Male fertility significantly decreased in the last 50 years, as showed in several studiesreporting a reduction of sperm counts per ml in the seminal fluid. Several “acute”pharmacological treatments, as antibiotics, could cause subclinical and temporaryreduction of male fertility; conversely, long-term medical treatment may severelyaffect male fertility, although this effect could be considered transient in most of thecases. Thus, nowadays, several long-term pharmacological treatments may represent a clinical challenge. The association between several kind of antihypertensivedrugs and reduction of male fertility has been showed in the mouse model, althoughthe modification(s) which may alter this fine-regulated machinery are still far tobe elucidated. Furthermore, well-designed observational studies and randomizedcontrolled trials are needed to accurately define this association in human model,meaning a narrative overview synthesizing the findings of literature retrieved fromsearches of computerized databases. We strongly solicit future human studies  on large cohorts with adequatestatistical power which may clarify this possible association and the effects (reversible orpermanent) of each drug. Furthermore, we suggest a close collaboration betweengeneral practitioners, cardiologists, and andrologists in order to choose the mostappropriate antihypertensive therapy considering also patient’s reproductive desireand possible risk for his fertility. Male fertility significantly decreased in the last 50 years, as showed in several studies reporting a reduction of sperm counts per ml in the seminal fluid . To dateIn this regards, Bechara et al.  have stu2+ plays a prominent role during fertilization in all animal species. On one hand, in mice, rats, pigs, hamsters and bovines, extracellular Ca2+ is necessary for epididymal acquisition of sperm motility  seem to play a detrimental role on male fertility, causing in several cases azoospermia and/or oligozoospermia. On the other hand, inhibitors of the funny channel, such as oral ivabradine, seem not to be associated with reduction of male fertility. In this regard, it was already demonstrated in the mouse model that CCBs, like amlodipine, can cause a reduction of testosterone, luteinizing hormone (LH) and follicular stimulating hormone (FSH), leading to affect spermatogenesis and sperm parameters ,13. Howemotility ,18. Furthermore, it is known to regulate both activated and hyperactivated motility ,21. In aThe average life expectancy is increasing and even more older male patients refer to fertility experts, the association between antihypertensive therapy and male infertility is a growing area of interest. Based on this scenario, we strongly solicit future human studies  on large cohorts and with adequate statistical power which may show this possible association and the effects (reversible or permanent) of each drug. Furthermore, we suggest a close collaboration between general practitioners, cardiologists, and andrologists in order to choose the most appropriate antihypertensive therapy considering also patient’s reproductive desire and possible risk for his fertility.


Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500–2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag–Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag–Gag interactions that occur during virus particle assembly and in released immature particles. Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified, and it is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) . It is eHTLV-1 is a deltaretrovirus and like other retroviruses, requires the expression of the Gag polyprotein—the main retroviral structural protein—to drive particle assembly and release . Gag traLipid rafts, i.e., tightly packed saturated lipids, are typically associated with virus budding sites , and itBudding and newly released particles are typically immature and are observed via thin section transmission electron microscopy as having an electron dense Gag layer along the inner leaflet of the viral membrane. The activation of the viral protease cleaves Gag to release the mature MA, CA, and NC proteins, which results in the conversion of an immature particle to a mature infectious particle. Following virus maturation, MA remains associated with the inner leaflet of the viral membrane, NC coats the viral RNA, and this complex along with reverse transcriptase and integrase are encapsidated by the CA core ,25. We previously reported a tractable model system for HTLV-1 Gag expression and the production of HTLV-1-like particles using yellow fluorescent protein (YFP)-tagged Gag . These vIn this study, we sought to investigate whether HTLV-1-like particle morphology would be perturbed as a result of co-packaging of Gag and Gag fused to a fluorescent protein . Gag co-packaging is biologically relevant in the retrovirus assembly pathway as HTLV-1 and retrovirus particles naturally co-package Gag and Gag-Pol. To help address this, we have used cryogenic transmission electron microscopy (cryo-TEM), scanning transmission electron microscopy (STEM), and fluorescence fluctuation spectroscopy (FFS) to investigate co-packaging of Gag and Gag-YFP. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 83 to 120 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, STEM and FFS analyses indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was in the range of 1500–2000 molecules per VLP. Thus, the HTLV-1 Gag copy number and particle size distribution observed with a 3:1 ratio of Gag:Gag-YFP was similar to that in authentic HTLV-1 particles  and with6 HEK 293T cells maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% Fetal Clone III  were co-transfected with 6 µg of pN3-HTLV-1 Gag-YFP or 6 µg total of pN3-HTLV-1 Gag and pN3-HTLV-1 Gag-YFP  along with an HTLV-1 envelope protein (10:1 ratio of Gag to envelope plasmids) expression construct using GeneJet  following the manufacturer’s instructions. Twenty-four hours post-transfection, 2 mL of fresh media was added to cells and incubated for another 24 h. Cell culture supernatants of transfected cells were collected 48 h post-transfection, clarified by centrifugation 3000× g for 5 min, and filtered through a 0.2 µm filter.A plasmid construct expressing HTLV-1 Gag with YFP fused to the carboxy-terminus   and a cog for 1.5 h using a 50.1 Ti rotor  at 4 °C. The VLP pellet was resuspended in 0.5 mL of 1× sodium chloride-Tris-EDTA (STE) buffer ), and overlaid onto a 4 mL 10–40% OptiPrep gradient and centrifuged to equilibrium in a SW55 Ti rotor (Beckman) at 250,000× g for 3 h at 4 °C. The fraction containing the concentrated particles was removed from the gradient using a hypodermic needle, diluted 10-fold in 1× STE buffer and pelleted at 195,000× g for 1 h in a SW55 Ti rotor at 4 °C through an 8% Optiprep cushion. Following centrifugation, the VLP pellet was resuspended in ~15 µL of 1× STE overnight at 4 °C. Samples were then used for cryo-TEM and STEM analyses. For FFS measurements, 293T cells were transfected and cell culture supernatant was collected in the manner described above. Supernatant was filtered through a 0.2 µm filter and 200 µL was added to 8-well Nunc Lab-Tek Chamber Slides . Slides were then sealed to prevent sample loss due to evaporation and used in FFS analyses.VLPs were then subjected to ultracentrifugation through an 8% OptiPrep  cushion at 109,000× 2) and 1 to 5 µm underfocus conditions using a Gatan 4 k by 4 k charged coupled device (CCD) camera . The fluorescence image and corresponding TEM picture were recorded using an FEI iCorr installed on a FEI Tecnai Spirit TEM.VLP samples were prepared for cryo-TEM analysis as previously described . BrieflyTwo perpendicular diameter measurements were done for each VLP, as previously described ,26. CryoVirus-like particles were analyzed on an inverted microscope  modified for two-photon FFS with an excitation wavelength of 1000 nm provided by a titanium-sapphire laser . FFS measurements were performed by focusing the laser inside the VLP solution and recording the fluorescence emission as the VLPs moved in and out of the observation volume via passive diffusion. Fluorescence emission was collected by a 63× water immersion objective , separated from excitation light by a dichroic filter, and detected by a single-photon counting module. The photon counts were recorded with a sampling frequency of 20 kHz and stored for later analysis with routines written in IDL 8.3 .N of the two brightness species. The normalized brightness b of the VLP sample was defined by the ratioYFPλ, was obtained from a separate series of FFS measurements where the excitation power was systematically varied and the brightness of a purified YFP solution was determined for each excitation power. A representative PCH of purified YFP measured at 0.47 mW excitation power and its corresponding fit to a model with a single fluorescent species is excerpted from this calibration series and shown in 2) of 1.7. This occupation number corresponds to a concentration of ~1 μM, which is far below the reported dissociation constant of YFP dimers (~100 μM) . B. B43]. Bterature , which iD byd of VLPs was obtained from the Stokes–Einstein relationThe diffusion time, The total mass of HTLV-1-like particles was determined (as previously shown ) by usinThe total RNA content for each sample of VLPs was determined by extracting the RNA from particle lysates with RNA columns, using Roche’s High Pure Viral RNA Kit . The extracted RNA was detected by UV absorption at 260 nm using a Beckman DU-65 spectrophotometer . A Thermo Scientific Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific) was used to estimate the protein content of the same sample used to determine VLP RNA content. A bovine serum albumin protein standard and measurement of UV absorption at 562 nm were used to establish an approximate absorbance-to-concentration curve and estimate the total protein concentration of VLP samples.We recently reported that HTLV-1-like particles produced by expression of HTLV-1 Gag have an electron dense ring of Gag along the inner leaflet of the viral membrane . The aveCorrelative light and electron microscopy demonstrated that the fluorescence signal co-localizes with the distribution of YFP-labeled VLPs on a frozen-hydrated TEM grid A,B. Figun = 9) of HTLV-1 Gag-YFP VLP samples resulted in a copy number of 370 ± 50.Fluorescence Fluctuation Spectroscopy (FFS) was used to measure Gag stoichiometry and determine an average Gag copy number. The experiment records the photon counts of the fluorescence emitted as VLPs diffuse through the observation volume. A representative photon count trace of HTLV-1 Gag-YFP VLPs is shown in VLPs were then harvested and analyzed from cells transfected with HTLV-1 Gag and HTLV-1 Gag-YFP at a ratio of 1:1. Copy number determinations by FFS represent only the Gag-YFP population packaged into the VLPs. Therefore, we estimated the total Gag copy number by using the assumption that the ratio of Gag to Gag-YFP within the VLPs is equivalent to the transfection ratio, resulting in a total copy number of 730 ± 130 Gag molecules per VLP. FFS measurements of VLP samples with 2:1 and 3:1 transfection ratios of Gag:Gag-YFP were then analyzed by PCH and the total Gag stoichiometry was inferred from the transfection ratio to be 990 ± 130 and 1600 ± 530 molecules of Gag per VLP, respectively. The FFS experiments identified an increase in the total Gag copy number as a function of the Gag:Gag-YFP ratio A, which In addition to PCH analysis, we also investigated the autocorrelation function (ACF) of the photon counts to estimate the size of VLPs by FFS. The diffusion time, mplitude C. The dimplitude B. The vamplitude B.