Here, we discuss the mechanism in which inflammasomes occur, as well as their roles in inflammatory kidney diseases, in order to shed light for discovering new therapeutical targets for the prevention and treatment of inflammatory kidney diseases and consequent end-stage renal disease.The immune system has a central role in eliminating detrimental factors, by frequently launching inflammatory responses towards pathogen infection and inner danger signal outbreak. Acute and chronic inflammatory responses are critical determinants for consequences of kidney diseases, in which inflammasomes were inevitably involved. Inflammasomes are closely linked to many kidney diseases such as acute kidney injury and chronic kidney diseases. Inflammasomes are macromolecules consisting of multiple proteins, and their formation initiates the cleavage of procaspase-1, resulting in the activation of gasdermin D as well as the maturation and release of interleukin-1 Tschopp et al. to describe protein complexes that form within activated immune cells and tissue-resident cells, leading to a series of inflammatory responses including cytokine production and cell death , 2. The assembly . Activatthe cell . Pyroptothe cell , 10. Theexplored –16.NLRP3 gene is linked to hereditary cryopyrin-associated periodic syndrome (CAPS), a spectrum of clinical manifestations including Muckle-Wells syndrome, familiar cold autoinflammatory syndrome, and neonatal-onset multisystem inflammatory disease [κB and thus increase the expression of NLRP3, pro-caspase-1, pro-IL-1β, and pro-IL-18. The second signal required is that NLRP3 molecules sense a variety of danger signals and recruit other components to form the macromolecular complex [Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Sendai virus, and Influenza virus, as well as DAMPs such as uric acid crystal, silica crystals, asbestos, alum, and X-ray. Previously, it seemed that potassium efflux was a downstream convergence point for the NLRP3 inflammasome assembly triggered by these diverse signals [+ signal relies on a series of events including the activation of purinergic receptor P2X7 via ATP from dying cells, unstable mitochondria, integration of lysosomes, and the production of reactive oxygen species (ROS) [+ signals are not necessary for the formation of NLRP3. NIMA-related kinase 7 can directly bind to the LRR domain of NLRP3 and controls the formation of the NLRP3 macromolecules [β and pro-IL-18 into mature IL-1β and IL-18, and cuts GSDMD into N-terminal and C-terminal fragments. GSDMD-N of GSDMD binds to phosphoinositides and cardiolipin presented in the mammalian cell plasma, creating extensive membrane pores with an inner diameter of 12-14 nm, causing leakage of IL-1β and IL-18 and other cell components [2+, efflux of K+, and finally NLRP3 inflammasome activation and pyroptosis [The NLRP3 inflammasome is the most studied type, and genome-wide associated studies show that the mutation in the human  disease . It is a disease . The NLR signals . The mololecules , 21. Recolecules . Also, colecules , 24. Aftmponents , 25, 26.roptosis , 27, 28.Bacillus anthracis lethal factor. Humans only have one NLRP1 protein, whereas mice have NLRP1a, NLRP1b, and NLRP1c paralogues, all of which lack a PYD domain. The human NLRP1 protein contains NBD, LRR, a function to find domain (FIIND), and C-terminal CARD regions. Recently, it was found that functional degradation of NLRP1 led to inflammasome activation by diverse pathogen enzymes. First, autoprocessing of FIIND domain generates two associated fragments. Secondly, NLRP1b is cleaved by the lethal factor of B. anthracis or ubiquitinated by Shigella lpaH7.8, targeting for proteasome degradation. Finally, the release of bioactive C-terminal of NLRP1b triggers inflammasome assembly [β, while Nlrp1a-deficient mice showed enhanced recovery from hematopoietic and infectious stress [B. anthracis, NLRP1 has also been implicated in the host response to protozoan Toxoplasma gondii. Mice deficient in Nlrp1b and Nlrp3 produced less IL-1β and IL-18 upon T. gondii infection, as they harbored increased parasitic loads [The NLRP1 inflammasome was first found responding to the assembly , 30. At s stress –34. Besiic loads .The NLRC4 inflammasome contains NLRC4 and NLR apoptosis inhibitory proteins (NAIPs). NAIP family proteins directly bind to a bacterium's type 3 secretion system (T3SS) and flagellin, and then become activated, allowing them to recruit and activate NLRC4. Humans only have one NAIP protein, whereas mice have several members, including NAIP1, NAIP2, and NAIP6. The ligand specificity differs in mouse and human NLRC4 inflammasomes. Mouse NAIP isoforms recognize flagellin, T3SS rod, and needle proteins, while the human NAIP is activated by T3SS needle proteins . The actListeria monocytogenes) and viruses , as well as endogenous DNA within the cells, triggering inflammasome activation and interferon synthesis [ALR proteins, which belong to the IFI family, have also been implicated in inflammasome activation and type I interferon response. Humans have 4 ALR genes, including AIM2, IFI16, pyrin-1, and myeloid cell nuclear differentiation antigen, whereas mice have 13 ALR genes . AIM2 reynthesis , 43. ALRynthesis , 45. Mouynthesis –49.MEFV gene, and its mature form includes a PYD, two B-boxes, and a coiled-coil domain. The human pyrin protein also includes a B30.2 domain. Pyrin can sense signals from Clostridium difficile TcdB, Clostridium botulinum C3, and Vibrio parahaemolyticus VopS proteins. Pyrin does not directly interact with the aforementioned signals; instead, these signals cause various modifications  of Rho GTPases, causing the rearrangement of the cytoskeleton and subsequent activation of pyrin inflammasomes [Pyrin is coded by the mmasomes –52.Streptococcus [β, high-mobility group box 1 protein, ATP, uric acid crystals, chromatin, and DNA) also activate cellular receptors, leading to downstream inflammation resembling PAMPs-triggered signaling pathways [Inflammatory responses exist in almost all kinds of kidney diseases, which are consequences of immune cells sensing PAMPs and DAMPs. It is believed that innate immune systems participate more actively than adaptive immune systems in recognizing and responding to PAMPs and DAMPs in kidney, maintaining homeostasis by regulating endogenous processes like inflammation and apoptosis . PAMPs, tococcus . Hepatittococcus . DAMPs dpathways .α and IFN-β, as well as proinflammatory cytokines TNF-α, IL-1β, IL-6, IL-18, etc. [β and IL-18 [β and IL-18 cause renal injury after binding to their receptors, which are abundantly expressed on infiltrated leukocytes, renal endothelial cells, and tubular epithelial cells [β and IL-18 can activate MAPK and NF-κB signaling pathways, thus leading to the production of nitric oxide, cyclooxygenase-2, inflammatory cytokines, and superoxide products [β and IL-18 promote the expression of adhesion molecules such as vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1 [β/IL-18 axis, other proinflammatory mediators  derived from inflammatory responses also contribute to ED, aberrant extracellular matrix metabolism, proliferation of renal resident cells, activation of coagulation system, and receptor-mediated vasoreactivity, finally leading to tubular injury, nephron dropout, and kidney function deterioration [The innate immune defense is characterized by PRR families of membranouse and cellular receptors  recognizing PAMPs and DAMPs, then initiating inflammation which primarily include interferon-(IFN-) 18, etc. . Within al cells –62. IL-1products , 63–65, lecule-1 , 66, andlecule-1 , which cioration .NLRP2, NLRP3, NLRP6, NLRP10, and NLRP12 in human kidney samples [NLRP3 was confirmed in both tubular cells and podocytes, and it was increased in biopsies of human kidney diseases  [Besides immune cells, resident kidney cells also take part in inflammation and the tissue repair process. Intrarenal cells respond to circulating proinflammatory mediators by amplifying production of ROS and other proinflammatory factors . Intrins samples . The expphritis) . Most ofκB via TLR2 and TLR4 in a MyD88-dependent pathway to promote the expression of NLRP3 and pro-IL-1β, therefore activating the inflammasome pathway [κB signaling and activity of cathepsins B and L, thus attenuating renal dysfunction in an ischemia-reperfusion (I-R) model [Nlrp3-/- mice demonstrated decreased mature forms of IL-1β, IL-18, and caspase-1, alleviated renal function damage, tubular necrosis, and leukocyte infiltration [Il-18-/- mice, Il-1r-/-mice, caspase1-/- mice, and Asc-/- mice to regular mice after I-R injury, which indicated a new functional role of NLRP3. They also found that in the I-R injury model, Nlrp3-/- mice had much less apoptosis than wild-type and Asc-/- mice [+ efflux-independent manner while the apoptosis signal can induce a K+ efflux-dependent inflammasome activation [Yersinia infection recruited caspase-8 to cleave GSDMD at Asp276, leading to cell pyroptosis but not apoptosis [β. In brief, apoptosis limits cells from further cytokine production and tissue injury, while inflammasome-induced pyroptosis destroys injured cells but releases proinflammatory cytokines. The regulation and switching of these events are fine-tuned and controlled under different pathological situations [Acute kidney injury (AKI) is acute, but reversible kidney function deterioration in a short period caused by ischemia, sepsis, and renal toxins. Research suggests that AKI accompanies large amounts of cell apoptosis and necrosis, as well as the release of cell debris. The heat shock proteins, histones, and high-mobility group B1 proteins in the cell debris can activate NF- pathway –72. HydrR) model . In the R) model . In the ltration . However-/- mice . All evi-/- mice . In humativation . Recentlpoptosis , 79. Thituations .Nlrp3-deficient mice demonstrated less damage regarding of tubular injury, inflammation, and fibrosis, as well as less activation of caspase-1 and release of mature IL-1β and IL-18. Furthermore, it has been proven that myeloid cells and nonmyeloid cells both play important roles in NLRP3-mediated renal fibrosis by chimeric mouse experiments [Aim2-/- mice exhibited attenuated renal injury, fibrosis, and inflammation compared with wild-type littermates in the UUO model. In terms of mechanisms, researchers found that DNA released from necrotic cells drives the activation of AIM2 inflammasomes in macrophages, thus promoting tissue injury in the kidney [Chronic kidney disease (CKD) develops over several years without obvious clinical symptoms, but results in irreversible kidney damage, ultimately leading to end-stage renal disease. CKD is characterized by leukocyte infiltration, proinflammatory cytokine secretion, cell damage/death, fibrosis, and renal function failure as a common end. Persistent, low-grade chronic inflammation has been recognized as an essential part of CKD. Biomarkers of inflammation were inversely correlated with the estimated glomerular filtration rate (eGFR) in CKD patients . The unieriments . Additioe kidney .Nlrp3-deficient mice demonstrated less leukocyte infiltration, better renal functions, and less caspase-1, IL-1β, and IL-18 synthesis; while their Th17 ratios were decreased, and Treg ratios were increased [β and IL-18. Hence, suppressing the activation of NLRP3 decreased inflammatory response [NLRP3 expression in the kidney was increased in patients of IgAN . In normncreased . The mecresponse . These rNLRP3, NLRP1, NLRC4, NAIP, AIM2, IFI16, PYRIN, ASC, caspase-1, IL-1β, and IL-18) from peripheral blood mononuclear cells (PBMCs) in IgAN patients, and we found that mRNA levels of NLRP3 were significantly increased in PBMCs of IgAN patients , NLRP3 inflammasomes promoted disease onset and progress under high-glucose conditions ; meanwhiseverity –89. Indets of DN . Moreovets of DN , 91. In ts of DN . Considets of DN , 94. Whets of DN . In addint of DN , 97. Recic model . NLRC4 immasomes .α [β and IL-18 [β release, and improved kidney functions [β release, anti-dsDNA antibody concentration, and symptoms of LN [Il-1r-deficient mice and caspase-1-deficient mice demonstrated major improvements in skin inflammation, with decreased expression of MCP-1 and TNF-α [lpr lupus model, the lack of NLRP3 and ASC did not deliver an expected effect on disease improvement, instead further damaging kidney function and causing exacerbated activation of lymphocytes [β receptor and downstream molecules which can suppress lupus progression [lpr lupus model is based on extensive cell apoptosis, and as mentioned before, the apoptosis signaling pathway interacted with the NLRP3 signaling pathway.As an autoimmune disease, systemic lupus erythematosus is composed of a series of immune aberrances, including abnormal T cell development, innate immune dysregulation, and increased B cell activity . These eα . Lupus nα . The rolα –103. Thend IL-18 , 105. Atnd IL-18 . New Zeand IL-18 , 108. NZnd IL-18 . Accordiunctions . At the ms of LN , 111. Moms of LN . In anotms of LN . In a mund TNF-α , indicatphocytes . Furthergression . AnotherNLRP1/IL-1β polymorphism was correlated with the pathogenesis of autoimmune diseases including lupus [On the other hand, complement components were also found capable of influencing the activation of inflammasomes in lupus. Genomics research found that polymorphism of C1q was closely related to the pathogenesis of lupus and the lack of C1q promoted the development of lupus-like autoimmune diseases . Evidencng lupus –119; howAIM2 expression was positively correlated with the disease's SLEDAI score and was regulated by body hormones [AIM2 in cells [AIM2 mRNA in macrophages was observed in male patients with lupus compared with female patients [AIM2 expression in macrophages showed a substantial increase, demonstrating a positive correlation to anti-dsDNA antibody titer. Injecting siAIM2 can reduce the activation of macrophages, thus diminishing renal inflammatory responses [Aim2 knockdown augmented type I IFN response induced by cytosolic DNA in macrophages [α, can influence expression/activity of both AIM2 and IFI16 [The importance of dsDNA-sensing inflammasomes  in LN is also worth mentioning. AIM2 expression was first found to be related with colorectal cancer and prostate cancer –122. Addhormones , 125. Mapatients . In the esponses . Howeverrophages . Inhibitrophages , which wrophages –131. IFIrophages , 49, 128nd IFI16 , 133. Coin vitro engineered cell line have also been used for hunting specific compounds for inflammasomes [β (canakinumab) is beneficial in the treatment of RA and CAPS [β antibody (gevokizumab) has been tested in patients with DN since 2015 [From all of the above, it is clear that inflammasomes play key roles in inflammatory kidney diseases. At present, the most current research is still focused on NLRP3 inflammasomes. Glomerular dysfunction associated with inflammatory microenvironments may benefit from inhibiting NLRP3 inflammasomes, and many compounds have shown this effect in murine models of kidney diseases . High-thmmasomes , 135. Commasomes . A monocand CAPS , 138. Annce 2015 . Also, ance 2015  and RA [nce 2015 , 142.Much of our knowledge about inflammasomes is limited to experimental animal models, and the role of inflammasomes in kidney diseases still requires more intensive research. For instance, the effects of AIM2 and IFI16 in kidney diseases are very intriguing, though there is still no direct evidence proving how they play roles in the pathogenesis of nephritis. As many signals may influence the activation of inflammasomes, any molecule involved in the inflammasome pathway could be the key to therapeutic intervention of kidney diseases. Analyzing how these signals influence inflammasomes will provide much-needed evidence in understanding and curing kidney diseases.


Ca2+ takes part in the regulation of neuronal excitability, and the imbalance of intracellular Ca2+ is a trigger factor for the occurrence of epilepsy. Several anti-epileptic drugs target voltage-dependent calcium channels (VDCCs). Intracellular Ca2+ levels are mainly controlled by VDCCs located in the plasma membrane, the calcium-binding proteins (CBPs) inside the cytoplasm, calcium channels located on the intracellular calcium store (particular the endoplasmic reticulum/sarcoplasmic reticulum), and the Ca2+-pumps located in the plasma membrane and intracellular calcium store. So far, while many studies have established the relationship between calcium control factors and epilepsy, the mechanism of various Ca2+ regulatory factors in epileptogenesis is still unknown. In this paper, we reviewed the function, distribution, and alteration of VDCCs and CBPs in the central nervous system in the pathological process of epilepsy. The interaction of VDCCs with CBPs in the pathological process of epilepsy was also summarized. We hope this review can provide some clues for better understanding the mechanism of epileptogenesis, and for the development of new anti-epileptic drugs targeting on VDCCs and CBPs.As an important second messenger, the calcium ion (Ca Alterations of VDCC functions can cause abnormality in cellular events, leading to pathological consequences. In neurons, VDCCs initiate synaptic transmission [Neuronal intracellular calcium increase plays an important role in the triggering and propagation of seizure activity ,2. Ca2+ ne cells ,3,4. It ne cells ,11,12,13smission ,15. Enhasmission ,17,18,19smission  suggestssmission , VDCCs asmission ,23,24.2+ dependent-signaling cascades are largely mediated by calcium-binding proteins (CBPs) [2+-dependent or Ca2+-independent signaling mechanisms [2+-dependent signaling transduction pathways and regulate Ca2+ influx via the VDCCs in Ca2+-dependent feedback mechanisms [2+ binding motifs are verified to regulate high voltage-activated VDCCs [2+ kinetics directly through the regulation of VDCC properties [2+ in these neurons is controlled by the synergy of VDCCs and CBPs. With the availability of human genetic databases and advanced molecular technologies, accumulated evidence suggest that dysfunctions in CBP-mediated VDCC regulation may be one of the mechanisms leading to human diseases [2+, and protect neurons from over-excitation and neuronal damage [Cas (CBPs) ,26, and chanisms ,28. CBPschanisms . CBPs coed VDCCs ,32,33,34operties ,32,33,34operties ,36, and diseases . CBPs arl damage .2+ influx and may be modulated by CBPs [The pathological process of epilepsy includes three periods: an acute period with initiating factors , including status epilepticus, a latent period , and a chronic stage with spontaneous recurrent seizures. VDCCs and CBPs have been reported to be involved in all stages of the pathogenesis of epilepsy. Epileptogenesis is mainly related to the latent period . Interictal spikes, as the omen of seizure activity, present during this period ,39,40. I by CBPs ,43,44, i2+ currents designated as L-, N-, P/Q-, R-, and T-type, based on the long-lasting current and intermediate voltage dependence, which were first recorded in Purkinje neurons and cerebellar granule neurons that were resistant to subtype-specific organic and peptide Ca2+ channel blockers, and transient current, respectively [v1.1), Cav1.2, Cav1.3, and Cav1.4. P/Q-, N-, and R-type are corresponding to Cav2.1, Cav2.2, and Cav2.3, respectively. T-type VDCCs include Cav3.1, Cav3.2, and Cav3.3. Electrophysiological studies reveal different Caectively ,46. The v1.2 and Cav1.3, shape neuronal firing and activate Ca2+-dependent pathways involved in the control of gene expression [v3.1, Cav3.2, and Cav3.3 are important for the repetitive firing of action potentials in rhythmically firing cells, and could be activated and inactivated more rapidly and at more negative membrane potentials than other VDCCs mentioned above [L-type VDCCs members, Capression . N-type,ed above . T-type ed above .The physiological function, and distribution of VDCCs are summarized in the 2+ concentrations might trigger seizures in the brain tissues [2+ may indicate at least in part, the Ca2+ influx through neuronal VDCCs [The evidence to show VDCCs in epileptogenesis has been reported four decades ago, which indicated that decreases in extracellular free Ca tissues ,73,74. Wal VDCCs . Pieces 2+ concentration in the cytosol, and participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e., decoding Ca2+ signals. The members of intracellular CBPs with EF-hand [2+ binding domains [Intracellular CBPs of the EF-hand superfamily are involved in the regulation of cell function and they contribute to the control of Ca EF-hand ,26,105,  domains , have im domains . 