICa or GIRK. NG-108-15 and N18 cells, with endogenous CB1, were used for the very earliest descriptions of CB1 receptor inhibition of ICa,76–78 however, receptor regulation was not examined. Recombinant CB1 receptors expressed in murine AtT-20 cells couple to both inhibition of ICa and activation of GIRK,79 and desensitization of GIRK activation has been reported.80 AtT-20 cells and Xenopus oocytes have been used to provide some insight into CB1 regulation of ion channels.81 In oocytes, desensitization of rCB1-mediated activation of GIRK was shown to be stimulated by coexpression of both GRK3 and β-arrestin-2, but not affected by either protein alone.80 The efficiency of other members of the GRK family, or of β-arrestin-1, was not addressed in the oocyte studies, nor were the effects of coexpression of these molecules on basal coupling of CB1 to GIRK.By contrast to traditional single-point assays of cAMP accumulation or ERK phosphorylation, GPCR modulation of ion channels provides a continuous and relatively direct readout of receptor activity, particularly when Gβγ inhibition of 1 was sufficient to block GRK3/β-arrestin-2-mediated desensitization of coupling to GIRK in oocytes.80 When CB1 missing the last 55 intracellular residues was expressed in AtT-20 cells, WIN55,212-2 desensitization was abolished suggesting a role for the putative GRK phosphorylation sites contained in the missing domain in desensitization. Unfortunately, coupling of the S426A/S430A mutant to native GIRK in AtT-20 cells was not measured, and the role of GRK3 (or other GRK family members) in regulation of CB1 in these cells has not been directly addressed.Mutation of two of six available serine/threonine residues in the C-terminal tail (S426/S430) of rCB1 has also been demonstrated in both AtT20 and HEK293 cells.82 In a detailed study of C-terminal mutants, Daigle et al.82 found that all internalization-competent CB1 mutants could recruit β-arrestin-2, although some mutants appeared to recruit β-arrestin-2 at a reduced rate, while mutation of all six serine/threonine residues in the rCB1 C-terminus (460–473) prevented internalization and also failed to recruit β-arrestin-2. A recent detailed bioluminescence resonance energy transfer (BRET)-based study of CB1 arrestin interactions, suggested a low-affinity, transient interaction between CB1 and β-arrestin-2, with no interaction in late endosomes consistent with a family-A interaction, while recruitment of β-arrestin-1 by orthosteric ligands was not observed.83 Structural studies have suggested an interaction between β-arrestin-1 with a synthesized CB1 C-terminus,85 and this has recently been observed in a whole cell.86Direct recruitment of β-arrestin-2 to activated CB1-mediated signaling. THC produced both greater antinociception and greater decreases in body temperature in β-arrestin-2 KO mice compared with wild-type mice, consistent with a role for arrestins in blunting receptor signaling, however, the action of a range of synthetic ligands was normal.87 Tolerance to THC antinociceptive effects was also reduced in KO mice and decreased downregulation of CB1 was observed.88 These studies could suggest substantial agonist differences in arrestin recruitment for different assays, which requires further study. Alternatively, as THC is a partial agonist at CB1, it may be more sensitive to subtle changes in receptor availability as presumably it requires full occupancy to exert maximum effect. Finally, as with all studies of global KO animals, it is possible that the signaling of many GPCRs in the circuits that mediate cannabinoid effects is altered, and has been since the start of the animals' life. Single-cell studies examining CB1 receptor function and regulation have not be done using cells from arrestin KO animals. Studies on Gαi/o-coupled μ-opioid receptors in single neurons from KO animals show that there is not always a clear correlation between changes in agonist actions at these neurons and with changes in behavior.90β-arrestin-2 KO mice revealed a role of arrestin in CB2-mediated arrestin recruitment. McGuinness et al.91 and Dhopeshwarkar and Mackie92 utilized the PathHunter DiscoveRx assay to investigate the ability of a range of cannabinoid ligands to recruit β-arrestin-2 at hCB2 and mCB2, respectively. This assay utilizes enzyme complementation to detect recruitment of tagged arrestin to the receptor. McGuinness et al. observed robust and potent recruitment of arrestin to a range of cannabinoid ligands, including CP55,940, JWH015, and WIN55,212-2.91 Dhopeshwarkar and Mackie found that nonclassic ligands  recruited arrestin, whereas classic cannabinoid ligands  did not, suggesting a strong functional bias with structure.92 Recently, Soethoudt et al.32 also investigated the ability of a wide range of ligands to activate arrestin utilizing this assay and found that all ligands modulated arrestin recruitment to some extent. CP55,940 was the most potent ligand in this assay, followed by WIN55,212-2. Only CP55,940 acted as full agonist, while WIN55,212-2, JWH133, JWH015, HU-308, HU-910 were partial (Emax 50–70%) and the endocannabinoids (Emax 40–80%) only partially recruited arrestin.32 Interestingly, JWH133, HU-308, and HU-910 were all significantly less potent on mCB2 than hCB2 in arrestin recruitment, strongly suggesting species differences. A potential confounding issue with all these assays is the protein modules added to the receptor and arrestin constructs to enable visualization of the interactions using optical techniques. The signaling capacity of these constructs is not usually assessed, and so it is not obvious that the conformational changes being reported faithfully reflect those of the native receptor and effector. In summary, while there is some evidence to suggest that arrestin-biased signaling is observed between cannabinoid ligands, the current lack of consistency between studies needs to be resolved to enable the design of genuinely biased ligands.Surprisingly, there has been little detailed study of CB1 undergoes rapid internalization following agonist stimulation.94 While early studies suggested that CB1 may recycle following internalization,95 subsequent detailed studies have suggested that the internalized receptor enters degradative pathways96 and is rapidly degraded, with resensitization requiring the delivery of newly synthesized receptors.93CB1 has generally been shown to be clathrin mediated,97 although in some systems, caveolae-mediated internalization has been observed.97 Mutation of a highly conserved aspartate residue in the second intracellular loop (D164 in rCB1) resulted in a loss of CB1 internalization in AtT20 cells.81 Intriguingly, receptors with this mutation demonstrated preserved binding, cAMP inhibition, and inhibition of Ca2+ currents but did not activate GIRK.81 This mutation has also been suggested to decrease the constitutive activity of CB1.98 Further studies identified the extreme carboxy-terminal tail of the rCB1 as a central mediator of agonist-induced internalization of the receptor in AtT20 cells.94 Subsequently, truncation of this region was also shown to prevent arrestin recruitment to CB1 in AtT20 cells.82 However, the truncated receptor internalized normally when expressed in HEK293 cells.82 Mutation of all of the serine/threonine residues in this region resulted in failure to recruit arrestin and a decreased extent of internalization, although the initial rates were normal.82 Changes in the extent of internalization are challenging to interpret, as higher expression levels frequently result in a decreased extent of internalization of receptors in transfected cell lines, likely due to saturation of the internalization machinery. Thus, the precise mechanisms controlling internalization kinetics are far from clear.Internalization of CB1 trafficking have been complicated by the presence of a large intracellular pool of receptors observed in both primary and recombinant cell lines, and native expressing