−1)  C,F. By a−1) C and by −1) F were deTo confirm the FFS measurements of Gag stoichiometry, we also conducted measurements using STEM. The total mass measurements of VLPs produced by expression of HTLV-1 Gag-YFP or HTLV-1 Gag:Gag-YFP particles at a ratio of 3:1 were used to estimate the average Gag copy number per VLP. The Gag:RNA mass ratio was determined to be 19.8:1 and 18.3:1 for VLPs produced with HTLV-1 Gag:Gag-YFP at a 3:1 ratio and HTLV-1 Gag-YFP, respectively, which yielded an RNA content of approximately 3% of the total VLP mass . The average VLP diameter of 83 nm and 113 nm for VLPs produced by expression in 293T cells of HTLV-1 Gag-YFP and HTLV-1 Gag:Gag-YFP at a ratio of 3:1, respectively, and the 5 nm thickness of the viral membrane were used to estimate the number of lipid molecules in the VLP membrane. The average molecular weight of membrane lipids was estimated to be 750 Da, and it was assumed that the distance between lipid molecules is 0.85 nm . Given tWhile HTLV-1 was the first human retrovirus discovered 35 years ago ,52, the HTLV-1-like particles were produced by transient transfection into 293T cells of an HTLV-1 Gag expression plasmid, an HTLV-1 Gag-YFP expression plasmid, or by co-transfection of these two plasmids at various ratios. VLP production was highly robust, due in part to the fact that the human-codon optimized versions of the Gag gene were used. We previously found that the HTLV-1 Gag-YFP construct can be used to produce VLPs ,26. InteFFS A and STEThe findings of this study support the conclusion that biologically relevant Gag–Gag interactions occur between Gag and Gag-YFP when they are at a ratio of 3:1 (Gag:Gag-YFP). Increasing the amount of Gag led to similar observations, while increasing the amount of Gag-YFP resulted in small VLPs, fewer Gag molecules per VLP, and an altered particle morphology . These findings are important for the analysis of Gag-YFP in living cells, particularly regarding the study of the processes involved in Gag oligomerization and Gag lattice formation. Furthermore, these studies are important for understanding the natural interactions and co-packaging between Gag and Gag-Pol, which is critical to ensure that the viral enzymes are packaged into virus particles. Proper formation of the immature Gag lattice is likely critical in order to ensure efficient Gag processing by the viral protease and formation of mature, infectious virus particles. Therefore, the ratio of co-packaging Gag and Gag-Pol is critical in order to ensure proper Gag stoichiometry as well as immature particle morphology (and likely particle infectivity). These observations support previous reports of the importance of Gag to Gag-Pol ratios in retroviral assembly ,56. Give


Neurotoxins such as amyloid-β, tau protein, and α-synuclein trigger abnormal calcineurin/NFAT signaling activities. Additionally increased activities of endogenous regulators of calcineurin like plasma membrane Ca2+-ATPase (PMCA) and regulator of calcineurin 1 (RCAN1) also cause neuronal and glial loss and related functional alterations, in neurodegenerative diseases, psychotic disorders, epilepsy, and traumatic brain and spinal cord injuries. Treatment with calcineurin/NFAT inhibitors induces some degree of neuroprotection and decreased reactive gliosis in the central and peripheral nervous system. In this paper, we summarize and discuss the current understanding of the roles of calcineurin/NFAT signaling in physiology and pathologies of the adult and developing nervous system, with an emphasis on recent reports and cutting-edge findings. Calcineurin/NFAT signaling is known for its critical roles in the developing and adult nervous system. Its role in physiological and pathological processes is still controversial. However, available data suggest that its beneficial and detrimental effects are context-dependent. In view of recent reports calcineurin/NFAT signaling is likely to serve as a potential therapeutic target for neurodegenerative diseases and conditions. This review further highlights the need to characterize better all factors determining the outcome of calcineurin/NFAT signaling in diseases and the downstream targets mediating the beneficial and detrimental effects.The ongoing epidemics of metabolic diseases and increase in the older population have increased the incidences of neurodegenerative diseases. Evidence from murine and cell line models has implicated calcineurin-nuclear factor of activated T-lymphocytes (NFAT) signaling pathway, a Ca NFATof NFAT5 , 75, 76.programs , 77, 78.2+-regulated NFATs (NFATc1–c4) are expressed in neurons [All Ca neurons . The pre neurons , 80, 81. neurons . In that3, which in turn leads to the release of Ca2+ from intracellular stores through IP3Rs [2+ release from the intracellular stores requires a stimulus capable of generation of second messengers that trigger Ca2+ release from intracellular stores in the endoplasmic/sarcoplasmic reticulum, via channels of IP3R1–3 and ryanodine receptors (RyR1–3) [2+/CaM-dependent enzymes, calcineurin activation requires increase in cytosolic Ca2+ levels. Such activation may result from the binding of Ca2+ to calcineurin B subunit or from the binding of Ca2+-activated CaM to calcineurin [2+ signaling and gene expression. Calcineurin also promotes nuclear retention of NFAT by masking nuclear export signals (NES) and preventing NES-dependent nuclear export [2+ released from intracellular stores is not sufficient for direct NFAT activation [The activation of calcineurin/NFAT signaling pathway involves three key steps: (i) NFAT protein dephosphorylation by calcineurin; (ii) nuclear translocation of NFATs; and (iii) increased affinity for DNA. Ligand-receptor interaction activates phospholipase C (PLC) resulting in the release of IPgh IP3Rs , 82. Not(RyR1–3) , 83–87. cineurin . In turncineurin . The depcineurin . In the r export , 89. Mortivation , 90.Regulators of calcineurin (RCANs) or modulatory calcineurin-interacting proteins  belonging to the calcipressin family of proteins play a pivotal role in regulating calcineurin activity. The RCANs are evolutionarily conserved proteins that can directly bind and inhibit calcineurin. The RCAN1 genes are found on chromosome 21. The RCAN gene encodes different isoforms of protein, namely, RCAN1, RCAN2, RCAN3, and RCAN4 where RCAN1 and RCAN4 are the main isoforms. Furthermore, it appears that RCAN1 has two RCAN1 isoforms: 1S with 197 amino acids and RCAN.1L with 252 amino acids. Previous studies have shown that an aberration in RCANs activities decreases calcineurin-NFAT signaling activities. Recent studies have shown that RCANs can show both inhibitory and facilitatory roles in activation of the calcineurin-NFAT signaling pathway. A mechanistic explanation on how RCAN proteins precisely modulate calcineurin function is still debatable. Overall RCANs have been implicated to function primarily as chaperones for calcineurin biosynthesis or recycling, requiring binding, phosphorylation, ubiquitylation, and proteasomal degradation for their stimulatory effect , 91, 92.2- terminal kinases (JNKs) and cellular stress-associated p38 kinase [The regulation of calcineurin/NFAT activity is achieved: (i) in the nucleus by activities of serine/threonine kinases promoting the export of nuclear NFAT; (ii) in the cytoplasm, through phosphorylation of NFAT serine (SP) repeats and N-terminal domain, which are critical for NFAT activation and nuclear import , 93, 94.8 kinase , 54. Alt8 kinase , 54. Oth8 kinase , 89, 95.Calcineurin/NFAT signaling blocking agents mainly act at calcineurin and NFAT levels. They include cellular (protein inhibitors) and pharmacological inhibitors. Although numerous endogenous proteins may have potential to inhibit calcineurin or NFAT activities, only four are well-characterized: (i) A-kinase anchoring protein 79 (AKAP79), a scaffold protein that prevents calcineurin-substrate interactions , 97; , and its ethyl analog ascomycin, are commonly used as immunosuppressants, particularly in organ transplantation –107. TheRecent studies in rat brain slices and cultured astrocytes revealed that cyclosporine A increases reactive oxygen species (ROS) formation and alters glucose and energy metabolism partly by Krebs cycle inhibition and anaerobic glycolysis activation . These dMoreover, experimental evidence has shown increases in spinal NMDA receptor activity as a result of calcineurin inhibitor induced pain syndrome (CIPS) –116. A w per se, but rather interfere with enzyme targeting of one or more of its substrates, have recently been developed and used. For instance, the inhibitors of NFAT-calcineurin association (INCA) compounds may interfere selectively with calcineurin/NFAT interaction without preventing dephosphorylation of other calcineurin substrates [Interestingly, inhibitors that do not block calcineurin enzymatic activitybstrates , 120. SuCalcineurin/NFAT signaling pathway is multifunctional. In the PNS, calcineurin/NFAT signaling was reported to have critical roles in the survival, proliferation, and differentiation of both neural and glial precursor cells , highlig2+ entry (SOCE) activation regulates gene transcription in the developing nervous system and mediates neural progenitor cell proliferation through calcineurin/NFAT signaling [Evidence supporting the involvement of the calcineurin/NFAT4 signaling pathway in corticogenesis includes a study in developing mouse cerebellar granule neurons that reported a pivotal role in controlling the temporal regulation of nuclear factor 1 occupancy. This serves as a key link between membrane potential and dendritic maturation, by a voltage-sensitive developmental switch . In addiignaling –125.The role of the calcineurin/NFAT signaling in corticogenesis is probably mediated by NFATc3, the predominant NFAT isoform in neural progenitor cell cultures, which is also a potent inducer of neural progenitor cell differentiation into neurons and astrocytes , 126. InThe complex interactions between inhibitory gamma-aminobutyric acid (GABA) and excitatory NMDA receptor activities are required during synaptogenesis. Furthermore, this interaction plays an important role in the induction of immediate early genes necessary for effective changes in synaptic plasticity and long-term memory formation through calcineurin-dependent transcription of the key brain-derived neurotrophic factor (BDNF) , 130. Of2+ signaling in rat hippocampal CA1 pyramidal neurons via a mechanism depending on calcineurin and on actin-binding protein cofilin [Moreover, GABA promoted the shrinkage and elimination of synapses by suppressing local dendritic Ca cofilin . Similar cofilin , 136.It is now widely accepted that calcineurin signaling is a major player in the control of the trafficking and signaling of endosomes performing the retrograde signaling, an event critical for the development, but also for the nervous system function , 138. Caβ, independent of Ca2+ and calcineurin signaling. For instance, a study assessing the physiological roles of estrogen-related receptor gamma (ERRγ), an orphan nuclear receptor highly expressed in the nervous system during embryogenesis and over lifespan, revealed its involvement in the regulation of dopaminergic neuronal phenotype. Such effect was mediated by GSK-3β-NFAT interactions, independently of Ca2+/calcineurin signaling [2+/calcineurin signaling. Instead, NGF effects were induced by phosphoinositide 3-kinase (PI3K)/Akt signaling triggered by TrkA receptor activation, which abrogated GSK-3β activity [Notably, as other tyrosine kinase receptors, the receptors of neurotrophins may also activate NFATs via inhibition of the promoter of NFAT nuclear export GSK-3ignaling . Similaractivity .