2+ sensor in all types of cells, it is assumed that CaM may be involved in many pathological processes, such as epilepsy [Among the EF-hand CBPs, the CaM family is very large and has been extensively characterized. CaM was firstly discovered in the bovine  and rat epilepsy ,111, PD,In the PV family of CBPs, PVs play an important role in cells by checking on calcium switching . BecauseCR, as a member of the EF-hand CBPs family, was first identified in the retina. It is involved in intracellular calcium buffering, messenger targeting, cell cycle arrest, and apoptosis . CR play2+ buffer [2+ transients and evoke Ca2+ signals in most neuronal groups [2+ mediated neurotoxicity by buffering intracellular Ca2+.As a high-affinity CBP, CB is expressed in the mammalian kidney, intestine, placenta, brain, peripheral nervous system, parathyroid gland, bone, and pancreas ,118. Whi+ buffer ,120, age+ buffer ,123,124.l groups ,125, and2+ concentration in the cytosol and participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e., decoding Ca2+ signals [Neuronal intracellular calcium increase plays an important role for the triggering and propagation of seizure activity . CBPs co signals . Therefo2+ are frequently mediated via its interaction with CaM, and strong evidence indicate that the effects of Ca2+/CaM are often achieved through the regulation of conductance of ion channels and the protein phosphorylation process [+ channel (Nav) intracellular C-terminal domains (CTDs), and can limit persistent Na+ current and accelerate inactivation across the Nav family. Mutations located in Nav1.2 channel CTDs can reduce CaM binding and result in increased persistent Na+ current and the occurrence of epilepsy [+ current and decreased neuronal activity. Meanwhile, CaM knockdown reduced the KCNQ2 current and increased neuronal activity. It suggests that CaM may regulate neuronal excitability by KCNQ2, and it could be a target of gene therapy in epilepsy [The effects of Ca process . A recenepilepsy , suggestepilepsy . CaM oveepilepsy . These depilepsy . CaM mayepilepsy . In variepilepsy ,132,133.epilepsy . These depilepsy . In the epilepsy . These repilepsy ,138,139 epilepsy ,141 are epilepsy  and betaepilepsy  subunits2+ to reduce the overload-induced excitotoxicity and prevent the pathological process of epilepsy. The loss of CB from granule cells markedly increased the Ca2+-dependent inactivation (CDI) of VDCCs currents, thereby diminishing Ca2+ influx during repetitive neuronal firing in epilepsy [By a gene knockout study, the vulnerability of hippocampal CA1 neurons to seizure-induced injury was shown to be modified by CB . In GEPRepilepsy  which maepilepsy . -/- mice show impaired LTP induction following tetanic stimulation of hippocampal inputs, which could be restored by using bicuculline (a GABA receptor blocker) [In the cerebellum, granule cells produce CR to excite Purkinje cells through parallel fibers. In vivo electrophysiological recording indicated that knocking out CR induced dramatic alterations in motor coordination and Purkinje cell firing . In acutblocker) . At a ceblocker) ,156. CR-blocker) ,158,159.blocker) ,164,165.blocker) ,161, andblocker) ,175,176 blocker) ,163,177.blocker) . Based o2+ buffering and GABA release regulation are two pathways through which PV regulates neuronal excitability and epileptogenesis , facilitation (CDF), and Ca2+-independent regulation (CIR) of the channels. Pathogenesis of human diseases may occur once CBPs-mediated VDCCs are impaired [v2.1 CDF was blocked in the familial hemiplegic migraine type 1(FHM-1). This disruption of Cav2.1 CDF may cause the cerebellar ataxia-associated FHM-1 due to an imbalance between the excitatory and inhibitory inputs to the cerebellar Purkinje cells [CBPs such as CaM do not act as solely Caimpaired . The besimpaired . CaM binimpaired ,215,216 impaired ,34,217. je cells . Therefov2.1 in vitro, co-expression of CR inhibits CDI and enhances CDF via a direct interaction with Cav2.1. The direct modulation of Cav2.1 by CR affects intracellular Ca2+ signaling, and probably also the neuronal excitability, via a mechanism that is different from Ca2+ buffering function [2+ entry into neurons  and the lack of isoform-specific inhibitors of VDCCs, clinical application of antagonists of VDCCs in patients with epilepsy has been compromised due to universal severe side effects. Recent studies have indicated that the redistribution of VDCCs may play an important role in the pathological process of epilepsy, understanding the trafficking mechanism of VDCCs becomes very important and may provide novel therapeutic targets for seizure control or prevention of epilepsy. CBPs  as Ca2+ buffers, can reduce Ca2+ overload and protect neurons from excitotoxicity, and regulate neuronal excitability through CDF and CDI of VDCCs, or other pathways. Further study to understand the molecular mechanisms of the interaction between CBPs and VDCCs in the pathological process of epilepsy will greatly facilitate the discovery of promising targets for the development of new antiepileptic drugs. By overexpression of CBPs to investigate their gain-function in reducing epileptogenesis, novel approaches in controlling or prevention of the pathological process of epilepsy may be developed. As important pathways for Ca


O/C-glycosides, seven flavanone-O-glycosides, and three limonoids were identified in the segments. The accumulation of these components was altered by light irradiation. Red and blue light resulted in higher levels of PMFs during exposure periods. The accumulation of PMFs was also significantly induced after white light, UVB and UVC irradiation were removed. Red and UVC irradiation induced the accumulation of flavone and flavanone glycosides throughout the entire experimental period. Single light induced limonoid accumulation during exposure periods, but limonoid levels decreased significantly when irradiation was removed. Principal component analysis showed a clear correlation between PMFs and white light, between flavonoid glycosides and red light and UVC, and between limonoids and UVC. These results suggest that the accumulation of flavonoids and limonoids in citrus is regulated by light irradiation. White light, red light and UVC irradiation might be a good potential method for improving the nutrition and flavor quality of post-harvest citrus.To investigate the effect of post-harvest light irradiation on the accumulation of flavonoids and limonoids, harvested Newhall navel oranges were continuously exposed to light-emitting diode (LED) and ultraviolet (UV) light irradiation for 6 days, and the composition and content of flavonoids and limonoids in the segments were determined using UPLC-qTOF-MS at 0, 6, and 15 days after harvest. In total, six polymethoxylated flavonoids (PMFs), five flavone- Many physical or chemical methods, such as cold storage , heat trCitrus unshiu Marc.), the combination of ethylene and red LED light irradiation on carotenoid metabolism +, 457.11142 +, 379.08057 +, 409.09124 [(M + H)-150-2·H2O]+ and 477.12020  (C-glucoside (vicenin-2). In total, twenty-one health-promoting phytochemicals were identified in the segments of Newhall navel oranges, including six polymethoxylated flavonoids, five flavone-O/C-glycosides, seven flavanone-O-glycosides, and three limonoids , 5,6,7,8,4′-pentamethoxyflavone (tangeretin), 5,7,8,4′-tetramethoxyflavone, 5,6,7,8,3′,4′-hexamethoxyflavone (nobiletin), 5,6,7,3′,4′-pentamethoxyflavone (sinensetin), and 3,5,6,7,8,3′,4′-heptamethphoxyflavone, were identified from the segments of Newhall navel oranges. The accumulation of these identified PMFs was altered in response to LED and UV light irradiation . In spitO/C-glycosides  and seven flavanone-O-glycosides . Among these compounds, two flavone glycosides  and four flavanone glycosides  were increased in response to red light irradiation throughout the entire experimental period  was performed based on the MS response value of 21 flavonoids and limonoids identified in response to different light treatments  and storage times . As shown in 2·s) induces some phenylpropanoids, such as scoparone [2) [2·s) and darkness, to identify the optimal light treatment for maintaining and improving health-promoting components in citrus fruits.Light irradiation has been developed as an efficient method with no pollution to extend the shelf-life and maintain the quality of vegetables and fruits. In general, visible and ultraviolet light are both intrinsic