tissues.93 While it was predicted that these receptors represented a pool of receptors from which CB1 could be rapidly mobilized to the cell surface,95 mobilization has not yet been observed, although the receptors do undergo constitutive synthesis and degradation.93 Recent studies have suggested that these intracellular receptors may be involved in signaling,100 a suggestion made more feasible by the high lipid solubility of the endocannabinoid ligands. Intracellular CB1 has recently been suggested to be present in mitochondria in neurons and astrocytes, suggested to signal through mitochondrial G proteins leading to inhibition of cAMP and mitochondrial respiration.102Studies on CB2 are limited. Early studies utilizing an antibody that failed to detect hCB2 when phosphorylated at serine 352 suggested that CB2 undergoes agonist-mediated phosphorylation in response to CP55,940. The phosphorylation was accompanied by decreased signaling in both CHO and HL60 cells.104 Agonist-mediated internalization has also been observed in response to CP55,940103,105 and 2-AG.106 Early studies suggested that CB2 was repeatedly phosphorylated and dephosphorylated by alternate stimulation with agonist and inverse agonist implying that CB2 recycling may occur,69 and this was more recently demonstrated to be the case, and to be Rab11 dependent.107 The effect of the inverse agonist SR144528 on CB2 internalization is unclear as some find a decrease in cell surface CB2,103 while others find no change.106 One recent study has suggested that chronic treatment with a cannabinoid agonist leads to CB2-mediated upregulation of GRK5 in rat prefrontal cortex via a β-arrestin-2-dependent pathway,108 but further molecular studies are required.Studies detailing the trafficking properties of CB109 The consequences of such bias are not straightforward to interpret. We can speculate that if a receptor remained on the cell surface longer in the presence of a particular ligand, it might exhibit prolonged signaling, however, equally, if the receptors are desensitized but remain on the cell surface, feedback mechanisms governing the recycling of the receptor (for CB2) or delivery of new receptor to the cell surface (for CB1) might be altered leading to an overall reduction in signaling.For many receptors, bias has been observed in the ability of ligands to induce internalization.The diverse chemical structures of commonly used cannabinoid ligands increase the likelihood of identifying biased signaling, and evidence of agonist bias has emerged from a number of studies.1 came from assays in which membranes of CB1 expressing Sf9 cells, stripped of their endogenous G proteins, were reconstituted with purified G proteins.33 These studies demonstrated that the relative activation of Gαi and Gαo is dependent on the agonist. HU-210, WIN55,212-2, and anandamide all elicited maximal Gαi activation, whereas THC caused only partial Gαi activation. In contrast, only HU-210 effected maximal CB1 stimulation of Gαo, with anandamide, WIN55,212-2, and THC all partially stimulating.33 This work was further extended by coimmunoprecipitation of activated G proteins in N18TG2 cells, which demonstrated that WIN55,212-2 behaved as a full agonist for all three Gαi subtypes, while methanandamide appeared to be an agonist at Gαi3 and an inverse agonist at Gαi1 and Gαi2.110 In addition, relative signaling efficacies and potencies of CB1 ligands differ in different brain regions,111–113 which may represent the different G protein compositions of different regions. Intriguingly, plasmon-waveguide resonance (PWR) spectroscopy has demonstrated that CP55,940 and WIN55,212-2 produced distinct spectral changes (PWR shifts in opposite directions) on binding to the hCB1 indicating that the two agonists produce qualitatively distinct active conformations of the receptor, which have differing affinity for Gαi.114 Differential signaling by WIN55,212-2 and CP55,940 is consistent with the suggestion that these ligands have overlapping but distinct binding sites,116 a finding supported by molecular docking in the recently described crystal structure of CB1.118The first evidence for biased signaling through CB20 investigated the biased signaling of WIN55,212-2, CP55,940, 2-AG, anandamide, THC, cannabidiol, and the combination THC+cannabidiol on several signaling pathways. The agonists were used on in vitro medium spiny projection neurons having a wild-type (STHdhQ7/Q7) or Huntington disease (STHdhQ111/Q111) background. The effect on a range of cannabinoid-dependent signaling pathways was measured via pERK1/2 (Gαi/o mediated), β-arrestin-1 recruitment to CB1 (by BRET), phosphorylation of CREB , phosphorylation of phospholipase C , and phosphorylation of Akt (pAkt) (Gβγ mediated). The signaling bias was calculated relative to WIN55,212-2 signaling. This study found that CP55,940 induced signaling biased toward Gαs and β-arrestin-1 compared to Gαi/o, while Gαi/o signaling was biased compared to Gαq and Gβγ in both cell types . 2-AG elicited signaling bias toward Gβγ compared to Gαi/o (in STHdhQ7/Q7 cells), and Gαi/o-biased signaling compared to β-arrestin-1 (in STHdhQ7/Q7 cells) and Gαq (predominantly in STHdhQ111/Q111) . Similar to 2-AG, anandamide signaling was biased toward Gβγ compared to Gαi/o (in STHdhQ7/Q7 cells), while Gαi/o signaling was biased compared to β-arrestin-1 and Gαq (mostly in STHdhQ111/Q111) . THC produced signaling biased toward β-arrestin-1, Gαq, and Gβγ compared to Gαi/o in both cell types . Since cannabidiol treatment only evoked significant Gαs-mediated pCREB, bias values could not be calculated for this ligand. The combination of THC+cannabidiol induced signaling biased toward Gαs compared with Gαi/o, while signaling was biased toward Gαi/o compared with β-arrestin-1, Gαq, and Gβγ (predominantly in STHdhQ7/Q7 cells) .Laprairie et al.60 investigated biased signaling and allosteric modulation between cAMP and ERK1/2 activation using CHO cells stably expressing CB1 treated with CP55,940, HU-210, WIN55,212-2, THC, methanandamide, anandamide, and 2-AG. pERK1/2 and cAMP levels were measured in response to the treatments. The signaling bias induced by each ligand was calculated relative to 2-AG. CP55,940, HU-210, WIN55,212-2, THC, methanandamide, and anandamide all showed a preference toward cAMP inhibition compared to pERK1/2. HU-210 and methanandamide displayed strong bias toward cAMP inhibition, whereas CP55,940, THC, and anandamide showed nonsignificant bias toward cAMP inhibition. As both these pathways are pertussis toxin sensitive in these cells, this may indicate bias between α- and βγ-mediated pathways . The mechanism by which α and βγ bias could be mediated is currently unclear.Khajehali et al.s coupling of CB1 has also shown potential for agonist bias. While equivalent rank order of potencies was observed for inhibiting or stimulating cAMP in CHO-hCB1 cells, anandamide and CP55,940 were significantly less efficacious in stimulating the accumulation of cAMP than in inhibiting its formation.119 Forskolin acts synergistically with CB1-activated Gαs at adenylyl cyclase.35 Cannabinoid receptor-mediated stimulation of cAMP also revealed differences among agonists in as much as forskolin enhanced the potency of HU-210 and CP55,940 by ∼100-fold but had no effect on the potency of WIN55,212–2 or anandamide.119The putative Gα1 is that observed in the proposed coupling of CB1 to Gαq. Lauckner et al.40 demonstrated that high concentrations of WIN55,212-2, but not THC, HU-210, 2-AG, or methanandamide resulted in increased release of [Ca]i through a Gαq pathway. More recently, rat hippocampal autaptic long-term potentiation has been suggested to be mediated by 2-AG-induced CB1 activation of Gαq, suggesting that biased signaling may not hold between different cell types.120N-arachidonoyl dopamine (NADA) represents an interesting case for a putative highly biased CB1 agonist as it is currently only known to affect very select pathways.121 NADA is an endocannabinoid