γ isoform of the calcineurin catalytic subunit, such as synaptic vesicle cycling, have been suggested to contribute to schizophrenia, where variations in calcineurin Aγ gene PPP3CC are common in neurons [2+ also associates with the disruption of fast axonal transport (FAT) in the pathogenesis of Alzheimer's disease (AD) [Furthermore, calcineurin is universally involved in vesicle endocytosis , and alt neurons . Deregulase (AD) –144.Calcineurin/NFAT has critical roles in neuronal and glial cell activity and survival, as well as resulting events, fundamental for nervous system function like neurotransmission and synaptic plasticity, and myelination as summarized in 2+ channel activation play critical roles in the shaping of synaptic connectivity of thalamocortical and nucleus reticularis thalami GABAergic neurons mediated by slow wave sleep [Calcineurin/NFAT signaling and other pathways activated by T-type Cave sleep , a proceve sleep , 147. PLve sleep . Furtherve sleep , 87 that+ channels, in dentate granule cells [2+ levels associated with prolonged blockade of synaptic activity resulted in the synthesis of retinoic acid, which triggered the related homeostatic synaptic plasticity, via a calcineurin-dependent mechanism in neurons [Experimental evidence also suggests that calcineurin is a regulator of synaptic plasticity. For instance, axon initial segment rapid shortening was partly mediated by calcineurin-dependent mechanisms, including phosphorylation of voltage-gated Nale cells . Rapid m neurons . Moreove neurons . Similar neurons .β substrate presenilin 1 (PS1) [β (Aβ) generation. In a study using a spinocerebellar ataxia type 3 (SCA3) transgenic mouse model, typical impairments of motor learning and cerebellar motor coordination resulted from altered long-term depression (LTD) of glutamatergic transmission in parallel fiber-Purkinje neurons [β4, IP3R1, and calcineurin B, suggesting that PLC/IP3-Rs/calcineurin signaling is required for cerebellar LTD induction, thus motor learning and coordination.Evidence of calcineurin/NFAT involvement in learning and memory processes was provided by experimental models of neurodegenerative disorders. A study in PS1-M146V knock-in FAD mice showed that decreased calcineurin activity is a common phenomenon in aging-related memory decline and may account for memory defects in AD, together with mutations in the gene encoding for GSK-3 1 (PS1) , which a neurons . Such al+ channels [γ2 subunit at inhibitory synapses [Calcineurin/NFAT signaling is also involved in neuronal excitability and neurotransmission. Under physiological conditions, AKAP79/150-mediated calcineurin/NFAT signaling may prevent neuronal hyperexcitability in hippocampal neurons by increasing the transcriptional expression of key regulators of neuronal excitability like M-type Kchannels . PKC/calchannels , and memsynapses .2+-dependent inactivation of L-type Ca2+ channels, while the activation of the phosphatase activity of AKAP79/150-anchored calcineurin reversed such PKA action, reducing Ca2+-dependent inactivation [2+ channel-calcineurin/NFAT signaling functional coupling by preserving the phosphorylation of these channels, contrary to anchored calcineurin [The interplay between calcineurin and PKA signaling plays a critical role in the negative-feedback mechanism driving homeostatic synaptic plasticity , that istivation , 159. Thcineurin . Interescineurin .Furthermore, PKA can induce NFAT nuclear export , 54. PKAOpposite effects of calcineurin and PKA were also reported as key events in (i) the dynamic fission-fusion events that determine the shape and function of mitochondria , 163, th2+-dependent NFAT isoforms; (ii) the promoter and upstream enhancer elements of the myelinating factor Krox-20 contain NFAT binding sites; and (iii) NFATs recruited by Ca2+-dependent signaling can make transcriptionally active complexes with Krox-20 [ in vitro model of myelination that promyelinating actions of calcineurin/NFAT signaling, including increases in the expression of the myelinating genes Krox-20, Periaxin, and P0, require cAMP elevation [2+ failed to induce Krox-20 expression. Furthermore, cyclosporine A and FK506 abrogated Krox-20 expression. Comparable observations were reported more recently by other authors [Available data suggest that calcineurin/NFAT pathway participates in signaling cascades pivotal for Schwann cell myelination. Early studies reported that (i) murine Schwann cells express all Ca Krox-20 , 93, 165 Krox-20 , 166. Mo authors .Experimental evidence also suggests that the activation of calcineurin/NFAT required for Schwann cell myelination occurs partly through neuregulin 1 (NRG1) stimulation , 168–170Unexpectedly, FK506 stimulated Schwann cell proliferation and promoted the survival of oligodendrocyte in murine models of traumatic spinal cord injury –174, sug2+ pumps (PMCAs), a suggested link between brain aging and the onset of neurodegenerative diseases [Increased calcineurin activity was reported in both aging and AD models , 178. Fodiseases , 180. Thdiseases –183. Apodiseases , 29.β oligomers resulted in calcineurin activation with transient changes in postsynaptic proteins and morphological in spines, while longer exposure resulted in NFAT activation and marked spine loss in primary cortical neurons of wild-type mice [β-treatment of murine hippocampal neurons also resulted in Ca2+ signaling-dependent defects in BDNF transport first in dendrites and then in axons [Overactivated calcineurin/NFAT signaling may contribute to synaptic plasticity affection in pathological conditions. Notably, in a postmortem study in human hippocampi, high nuclear levels of NFATs observed at the early stage of AD increased with cognitive decline severity . Additioype mice . Aβ-treain axons . Studiesin axons .β-induced neurodegeneration [β levels in postmortem hippocampus, while oligomeric Aβ strongly stimulated NFAT activation in primary rat astrocyte cultures. In another study, NFATc4 levels were significantly increased in brains of APP/PS1 transgenic mice (AD model) and NFATc4 overexpression increased Aβ production in human myeloid leukemia SAS-1 cells [β and Aβ precursor protein (APP) trafficking [NFAT signaling alterations in neurodegenerative diseases are selective. A report by Abdul and colleagues strongly suggested that such selective alteration may play a key role in Aneration . These a-1 cells , suggestfficking –190.α-synuclein-mediated midbrain dopaminergic neuron toxicity, a hallmark of Parkinson's disease (PD), is controversial. In a study addressing the underlying intracellular mechanisms driving α-synuclein-mediated neurodegeneration, transgenic expression of PD α-synuclein A53T missense mutation promoted calcineurin/NFAT signaling, suggesting that this signaling pathway may contribute to the neurotoxic effects of aberrant α-synuclein [α-synuclein also seemed to induce cellular toxicity via overactivation of Ca2+-dependent signaling pathways, calcineurin inhibition with FK506 also resulted in toxicity [α-synuclein. Characterizing the precise downstream targets mediating calcineurin beneficial or toxic effects may provide more insights of the novel therapeutic targets for synucleinopathies.The role of calcineurin in aberrant ynuclein . Surpristoxicity , suggestβ treated in cultured rat hippocampal neurons and cultured hippocampal neurons from APPSwe AD-transgenic mice suggested that calcineurin signaling mediates AD-like synaptic dysfunction induced by tau protein partly via AMPA receptor downregulation [β oligomer-induced deficits in AMPA receptor trafficking were mediated by tau phosphorylation and mislocalization to dendritic spines. FK506 abrogated all these alterations. Concomitant tau hyperphosphorylation and calcineurin overactivation were also reported in mouse models of Huntington's disease [2+ channel activity and intracellular Ca2+ store release [Calcineurin also mediates some beneficial and toxic effects of AMPA and NMDA receptors, which are postsynaptic site-located glutamate-gated ion channels critical for synaptic plasticity. Calcineurin translocation to synapses and increases in its activity mediated by NMDA receptor trafficking were reported as critical components of mechanisms driving rapid compression-induced dendritic spine plasticity in cortical pyramidal neurons, that is, the rapid trimming of dendritic spines occurring about 12 hours after mechanical compression . A studygulation . In that disease . On the  release .β/calcineurin-induced release of IGF-1-binding protein 3 (IGFBP-3); but intriguingly, Aβ directly induces increases in tau phosphorylation, and resulting neuronal death, via a mechanism involving the silencing of NFAT export kinase GSK-3β [α-induced nuclear translocation of NF-κB [α, resulting in detrimental neuroinflammation and functional alterations in neurons, is mediated by astrocytic and microglial calcineurin/NFAT signaling [ in vitro models of neuroinflammatory diseases provided mechanistic insights into the context of these opposite roles. These studies showed that TNF-α activate calcineurin/NFAT/NF-κB canonical inflammatory pathway in quiescent astrocytes, while in activated astrocytes, IGF-1 released locally recruited calcineurin signaling to inhibit NF-κB-NFAT transcriptional activity through activation of the purinergic receptor P2Y6 [Moreover, in AD pathogenesis, insulin-like growth factor 1 (IGF-1) that acts as a regulator of tau phosphorylation is silenced in activated astrocytes by Ae GSK-3β , suggeste GSK-3β . Althougof NF-κB –199, it ignaling –202. Stutor P2Y6 , 204, suβ plaques, reduced glial activation, alleviated both Aβ synaptotoxicity and neurotoxicity, and improved synaptic function [2+-permeable AMPA receptors, and promoted synaptic trafficking of Ca2+-permeable AMPA receptors, as well as the resulting improvement in animal cognition [2+-permeable AMPA receptor-mediated hippocampal LTP [γ-8, which can stop the trafficking of both AMPA and to a lesser extent NMDA receptors [The pharmacological inhibition of calcineurin/NFAT signaling improved animal condition in a number of studies in neurodegenerative diseases and models. Examples include AD mouse models where pharmacological inhibition of this signaling pathway decreased Afunction , 205–207function , 208. Thognition . Such imeceptors –211, mayeceptors  and calceceptors , 213.2+ overloading-induced neuronal apoptosis [2+ overloading and oxidative stress in neurodegenerative disorders  protected these cells against NMDA receptor-induced excitotoxicity, through metal chaperone PBT2-induced calpain cleavage of calcineurin [Altogether, these observations suggest a role for calcineurin/NFAT signaling in astrocyte and neuronal losses observed in nervous system injury. Unexpectedly, sublethal ischemia increased neuronal resistance to excitotoxicity via calcineurin-dependent mechanisms including cyclin E1 protein increased expression and declustering of the delayed rectifying Kclusters . In addicineurin , 112. Th2+ signaling [2+ channels, suggested