parts of sunlight, while in the natural environment, all solar UVC (<280 nm) and most UVB (280–315 nm) rays are absorbed by the stratospheric ozone layer before reaching the Earth’s surface . Artificcoparone , and, asrone [2) . In thisCitrus sinensis Osbeck) and further identified six PMFs, twelve flavonoid glycosides, and three limonoids, which are almost identical to citrus characteristic compounds reported previously [Saussurea [2·s) irradiation specifically enhanced the accumulation of PMFs when light was removed, suggesting that each compound responds differently to a single light source. In Arabidopsis thaliana, key enzymes of anthocyanin biosynthesis are strongly induced by blue light, and the induction intensity decreases in the order of UVA, UVB, and red light [C-glucoside, diosmin, hesperidin, neoeriocitrin, narirutin, and didymin. Furthermore, UVC remarkably elevated levels of vicenin-2, rhoifolin-4′-O-glucoside, narirutin-4′-glucoside, and stellarin-2 in fruit segments, and the accumulation effects remained after removal of the light source until the last day of observation, which was on day 15 of storage. Therefore, red light and UVC irradiation might represent a more efficient method for improving flavonoid content after harvest, which is an essential demand of post-harvest logistic systems of fresh fruits.Herein, we detected 21 phytochemicals in the segments of Newhall navel oranges  freeze-dried grapefruit pulp . Our resA previous report found that irradiation of pear with UVB 280–315 nm) and/or fluorescent lamps for 10 days after harvest not only results in a significant increase in the anthocyanin, flavonoids, chlorophyll, and carotenoid concentration in the peel, but also leads to a slight increase in soluble solid and organic acid concentration and a significant increase in total sugars in the flesh [ nm and/oR2R3 MYB transcription factors. Constitutive photomorphogenic 1 (COP1) acts as a central repressor in the light signaling pathway by interacting directly with photoreceptors to mediate flavonoid biosynthesis [R2R3 MYB transcription factors which may be regulated by basic leucine zipper (bZIP) transcription factors such as HY5. Ultraviolet B radiation is strongly absorbed by tryptophan (Trp) amino acid residues in the dimeric form of a UV resistance locus 8 (UVR8) photoreceptor leading to the monomerization of UVR8. Monomeric UVR8 and COP1 form a complex that accumulates in the nucleus of the cells [HY5, promoting the activity of different R2R3 MYBs for the transcription of specific flavonoid biosynthesis genes. However, the possible mechanism of limonoids in response to light irradiation needs to be further studied in the future.Light-controlled flavonoid biosynthesis in fruits is known to be regulated by ynthesis . In visihe cells ,49. The The HPLC-grade acetonitrile and methanol were purchased from Sigma–Aldrich . The HPLC-grade formic acid was purchased from Fisher Scientific . Ultrapure water was acquired using the Millipore water purification System .2·s), UVB , UVA , blue light , red light , and white light (100 μm/m2·s) at 20 °C and 90–95% relative humidity (RH) for 6 days. On day 6, all lights were removed, while the same temperature and relative humidity conditions were maintained for 10 days. Fruits from each group were sampled at 0, 6, and 15 days. Sample fruits were peeled to retain only the edible portion (segments) and were immediately frozen in liquid nitrogen, powdered, and stored at −80 °C until analysis.Newhall navel oranges were collected in December 2018 from the National Citrus Germplasm Repository, Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing, China. Ten fruit trees with the same age and similar growth condition were marked at the same orchard before sampling. Fully ripe fruits were picked randomly from four directions on these marked trees. Then, the fruits were randomly divided into 7 groups, and 180 fruits were included in each group, and these were then treated with different wavelengths of light. For each sampling, 60 fruits were taken out, 20 fruits each as biological replicates. Treatment conditions were set using the RXZ-300D intelligent growth cabinet with adjustable light irradiation parameters, purchased from Southeast Instrument Company , and all the UV TL-D36/16 lamps were purchased from Philips Company (Netherland), and the LED RDN-500B-C lamps were purchased from Ningbo Southeast Instrument Co., Ltd. . The control group was stored at 20 °C and 90–95% relative humidity (RH) in the dark. The other groups were irradiated with UVC  was used as the lock-mass compound at a concentration of 200 ng/mL and flow rate of 10 μL/min. Mass spectrometry data were collected between m/z 100 and 1200. The identification and quantification of target compounds were performed according to the previous methods [2 ion, and MS responses represent the relative content of the compounds.Samples were analyzed using UPLC/Xevo G2-S Q-TOF MS , equipped with an ACQUITY UPLC BEH C18 column  coupled with the UNIFI Platform in triplicate. The analysis method was performed according to Zhao et al. . Briefly methods ,51. The The peak area of the quasi-molecular ion for each identified compound was integrally computed using UNIFI software . Statistical differences of compound contents for each treatment point were evaluated by Fisher’s protected least squares difference (LSD) test at a 0.05 probability. Data were expressed as the mean of three biological replicates ± standard deviation (SD), using IBM SPSS Statistics software v.23 for analyses. To determine all the variables attributed to differentiate different samples, a PCA model was developed using the contents of all target compounds as the independent variables and the treatment times as the dependent variables, respectively. Three line charts and principal component analysis (PCA) were plotted by Origin Pro 2018 .C-glucoside, diosmin, hesperidin, neoeriocitrin, narirutin, and didymin throughout the entire experimental period with light irradiation, whereas UVC enhanced the accumulation of vicenin-2, rhoifolin-4′-O-glucoside, narirutin-4′-glucoside, and stellarin-2. Three limonoids, limonin, epilimonin, and 7α-limonyl acetate, were induced by UVC during the irradiation period but decreased when the irradiation was removed. These results suggest that single light sources, such as red light and UVC, have great potential for improving the nutrition and flavor of citrus fruits, which can be used for post-harvest logistic systems and lighting conditions for supermarket sales. However, the specific molecular mechanisms underlying these effects require further investigation.Twenty-one flavonoids were identified in the segments of Newhall navel oranges, specifically six PMFs, five flavone glycosides, seven flavanone glycosides, and three limonoids. The accumulation of these compounds was altered in response to exposure to different LED and UV light irradiation. The levels of PMFs decreased significantly during the irradiation period, and red and blue light maintained higher levels of PMFs. White light and UVB significantly induced the accumulation of PMFs after the fruits were removed from irradiation. Red light enhanced the content of diosmetin 6,8-di-


These bioactive lipids belong mainly to two classes of compounds: N-acylethanolamines and acylesters, being N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), respectively, their main representatives. During the last twenty years, an ever growing number of fatty acid derivatives (endocannabinoids and endocannabinoid-like compounds) have been discovered and their activities biological is the subject of intense investigations. Here, the most recent advances, from a therapeutic point of view, on endocannabinoids, related compounds, and their metabolic routes will be reviewed.Endocannabinoids are lipid mediators able to bind to and activate cannabinoid receptors, the primary molecular targets responsible for the pharmacological effects of the Δ Remarkably, during the last few years multiple pathways have been described for eCBs metabolism , and will be described later in this review.The biological actions of eCBs and congeners are controlled through not yet fully characterized cellular mechanisms, that include key agents responsible for: (i) AEA and 2-AG synthesis, like the Strong pharmacological and biochemical evidence has demonstrated that eCBs and related molecules are also able to interact with non-CBR targets, increasing the complexity of the ECS and of the molecular pathways trigged thereof. In particular, the best known non-CBR target of eCBs is the transient receptor potential vanilloid type 1 (TRPV1) channel, which is activated by both AEA  and 2-AGOther potential receptors activated by eCBs are peroxisome proliferator-activated receptor (PPAR) α and γ , and theIn this review, we present the state of the art on eCBs, related compounds and their metabolic routes with a mention about their potential therapeutic role.Although many GPCRs have endogenous ligands that are hydrophilic cations, CBRs have neutral, highly lipophilic ligands derived from fatty acids. The differences in reciprocal endogenous concentration of eCBs, receptors affinity, and asymmetrical localization  of their metabolic enzymes support distinct roles for these molecules under various physiological conditions  ,73,74,75N-arachidonoylethanolamine, termed “anandamide” from the Sanskrit word “ananda” for inner bliss . Ev. Ev39]. Furthermore, n-3 PUFAs and their natural derivatives resolvins can modulate TRPV1 activity ,101,102.in vitro fertilization treatments . In. InN-acy-/- mice .in vivo (Solid evidence demonstrates that monoacylglycerol lipase (MAGL) is the main