agonist of both CB1 and TRPV1.123 NADA binds orthosterically to CB1 with high nanomolar affinity but has no substantial effect on GIRK-mediated hyperpolarization, cAMP levels, pERK, or adenylyl cyclase activity. At concentrations above 30 μM, NADA elevates [Ca]i levels from intracellular stores in cell cultures and causes a slow internalization of CB1 from the cell surface. The data strongly suggest that NADA signals via a Gαq-mediated pathway. Although NADA did not potently induce [Ca]i, it has been demonstrated to be more potent under different in vitro assay conditions122 and in brain slices.125 In summary, significant bias in activating Gαq-mediated responses occurs, with only a small subset of ligands reporting this , and then only at high concentrations and in a tissue-dependent manner.Perhaps the most extreme example of agonist bias for one G protein over another through CB1 has come from allosteric modulators. While ORG27569 has been suggested to generally inhibit agonist-mediated G protein activation on its own,112 it has been suggested to exhibit biased signaling toward pERK1/2 pathways via β-arrestin-1.58 This finding is not consistent across all reports, however, as others60 did not find that treatment with ORG27569 alone induced pERK1/2. The different findings could arise from differences in assay design or differences in the relative receptor expression, and signaling systems between the different cells utilized.126 In particular, time-dependent effects on CB1 signaling have been reported in several studies of ORG27569 and functionally related molecules,128 which may account for some variability.For most GPCRs, interest in biased signaling has focused predominantly on the ability of agonists to differentially modulate G protein and arrestin pathways. The greatest promise for this to date through CB1 function, modifies agonist signaling in a biased or selective manner, inhibiting pERK1/2 signaling while not modifying cAMP signaling.129 Interestingly, recent studies have suggested a similar profile for Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1), a CB1 interacting protein expressed in HEK293 cells.130 These findings emphasize the challenges in interpreting pERK1/2 studies when the pathway regulating it (G protein versus arrestin mediated) is generally not well defined. An understanding of the conformation of the receptors driven by the presence of allosteric modulators may help guide the development of highly biased agonists as it is likely that a greater range of conformations may be achieved by targeting regions outside of the relatively constrained orthosteric binding site.Pregnenolone, a putative endogenous allosteric modulator of CB1 (S426A/S430A) drive preferential signaling via β-arrestin-1,55 while different mutations in the DRY motif drive conformations to preferentially signal through either G proteins or β-arrestins.131Similarly, receptor mutation studies may enhance our understanding of the molecular drivers for bias. A few examples of this are currently emerging, for example, mutations in potential phosphorylation sites in rCB2, although some signaling biases have been described. Shoemaker et al.105 compared CP55,940, 2-AG, and 2-arachidonoylglyceryl-ether using MAPK activation, stimulation of calcium transients, and inhibition of adenylyl cyclase as the signaling pathways. Each ligand differed in its rank order of potency in the three assays despite similar efficacies. Schuehly et al.132 found that AM630 displayed inverse agonist/antagonist actions on CB2-mediated inhibition of cAMP production and was silent in its effects on [Ca]i transients. On the contrary, a novel CB2 ligand, 4-O-methylhonokiol, was an inverse agonist/antagonist with regard to cAMP production but potentiated the effects of 2-AG on calcium transients. Atwood et al.31 have further demonstrated that CP55,940, but not WIN55,212-2, leads to inhibition of voltage-gated calcium channels through CB2, in addition to different actions on receptor trafficking.Comparatively, there is little evidence for agonist bias through G proteins for CB2 internalization. Atwood et al.31 found that while CP55,940 induced robust internalization of rCB2, WIN55,212-2 failed to promote receptor internalization, despite both agonists activating pERK, and arrestin recruitment. Extending these studies, Dhopeshwarkar and Mackie92 examined a range of ligands in inhibiting adenylyl cyclase, internalization, and arrestin recruitment to mCB2. Of the most commonly utilized ligands, CP55,940 and JWH015 were the most balanced compounds evaluated although JWH015 had lower efficacy. The majority of the other compounds screened were G protein biased, while a few less commonly utilized ligands  were more arrestin biased. It will be interesting to see if this difference results in different tolerance profiles to these agonists in CB2-mediated effects.Recent studies have suggested agonist bias in CB2, Soethoudt et al.32 recently analyzed the ability of a wide range of ligands to signal through hCB2 and analyzed the data with operational analyses. This analysis suggested that THC showed bias toward pERK signaling compared to arrestin and GTPγS, and intriguingly, THC did not activate GIRK, indicative of high bias against this pathway. -AM1241 was biased toward arrestin coupling and pERK signaling compared to GIRK channel activation. JWH133 was moderately biased toward arrestin compared to GIRK, whereas both WIN55,212-2 and JWH015 showed preference for GIRK compared to cAMP signaling. Anandamide showed preference for pERK and GIRK signaling compared to cAMP, whereas 2-AG was significantly biased toward GIRK compared to cAMP and G protein signaling. On comparison between arrestin coupling and cAMP signaling, all ligands appear to be significantly biased, however, this may reflect the choice of CP55,940 as the reference ligand, as this ligand appears itself biased toward cAMP signaling. The authors of this study concluded that HU-910 and HU-308 were well-balanced ligands without significant bias toward any signal transduction pathway on hCB2, but this study also highlighted species differences, as at mCB2, HU-910 and HU-308 were significantly biased toward G protein signaling over arrestin coupling. A limitation of this study is that the different assays were conducted in different cells lines, and thus, some differences may represent different expressions of signaling molecules , which requires further investigation. The study concludes that THC, 2-AG, and -AM1241 are highly biased CB2 agonists and underscores that biased signaling at CB2 is subject to species variation.In a highly detailed analysis of functional selectivity of CB1- and CB2-biased signaling can be different between tissues and species, which might prove an issue for translating results from in vitro studies to in vivo. As a large body of the work describing biased signaling of the cannabinoid receptors is performed using heterologously expressed receptors, future work should include endogenously expressed receptors to determine if previous observations are relevant.The concept of agonist bias provides an exciting new direction for developing therapeutics with less adverse effects. However, for the cannabinoid receptors, this field is still in its infancy. The influence of different cell types, with different receptor numbers and second-messenger expressions in the signaling pathways measured, remains to be fully elucidated. This review highlights that CB133 Finally, and most importantly, we do not yet know which pathways are mediating desired therapeutic effects, and it remains an open question whether or not these can be clearly defined or if the therapeutic effects are mediated through a combination of signaling pathways.Importantly, almost all of the studies described here measured single time points for each signaling assay, rather than clearly defining and comparing the kinetics of each assay and it is clear that detection of bias can change across time.