that the disease may be caused by disturbances in transcriptional activities of Ca2+-dependent signaling molecules like FOX proteins, MEF2, CREB, and NFATs [Early genetic studies showed that polymorphisms of the genes coding for either the catalytic or regulatory subunit of calcineurin isoenzymes are strongly associated with the risk for developing schizophrenia and other psychotic disorders whose pathological features include disturbances in Caignaling , 243–246nd NFATs .Furthermore, GABA A receptor activation promoted a decrease in anxiety indicators and hippocampal neurogenesis via the calcineurin/NFAT4 signaling in mice, suggesting that pharmacological targeting of this signaling pathway may improve emotional disorders . SimilarMany lines of evidence also support calcineurin involvement in nervous system diseases. Notably, various reports have suggested that calcineurin is likely to mediate physiological and pathological activities of GABA receptors. For instance, complex interactions between PKC and calcineurin may play a key role in GABA B autoreceptor-mediated functional regulation of nicotinic acetylcholine receptors (nAChRs), whose activation triggers the release of neurotransmitters from presynaptic nerve terminals, in mouse striatal GABAergic nerve terminals . SomaticCertainly, calcineurin/NFAT signaling involvement was also shown in pathogenic processes of other models of status epilepticus, including intracerebral injection of kainic acid , bicucul2+-dependent and other signaling pathways from currently used models of neurodegenerative diseases, in particular transgenic animals and cell lines. A study addressing the suitability of rat striatal primordia-derived ST14A cell line for the study of voltage-gated Ca2+ channel of striatal medium spiny neurons called for serious caution on the assumption of the presence of complete signaling cascades of G-protein coupled receptors in cell lines [β1, a major effector of G-proteins for Ca2+ release from intracellular stores [2+ flux required for mammalian sperm acrosome reaction [A growing number of studies are raising concerns about mechanistic reports of Call lines . Notablyr stores , 82, whor stores ; (ii) threaction ; and (iireaction . In addireaction –266. Simreaction –269. In Calcineurin/NFAT pathway is pivotal during nervous system development and in various functions of mature central and peripheral nervous system. Notably, this signaling pathway is involved in myelination, corticogenesis, synaptogenesis, neuritogenesis, endosome trafficking, homeostatic synaptic plasticity, learning, and memory. Experimental evidence also shows that alterations in the activity of calcineurin/NFAT pathway and in activities of its endogenous regulators in the nervous system microvascular endothelial cells, astrocytes, microglia, Schwann cells, oligodendrocytes, and neurons participate in the pathogenesis of neurodegenerative diseases and conditions, but also psychotic disorders. Studies in transgenic animals and in cell lines also suggested that neurodegeneration-associated detrimental changes in calcineurin/NFAT signaling are NFAT isoform-selective as changes in NFATc3 and NFATc4, but not NFATc1 or NFATc2, are usually common. In addition pharmacological inhibition mitigated neuronal and astrocyte loss and improved cognitive functions in many models. However, other studies reported beneficial roles of calcineurin/NFAT in neurodegenerative diseases and conditions, in particular those reporting neurotoxic effects of pharmacological inhibition and increased endogenous regulation. These studies suggested that calcineurin can mediate both neuroprotective and neurodegenerative signals according to poorly understood determinant factors, which included the activation status of astrocytes in the central nervous system. Future studies should be devised to characterize better the factors determining the outcome of calcineurin/NFAT signaling in neurodegenerative diseases and conditions, as well as the downstream targets mediating the beneficial and detrimental effects of this signaling pathway, considering the implications for therapy.


In their recent study, Lowengrub, Waterman, and colleagues (Lee  PreviouThe inclusion of both Wnt signaling and glycolytic patterns in the mathematical model allows for a deeper characterization of how Wnt signaling could affect spatial patterning of metabolism in tumors. For instance, the model indicated that simply decreasing the level of Wnt signaling leads to a reduced number of glycolytic cells without affecting the location of the overall pattern. However, interfering with Wnt signaling by expressing a dominant‐negative LEF‐1 (dnLEF‐1) or a dominant‐negative TCF‐1 (dnTCF‐1) in xenografts resulted in both reduction in glycolysis activity and alterations in metabolic patterning that was marked by formation of larger and sparser clusters of glycolytic cells. In order to resolve this discrepancy, the authors hypothesized that the dnLEF‐1 and dnTCF‐1 models were eliciting effects more complicated than inhibiting Wnt signaling. Parameter sensitivity analysis of the model indicated that the change in spot distribution was likely to be the consequence of other factors that increase the diffusion length scale of both the activator and the inhibitor of Wnt signaling. One possible mechanism of increased diffusion length could be the enhanced expression of factors known as Wnt diffusers. By analyzing expression of candidate genes in dnLEF‐1 and dnTCF‐1 xenograft tumors, the authors identified the Wnt diffusers SPOCK2, GPC4, and SFRP5 as increased in response to interference of Wnt signaling, thus supporting the prediction of the model. Interestingly, increased expression of Wnt diffusers was also observed in tumor samples from colorectal cancer patients treated with radio‐ and chemotherapy, suggesting that increasing the diffusion length of Wnt signaling molecules might be a general strategy for cancer cells to respond to stress. This would provide an example of the role of the physical properties of a tumor in determining its phenotype. Finally, the model was further applied to simulate therapeutic interventions in tumors with metabolic heterogeneity and it demonstrated potential synergy between inhibiting Wnt signaling and selectively targeting glycolytic cells.et al (et al (As the authors mention, it is not entirely possible to exclude alternative models that could produce similar outcomes and it is also unclear whether pPDH is an actual measure of glycolytic metabolism and thus metabolic heterogeneity. Nevertheless, the study by Lee et al  identifil (et al  provides


Through mutation, selection, and drift, viruses can optimize their replication efficiency and produce more offspring, which could increase the chance of secondary transmission. To evaluate how higher CAI towards the host has been associated with higher viral titers, we explored temporal trends of several historic and extensively sequenced zoonotic flaviviruses and relationships within the genus itself. To showcase evolutionary and epidemiological relationships associated with silent, adaptive synonymous changes of viruses, we used codon usage tables from human housekeeping and antiviral immune genes, as well as tables from arthropod vectors and vertebrate species involved in the flavivirus maintenance cycle. We argue that temporal trends of CAI changes could lead to a better understanding of zoonotic emergences, evolutionary dynamics, and host adaptation. CAI appears to help illustrate historically relevant trends of well-characterized viruses, in different viral species and genetic diversity within a single species. CAI can be a useful tool together with  When a virus first jumps into a human host from a non-human vector or reservoir host, conserved changes in viral population sequences can occur that could allow the virus to adapt to a new host ,2. Part i.e., years), allowing side by side comparisons with their epidemic history. There is plausibility for increased CAI and the occurrence of human disease from acutely infecting zoonotic viruses. It can be argued that a virus, whose codon usage is calibrated to the host’s usage, may have the capability to translate and replicate faster than a virus that has a lower codon adaptation to a specific host [The availability of codon usage tables for a host’s constitutively expressed or highly expressed genes could allow analysis of adaptive changes of acutely infecting zoonotic viruses , as theyfic host ,11. Becafic host .A requirement for flaviviruses, and arborviruses in general, is the initial need to adapt to the codon choices of a vector species before the virus adapts to the codon usage choices of humans . The alti.e. sylvatic and endemic strains of DENV and YFV, or the distinct genetic lineages of WNV [Yellow Fever virus (YFV), Dengue virus (DENV) and West Nile virus (WNV) are globally prevalent flaviviruses that cause morbidity and mortality in humans. These three flaviviruses have extensive genetic and biological diversity within themselves, s of WNV ,18, whics of WNV –29. To pwww.geneious.com). See For each virus , all available complete genome sequences with collection date and country of isolation information were downloaded from Genbank. Open reading frames of sequences were exported aligned using MAFFT 7.222  and thenTo investigate if our selected viruses have evidence for codon adaptation in humans, we calculated the codon adaptation indices (CAI) for each coding region of each respective virus. For Yellow Fever virus, Dengue 2 virus, and West Nile virus, the complete polyprotein was used to calculate CAI. For Tobacco Mosaic virus, coding regions were concatenated into frame and then used to generate CAI values.To calculate normalized CAI, we first used the CAIcal program  to obtaiCulex pipiens, Aedes aegypti, Macaca mulata, Columba livia and Rhipicephalus microplus were downloaded directly from the publically available Codon Usage Database (www.kazusa.or.jp/codon).The codon usage tables for Nicotiana tabacum (tobacco) house-keeping genes. The human codon usage table available on the Kazusa Codon Usage Database was last updated in 2007. Since highly or constitutively expressed genes will manifest greater codon preference biases [https://github.com/CaioFreire/CUB).We used 3 in-house codon usage tables that aimed to represent human house-keeping genes, highly expressed human antiviral immune genes, and e biases ,2,9, we e biases  were sele biases ,34, varie biases –38, and e biases ,40. Ultie biases . Variant4) was estimated from the data. Tip times corresponding to the year of virus sampling, a lognormal uncorrelated relaxed clock using continuous quantile parameterization [Wm) for the polyprotein, which was approximated by the ratio of the population size in sequential time points (Wm = Ne.gt/Ne.gt−1) [First, 790 West Nile Virus complete polyprotein sequences from Genbank were downloaded. Sequences were aligned and curated by coding sequences using Muscle and Geneious respectively. Using FastTree.v2 , a maximrization , and a Grization  was usedrization . The MCMrization  and prevNe.gt−1) .s.d. = ±4.104) with a max difference of 18.1 (All extended codon table comparisons can be found in Supplementary Data).House-keeping genes are constitutively expressed in all human cells ,48; beinNicotiana tabacum) housekeeping genes [We first wanted to show that there are measurable and meaningful viral-host adaptations that could be observed using CAI. As a