responsible for 2-AG degradation in vivo . Indeed,Interestingly, MAGL exhibits higher specificity than FAAH-1, because treatment with its potent and selective inhibitor JZL-184 rises only the levels of 2-AG, without affecting those of any other MAG  . MAGL isTwo additional serine hydrolases, α/β-hydrolase domain 6  and 12 , are involved in 2-AG hydrolysis  31,32].,32.31,32abhd12 gene are associated with the neurodegenerative disease called PHARC  . T. T450  as ligands for CBeceptors ,180,181,eceptors ,182, as eceptors ,184.N-arachidonoyltaurine undergo LOX-catalyzed oxygenation [In addition to AEA, other fatty acid derivatives like DHEA, NArGly and genation ,186,187.genation .in vivo. Interestingly, an increase of prostamide F2α (PMF2α) was found in the spinal cord in mice, after induction of inflammation, and was found to exert a proalgesic effect, supporting the relevance of endocannabinoid oxidation in vivo [2α analogue bimatoprost is currently used for the treatment of glaucoma [2-glycerol ester was found to decrease macrophage activation, and this effect was dependent on ABDH6 activity [COX-2, but not COX-1, can oxygenate the main eCBs generating a number of oxygenated molecules . By anal in vivo . Furtherglaucoma . More reactivity .450, to form a number of structurally related epoxyeicosatrienoic ethanolamides (EETs-EA) [450, and acts as a potent and selective agonist of CB2 receptors [450 4X1 was found to produce 14,15-EET-EA, whose (patho)physiological relevance remains to be clarified [Finally, AEA can be metabolized by several different human cytochromes PEETs-EA) . In parteceptors . Insteadlarified .® (rimonabant), a CB1 receptor antagonist/inverse agonist that was licensed and marketed as an anti-obesity agent [The ubiquity of eCBs and their multiple (patho)physiological implications has allowed to identify novel targets for next generation therapeutics; yet, the numerous side effects at the central and peripheral levels may dampen enthusiasm towards these new targets. Indeed, recently they led to the withdrawal of Acompliaty agent .®  and Marinol® , that are preparations used to treat nausea and vomiting associated to cancer chemotherapy. The latter drug is also prescribed to manage the loss of appetite in people with acquired immunodeficiency syndrome (AIDS). In addition Sativex®, a medicine that contains THC and cannabidiol at a 1:1 ratio, has been licensed for the symptomatic treatment  of multiple sclerosis patients in a (ever-growing) number of countries . Furthermore, PEA is currently marketed to cure neuropathic (Normast®) and pelvic (Pelvilen®) pain, and is one of the main components of a cream (Physiogel®) used for inflamed or irritated skin of subjects with atopic dermatitis.However, over the last 20 years eCBs-related drugs have indeed been commercially available, such as Cesamet1 receptors [1 receptor function [To this aim, modulators of endocannabinoid metabolic routes have been tested, with a promise to be free of unwanted side effects typical of compounds that activate eCBs-binding receptors. These studies have led to opposite results, the FAAH blockade did not lead to desensitization of CBeceptors , while tfunction . In conc


FT/TFL1 and the micro RNA (miR)156-regulated SPL family members. Despite substantial progresses recently achieved in Arabidopsis and other plant species, information regarding the involvement of these genes during orchid development and flowering competence is still limited. Dendrobium catenatum, a popular orchid species, exhibits a juvenile phase of at least three years. Here, through whole-genome mining and whole-family expression profiling, we analyzed the homologous genes of FT/TFL1, miR156, and SPL with special reference to the developmental stages. The FT/TFL1 family contains nine members; among them, DcHd3b transcribes abundantly in young and juvenile tissues but not in adult, contrasting with the low levels of others. We also found that mature miR156, encoded by a single locus, accumulated in large quantity in protocorms and declined by seedling development, coincident with an increase in transcripts of three of its targeted SPL members, namely DcSPL14, DcSPL7, and DcSPL18. Moreover, among the seven predicted miR156-targeted SPLs, only DcSPL3 was significantly expressed in adult plants and was associated with plant maturation. Our results might suggest that the juvenile phase change or maturation in this orchid plant likely involves both the repressive action of a TFL1-like pathway and the promotive effect from an SPL3-mediated mechanism.Time to flower, a process either referring to juvenile–adult phase change or vegetative–reproductive transition, is strictly controlled by an intricate regulatory network involving at least both  Arabidopsis and rice [Flowering, the reproductive process of higher plants, is an important trait vital to agriculture practice. The timing of flower initiation is determined by measuring the day-length (photoperiod) and/or low temperature  in many annual plants, including the model plants and rice . These aand rice ,2. Arabidopsis flowering locus T (FT) [Hd3a [TFL1) [FT/TFL1-mediated photoperiod flowering pathway is widespread in angiosperm species [FT/TFL1 family can be divided into three subfamilies: FT-like, TFL1-like, and MFT-like. The MFT (mother of FT and TFL1) homologs were identified in genomes of some bryophytes, which lack the other FT/TFL1-like genes [MFT-like genes, together with mixed FT/TFL1-genes, also present in gymnosperm species [MFT-like genes are ancestral to both FT-like and TFL1-like genes and that the separated functions of FT and TFL1 were evolutionarily attained after divergence of gymnosperms and angiosperms  [AP1), Fruitfull (FUL), suppressor of overexpression of Constans 1 (SOC1) and the plant-specific transcription factor Leafy (LFY) to initiate vegetative to floral transition [The s T (FT)  and its T) [Hd3a  induce fa [TFL1)  from thea [TFL1) ,7,8,9. T species ,10, and ke genes . MFT-lik species ,12. It ieference  for a re factor)  and a bZansition ,16. The ansition ,17, althansition ,19.FT/TFL1 pathway, several of the miRNA156-regulated squamosa promoter binding protein-like (SPL) factors that specifically expressed at the shoot apex also act upstream of some of these MADS-box-containing floral regulators, albeit in an age-dependent manner [Arabidopsis, mature miR156 may be produced from eight precursor genes, namely miR156A through miR156H. By targeting the expression of several SPL genes, miR156 plays pivotal roles in vegetative phase change and shoot development as first demonstrated in Arabidopsis, e.g., overexpression of miR156 led to a prolonged juvenile to adult phase [In parallel to and independent of the t manner ,21,22. Mt manner ,24,25. Mt manner . In Arablt phase ,28,29. SPL mRNAs are necessary to induce vegetative phase change and flowering in Arabidopsis, maize, perennial A. alpina, and Brassicaceae species [SPL genes that are targeted by miR156 in Arabidopsis, SPL9/SPL15 and SPL13 are more relevant to both the juvenile-to-adult vegetative transition and shoot growth, while SPL3/SPL4/SPL5 do not play a major role in vegetative phase change or floral induction, but do promote the floral meristem identity transition through synergistic action with the FT–FD module under long days [AP1, whereas AtSPL15 is responsible for floral induction in short days through activation of the MADS-box gene FUL and another microRNA gene miR172b, which targets and inhibits several flowering repressor genes such as AP2, Schlafmutze (SMZ), Schnarchzapfen (SNZ), target of EAT1 (TOE1), TOE2, and TOE3 [Arabidopsis represents the endogenous flowering control axis. Nevertheless, fewer studies addressed the molecular pathways controlling flowering induction and competence to flower in most perennials.An age-associated reduction in the abundance of miR156 and the resultant increased levels in its target  species ,27,28,30ong days ,32. Exprand TOE3 . Both SPand TOE3 ,35,36,37FT and of other floral meristem identity genes were cloned and functionally studied in Dendrobium Chao Praya Smile [Oncidium Gower Ramsey [FT homologs were shown to correlate with flower organ development and accelerated flowering when overexpressed in Arabidopsis [FTs have a role in regulation of flowering time per se is still elusive, and information regarding the involvement of miR156/SPL in these unique species is lacking. Here, we took advantage of a currently available version of the Dendrobium catenatum genome sequence [FT/TFL1 and the miR156/SPLs gene families. The genome of D. catenatum encodes nine FT/TFL1 members and 12 SPL genes, where seven of the latter are predicted targets of miR156. A BLAST search of the whole-genome sequences resulted in only a single locus of the putative miR156 precursor gene. We further determined the expression profiles of these genes in the three developmental stages: protocorms, one-year-old young (juvenile) plants, and three-year-old (adult) plants. We found in this orchid that a TFL1-like gene, DcHd3b, is highly expressed in the seedling and juvenile phase, while the DcSPL3 is predominant in the adult phase.Orchids are important horticultural plants with high commercial value. Many orchids have long life cycles, as well as characteristic slow-growing habits, and take more than two years to achieve flowering . Recentlya Smile , Oncidiur Ramsey , and othr Ramsey . In thesbidopsis  and Chaobidopsis . Howeversequence  and utilFT/TFL1 family, we used the AtFT and AtTFL1 sequences to BLAST against the genome of D. catenatum and ended up with nine homologous loci, which were annotated in the database either as heading date 3A-like or FT-like genes. The deduced amino-acid sequences