The rapid population growth of many African cities has important implications for population health, yet little is known about factors contributing to increasing population, such as the fertility of internal migrants. We examine whether in-migrants to Kinshasa have different fertility patterns than lifetime Kinshasa residents, and identify characteristics of migrants that may explain differences in fertility. We also use detailed migration histories to examine whether fertility differs by features of migration. We use representative data from the PMA2020 Project for 2197 women in Kinshasa, including 340 women who moved to Kinshasa. We examine differences between migrants and non-migrants in fertility and other fertility-related characteristics. We also examine whether fertility differs by duration of residence in Kinshasa, number of lifetime moves, age at first migration, urban/rural classification of birthplace, and the distinction between intra-Kinshasa migration and migration to Kinshasa.. Migrants have significantly higher fertility than permanent Kinshasa residents, but the difference is relatively small in magnitude. This higher fertility appears due in part to patterns of contraceptive use among migrants. There is noteworthy heterogeneity among migrants: higher fertility among migrants is associated with longer duration in Kinshasa, more lifetime moves, urban-Kinshasa migration, older age at first migration, and moving to Kinshasa from outside (as opposed to intra-Kinshasa migration). The rapid urbanization of sub-Saharan Africa (SSA) has important implications for health in the region. The crowding and unsanitary conditions in SSA cities facilitate the proliferation of infectious and vector-borne diseases , Kinshasa, is one of the world’s “megacities.” With a population of over 11 million, Kinshasa is Africa's third largest city (after Lagos and Cairo) and one of the continent’s most rapidly growing urban areas  and in-migration. Regarding the former, most research on fertility in Kinshasa was conducted before the period of prolonged conflict  are most likely to migrate in their peak reproductive years  selection 2) adaptation, 3) socialization, and 4) disruption. Regarding selection, migrants are a self-selected group for whom relatively lower fertility preferences may be part of the motivation to move to a new area Kulu . MigrantOn the other hand, migrants may have higher fertility than non-migrants. The socialization hypothesis suggests that the fertility of migrants will primarily reflect fertility preferences dominant in their place of origin, with the assumption that norms about fertility are learned at a young age, and any changes in fertility behavior among migrants will only occur over the longer-term Kulu . If migrThe reason for moving to an urban area may also provide insight into the relationship between fertility and migration. Some reasons for internal urban migration involve characteristics that are typically associated with lower fertility, like attending school or starting a job Romaniuk . Other rMuch remains unknown about the migration/fertility association in most of SSA, due largely to limited data on migration. Migration differs by destination and origin , distance travelled, permanence, duration, and motivation, among other characteristics. Yet the demographic literature on fertility and migration has often neglected to account for variation in migration characteristics in developing countries and often uses a simple binary measure of migrant/non-migrant  duration of residence in Kinshasa, (2) number of lifetime moves, (3) age at first migration, (4) urban/rural classification of birthplace, and (5) the distinction between intra-Kinshasa migration and migration to Kinshasa. Using these measures, this research expands on previous analyses of migration and fertility. It is important to note that we do not intend to estimate the effect of migration on fertility in this research. Instead, we aim to identify differences in fertility between migrants and non-migrants in Kinshasa, and explore possible explanations for these differences.The DRC is Africa’s third most-populous country, with an estimated population of just over 77 million people according to UN estimates for 2015 . To achieve this aim, PMA2020 collects representative data in these countries on an annual basis for a range of fertility and family planning-related measures. This study has received approval to collect data from Institutional Review Boards at Johns Hopkins University, Tulane University, and the University of Kinshasa. PMA2020 was supported by the Bill and Melinda Gates Foundation, Seattle, WA; under grant # OPP1079004.Our data comes from Performance, Monitoring, and Accountability 2020 project (PMA2020). PMA2020 currently operates in eleven countries worldwide and was established in part to measure uptake of contraceptive use in many of the world’s most populous countries  of their two most recent births, and child mortality. Because we do not have complete birth histories, we cannot establish the order of events between all births and moves, and our analysis therefore does not seek to measure the effect of migration on fertility.A goal of this research is to address some of the limitations in common measures of migration, such as those collected by DHS. The DHS asks two questions about migration: “How long have you been living continuously in (NAME OF CURRENT PLACE OF RESIDENCE)?” and “Just before you moved here, did you live in a city, in a town, or in a rural area?” As suggested by theories on the relationship between internal migration and fertility, there are important features of the migration process that are associated with fertility but not captured by these questions. While someone may have moved from another urban area in the most recent migration, they may originally be from a rural area, and the first move from rural to urban, for example, would therefore be missed by the DHS questions. This is important in the context of the “socialization” hypothesis, which suggests that migrants are influenced by the fertility norms of their birthplace. Under the adaption theory, the fertility norms of migrants may eventually conform to those of the destination with longer durations at destinations and away from areas of origin. As a result, the age at first migration and duration at current residence may be associated with fertility. DHS collects information only on the latter. In the DHS survey, the household is considered the “current place of residence”. As a result, it is not possible to distinguish between migrants who moved within Kinshasa from those that moved to Kinshasa from another urban area of DRC in the DHS data. Since Kinshasa has lower fertility than all other provinces in DRC, one would expect that intra-Kinshasa migrants do not have higher fertility than Kinshasa non-migrants, but those moving to Kinshasa may have higher fertility than both. Since theory suggests that migration may disrupt fertility, the number of lifetime moves is relevant to fertility. Finally, it is important to note that, the relatively few questions on migration from the DHS survey are not included in every round or country; the DHS Phase 6 survey, the most recent DHS in DRC (2013–14), did not include these questions.The PMA2020 data offer several important advantages over migration data collected by DHS. The first round of the PMA2020 study in Kinshasa included an extensive residence/migration history, which contains information on all locations where respondents previously lived for 6 months or more, and selected characteristics of these former residences. The survey captures the year and month in which the respondent moved to a particular area, along with reasons for the migration, and urban or rural classification for each place of residence. We use this information to categorize migrants as women who moved to Kinshasa from another location (women who were born in and later moved within Kinshasa are not considered migrants). Using the PMA2020 residence histories, we are able to create several characteristics of migration, including the duration lived in Kinshasa, number of lifetime moves, age at first migration, urban/rural classification for birthplace, and the distinction between intra-Kinshasa migration and migration to Kinshasa.We begin by tabulating background characteristics for our study population, including age, marital status, level of education, number of lifetime births, and household wealth. Household wealth is measured using a constructed wealth index based on ownership of 25 household durable assets, house and roof material, livestock ownership and water source. Here again, PMA2020 uses measures similar to those in the most recent DRC DHS survey. A wealth index was created using principal component analysis , and the age at migration to Kinshasa.We then use multivariate Poisson regressions with the number of cumulative births at the time of the survey as the dependent variable. The main independent variable of interest is the binary category of migration, compared to non-migrants. To explore whether any fertility differences among migrants are related to differences in age, education