proof of concept for virus-host codon adaptation, we calculated the CAI of Tobacco Mosaic virus (TMV) using complete coding sequences against the codon preferences of tobacco , and no known-vector flaviviruses [et al.’s 2015 paper [i) human housekeeping, ii) highly expressed human antiviral immune genes, iii) Aedes aegypti, iv) Culex pipiens, v) Rhipicephalus microplus (tick), vi) Macaca mulata (rhesus monkey) and vii) Columba livia (rock dove pigeon)  .in vitro and in vivo [p-values ranged from 1.985x10-5 to 0.0457). Although all CAI results for vertebrate CAIs were significantly lower to the Culex and Aedes tables, we did not find any significant difference between tick and monkey codon adaptation within the group A clade.Insect-specific groups A includes Cell fusing agent virus (CFAV), Culex flavivirus (CxFV), Aedes flavivirus (AEFV), Kamiti River virus (KMV) and seven others : all vir in vivo ,64–70. BAedes and Culex-borne viruses. The general equality across all of the CAIs of group F is different than the other insect-specific group (A), where the CAI of vector species is visibly greater than vertebrate species. The group F species showed no clear relationship between vertebrates and vector species codon usages.The other insect-specific flavivirus group F clade includes 5 species (Chaoyang virus (CHAOV), Donggang virus (DONV), Lammi virus (LAMV), Barkedji virus (BJV) and Ilomantsi virus (ILOV) paralogous to mosquito-borne flaviviruses, supporting a more recent emergence and closer relation to -7 to 0.0192). Additionally, we found that CAI values were on average higher in monkeys than in human genes for vertebrate-associated flavivirus groups, and in some cases differences were significant. A higher CAI average in vertebrates compared to vectors was universal in all vertebrate-associated groups. Interestingly, the group B clade (no known-vector) shared a similar trend to mosquito-borne and tick-borne CAI box plots, where mammalian vertebrate CAIs were significantly higher than vector species .Mosquito-borne and tick-borne groups that have known associations with a vertebrate host revealed a different codon bias. In groups C (tick-borne), E (Yellow fever virus complex), G (Dengue virus complex) and H (West Nile virus complex), viral codon adaptation indexes were significantly higher in mammalian vertebrates than in vector species  emerged from Aedes-borne flaviviruses thousands of years ago [Culex-borne species have a life cycle involving primarily avian species and a few species have even been isolated from rodents. For example, the rock dove pigeon (Columba livia) is an avian species with prior associations to Culex-borne viral infections i.e. West Nile virus [Aedes-borne flaviviruses species, CAI values to mammalian vertebrate tables were significantly higher than in mosquito species . In the case of the rock pigeon, the Culex-borne flaviviruses had a lower CAI distribution to pigeons than to mammalian vertebrates . Furthermore, Culex-borne species had a significantly higher CAI to pigeons than to Culex pipiens  but only averaged higher than Aedes aegypti and tick CAI values. Across flavivirus subgroups that are known to harbor viral species with known capability of infecting mammalian vertebrates , the CAI to pigeons rests in the middle of vector and mammalian CAI values.The ears ago ,71. Culele virus . Like thAedes-borne flaviviruses who boast century-long interactions with human hosts [After observing CAI differences between the flavivirus genus subgroups, we explored if changes in CAI over time could be seen in specific viral species. Yellow fever virus  and Dengue 2 virus  are paralogous an hosts ,73,74. BMacaca mulata) Culex pipiens and the Aedes aegypti codon usage tables. For the YFV dataset . YFV demonstrated codon adaptation for both vectors and humans across all sampled strains. YFV CAI to Rhesus monkey codon usage was significantly higher than human and vector tables . We also observed significantly higher CAI for Aedes aegypti than in Culex pipiens . Across all three datasets, YFV had a higher CAI to the mammalian codon usage tables, followed by DENV-2 endemic and sylvatic strains respectively.To start, we calculated their CAI to human gene codon usage datasets, the Rhesus monkey  . Since we were more interested in the interplay of DENV-2 endemic strains with humans, particularly when transmission alters between mosquitoes and humans in an urban setting, we removed the CAI data of DENV-2 endemic strains to the Rhesus monkey table. DENV-2 endemic strains showed a significantly higher CAI to housekeeping genes over time . This is divergent to YFV, where CAI over time was greater to the immune genes codon usage table .A higher CAI to its 11x10-5) . DENV-2 -5). Furthermore, sylvatic strains did not show any evidence for codon adaptation towards Culex pipiens, as the CAI for all sequences was below 1. Interestingly, codon adaptation to the Rhesus monkey shows an increase in CAI from 1995 to the 2008 sequences, positively correlating with the same increase in CAI that takes place later in DENV-2 endemic strains to human genes from 2003 to 2016 . In fact, this same positive correlation was shared in DENV-2 sylvatic strains codon adaptation to Aedes aegypti. .In DENV-2 sylvatic strains, we saw a different pattern . DENV-2 Aedes-borne flaviviruses, we then asked if a similar result could be found in a Culex-borne flavivirus such as West Nile virus (WNV). WNV is transmitted primarily by the Culex pipiens mosquito and can cause severe neurological disease in vertebrates: including humans, horses and birds [Accipiter gentilis) had neurological symptoms that were later identified as WNV disease from a L2 strain [After exploring the CAI trends of YFV and DENV-2, both nd birds . Furthernd birds . In addind birds . WNV linnd birds ,77. In 22 strain . L2 was 2 strain . In 20102 strain . From 202 strain .p-values ranged from 0.001 to 0.02). We then calculated the Malthusian fitness (Wm) for L2 from 2004 to 2014, a time period that coincided with L2 strains isolated and sequenced in Europe  and the pigeon (Columba livia) codon usage tables. With Wm, a measure of relative fitness over time, we were able to compare two independent measurements of viral fitness that span before and during the European WNV L2 epidemic . Notably, the joint increase of Wm and mosquito CAI during the 2010 to 2012 period was highly correlated . Interestingly, an increase of CAI in pigeon and human tables followed in 2012 to 2014 that was highly correlated to the earlier increase in mosquitoes and Wm . The increase in vector CAI that precedes the increase in human CAI was similar to what we observed in sylvatic DENV-2 CAI to Aedes aegypti and rhesus monkeys, followed by a later increase in the CAI of endemic DENV-2 strains in humans.We then calculated the CAI of L2 sequences against human, mosquito  table did not support the biological and epidemiology of tick-borne viruses Click here for additional data file.S2 DatasetGenes used in the creating of in-house codon usage tables, including tobacco house-keeping genes and human immune/antiviral genes.(XLSX)Click here for additional data file.S1 FigTaxon are included and all nodes had a posterior probability > 0.9. Associated viral vectors and vertebrate host groups are colored. The scale bar represents 0.06 mutations per site.(EPS)Click here for additional data file.S1 Textcodcmp program from the EMBOSS package, raw outputs from relevant comparisons between human, tobacco, and vector species codon usage tables are shown.Using the (TXT)Click here for additional data file.


G-protein-coupled receptor (GPCR)-interacting proteins likely participate in regulating GPCR signaling by eliciting specific signal transduction cascades, inducing cross-talk with other pathways, and fine tuning the signal. However, except for G-proteins and β-arrestins, other GPCR-interacting proteins are poorly characterized. 14-3-3 proteins are signal adaptors, and their participation in GPCR signaling is not well understood or recognized. Here we demonstrate that GPCR-mediated 14-3-3 signaling is ligand-regulated and is likely to be a more general phenomenon than suggested by the previous reports of 14-3-3 involvement with a few GPCRs. For the first time, we can pharmacologically characterize GPCR/14-3-3 signaling. We have shown that GPCR-mediated 14-3-3 signaling is phosphorylation-dependent, and that the GPCR/14-3-3 interaction likely occurs later than receptor desensitization and internalization. GPCR-mediated 14-3-3 signaling can be β-arrestin-independent, and individual agonists can have different potencies on 14-3-3 and β-arrestin signaling. GPCRs can also mediate the interaction between 14-3-3 and Raf-1. Our work opens up a new broad realm of previously unappreciated GPCR signal transduction. Linking GPCRs to 14-3-3 signal transduction creates the potential for the development of new research directions and provides a new signaling pathway for drug discovery. Upon agonist stimulation, GPCRs activate heterotrimeric G-proteins, leading to the dissociation of G-proteins and the generation of second messengers. G-protein-coupled receptor kinases (GRKs) then phosphorylate activated receptors, leading phosphorylated receptors to recruit signal adaptors or cellular effectors. Interactions between GPCRs and an array of signal adaptors or cellular effectors modulate both G-protein-dependent and G-protein-independent signaling pathways, and offer the possibility of eliciting specific spatiotemporal signaling cascades, inducing cross-talk with other signaling pathways, and fine tuning and specifically regulating GPCR signaling at multiple levels.2-adrenergic receptors (α2ARs).3 Binding of 14-3-3 to the third intracellular loop of α2ARs regulates receptor cellular localization and coordinates signal transduction. Interaction of 14-3-3 proteins with other GPCRs such as thromboxane receptors,4 parathyroid hormone receptor,5 cannabinoid receptor6 and others7–9 has been associated with various cellular functions. 