showed well-conserved PEBP (phosphatidylethanolamine-binding protein) signature motifs and could be assigned within the three subfamilies: FT-like, TFL1-like, and MFT-like with those from Arabidopsis, rice, and two other orchid species, Phalaenopsis equestris and Apostasia shenzhenica  were roughly conserved among Dendrobium sequences  and one precursor gene of miR156 in D. catenatum  is annotated as uncharacterized and spans a region of 29,481 bp, which produced three non-coding RNA (ncRNA) variants with lengths of 1194 bp, 1332 bp, and 902 bp [DcMIR156 and its transcription to pre-miRNA156 X1/X2/X3  and DcFTL5 (~17-fold lower than DcHd3b). Transcripts of other FT/TFL genes were barely detectable , and declined by a factor of ~4.98 at 40 days following germination and differentiation. Mature miR156 level was further reduced by 33.50- and 193.28-fold in one-year-old leaves and stems, and by 22.35- and 52.58-fold in three-year-old leaves and stems, respectively  under in vitro culture. The one-year-old seedlings represent the juvenile stage, and the three-year-old plants are developmentally ready to reproduce. These three stages showed divergent gene expression patterns for both family members for FT/TFL1 and SPL.Time to flower, a process either referring to juvenile–adult phase change or vegetative–reproductive transition, is strictly controlled by an intricate regulatory network involving at least both  success . Like moTFL1-like gene DcHd3b from the FT/TFL1 family was predominantly expressed  were able to complement the respective mutant phenotypes of Arabidopsis. Both OnTFL1 and OnFT were expressed highly in pseudobulbs in the reproductive stage and juvenile axillary bud. However, the OnFT mRNA was more abundant than OnTFL1 and it also accumulated in reproductive buds and leaves [Dendrobium Chao Praya Smile, an ortholog of FT was upregulated in reproductive organs, including inflorescence apices, stems, floral buds, and open flowers. Overexpression of DoFT promoted flowering and notably pseudobulb formation, while suppressing the endogenous DoFT transcripts delayed flowering in the orchid plants [TFL-like gene(s) in the adult stage suggests that, in D. catenatum, there might exist a repressed mechanism to prevent flowering during vegetative growth. However, more work is needed to evaluate the functions of DcHd3b.It was shown in an orchid plant, d leaves . In Dendd plants . HoweverArabis alpine, downregulation of miR156 by age and de-repression of its target SPL transcription factors in the apices account for its competence to flower under vernalization [MiR156 is a “negative” regulator in many developmental processes by repressing specific SPL transcription factors at the mRNA level ,49,50. Tlization .Dendrobium protocorm differentiation or early seedling development, the increase in DcSPL14/DcSPL7/DcSPL18 transcript levels is associated with the reduced abundance of miR156  was actively associated with adult stage in both leaves and stems, where its transcript level exceeded that of miR156 , Leafy (LFY), Apetala1 (AP1), and FT [A. alpine suggested that, in young plants, TFL1 is required to delay flowering through repression of the floral meristem identity gene LFY. Until in the later developmental stage, this blockage of flowering competence was overcome by the upregulated activity of SPL transcription factors which targeted to the common floral meristem identity genes [D. catenatum, given that the opposite pattern of expression exists between DcHd3b and DcSPL3 in young and adult. It is, thus, possible that the convergent antagonistic action of both pathways determines the timing of juvenile phase change in D. catenatum. Of course, involvements of other members from the FT/TFL or SPL family are not exclusive.The stage-specific expression patterns of f miR156 . In turn, and FT ,22. Evidty genes ,52. AlthD. catenatum were germinated aseptically in half-strength MS medium solidified with 3 g/L agar and supplemented with hormones (0.22 μM 6-benzyladenine (BA) + 0.027 μM α-naphthalene acetic acid (NAA) + 0.05 μM indole-3-butyic acid (IBA)). Seed-derived protocorms were proliferated in the liquid proliferation medium (½ MS + 0.269 μM NAA + 2.22 μM BA + 5% potato pulp + 2.5% sucrose) and developed on differentiation medium (½ MS + 2.69 μM NAA + 5% banana pulp + 3% sucrose + 3 g/L agar), both cultured in 25 ± 2 °C, with a 12-h photoperiod and 2000 lux from an LED light source. Seedlings developed from germinating protocorms after ca. four months were grown in pots and maintained in a climate room under long days (14-h light/10-h dark) and 26 °C/22 °C. In our conditions, one-year-old plants were unable to flower; only after three years of growth can plants set flowers. In addition to protocorms at either proliferation or the 40-day differentiation stage, these one-year-old and three-year-old plants were used as materials for RNA isolation.Seeds of D. catenatum was used for BLAST search homologous sequences of FT/TFL1 and SPL families using known proteins from Arabidopsis, rice, and tomato. A BLAST search was also conducted with Apostasia shenzhenica [Phalaenopsis equestris [D. catenatum’s DNA and RNA databases in NCBI to include all putative sequences. All resulting sequences and annotations were manually filtered, selected, and retrieved. For alignments, protein sequences were submitted to MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/), and the phylogenetic trees were constructed by MEGA X (https://www.megasoftware.net/) using the maximum-likelihood method with a 1000 bootstrap number. The neighbor-joining trees were generated via Figtree version 1.4.4 downloaded from https://github.com/rambaut/figtree. The NCBI genome assembly (ASM160598v2) of nzhenica  and Phalquestris  database2, and about 200 mg of the frozen powder was extracted in 500 mL of extraction buffer. Finally, we merged four extractions from the same sample together. These RNA isolations were kept at −80 °C or used immediately for quantitative RT-PCR measurements of miR156 and FT/TFL1 or SPL genes. Usually, 2 μg of total RNA was used in one RT reaction.For RNA isolation, protocorms, leaves, and stems were collected and frozen in liquid nitrogen. We pooled two plants for leaf and stem samples as one biological replication, and six to 10 protocorms as one. Three biological replications were used. Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Ambion) following the manufacturer’s instructions. During RNA preparation, approximately 1 g of fresh tissue was ground into fine powder in liquid NTo measure the abundance of miR156, the stem–loop qRT-PCR method with SYBR Green was employed as detailed in Reference , using aFT/TFL1 and SPLs, the first-strand complementary DNA (cDNA) was synthesized with random primes using the Prime Script™ First Strand cDNA Synthesis Kit  Co., Ltd.). Gene-specific primers were designed with the Primer3+ program (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) . and are listed in For qPCR analysis of transcript levels of All qPCR reactions were performed in triplicate for each biological replicate on a Roche Light Cycler 96, using a PCR program set as an initial step of 30 s at 94 °C, followed by 40 cycles of 15 s at 94 °C, and 30 s at 60 °C. The relative expression levels of genes were calculated by the 2 (−ΔΔ C (T)) method , using 1t-tests on means of at least three biological replicates was performed using the GraphPad Prism software (version 7). The adjusted p-values were presented in the figures.For statistical analysis, either two-way ANOVA or pair-wide multiple


Pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of <10% due in part to a lack of effective therapies. Pan-histone deacetylase (HDAC) inhibitors have shown preclinical efficacy against PDAC but have failed in the clinic due to toxicity. Selective HDAC inhibitors may reduce toxicity while retaining therapeutic efficacy. However, their use requires identification of the specific HDACs that mediate the therapeutic effects of HDAC inhibitors in PDAC. We determined that the HDAC1/2/3 inhibitor Mocetinostat synergizes with the HDAC4/5/6 inhibitor LMK-235 in a panel of PDAC cell lines. Furthermore, while neither drug alone synergizes with gemcitabine, the combination of Mocetinostat, LMK-235, and gemcitabine showed strong synergy. Using small interfering (si)RNA-mediated knockdown, this synergy was attributed to inhibition of HDACs 1, 2, and 6. Pharmacological inhibition of HDACs 1 and 2 with Romidepsin and HDAC6 with ACY-1215 also potently synergized with gemcitabine in a panel of PDAC cell lines, and this drug combination potentiated the antitumor effects of gemcitabine against PDAC xenografts in vivo. Collectively, our data show that inhibition of multiple HDACs is required for therapeutic effects of HDAC inhibitors and support the development of novel strategies to inhibit HDACs 1, 2, and 6 for PDAC therapy. Pancreatic ductal adenocarcinoma (PDAC), the third leading cause of cancer death in the United States, is one of the most deadly forms of cancer, with a five year survival rate of less than 10% and a median survival following diagnosis of 6–12 months . This di2+-dependent HDACs 1–11  and the NAD+-dependent sirtuins (Class III) [Histone deacetylase (HDAC) inhibitors are novel drugs that have attracted considerable attention for the treatment of cancer . The HDAass III) . HDACs oass III) . As suchass III) . Expressass III) . Intraceass III) ,16.Consistent with the biological roles of HDACs, HDAC inhibitors have shown preclinical efficacy against a broad spectrum of cancer types . They haAs with other tumor types, preclinical studies have pointed to the potential of HDAC inhibitors in the treatment of