or other background characteristics, we also control for characteristics that significantly differ by migration status and likely affect fertility, specifically: age, level of education, marital status, and household wealth. Next, to examine whether differences in fertility between migrants and non-migrants are due to differences in contraceptive use between these groups, we add a measure of contraceptive use to this model, measured as a three-category variable for (0) not using any contraceptive method, (1) using a traditional contraceptive method, and (2) using a modern contraceptive method.We next measure differences in family planning and fertility preferences by migration status by running three logistic regressions where the dependent variables are overall contraceptive use, traditional contraceptive use, and modern contraceptive use. Then, we examine fertility-related attitudes, specifically unmet need for family planning, desire to have another child, and whether the most recent birth was unintended. We run separate logistic regressions to examine whether these measures differ by migration status.Finally, we examine whether fertility differs by particular characteristics of migration, specifically (1) the duration of time living in Kinshasa, (2) the number of lifetime moves, (3) whether their birthplace was urban or rural, (4) their age at first migration, and (5) a distinction between migration within Kinshasa and migration to Kinshasa. Number of lifetime moves is measured as categorical, with those moving from one, two, and three or more times compared to the reference group of non-migrants. Duration of time in Kinshasa is categorical, according to short-term, medium-term and longer-term residence in Kinshasa: 0–5 years, 6–20 years, and 21 or more years resided in Kinshasa. Age at first migration is measured in 10-year age intervals. Alternative categorizations yielded results that were not substantively different. The same regression controls from previous models are included here.Table Characteristics related to family planning and fertility preferences show greater differences between the two groups. Although levels of contraceptive use are similar (32.3% for migrants and 35.0% for non-migrants), traditional method use is higher and modern method use is lower among migrants. While nearly a quarter of migrant women (24.1%) have an unmet need for contraception, only 16.5% of non-migrants do. Notably, 39.2% of migrants say they want no more children while only 26.7% of non-migrants expressed the same desire.According to migration characteristics Table , most miWe find that fertility is significantly higher for migrants compared to non-migrants in Kinshasa. Migrant women had on average cumulative fertility of at the time of the survey of 4.76 children compared to 4.00 for non-migrants in Kinshasa. However, the difference in cumulative fertility between migrants and non-migrants is relatively small and perhaps less than would be expected given the much higher fertility from the migrants' places of origin: the TFR in other (non-Kinshasa) urban areas of DRC is 6.03, and is 7.25 in rural DRC (using DHS data using two-year ASFRs) .The fertility differences between migrants and non-migrants in Kinshasa, however, are nonetheless significant, leading us to ask: is the higher fertility among migrants due to the differences in age, education or other background characteristics? Results shown in Table Results for fertility preferences and family planning-related characteristics may yield some insight into the differences in fertility between migrants and non-migrants. First, we find no significant differences in overall contraceptive use between migrants and non-migrants Table . When thp < 0.10) and the coefficient was smaller (0.09) than results for birthplace. We also find statistically significant differences by age at first migration, in which women who first moved by age 20 had significantly higher fertility than women who did not move  and indirectly (through their relatively higher fertility).However, the difference in fertility between migrants and non-migrants is relatively small in magnitude when considering the difference in fertility levels between Kinshasa and the rest of the country. Our results show a TFR for migrants is 4.76, compared to 4.00 for non-migrants, compared to a TFR in other (non-Kinshasa) urban areas of DRC of 6.03, and 7.25 in rural DRC (with DHS data using 2-year ASFRs) , migrants may face more barriers to accessing family planning services  and have low fertility while in Kinshasa but are also more likely to return to their villages during peak childbearing years while migrants from urban areas are more likely to stay in Kinshasa permanently. Higher fertility among migrants is concentrated among those who first migrated at relatively older ages (by age 20), which provides some evidence for the socialization hypothesis. Finally, we show that the distinction between intra-Kinshasa migration and moving to Kinshasa is important, as migrants to Kinshasa have significantly higher fertility than non-migrants, but intra-Kinshasa migrants are not different. It is important to note that the order of events between migration and fertility cannot be accurately measured with this dataset , but our nuanced measures, which are also seldom available, nonetheless yield insight into migration processes in SSA.We also tested other possible explanations for higher fertility among migrants for which no significant associations were found. There did not appear to be differences in geographic access to family planning: migrants were no different from non-migrants in knowledge of a location to obtain family planning methods, although it is possible they may face other barriers to access. Similarly, as mentioned above, there was no statistically significant relationship between age at most recent migration and cumulative fertility.Lower fertility among urban migrants compared to non-migrants in the places of origin has been found in much of the recent literature on SSA , compared to other urban areas of the DRC (8.0) and rural DRC (4.5) . In addition, we do not find evidence of adaptation; fertility does not decline with greater durations of time in Kinshasa; in fact we find that fertility is highest among longer-term migrants. In addition, migration does not appear to disrupt fertility, as we also find higher fertility with more moves.We do not intend to estimate the effect of migration on fertility in this research. Instead, we seek to identify differences in fertility between migrants and non-migrants in Kinshasa, and explore possible explanations for these differences. Establishing a causal relationship between migration and fertility would require longitudinal data that includes complete birth histories and precise timing of births and migration—a rarity for SSA. Without complete birth histories for respondents, we cannot identify the order of events between births and migration and thus cannot determine whether the higher fertility found for migrants is a result of higher fertility prior to migration or higher levels of fertility by migrants after re-locating to Kinshasa. Similarly, without longitudinal data, it is not possible to establish the order of events; for example, was economic status affected by migration to Kinshasa, and/or did migration select individuals of differing economic status? In addition, the survey did not measure some factors that may be associated with fertility and differ by migration status, such as employment, which research in Kinshasa prior to the period of conflict found to be highly correlated with contraceptive use and access to abortion , making it unlikely that migrants are continuing the preferred method from their places of origin. Rather, this may signal migrant adaptation to traditional methods in the absence of easy access to modern methods, meaning migrants currently using traditional methods may represent a segment of women eager to transition to modern methods. The complexity of our results also reinforces the importance of acknowledging diversity among migrants. Different hypotheses may apply for different migrant groups at different times and in different contexts, and the collection of more detailed data on both migration and birth histories is necessary to better identify dominant theories of fertility and mobility in different contexts.Our findings suggest that rather than fuel rapid increases in Kinshasa’s urban growth, continued migration to Kinshasa may also have a positive contribution to the process of the fertility transition throughout the country, if the city continues to absorb high numbers of migrants who ultimately have significantly and substantially lower fertility than they would likely have had if they remained in their urban or rural places of origin. While more complete data on the timing of migration and fertility is needed to tease out the dominant mechanisms at play, our findings strongly suggest that residence in a new urban area may play an important role in lowering fertility rates of the substantial numbers of women who are expected to migrate to Kinshasa from other areas of the DRC in the coming years.