14-3-3 has been found to interact with the C-terminal domain of GABAB1 and to inhibit the heterodimerization of GABAB1 and GABAB2 at their C-terminal domains.10 Proteomic analysis has also indicated an association between 14-3-3 and angiotensin II type 1a receptor (AT1aR).11 Recently, bioinformatic analysis has predicted that 68% of neurotransmitter GPCRs have 14-3-3-binding motifs.12In addition to well-documented G-protein-dependent and β-arrestin-dependent GPCR signaling pathways, other cellular effectors are recruited to GPCRs. One of them is the multifunctional signal adaptor protein 14-3-3. The first evidence of 14-3-3 as a GPCR cellular effector was demonstrated with the α13 The remarkable feature of 14-3-3 proteins is the number and diversity of their protein interaction partners. Bioinformatic and proteomic analyses predict that >2000 phosphoproteins interact with 14-3-3 proteins.14 Their interaction partners include kinases, phosphatases, ubiquitin ligases, transcription factors, scaffold proteins, cytoskeletal proteins and membrane proteins, including GPCRs, receptor tyrosine kinases, cytokine receptors and ion channels.15 These interactions facilitate the formation of large molecular complexes that coordinate the responses of multiple signaling pathways to incoming stimuli, allow signal transduction among different cellular compartments and carry out a variety of physiological functions. Not surprisingly, 14-3-3 proteins are also associated with human diseases, most notably with cancers and with neurological disorders such as Alzheimer’s disease, Parkinson’s disease, spinocerebellar ataxia type 1, schizophrenia and bipolar disorder, based on the evidence from both clinical and laboratory studies.1914-3-3 proteins are ~30 kDa proteins that are ubiquitously expressed in eukaryotic cells but that are most highly expressed in the brain, where they make up ~1% of total soluble protein. Although they lack enzymatic activity, 14-3-3 proteins bring two or more proteins together to form signal complexes.21 to assess GPCR/14-3-3 protein interactions, we can pharmacologically characterize GPCR-mediated 14-3-3 signaling pathways.Although evidence shows that 14-3-3 forms complexes with some GPCRs, investigation of GPCR-mediated 14-3-3 signaling has been largely ignored. The inability to assess specific 14-3-3 signaling is a major reason for such studies to lag behind studies of G-protein and β-arrestin signaling pathways. Significant progress in pharmacologically characterizing GPCR-mediated 14-3-3 signaling depends on the availability of research tools and methods. Here we show that, like β-arrestins, 14-3-3 proteins interact with activated GPCRs to form signaling complexes that mediate signal transduction. By applying LinkLight assay technology3AR, which lacks a β-arrestin interaction motif, as an example, we demonstrate that GPCR-mediated 14-3-3 signaling can be β-arrestin-independent. Furthermore, we compare assays of GPCR-mediated 14-3-3 and β-arrestin signaling pathways by profiling a panel of agonists. The data indicate that a ligand can exert different activity in different signaling pathways. Finally, we investigated the interaction between 14-3-3 and the signal effector Raf-1. The Raf-1/14-3-3 interaction can be stimulated by serum and GPCR agonists through endogenous receptors, and this interaction is blocked by staurosporine. Linking GPCR signaling to the vast 14-3-3 protein interaction networks provides the potential for understanding GPCR signaling at a more complex level, as well as for the development of new approaches that target specific protein–protein interactions that may aid GPCR drug discovery.We show for the first time that several GPCRs signal through 14-3-3, and that GPCR antagonists can specifically block agonist-induced 14-3-3 signaling. And, we find out that GPCR-mediated 14-3-3 signaling is phosphorylation-dependent. The pan-kinase inhibitor staurosporine blocks GPCR-mediated 14-3-3 signaling, whereas the GRK2/3 inhibitor CMPD101 does not. Moreover, we demonstrate that the GPCR/14-3-3 interaction occurs on a slower timescale than the GPCR/β-arrestin interaction. In addition, using the βCompounds and chemicals were purchased from Sigma-Aldrich  and Tocris Biosciences .2. Cell culture medium was replaced every 3–4 days, and cells were passaged at 90% confluence.HEK293 cells were routinely maintained and passaged in standard Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin/streptomycin . U2OS cells were purchased from the American Type Culture Collection  and were cultured with McCoy’s 5A medium  supplemented with 10% FBS  and 1× penicillin/streptomycin . Cells were cultured in a 37 °C incubator with 5% CO21 The β-arrestin-2-permuted luciferase (β-arr-2-pLuc) expression plasmid was also previously described.21 14-3-3ɛ and 14-3-3ζ full-length complementary DNAs without stop codons were used to replace β-arrestin-2 in the β-arr-2-pLuc for construction of the 14-3-3ɛ-pLuc and 14-3-3ζ-pLuc expression plasmids. Transfections of HEK293 and U2OS cells were performed with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions . Monoclonal cell lines stably expressing both GPCR-TEV and the 14-3-3ɛ-pLuc fusion protein were selected using 400 μg ml−1 G418  and 100 μg ml−1 Hygromycin B . Multiple stable clones were selected for evaluation.Full-length complementary DNAs of human GPCRs without stop codons were subcloned in frame with the tobacco etch virus (TEV) protease vector as previously described.m dimethylsulfoxide stocks. For antagonist inhibition assays, the medium was replaced with 10 μl of serum-free DMEM. Antagonists or inhibitors were added at 5 μl per well and incubated with the cells for 15 min. Then, the stimulant or agonist was added at 5 μl per well and incubated for 240 min or for other times as indicated. An equal volume of Bright-Glo , One-Glo (Promega), BriteLite  or NeoLite (PerkinElmer) luciferase detection reagent was added for a 5-min incubation. The luminescence signals were determined using a luminescence plate reader (EnSpire or TopCount). Alternatively, after compound incubation, the culture medium was replaced with 10 μl of luciferase detection reagent, and luminescence signals were recorded.The LinkLight cells stably expressing both GPCR-TEV and 14-3-3ε-pLuc were seeded into a 384-well white plate  at 20 000 cells per well with 30 μl of regular medium. Cells were cultured overnight. The following day, the culture medium was replaced with 15 μl of serum-free DMEM, followed by the addition of 5 μl of compounds/drugs. Agonists were added as serial dilutions in serum-free DMEM from 10 mm agonists for 240 min before proceeding to the luciferase assay.The GPCR/14-3-3 LinkLight cells were seeded in white 384-well plates. After overnight culture, the culture medium was replaced with serum-free DMEM, and a serial dilution of GPCR ligand was added to the cells. After agonist incubation (240 min unless otherwise specified), a luciferase detection reagent was added to the cells, and luminescence signals were recorded. The antagonist effects were measured after the cells were preincubated with the compounds for 15 min, followed by the stimulation with 1 μThe GPCR/β-arrestin-2 LinkLight cells were seeded in white 384-well plates. After overnight culture, the culture medium was replaced with serum-free DMEM, and a serial dilution of GPCR ligand was added to the cells. After agonist incubation (120 min unless otherwise specified), a luciferase detection reagent was added to the cells, and luminescence signals were recorded.The Raf-1/14-3-3ε LinkLight cells were seeded in white 384-well plates. After overnight culture, the culture medium was replaced with 15 μl of serum-free DMEM/ per well, and a serial 1:3 dilution of adrenaline, dopamine or 5'-N-ethylcarboxamidoadenosine (NECA) (5 μl per well) was added to the cells. After 180 min of ligand incubation, a luciferase detection reagent was added to the cells, and luminescence signals were recorded.The Raf-1/14-3-3ε LinkLight cells were seeded in white 384-well plates. After overnight culture, the culture medium was replaced with 15 μl of serum-free DMEM per well, and a serial 1:2 dilution of FBS (5 μl per well) was added to the cells. After 180 min of FBS incubation, a luciferase detection reagent was added to the cells, and luminescence signals were recorded.The LinkLight cells were cultured in a white 384-well plate overnight. The culture medium was then replaced with serum-free DMEM. Serial dilutions of kinase inhibitors were added to cells for 15 min, followed by agonist stimulation for 240 min and then by the luciferase assay as described above. For Raf-1/14-3-3 kinase inhibition assays, cells were preincubated with a kinase inhibitor  for 15 min, followed by stimulation with 7.5% FBS for 180 min and then by the luciferase assay as described above.n=3).Concentration–response curves were analyzed using Prism software . All values are expressed as mean±s.d.  as a prototype for developing GPCR/14-3-3 signaling assays. In humans, there are seven 14-3-3 proteins; all the isoforms contain the K49, R56 and R127 residues that are critical for the binding of 14-3-3 to different partners.22 We selected 14-3-3 epsilon (ε) and 14-3-3 zeta (ζ) for the assays, and we constructed 14-3-3ε-pLuc and 14-3-3ζ-pLuc expression plasmids. We transiently expressed ADRB2-TEV with 14-3-3ε-pLuc or ADRB2-TEV with 14-3-3ζ-pLuc in HEK293 cells, and we then looked for luciferase signals induced by stimulation with the ADRB2 agonist isoproterenol. Cells transfected with either construct produced concentration-dependent signals in response to isoproterenol, indicating that agonist-activated ADRB2 interacted with 14-3-3 proteins. To further demonstrate ligand-induced formation of ADRB2/14-3-3 signal complexes, we also tested assay design B (reversing TEV and pLuc) by co-expressing ADRB2-pLuc and 14-3-3ε-TEV in HEK293 cells. The result of assay design B confirmed ADRB2/14-3-3 protein interaction in an agonist-dependent manner , adenosine receptor A2a (ADORA2A), dopamine receptor D2 (DRD2), muscarinic cholinergic receptor 3 (CHRM3) and 5 (CHRM5) all showed agonist-stimulated 14-3-3 signaling , DRD2 , CHRM3 , and CHRM5  are comparable to the reported values from G-protein signaling assays. We treated stable CHRM5/14-3-3 LinkLight cells with the agonists oxotremorine, pilocarpine and carbachol to produce concentration-dependent 14-3-3 signals. Oxotremorine and carbachol are full agonists, and pilocarpine behaves as a partial agonist for 14-3-3 signaling. Next, we investigated whether antagonists could inhibit agonist-induced GPCR-mediated 14-3-3 signals. Preincubation of the cells with various concentrations of antagonists (scopolamine or atropine) followed by treatment with 3 μm carbachol produced antagonist concentration-dependent inhibition signals  inhibitor, CMPD101, or a PKA inhibitor, H-89. Preincubation of α1AR/14-3-3 cells with CMPD101 or H-89 did not inhibit signals stimulated by the agonist cirazoline, suggesting that GRK2/3 and PKA do not contribute to promoting the α1AR/14-3-3 interaction. We then used the pan-kinase inhibitor staurosporine in a similar experiment. Pretreatment of α1AR/14-3-3 cells with staurosporine (IC50: 19.7 nm) blocked signals stimulated by the agonist cirazoline (3 μm). These data indicate that the GPCR/14-3-3 interaction is phosphorylation-dependent, and that one or more kinases participate in the GPCR-mediated 14-3-3 signaling pathway (GRKs and cyclic AMP-dependent protein kinase (PKA) phosphorylate agonist-occupied receptors on serine or threonine residues within the C terminus or the third intracellular loop. pathway .3 adrenergic receptor (ADRB3) as an example to investigate this question. ADRB3 lacks consensus sequences for GRK phosphorylation and therefore does not recruit β-arrestins, and it does not display short-term agonist-induced functional desensitization or sequestration.28 We generated HEK293 cells stably expressing ADRB3-TEV and 14-3-3-pLuc fusion proteins and assessed a panel of adrenergic agonists for their ability to promote an interaction between ADRB3 and 14-3-3. The ADRB3 agonist BRL37344 and the βAR agonist isoproterenol promoted ADRB3/14-3-3 interaction signals in a concentration-dependent manner, whereas the α1AR agonist cirazoline and the α2AR agonist guanfacine showed very weak signals even at 10 μm >adrenaline (9.6 nm)>methoxamine (11.1 nm)>guanfacine (12.4 nm)>UK14304 (55.2 nm)>BRL37344 (386 nm)>isoproterenol (995 nm). The rank order of potency for those agonists in β-arrestin signaling is cirazoline (18.4 nm)>adrenaline (152 nm)>methoxamine (318 nm)>UK14304 (780 nm)>guanfacine (1610 nm). BRL37344 and isoproterenol showed much lower signal strength and behaved as much weaker agonists of β-arrestin signaling than of 14-3-3 signaling. These data suggest that individual ligands may not exert equal potency on different signaling pathways.Having demonstrated