PDAC. Multiple HDAC inhibitors have antiproliferative and cytotoxic activity as single agents and synergize with clinically relevant therapeutics  to induce apoptosis in PDAC cell lines, both in vitro and as xenografts in mice ,21,22,23Historically, the HDAC inhibitors used in clinical trials have been broad-spectrum pan-HDAC inhibitors, such as Panobinostat, Vorinostat, and Belinostat. More recently, considerable effort has been dedicated to the development of more selective inhibitors of HDACs with the goal of retaining the antitumor effects of the pan-HDAC inhibitors while reducing toxicity associated with broad inhibition of multiple HDAC family members . However50 values ranging from 0.75 to 5.7 µM and 0.47 to 1.8 µM, respectively . Notably, while individual knockdown of these HDACs was less effective in enhancing the response of cells to LMK-235 (50 of Mocetinostat in Capan-1 cells (p = 0.01). Strikingly, the effects of knocking down HDAC6 alone on the ED50 of Mocetinostat (p = 0.01) recapitulated the effects of combined knockdown of HDACs 4, 5, and 6  or HDACs 4, 5, and 6 (the targets of LMK-235) was not toxic to the cells C. These  LMK-235 A, combin LMK-235 B. Thus, 5, and 6 C, indica5, and 6 B. Collec50 (p < 0.01).Having identified HDACs 1, 2, and 6 as relevant targets in the synergistic effects of Mocetinostat and LMK-235 in PDAC cells, we tested the effects of HDAC1, 2, and 6 knockdown on the cytotoxicity of gemcitabine, which continues to be a mainstay in the treatment of pancreatic cancer ,10. As sTo further explore the therapeutic potential of the interaction between selective HDAC1, 2, and 6 inhibition and chemotherapeutic agents in PDAC, we evaluated the synergy between gemcitabine and combinations of the Food and Drug Administration (FDA)-approved HDAC1/2 selective inhibitor, Romidepsin ,34, and Since ACY-1215 is not cytotoxic at concentrations that inhibit HDAC6 activity, the mass action formulas underlying the Chou–Talalay method could not be directly used to examine the interaction between pharmacological inhibition of HDACs 1, 2, and 6 and gemcitabine in reducing PDAC cell viability. Therefore, the effects of ACY-1215 on the interaction between Romidepsin and gemcitabine were evaluated by treating PDAC cells with varying concentrations of Romidepsin and gemcitabine at a constant ratio in the presence or the absence of 1 µM ACY-1215. As seen with the HDAC1/2/3 inhibitor Mocetinostat, Romidepsin had an additive to mildly synergistic effect on cell viability when combined with gemcitabine in MiaPaCa-2 and Capan-1 cells C. HoweveTo determine the ability of selective HDAC 1, 2, and 6 inhibition to augment the therapeutic effects of gemcitabine in vivo, the impact of combined Romidepsin/ACY-1215 treatment on effects of gemcitabine was examined in mice bearing PDAC xenografts . The use of this combination in mice was further supported by in vitro analysis of the effects of Romidepsin, ACY-1215, and gemcitabine on apoptosis in PDAC cells. As shown in To model the use of gemcitabine in the clinic, mice were treated with gemcitabine at 640 mg/kg twice weekly, which is 80% of the maximum tolerated dose. The Romidepsin/ACY-1215 combination had a mild inhibitory effect on the growth of Capan-1 xenografts B. AlthouThe dose limiting toxicities of pan-HDAC inhibitors in patients have led to the development of selective inhibitors, based on the concept that targeting a subset of HDACs would reduce toxicity while retaining the promising anti-tumor activity of these agents . HoweverOur identification of HDACs 1, 2, and 6 as critical players in the therapeutic effects of HDAC inhibitors likely explains the lack of success of selective HDAC inhibitors in clinical trials for PDAC. The selective class I HDAC inhibitors CI-994  and Mocetinostat have been tested in combination with gemcitabine in phase II trials for the treatment of patients with advanced PDAC; however, the combinations were found to offer no advantage compared with gemcitabine alone, and further trials were not recommended ,44. A phAlthough the molecular factors underlying the requirement for simultaneous inhibition of HDACs 1, 2, and 6 in PDAC cells are not known, several studies point to a possible basis for the therapeutic advantage of this combination. HDAC1 is overexpressed in 40% of PDACs and is associated with a significantly worse prognosis ,47. SimiIn contrast to the effects of many HDAC inhibitors, knockdown of HDACs 1–6 alone or in combination was not toxic to PDAC cells. Although this may reflect lower levels of HDAC inhibition than can be achieved with pharmacological agents, off-target effects of the HDAC inhibitors are also likely to contribute to the difference. This can be seen with ACY-1215—consistent with findings that HDAC6 inhibition is not inherently cytotoxic to PDAC cells ,36, ACY-2. Where indicated, cells were treated with Panobinostat , Mocetinostat (Sellechem), LMK-235 (Sellechem), ACY-1215 , Romidepsin (MedChem Express), and/or gemcitabine  in DMSO . For controls, cells were treated with vehicle; DMSO concentrations in the final culture medium were ≤ 0.001%.Human PDAC cell lines, MiaPaCa-2 (ATCC CRL-1420), Capan-1 (ATCC HTB-79), BxPC-3 (ATCC CRL-1687), CFPAC-1 (ATCC CRL-1918), T3M-4 (Riken RCB1021), and PANC-1 (ATCC CRL-1469) were maintained in McCoy’s 5A medium supplemented with pyruvate, vitamins, amino acids, antibiotics, and 10% fetal bovine serum at 37 °C in 5% COCells were lysed in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 0.1% SDS, 50 mM NaF, 10 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 μg/mL β-glycerophosphate, and one protease inhibitor mixture tablet from Roche, and extracts (30 μg protein) were analyzed by Western blotting as described . Primary3 (MiaPaCa-2) or 3 × 103 (Capan-1) cells per well in 96-well plates and treated with the indicated concentrations of drug (or vehicle) after 24 h. Cell viability was assessed after 72 h of drug treatment using 3--2,5-diphenyltetrazoliumbromide (MTT) as described previously [Cells were plated at 2 × 10eviously .plus SMARTpool siRNAs against HDACs 1, 2, 3, 4, 5, and 6 and non-targeting siRNA were from Dharmacon. ON-TARGETplus SMARTPool siRNAs consist of four siRNAs that are designed and modified to increase specificity and reduce off-target effects. MiaPaCa-2 and Capan-1 human PDAC cells were transfected with 37.5 pmol each of targeted and non-targeting siRNA using Lipofectamine 3000  according to the manufacturer’s protocol. After 48 h, cells were analyzed by Western blotting or subjected to drug treatment and MTT assay.Human ON-TARGET5 cells in 10 cm culture plates and treated with HDAC inhibitors and/or gemcitabine after 24 h. Following 30 h of drug treatment, apoptosis (cell surface phosphatidylserine) was assessed using the Annexin V-FITC Apoptosis Detection Kit  as prescribed by the manufacturer. Flow cytometry was performed in the Flow Cytometry Research Facility at the University of Nebraska Medical Center (UNMC).Cell lines were plated at 2.5 × 106) were injected into the flanks of athymic NCr-nu/nu mice . After tumors had reached ~1000 mm3, 16 mice per group were treated with vehicle, 2 mg/kg Romidepsin plus 40 mg/kg ACY-1215, 640 mg/kg gemcitabine, or 2 mg/kg Romidepsin plus 40 mg/kg ACY-1215 plus 640 mg/kg gemcitabine for 3 weeks. Treatment schedules were as follows: 40 mg/kg ACY-1215, daily; 2 mg/kg Romidepsin, twice weekly; 640 mg/kg gemcitabine, twice weekly. Tumors were measured daily using calipers, and volumes were estimated using the formula 3.14/6 × length × width2.All animal experiments were performed under a protocol approved by the IACUC at UNMC . Capan-1 cells . CI > 1 indicates antagonism between drugs, CI = 1 indicates additive effects, CI < 1 indicates drug synergy.Analysis of drug combination effects and combination index (CI) was performed using the method of Chou and Talalay ,31. Cell50 values for drug treatments were performed using GraphPad Prism Software . Statistical differences were determined by Student’s t test using Microsoft Excel . For xenograft growth, descriptive statistics and mean plot  were used to summarize the tumor growth for treatment days and groups. Measurements of each outcome observed over treatment days were considered as repeated measures and fit with a linear mixed effect model with AR(1) structure for correlations to evaluate the change of the outcome measurement values (tumor growth) between treatment groups. In addition, pairwise comparisons of outcome measurements in treatment groups were compared in each measurement time point and adjusted for multiple comparisons with Tukey’s method. Significance was determined by including interaction terms of treatment day and group in the mixed effect models. All analyses were done using SAS, Version 9.4 . p < 0.05 was considered statistically significant.Curve fitting and estimation of EDUsing selective HDAC inhibitors and siRNA-mediated knockdown in PDAC cells, this study demonstrates that the therapeutic effects of pan-HDAC inhibitors result from combined repression of the activity of multiple HDACs from different classes. Therefore, development of treatments based on selective HDAC inhibitors requires detailed knowledge of the relevant HDACs in a particular cancer type. Our analysis determined that HDACs 1, 2, and 6 are major targets of pan-HDAC inhibitors in PDAC and that non-toxic levels of inhibition of selective HDACs can augment the effects of chemotherapeutic agents. Inhibition of HDACs 1, 2, and 6 cooperates with the anticancer effects of gemcitabine both in vitro and in vivo. Thus, our findings point to development of treatment regimens that target HDACs 1, 2, and 6 as a therapeutic option for management of PDAC.