Wnt signaling is an important pathway in health and disease and a key regulator of stem cell maintenance, differentiation, and proliferation. During heart development, Wnt signaling controls specification, proliferation and differentiation of cardiovascular cells. In this regard, the role of activated Wnt signaling in cardiogenesis is well defined. However, the knowledge about signaling transmission has been challenged. Recently, the packaging of hydrophobic Wnt proteins on extracellular vesicles (EVs) has emerged as a mechanism to facilitate their extracellular spreading and their functioning as morphogens. EVs spread systemically and therefore can have pleiotropic effects on very different cell types. They are heavily studied in tumor biology where they affect tumor growth and vascularization and can serve as biomarkers in liquid biopsies. In this review we will highlight recent discoveries of factors involved in the release of Wnts on EVs and its potential implications in the communication between physiological and pathological heart cells. AdditioCurrently, increasing evidences show the importance of different Wnt signaling branches and their crosstalk among different cell types. Secreted Wnts are lipidated and this hydrophobic moiety hinders them to freely move in the extracellular space. Different experimental proofs showed how their biochemical properties would fit to the idea of “diffusible” morphogens . LipoproDrosophila Wnt, Wingless. In Drosophila, exosomes, containing the Wnt secretion factor Evi, transport Wnts across the Drosophila neural-muscular junctions (NMJ) and in the wing imaginal disc  functions as an extracellular inhibitor of non-canonical Wnt signaling  that antin vitro . Anotherin vitro . Of notein vitro . Exosomein vitro . The mecin vitro .Hypoxic cardiomyocytes upregulate Wnt Inhibitory Factor 1 (WIF1) , which iTaken together, Wnt signaling activation is key in pathological heart remodeling and EV-mediated signaling may participate in this activation. The contribution of EV-mediated Wnt signaling to block tissue regeneration needs further investigation in order to engineer EV-modifications allowing the recovery of developmental plasticity.in vitro  and induced pluripotent cells (iPSCs) have been widely used in translational medicine for their enormous therapeutic potential in tissue repair and regeneration. Isolated exosomes secreted from iPSCs showed protective effects on ischemic myocardium by transferring the endogenous molecules to salvage the injured neighboring cells . In this in vivo environment will allow cell-type and cargo-specific EVs and will enormously advance the field of EV-mediated signaling.Cell therapies can directly support regenerative processes by forming new functional tissues or supporting tissue generation via paracrine mechanisms. Dissecting the precise role of Wnt signaling in cardiac tissue regeneration and the potential use of synthetic EVs may help tailor therapeutic approaches aiming to restore tissue functionality in a non-regenerative environment such as the heart. Moreover, human PSCs provide an excellent tool to address EV-mediated signaling in the context of early cardiogenesis. Developing protocols for exosomes isolation in theirModulation of Wnt signaling is crucial for tissue homeostasis in the developing and postnatal heart. However, the role of Wnt/β-catenin-dependent and -independent pathways in the intercellular crosstalk of heart cells is not fully understood. Activating or inactivating branches of the Wnt-network in specific target cells may be attractive to modulate pathological processes in the cardiovascular system or to enhance regenerative capacities of stem cell therapies. Many of these mechanisms might be mediated by EVs. Hence, understanding Wnt signal transduction via EVs between cell populations and tissues will advance our strategies for therapeutic modulation of these pathways.Both authors conceived, discussed and wrote the review.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 reviewer PY and handling Editor declared their shared affiliation.


Uric acid (UA) is an antioxidant found in human serum. However, high UA levels may also have pro-oxidant functions. According to previous research, aldose reductase (AR) plays a vital role in the oxidative stress-related complications of diabetes. We sought to determine the mechanism by which UA becomes deleterious at high concentrations as well as the effect of AR in this process.2O2 and vWF levels were measured in vivo.Endothelial cells were divided into three groups cultured without UA or with 300 μM or 600 μM UA. The levels of total reactive oxygen species (ROS), of four ROS components, and of NO and NOX4 expression were measured. Changes in the above molecules were detected upon inhibiting NOX4 or AR, and serum H2•-, •OH, and 1O2. However, when the UA concentration was increased, both O2•- levels and downstream H2O2 production significantly increased. Finally, an AR inhibitor reduced H2O2 production in hyperuricemic mice and protected endothelial cell function.Increased AR expression in high UA-treated endothelial cells enhanced ROS production by activating NADPH oxidase. These effects were blocked by the AR inhibitor epalrestat. 300 μM UA decreased the levels of the three major reactive oxygen species (ROS) components: O2O2 could protect endothelial function and maintain the antioxidant activities of UA. These findings provide new insight into the role of UA in chronic kidney disease.Our findings indicate that inhibiting AR or degrading HThe online version of this article (doi:10.1186/s12964-016-0158-6) contains supplementary material, which is available to authorized users. UA also chelates transition metals to reduce ion–mediated ascorbic acid oxidation [+/Ca2+ exchange-mediated mitochondrial calcium overload [Uric acid (UA) is the final enzymatic product in the degradation of purine nucleosides and free bases in humans and the great apes –3. UA isxidation –7. UA isxidation . Howeverxidation . Strong xidation . High UAxidation , 11. Altxidation . UA dosexidation , and hypoverload . Howeveroverload , 16, it Aldose reductase (AR) is the rate-limiting enzyme in the polyol pathway, and NADPH acts as a cofactor . AR playIn this study, we detected the different ROS types generated in HUVECs cultured with different UA concentrations as well as the changes in ROS production upon transfecting HUVECs with siRNA against AR. We then investigated the role of AR in UA-induced endothelial injury in vitro and in vivo. Our results suggest a novel mechanism underlying the endothelial dysfunction caused by high UA levels.2 atmosphere.Human umbilical vein endothelial cells (HUVECs) were purchased from YRbio  and cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator in a 5% COUric acid was purchased from Sigma–Aldrich . Uric acid powder was dissolved in a 1 mol/L NaOH solution at a concentration of 40 mmol/L. Then, the uric acid solution was added to the serum containing medium at a final concentration and at a pH 7.2–7.4.n = 3). Cells in the UA and HUA groups were cultured in medium containing 300 or 600 μM uric acid, respectively, for 24 h followed by incubation with the total oxidative stress indicator chloromethyl derivative dichlorodihydrofluorescein diacetate  for 30 min in the dark at 37 °C. After three washes in PBS, green fluorescence was visualized using a laser scanning confocal microscope at an excitation wavelength of 488 nm and an emission wavelength