that GPCRs interact with 14-3-3, we wanted to compare the GPCR-mediated 14-3-3 and GPCR-mediated β-arrestin signaling pathways. We used α1AR as an example. A panel of adrenergic agonists, including the α1AR agonists cirazoline and methoxamine, the αignaling . The ran29 Activated Ras recruits Raf-1 to the plasma membrane, where it becomes activated and in turn activates the Raf/MEK/ERK pathway. Binding of 14-3-3 is required for the maintenance of Raf-1 phosphorylation and kinase activity.30 Thus, we can utilize Raf-1/14-3-3 protein interaction signals as an indicator of Raf-1 activation. U2OS cells contain several endogenous GPCRs, including adrenergic, dopamine and adenosine receptors. Previously, we showed GPCR agonist-induced ERK2/β-arrestin-2 interaction signals in U2OS cells.21 We generated U2OS cells stably co-expressing Raf-1-TEV and 14-3-3ɛ-pLuc, and used those cells to examine the Raf-1/14-3-3 interaction in response to the treatment with GPCR agonists. Stimulation of the cells with adrenaline, dopamine or NECA produced concentration-dependent Raf-1/14-3-3 interaction signals . Therefore, it is likely that simultaneously inhibiting Raf-1 and BRAF blocked Raf-1/14-3-3 interaction signals. Potentially, the Raf/14-3-3 LinkLight assay could be used for identifying new Raf inhibitors or modulators. Activation of the Raf/MEK/ERK signaling pathway can be either Ras-dependent or Ras-independent.34 To identify which pathway is utilized for the Raf-1/14-3-3 interaction, a Ras/Raf-1 direct interaction assay needs to be developed. Such an assay could also be a useful tool for identifying blockers or modulators of Ras/Raf-1 interactions.In addition to the agonist-promoted GPCR/14-3-3 protein interaction, we also investigated whether Raf-1 interacts with 14-3-3. Ras, in response to receptor activation, switches from its inactive (GDP-bound) form to its active (GTP-bound) form, and it then recruits Raf to the membrane and promotes Raf dimerization and activation. Raf-1 kinase activity is known to require 14-3-3 protein binding.35 These biased ligands may maximize clinical effectiveness and minimize unwanted side effects. The identification of signaling pathway-dependent ligands challenged the early conventional views of GPCR activation and suggested the existence of multiple conformations induced by different ligands.36 Conceptually, a ligand may bind and stabilize a receptor in a conformation that may exert differential activity on the 14-3-3 signaling pathway in addition to the G-protein and β-arrestin signaling pathways. We profiled the effects on the α1A receptor of a panel of adrenergic agonists in both the 14-3-3 and β-arrestin signaling pathway assays. We found that the rank of order of potency, as well as the absolute potency, of these agonists differs between the two different signaling pathway assays, suggesting that a single ligand could possess different activity on different pathways. Signaling pathway-biased ligands that may have differential activity for 14-3-3, G-protein and β-arrestin signaling pathways have yet to be identified. Such biased ligands would produce unequal activation of specific signaling pathways and would produce distinct physiological outcomes. Using the new technique described here, a high-throughput screening campaign to identify ligands biased towards 14-3-3 signaling is now possible. Demonstration that GPCR-mediated 14-3-3 signaling can be directly measured, and that we can pharmacologically characterize ligands modulating this pathway opens up a new realm of previously unappreciated GPCR signal transduction. Consequently, the GPCR-mediated 14-3-3 signaling pathway provides the potential for understanding GPCR signaling pathways more deeply, as well as for the development of novel strategies that target specific GPCR intracellular signaling pathways.Historically, GPCR drug discovery research has relied on G-protein signaling pathways for assessing the activity of various compounds. The discovery of G-protein-independent and β-arrestin-dependent signaling pathways created an opportunity for identifying pathway-selective or signaling-biased ligands.


Retinoic acid-inducible gene-I (RIG-I) is critical in triggering antiviral and inflammatory responses for the control of viral replication in response to cytoplasmic virus-specific RNA structures. Upon viral RNA recognition, RIG-I recruits the mitochondrial adaptor protein mitochondrial antiviral signaling protein, which leads to a signaling cascade that coordinates the induction of type I interferons (IFNs), as well as a large variety of antiviral interferon-stimulated genes. The RIG-I activation is tightly regulated via various posttranslational modifications for the prevention of aberrant innate immune signaling. By contrast, viruses have evolved mechanisms of evasion, such as sequestrating viral structures from RIG-I detections and targeting receptor or signaling molecules for degradation. These virus–host interactions have broadened our understanding of viral pathogenesis and provided insights into the function of the RIG-I pathway. In this review, we summarize the recent advances regarding RIG-I pathogen recognition and signaling transduction, cell-intrinsic control of RIG-I activation, and the viral antagonism of RIG-I signaling.Innate immunity is the first line of defense against invading pathogens. Rapid and efficient detection of pathogen-associated molecular patterns  Eukaryotic organisms rely on the host innate immune system to defend against viruses or other pathogenic microbes in early phases of infection. The innate antiviral immune response starts with the detection of evolutionarily conserved structures, termed pathogen-associated molecular patterns (PAMPs), by a set of germline-encoded pattern-recognition receptors (PRRs). With respect to their cellular localization, ligand specificity, and functions, PRRs are categorized into distinct families including the toll-like receptors, nucleotide-binding oligomerization domain-like receptors, C-type lectin receptors, retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) –5, as weThe three members of the RLR family: RIG-I, MDA5 (melanoma differentiation factor 5), and LGP2 (laboratory of genetics and physiology 2) are expressed in most cell and tissue types. They function as cytoplasmic sensors for the recognition of a variety of RNA viruses and subsequent activation of downstream signaling to drive type I IFN production and antiviral gene expressions. These three RLR proteins are RNA-dependent ATPases belonging to the DExD/H-box family of helicases . Structuin vitro transcription, or via chemical synthesis, were all shown to bind to RIG-I and were sufficient to activate RIG-I , and the activation of nuclear factor-κB (NF-κB) , vesicular stomatitis virus, measles virus (MV), Newcastle disease virus, Ebola virus (EBOV), dengue virus (DENV), and hepatitis C virus (HCV) –19. The te RIG-I , 23. Phyionality . It is wtivation . These ativation –30. Furtependent , 32. Intependent . Notably (NF-κB) , 35. UpoActivated RIG-I recruits its downstream adaptor molecule MAVS  through CARD–CARD-mediated interactions , 37. TheStructural and biochemical studies have demonstrated that the activation of RIG-I is a multi-step process and is primarily regulated by conformational changes and PTMs. When initially identified as a dsRNA sensor, it was hypothesized that RIG-I was under negative regulation in physiological conditions. The over expression of the CARD domain of RIG-I alone demonstrated superior signaling activity than full length RIG-I in absence of viral PAMPs . StudiesOne of the first PTMs of RIG-I following the initial ligand recognition is performed by the robust ubiquitination machinery Figure . Mass spOn the other hand, several deubiquitinating enzymes (DUBs) were identified to remove K63-linked pUb chains from RIG-I, thus dampening RIG-I signaling. The tumor suppressor protein cylindromatosis (CYLD) removes K63-linked pUb chains from RIG-I as well as TBK1 and IKKϵ to inhibit the IRF3 response, serving as a pathway negative regulator . SyndecaIn contrast to K63-linked ubiquitination, which promotes protein activation, K48-linked ubiquitination triggers proteasomal degradation of its target. For instance, the RING-finger protein 125 (RNF125), together with the ubiquitin E2 ligase UbcH5, conjugate K48-linked ubiquitin to RIG-I and MAVS, targeting them for proteasomal degradation and thereby inhibiting downstream signaling . SimilarIn parallel with ubiquitination, phosphorylation has emerged in the past several years as a critical regulator of the RIG-I signaling transduction Figure . ProteinIn addition to the ubiquitination and phosphorylation described above, acetylation modulation has recently started to gain more acknowledgment for controlling RIG-I activity Figure . Mass spRIG-I signal transduction is further regulated by additional PTMs, regulatory proteins, and other cellular processes Figure . It is wIn order to establish infections, viruses have developed sophisticated mechanisms to counteract host immune responses. With regard to RIG-I signaling, these include mechanisms such as altering viral genomes and their intermediate transcripts to avoid detection, manipulating the activation and degradation of RIG-I and MAVS, as well as modulating downstream signaling cascades. Studying these antagonistic viral strategies has greatly broadened our understanding of RIG-I activation and regulation.O position makes viral RNA non-distinguishable from the host mRNAs, and therefore does not stimulate RIG-I (Since 5′ triphosphate (5′ppp) is an important feature recognized by RIG-I, modification of this motif has long been described as one of the major mechanisms for viruses to antagonize RIG-I signaling. Crimean–Congo hemorrhagic fever virus, Borna disease virus (BDV), and hantavirus (HTNV) remove the 5′ppp group on their genome posttranscriptionally, make RIG-I unable to bind to viral RNA, and therefore incapable of triggering RIG-I activation . Mechanite RIG-I , 92.via its viral polymerase subunit PB2. PB2 position 627K in the mammalian strain increases PB2-nucleocapids binding affinity, thus inhibiting RIG-I interaction with the nucleoprotein-encapsidated 5′ppp RNA , and Golgi, to avoid cytosolic surveillance by RIG-I. For instance, the DENV conceals dsRNA in the intracellular membrane as an escape strategy . ER is aAs reviewed above, ubiquitination represents one critical PTM mechanism of RIG-I activation and, not surprisingly, is an attractive target for viral manipulation Figure A. ViruseOn the other hand, some viruses encode enzymes that directly deubiquitinate RIG-I. For instance, KSHV encoded deubiquitinase ORF64 cleaves Lys63-ubiquination chains on CARDs, blocks CARDs interaction between RIG-I and MAVS, thereby downregulating RIG-I signaling . Other vvia suppressing RIG-I dephosphorylation in dendritic cells (DCs). In this study, the growth arrest and DNA damage protein (GADD34) was shown to form complexes with PP1 to facilitate RIG-I activation. The MV infection induced DC-SIGN signaling results in an inhibition of GADD34-PP1 phosphatases activity and thereby impairs RIG-I activation , and enterovirus 71 (EV71)  , 110. Th1 (EV71) . Intrigu1 (EV71) .pro  cleaves MAVS for proteolysis by its protease 3Cpro . Both CVactivity , 114. Inactivity , 115. Inactivity . Anotheractivity . Additioactivity –119.It is important to note that the proper localization of RIG-I and MAVS is a prerequisite for