Activated by AMP-dependent and -independent mechanisms, AMP-activated protein kinase (AMPK) plays a central role in the regulation of cellular bioenergetics and cellular survival. AMPK regulates a diverse set of signaling networks that converge to epigenetically mediate transcriptional events. Reversible histone and DNA modifications, such as acetylation and methylation, result in structural chromatin alterations that influence transcriptional machinery access to genomic regulatory elements. The orchestration of these epigenetic events differentiates physiological from pathophysiological phenotypes. AMPK phosphorylation of histones, DNA methyltransferases and histone post-translational modifiers establish AMPK as a key player in epigenetic regulation. This review focuses on the role of AMPK as a mediator of cellular survival through its regulation of chromatin remodeling and the implications this has for health and disease. O-GlcNAcylation, ribosylation, and methylation. Such signature modifications or marks characterize nucleosome remodeling and determine the degree of gene activation or silencing. At a fundamental level, stressors such as nutrient deprivation or heightened physical activity trigger dynamic epigenetic markings that orchestrate adaptive gene regulation to improve survivability . I. Iperoxifunction B 43]..peroxisoAlthough less characterized, it is likely that AMPK phosphorylates an array of HATs including transcription regulator family member A (SIN3), CREB binding protein (CREBBP), elongator acetyltransferase complex subunits (ELP) 2, 3, and 4, and K(lysine) acetyltransferases (KAT) 2A, 2B, 6A, 6B, 7, and 8, and HDACs 1–5, 8–9, and 10, 11, as well as SIRTs 2, 3, 4, 5, 6, and 7; all of which contain AMPK consensus sequences and are opportune for future study .K4, H3K36, and H3K79 promotes an active euchromatin state; while methylation of H3K9 and H3K27 promotes a silent heterochromatic state . H. Hcytochromoters . This reromoters ,70.In addition to the DNMTs, AMPK also regulates TETs, which, in turn, govern loci-specific CpG methylation. The effect of AMPK on TETs occurs through its regulation of TCA cycle intermediates. For example, AMPK regulates isocitrase dehydrogenase 2 (IDH2) to increase the levels of α-ketoglutarate, an activator of TET1–TET4 resulting in CpG demethylation ,71. ActiAlthough biochemical studies have resolved a number of mechanistic insights into the role AMPK plays in the epigenetic regulation of chromatin structure, the study of AMPK on epigenetic function is still in its infancy. A number of predictive algorithms and studies have identified a potential role of AMPK on a number of epigenetic regulators. However, the role they may play under different physiological stimulations and their tissue specificity are still unknown. Because of the interconnected nature between regulatory cascades, the study of AMPK on epigenetic function in a disease-relevant capacity requires systems biological and bioinformatics approaches.K27 acetylation H3K4 and monomethylation (H3K4me1) immunoprecipitation sequencing (IP-Seq); assay for transposase-accessible chromatin using sequencing (ATAC-seq); assay for transposase-accessible chromatin using sequencing (ATAC-seq); and Hi-C [Scientific advances in the computational and Big Data arenas have resulted in novel experimental approaches, databases, and bioinformatics techniques that can be used in the exploration of AMPK epigenetic-regulated signaling pathways. Examples of tools used to explore chromatin characteristics include H3and Hi-C . The reland Hi-C ,73,74,75and Hi-C ,77,78,79O-GlcNAcylation, and ribosylation. Additional, AMPK regulates DNA methylation mediated by influencing the SAM/SAH ratio, TET regulation and inhibition of DNMT1. Technological advances in data analysis continue to reveal the remarkable and diverse roles of AMPK in signaling pathways and epigenetic regulation, providing opportunities to exploit novel therapies relating to health and disease.AMPK’s role as an epigenetic landscape modulator is underscored by its multifunctional kinase effects that regulate histones and epigenetic enzymes to mediate histone and DNA modifications. AMPK is crucial for cell survival and adaptation as evident by its activation upon nutrient depletion and stressors resulting in histone phosphorylation, acetylation, methylation,


The capability of cancer cells to manage stress induced by hypoxia, nutrient shortage, acidosis, redox imbalance, loss of calcium homeostasis and exposure to drugs is a key factor to ensure cancer survival and chemoresistance. Among the protective mechanisms utilized by cancer cells to cope with stress a pivotal role is played by the activation of heat shock proteins (HSP) response, anti-oxidant response induced by nuclear factor erythroid 2-related factor 2 (NRF2), the hypoxia-inducible factor-1 (HIF-1), the unfolded protein response (UPR) and autophagy, cellular processes strictly interconnected. However, depending on the type, intensity or duration of cellular stress, the balance between pro-survival and pro-death pathways may change, and cell survival may be shifted into cell death. Mutations of p53 (mutp53), occurring in more than 50% of human cancers, may confer oncogenic gain-of-function (GOF) to the protein, mainly due to its stabilization and interaction with the above reported cellular pathways that help cancer cells to adapt to stress. This review will focus on the interplay of mutp53 with HSPs, NRF2, UPR, and autophagy and discuss how the manipulation of these interconnected processes may tip the balance towards cell death or survival, particularly in response to therapies. TP53 is the most frequently inactivated tumor suppressor gene in tumors, being mutated in over 50% of human cancer types and indirectly inactivated in many others [An important protagonist in cellular network preventing human cancer development by protecting the cells from DNA damage and oncogenic signals is p53 tumor suppressor, a DNA sequence-specific transcription factor. Following stress stimuli, such as hypoxia or DNA damage, p53 accumulates in the nucleus and becomes activated inducing target genes involved in several different cellular responses, such as growth arrest, senescence, apoptosis, and DNA repair. These activities prevent the establishment of mutations in the future generation of cells, highlighting the role of oncosuppressor p53 as “guardian of the genome” ,2. For ty others ,7. Majory others . In addiy others . Mouse my others ,11. The y others , and thiy others ,14,15. Ay others . Componey others . In thisHsf1-knockout, Trp53R172H  mice that do not develop cancer [Under normal conditions, wtp53 is targeted by the E3-ubiquitin ligases MDM2, COP1, Pih2, CHIP for ubiquitination and rapid degradation via proteasome. After DNA damage wtp53 undergoes posttranslational modifications-induced stabilization and nuclear translocation for transcriptional activity . Differep cancer . Inhibitp cancer .ABCA1 cholesterol transporter gene [Mutp53 stabilization is also achieved by the interaction with chaperone DNAJA1, an Hsp40 family member that protects mutp53 from CHIP-mediated degradation . Both Hster gene . These fter gene . Therefoter gene ,22,24,45The endoplasmic reticulum (ER) is the principal intracellular organelle responsible for protein folding, localization and post-translational modifications . Under nInterestingly, HSP and UPR are inter-connected responses . Indeed NFE2L2 gene, considered the major regulator of the antioxidant response [KEAP1 gene or gain-of-function mutations on the NFE2L2 gene, that occur in several different kinds of tumors , may act as a driver of cancer progression, metastasis, and resistance to therapies [Cip1/WAF1 that directly interacts with NRF2 interrupting the KEAP1/NRF2 complex, leading to NRF2 stabilization and upregulation of the NRF2 signaling pathway, under both basal and induced stressful conditions [Cancer cells adapt and survival to various forms of oxidative, electrophilic, thermal and inflammatory stress by activating not only the HSF/HSP response and UPR but also the nuclear factor erythroid 2-related factor 2 (NRF2) encoded by the response . Both HSresponse . Althougresponse . NRF2 maresponse  and, forresponse ,75,76. Bresponse . Constitherapies . Therefoherapies . Among therapies ,80 and Nherapies ,82. The herapies . In partherapies ,85. Amonherapies  . HIF-1 is a heterodimeric transcription factor that consists of the HIF-1β subunit, constitutively expressed in cells, and the HIF-1α subunit whose stability is stimulated by low intracellular oxygen or genetic alterations . HIF-1 iIn this context, it is worth to discuss that one of the main tumor promoter roles of NRF2 is its ability to induce cancer cell proliferation and aggressiveness by affecting cancer cell metabolism. Thus, NRF2 redirects glucose and glutamine into anabolic pathways, in particular under the sustained activation of PI3K-Akt signaling . In addicis with p53 at canonical, as well as noncanonical p53 target sequences to enhance transactivation and this synergy applies to cancer-associated p53 mutants, greatly expanding the transcriptional master network regulated by p53 in terms of genes affected and levels of expression [To further link these pathways together, a reciprocal interplay has been shown between HIF-1 and Estrogen receptor-α (ERα). Thus, HIF-1α is a direct transcriptional target of ERα . A majorpression . These fpression . It woulAltogether, these findings underline the strict relationship between mutp53, HSF1, NRF2 and HIF-1/ERα signaling pathways that reinforce each other, increasing their oncogenic potential. Given the interplay among them, it is conceivable that blocking one pathway may influence the other pathways and that synchronously blocking them may have greater success in anticancer therapy.DRAM (damage-regulated autophagy modulator) promoting autophagy-induced degradation of mutp53 (R175H and R273H mutants) proteins and restoring cancer chemosensitivity [In the last years, direct mup53 protein-targeting drug strategies have been developed at various preclinical levels for mutp53 degradation and/or wtp53 reactivation. Pleiomorphic degradation is the most advanced strategy that shows impressive survival effects in mutp53 knock-in mouse models . We havesitivity ,115,116.sitivity . Other nsitivity . Gambogisitivity , althougsitivity . Similarsitivity . We founsitivity  and, intsitivity . Anothersitivity  and longsitivity . Finallysitivity .Interestingly, a mutual interplay between autophagy and mutp53 occurs, as on one hand autophagy degrades mutp53 rendering the cancer cells free from this oncogene and, on the other hand, mutp53 inhibits autophagy counteracting its own elimination, and promoting chemoresistance  Figure a. Thus, Given the interplay between mutp53 and NRF2/HIF-1, between ERα and HIF-1, between mutp53 and autophagy, and between NRF2/HIF and autophagy, it is conceivable to hypothesize that mutp53 stabilization could promote NRF2 and ERα/HIF-1 activation also by reducing autophagy. In the case of NRF2, because it is stabilized by p62 that accumulates following autophagy inhibition by mutp53 and, in the case of HIF-1, because its lysosomal degradation may be reduced by mutp53. On the other hand, the oncogenic potential of these partners in crime may be dismantled by induction of autophagy to degrade mutp53 and disrupt the connected complexes like falling dominoes. Of note, differently from the other pro-survival pathways that are activated by mutp53, autophagy seems to be negatively regulated by this oncogenic protein. This may represent a self-protective mechanism, as autophagy contributes to mutp53 degradation. For this effect, for the autophagy capacity to degrade other oncogenic proteins other than mutp53, such as HIF-1 and c-myc  and for TP53 mutations and some of them may acquire oncogenic functions that underline the aggressive phenotype of breast cancer, pancreatic cancer, and non-small cell lung cancer, therefore providing numerous exciting therapeutic possibilities [Many different types of cancer show a high incidence of bilities . Given ibilities ,139. How