of 515 nm.Cells were seeded onto 35-mm confocal dishes (with a cover glass) and classified into control, normal uric acid (UA), and high uric acid (HUA) groups  or 0.1 μM epalrestat (Santa Cruz Biotechnology). Pyocyanin  was used as a positive control in the comparisons of ROS production.For the total intracellular ROS levels, method was referenced previously by the oxidant-sensing fluorescent probe CM-H 2 DCFDA . Briefly2•−: cells were incubated with 4 μmol/L Mito-SOX Red (Invitrogen) in the dark at 37 °C for 10 min, and red fluorescence was observed at an excitation wavelength of 510 nm and an emission wavelength of 580 nm. For H2O2: cells were incubated with 30 μmol/L BES-H2O2  in the dark at 37 °C for 1 h, and green fluorescence was observed at an excitation wavelength of 485 nm and an emission wavelength of 515 nm. For · OH: cells were incubated with 100 μmol/L proxylfluorescamine (Invitrogen) in the dark at 37 °C for 30 min, and green fluorescence was observed at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. For 1O2: cells were incubated with 20 μmol/L trans-1-(2′-methoxyvinyl)pyrene (Invitrogen) in the dark at 37 °C for 10 min, and blue fluorescence was observed at an excitation wavelength of 405 nm and an emission wavelength of 460 nm. For ONOO−: cells were incubated with 10 μmol/L dihydrorhodamine123 (Santa Cruz) in the dark at 37 °C for 30 min, and green fluorescence was observed at an excitation wavelength of 488 nm and an emission wavelength of 520 nm.The detection of intracellular ROS components occurred as follows. For OReverse transcription was carried out on human kidney RNA with Superscript II reverse transcriptase, according to the manufacturer’s instructions. The full-length human NOX4 was amplified by PCR using the primers NOX4_F, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGCTGTGTCCTGGAGG-3′, and NOX4_R, 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCA GCTGAAAGACTCTTTATTGTATTC-3′. The PCR product was cloned into a pcDNA3.1 vector according to the manufacturer’s instructions, obtained pcDNA-NOX4. HUVECs were transfected with the pcDNA-NOX4 plasmid using Lipofectamine 2000 (Invitrogen). Clones were selected 10–16 days after transfection using 400 μg/ml neomycin (G418) to obtain HUVECs stably expressing NOX4.2−..Before treatment with chemicals or UA, media were replaced with Dulbecco’s modified Eagle’s medium (DMEM). The supernatant or serum was centrifuged and subjected to NO level evaluation using the Nitric Oxide Assay Kit  according to the manufacturer’s instructions. The end-point measured formula was NONox4 mRNA sequences and synthesized by SBS Biotechnology Corporation : AR sense, 5′- CCTATGGCCAAGGACACACT-3′ and antisense, 5′-CTGGTCTCAGGCAAGGAAAG-3′; NOX4 sense, 5′-TTGCCTGGAAGAACCCAAGT -3′ and antisense, 5′- TCCGCACAATAAAGGCACAA-3′. As an internal control, mouse GAPDH was amplified using the following primers: sense, 5′-GGCATGGACTGTGGTCATGAG-3′ and antisense, 5′-TGCACCACCAACTGCTTAGC-3′. Relative expression (fold change vs. control) was quantified using the 2-ΔΔCt method.RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen). The cDNA was used as a template in quantitative real-time PCR reactions performed using SYBR green I PCR Master Mix and an ICycler system (Bio-Rad). The following primers were designed from the full-length AR and For Western blotting, proteins were extracted from tissues or cells using RIPA lysis buffer . Protein concentrations were measured using a BCA kit (Pierce). Protein samples (60 μg per lane) were separated by 12% SDS-PAGE and transferred to nitrocellulose (NC) membranes. After staining with Ponceau S, the membranes were incubated overnight at 4 °C in 5% non-fat milk followed by incubation with a primary antibody against AR (Santa Cruz Biotechnology) or β-actin (Sigma). Immunoreactive bands were visualized using ECL reagent (Santa Cruz Biotechnology) according to the manufacturer’s instructions and were then exposed to X-ray film. Protein band intensities were quantified using the Quantity One software (Bio-Rad). The assay was repeat 3 times.AR activity was measured spectrophotometrically as previously described , 36. Brin = 6) and hyperuricemic model (n = 24) groups. Mice were intraperitoneally injected with 250 mg/Kg · d oxonic acid potassium salt (Sigma) and 250 mg/Kg · d uric acid (Sigma). After receiving intraperitoneal injections for 3 days, the hyperuricemic model group was sub-classified into hyperuricemic mice (n = 6), hyperuricemic mice treated with epalrestat  (n = 6), and hyperuricemic mice treated with polyethylene glycol catalase  (n = 6). The antioxidant treatments PEG-catalase and epalrestat were intragastrically administered. After 10 days of modeling, the levels of UA, NO, H2O2, and von Willebrand factor (vWF) in the blood were evaluated.Hyperuricemic mouse models were established as described by Yang et al.  with sli2O2 levels were assayed using a hydrogen peroxide assay kit  with the end formula Mn2+. vWF was detected using a von Willebrand Factor ELISA kit.The serum UA level was assayed using an enzymatic method that measures the end production of quinonimine using an automatic biochemical analyzer . Serum Ht-tests. A p value < 0.05 was considered statistically significant.All data are expressed as the means ± SD. Mean comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). Comparisons of the means between two groups were conducted using randomized controlled To confirm the impairment of endothelial cells by UA treatment, we evaluated the effect of different UA concentrations on ROS production and NO release in HUVECs UA (300 μM) reduced total ROS levels in endothelial cells, whereas high UA (600 μM) treatment increased intracellular ROS production Fig. . NO releIn order to assay how UA trigger AR expression. P38 and extracellular signal–regulated kinase (ERK) 42/44 MAPK phosphorylation are involved in the UA-induced cell proliferation and activation in the UA-induced HUVEC . TherefoNOX4 is the main NOX as well as the main source of ROS in endothelial cells under oxidative stress –40. NOX42•−), 1O2, and hydroxyl radical (·OH) production and subtly increased H2O2 production. However, high UA treatment increased O2•− and H2O2 production but reduced 1O2 and · OH production , which is generated in mammalian systems as an end product of purine metabolism, is the most abundant antioxidant in human plasma and possesses free radical scavenging properties. In humans and other higher primates, uric acid is the final compound of purines catabolism, but all other mammals converts uric acid to allantoin with enzyme uricase which is deficient in humans and other higher primates , and is 2•-, ·OH, 1O2, and H2O2, all of which can be interconverted [2•-, ·OH, and 1O2 release and slightly increases H2O2. The slight increase in H2O2 levels , high UA levels likely did not suppress O2•- release but rather stimulated O2•- generation. This result is similar to previous reports [− levels, − which is a highly toxic molecule that can cause harmful effects. Under normal conditions, the ONOO− level remains low due to the low level of O2•- generation. However, if O2•- generation is enhanced, ONOO− production also increases [−, ONOO− levels decreased in the presence of normal UA concentrations. The increase in ONOO− levels resulting from high UA treatment was likely due to elevated O2•- levels. These results suggest that enhanced O2•- generation plays a central role in high UA-induced endothelial dysfunction.Mounting