effective signaling transduction. MAVS resides on the mitochondrial membrane, peroxisomes, and mitochondria-associated membranes for antiviral signaling. In fact, a RIG-I translocon has been identified to direct RIG-I redistribution from cytosol to membranes during viral infection . Studiesvia various regulatory mechanisms and are common targets of viruses  of FMDV (via the formation of inclusion bodies, causing signaling cascade termination (To ensure successful RIG-I signaling transduction, the kinase activities of TBK1 and IKKϵ are tightly controlled s Figure B. For ex of FMDV  and the  of FMDV  inhibit  of FMDV , 125. Se of FMDV . Two oth of FMDV , 128. Momination .Viral regulation of the transcription factors, IRFs and NF-κB, further serve as points of control in RIG-I signaling Figure B. One ofvia distinct mechanisms. For example, MNV NS5 inhibits STAT1 phosphorylation, whereas DENV NS5 interacts with UBR4 to promote STAT2 degradation (Viruses have also developed multiple inhibitory mechanisms to counteract the IFN stimulation of ISGs by targeting STAT1 and/or STAT2 Figure B. For exradation , 148. Byradation . Furtherradation , RSV (15radation , and parradation , also deStudies from the past decade have well established RIG-I as one of the principal PRRs for the recognition of cytoplasmic viral RNA, as well as defining its critical role in the induction of IFNs during viral infections. Our understanding of the RIG-I-mediated antiviral response has been greatly expanded with the key discoveries made regarding the molecular mechanism of RIG-I regulation, such as ubiquitination, phosphorylation, and acetylation. Meanwhile, investigating viral strategies to manipulate RIG-I responses not only allow us to understand the viral pathogenesis, but also significantly contributed to our knowledge of how RIG-I is activated and regulated. These new insights into the viral-mediated RIG-I regulations are important for vaccine and drug development aiming to suppress infectious diseases and enhance immune responses.YL wrote the manuscript. RL and DO revised and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The kinase Mps1, long known to be the ‘boss’ in mitotic checkpoint signaling, phosphorylates multiple proteins in the checkpoint signaling cascade. Related research article Ji Z, Gao H, Jia L, Li B, Yu H. 2017. A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling. eLife6:e22513. doi: 10.7554/eLife.22513Micromanagement has a bad reputation. It is frowned upon in the workplace, but it may have benefits in cellular signaling because a single regulator that interferes at multiple steps of a signaling cascade can lead to more reliable signaling. Now, in eLife, Hongtao Yu and colleagues at the University of Texas Southwestern Medical Center – including Zhejian Ji and Haishan Gao as joint first authors – report that a kinase called Mps1 acts as a micromanaging boss of a checkpoint signaling pathway that regulates cell division .When cells divide, their chromosomes duplicate and a protein complex called the kinetochore assembles on each chromosome copy. Microtubules then attach to the kinetochores to pull the copies apart and segregate them between the newly forming cells. The mitotic checkpoint is a cellular safeguard that triggers the checkpoint signaling cascade if the microtubules do not attach properly to the kinetochores. In particular, this cascade leads to the formation of the “mitotic checkpoint complex”, which inhibits another multi-protein structure called the anaphase-promoting complex. This inhibition prevents chromosome segregation and the final stages of cell division .Hints that Mps1 oversees and controls checkpoint signaling were uncovered decades ago. The overexpression of Mps1 was found to trigger checkpoint signaling even when the microtubules were all properly attached to kinetochores . In ordeOver the years, it became clear that Mps1 phosphorylates multiple checkpoint proteins, and also the kinetochore protein KNL1, but the mechanistic details of these events have only recently started to emerge. Phosphorylation of KNL1 leads to the recruitment of the checkpoint protein Bub1 to kinetochores. And work in budding yeast subsequently revealed that Mps1 phosphorylates Bub1 to enable it to bind to another checkpoint protein called Mad1. This interaction was crucial for checkpoint signaling in budding yeast , but effThe work of Ji et al. goes further by showing that Mps1 also phosphorylates Mad1 (as opposed to just phosphorylating Bub1 so that it can bind to Mad1). The region of Mad1 that is phosphorylated was known to have an essential role in checkpoint signaling, but its precise function had remained unclear . Ji et aFurther support for this model comes from a recent study that used a technique called FRET (which probes the distance between fluorescently labeled molecules) to follow the assembly of the mitotic checkpoint complex over time . This woBut back to micromanagement. Is it significant that Mps1 influences multiple interactions throughout the checkpoint signaling pathway? The phosphorylation of multiple substrates in a single pathway conceptually resembles the phosphorylation of a single substrate at multiple sites. As opposed to a single phosphorylation event, multi-site phosphorylation can lead to more interesting behaviors . In partLastly, if you have a micromanaging boss, you want to keep her or him in check. Close contact between Mps1 and the kinetochore protein KNL1 is sufficient to trigger checkpoint signaling, even if there are no unattached kinetochores in the cell . Hence,


Cell death isn’t just about apoptosis anymore. In the last decade, numerous alternative mechanisms have emerged, including regulated forms of necrosis, as well as entosis, a unique mechanism that is executed as a cell murder rather than cell suicide . Hamann Cells that undergo entosis are killed as a result of ingestion into their neighbors. They are not phagocytosed, but rather form junctions with their neighbors and then invade into them, ultimately becoming killed through a mechanism involving autophagy proteins and lysosomal enzymes , 3. ThisDuring entosis, the nutrients derived from ingested loser cells are scavenged by winners and used in metabolism . This prHow does nutrient starvation induce entosisŒ Biophysical studies indicated that glucose withdrawal leads to a bimodal mechanical response in starved cell populations, where some cells become stiffer, and others softer, than normal, resulting in a mechanical differential between neighboring cells. Activity of the energy sensor AMP-activated protein kinase (AMPK), a known activator of autophagy, is required for the appearance of stiffer cells, and also for entosis, specifically in loser cells. Long-term glucose starvation selects for winners with lowered levels of AMPK activity, and these cells are capable of ingesting naïve cells at high rates in mixed populations, demonstrating selection for the ability to scavenge nutrients through this competitive mechanism.These findings link a key nutrient signaling pathway and known inducer of autophagy to regulation of entosis. While autophagy promotes the recycling of intracellular nutrients to support cell survival, entosis allows winner cells in a population to scavenge extracellular nutrients and accumulate the biomass necessary to support proliferation Figure . MacropiOur findings identify entosis as a population-scale starvation response with parallels to cell competition occurring in developing tissues . As mechWhile we show that entosis is required for population re-growth, how this process links to the long-term proliferative capacity of winners is unknown, and may involve additional adaptations such as gluconeogenesis or other altered metabolic states that await discovery. Also, how AMPK signaling and autophagy pathway proteins coordinately regulate cell-autonomous (autophagy) versus non-cell-autonomous (entosis) processes will be interesting to explore in future studies. Autophagy proteins in this context control the digestion of extracellular nutrients through a pathway resembling LC3-Associated Phagocytosis, or LAP, first identified in macrophages . We findWe have found that entosis is one of numerous non-apoptotic cell death mechanisms that has a unique property to promote competition between cells in a population. Recently, a regulated form of necrosis called ferroptosis was shown to have a different non-cell-autonomous effect, to spread from cell-to-cell . How cel


During the early periods of evolution, as well as in niche environments today, organisms have had to learn to tolerate the presence of many reactive compounds, such as reactive oxygen species, nitric oxide, and hydrogen sulfide. It is now known that such compounds are instrumental in the signaling processes in plant cells. There are enzymes which can make them, while downstream of their signaling pathways are coming to light. These include the production of cGMP, the activation of MAP kinases and transcription factors, and the modification of thiol groups on many proteins. However, organisms have also had to tolerate other reactive compounds such as ammonia, methane, and hydrogen gas, and these too are being found to have profound effects on signaling in cells. Before a holistic view of how such signaling works, the full effects and interactions of all such reactive compounds needs to be embraced. A full understanding will be beneficial to both agriculture and future therapeutic strategies. Reactive compounds such as those derived from oxygen, nitrogen, and sulfur are instrumental in cell signaling pathways . It appe2O2) and the hydroxyl radical. The issue for newly evolving organisms as oxygen levels rose was that many of the ROS are toxic (The atmosphere during the history of the Earth has not been unchanging. Four billion years ago the atmosphere would have been approximately one part per million oxygen  and yet re toxic . Therefore toxic . These ire toxic  showing re toxic . It appere toxic . During Similar tolerance of toxic compounds can be seen with nitrogen- and sulfur-based compounds. The most commonly studied compound here is nitric oxide (NO). This was found to be instrumental in the control of vascular tone in mammals, where it was originally known as endothelial-derived relaxing factor EDRF:  but has 2S). H2S is produced at thermal vents  and so inhibits ATP production , be able to move the message to a new position in the cell (or to another cell), and be removed when no longer needed . Looking2S can be generated by desulfhydrases in plants (2S can be removed through the action of O-acetylserine (thiol) lyase  in higher plants  but plann plants . Removall) lyase .2S are all diffusible so they are all able to move their message through, or between, the cells. However, some care is needed when discussing if membranes can be traversed. For example, NO can be a radical and uncharged but the loss or gain of an electron will yield NO+ and NO-; both are hydrophilic. In a similar manner, the ROS H2O2 is neutral and can move across the lipid bilayer but ROS, NO, and Hignaling .2S can partake in signaling, that is, so long as their concentrations do not rise to toxic levels. One of the common themes of their use in plants is in response to stress . I have written a mini-review with an opinion build in so have suggested that it should be a Perspective – hope this is correct. I am the sole author.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.