evidence indicates that hyperuricemia induces heart and kidney injury by promoting free radical generation and subsequent endothelial dysfunction , which aonverted , 60–63. els Fig.  may not function , 65 and function . When th reports . Althougncreases –70. BecaOur previous study demonstrated high concentration uric acid could induce AR mRNA and protein expression level of HUVECs . Johnson2O2 but not ONOO− levels and increased NO levels. These results confirm that AR activation plays an important role in high UA-stimulated HUVECs.When an AR inhibitor was added to the high UA-treated HUVECs, total ROS levels significantly decreased, and NO levels recovered. AR-induced ROS production is associated with NADPH , 33; our2•- generation was associated with the switch of UA functioning as an antioxidant to a pro-oxidant in vitro. Because O2•- is catalyzed to H2O2 by SOD in vivo, H2O2 is likely the major contributor to endothelial dysfunction. Additionally, serum UA levels correlate with plasma H2O2 in preeclampsia [2O2, or decreasing O2•- would protect the endothelium from high UA-induced injury. In this study, epalrestat and PEG-catalase recovered NO secretion levels and decreased vWF levels.The above results suggest that the high UA-induced increased Oclampsia . TherefoPreviously, we measured blood pressure in hyperuricemic wild-type mice and reported no difference between the two groups (data not shown). Endothelial dysfunction resulting from hyperuricemia would not impact blood pressure in the two-week model. However, if the duration of exposure to high UA concentrations was extended, the accumulation of endothelial dysfunction would cause artery dysfunction and dysarteriotony. These data are similar to the report by Johnson et al. , 76.2O2 significantly increased in the high UA group compared with the control group. The elevated ROS levels were reversed when AR was inhibited in vivo or in vitro. Thus, the pro-oxidant activity of UA when present at high concentrations likely plays an important role in endothelial dysfunction via AR.Our study confirmed that the levels of ROS components changed in HUVECs cultured in media containing different UA concentrations. In particular, H


Cedrus libani A. Rich.) for three consecutive years. The averages of cone number were 19.4, 47.2, and 75.5 for the years. It was the highest in flat (23.5) for 2013, in south (92.1) for 2014, and in flat (95.7) for 2015, while it was lowest in south (16.3), in east (18.2), and in north (39.4) for the years, respectively. Significant correlations (p ≤ 0.01) were estimated among years for cone production in polled aspect. Estimated fertility variations changed for the years and stands. It could be generally acceptable level for typical natural stands except of west of 2014. Fertility variations were 1.55, 3.05, and 1.64 in polled stands for the years. Gene diversity was 0.99 for the years in polled stands. North aspect could be taken into consideration in establishment and selection of seed sources and gene conservation areas based on fertility variation and gene diversity.There are many environmental and biological factors on forestry practices as known. Aspect called as slope faces is one of the most important environmental factors in these practices because of its easy application for managers. Fertility variation defined as an individual ability to give progeny and gene diversity estimated based on effective number of parents were investigated as the proportion of numbers of cones counted from individuals in natural stands sampled aspectual of Taurus cedar ( There are many environmental  and biological  factors on forestry practices. Aspect defining the compass direction that a slope faces is one of the most important environmental factors because of its easy application and cheap determination for these practices such as silvicultural, watershed, and other forestry operations for managers because of its effect on soil and forest formation and habitat. Aspect can have a strong influence on temperature. This is because of the angle of the sun in the northern and southern hemispheres which is less than 90 degrees or directly overhead. For instance, aspect is a criterion in selection and establishment of seed sources and selection of gene conservation areas  and theiThe main objective of the present study is to evaluate the fertility variation in selected aspectual natural stands of Taurus cedar basis of cone production over a period of three consecutive years and to estimate the effective number of parents and gene diversity in the stands. The impacts of aspect on fertility variation and gene diversity were also discussed for future forestry practices of the species.Five different natural stands called also aspectual populations located at latitude between 37°49′ and 37°53′ N, longitude between 31°15′ and 31°20′ E, and average elevation of 1600 m of Taurus cedar were sampled aspectually  in Isparta region at southern part of Turkey. Numbers of mature cone were counted from the same numbered thirty trees selected phenotypically during three consecutive years (2013–2015).C) was estimated based on individual cone production as [N is the census number; Coni is the cone fertility of the ith individual. CVC is the coefficient of variation in total fertility. In this paper, the fertility of ith individual was estimated by the proportion of cone production in the population. Therefore, the cone fertility represents the total contribution as zygotic parents.Cone fertility variation (Ψction as  (1)ΨC=N∑Np) was estimated as [Nr) was estimated relatively compared to the census number as Nr = Np/N.The effective number of parents (mated as  (2)Np=NΨNp) as [Yij is the observation from the jth tree of the ith population/population, μ is overall mean, Pi is the random effect of the ith population, and eij is random error.Gene diversity (GD) was estimated based on effective number of parents (Np) as (3)GD=1−0rp) [f is the covariance between the characters x and y; σ2f(x) and σ2f(y) are the phenotypic variances of character x and character y, respectively.Phenotypic correlations (rp)  among yeThere were large differences among aspects and years and also within aspect and year for cone production .p ≤ 0.05) were found among stands for cone productions of 2014 and 2015 based on results of analysis of variance, while it was opposite for 2013. Large differences in fertility were reported among trees in natural populations of different forest tree species  and andp ≤ 0s  correlations among years for cone production in the stands  was mirrored to the fertility variation (The effective number of parents (ariation . The effariation . Besidesariation , fertiliariation . The resariation . It emphAspect is one of the most important environmental factors for forestry practices because of its easy application for managers. It is used in many purposes in forestry from selection of seed sources and nursery practice to natural regeneration or forest establishment. Results of the study also emphasized importance of aspect in forestry practices such as preparing of silvicultural planning aspect level and management of gene conservation areas in watershed.


A case report of transverse myelitis and optic neuritis is presented in which the latter occurred following a myelogram with iopamidol. The literature relating to neurotoxicity of contrast agents is reviewed, and it is suggested that the optic neuritis may have been a neurotoxic effect of iopamidol.