In the natural environment, plants are often bombarded by a combination of abiotic  and biotic (necrotrophic and biotrophic pathogens) stresses simultaneously. It is critical to understand how the various response pathways to these stresses interact with one another within the plants, and where the points of crosstalk occur which switch the responses from one pathway to another. Calcium sensors are often regarded as the first line of response to external stimuli to trigger downstream signaling. Abscisic acid (ABA) is a major phytohormone regulating stress responses, and it interacts with the jasmonic acid (JA) and salicylic acid (SA) signaling pathways to channel resources into mitigating the effects of abiotic stresses versus defending against pathogens. The signal transduction in these pathways are often carried out via GTP-binding proteins (G-proteins) which comprise of a large group of proteins that are varied in structures and functions. Deciphering the combined actions of these different signaling pathways in plants would greatly enhance the ability of breeders to develop food crops that can thrive in deteriorating environmental conditions under climate change, and that can maintain or even increase crop yield. Intracellular Ca2+ level is closely influenced by abscisic acid (ABA) signaling, which is an important plant hormone with multiple roles in abiotic and biotic stress responses [2+ sensors, ABA-mediated signaling events, and stress-responsive G-proteins, will be discussed to spotlight the convergent points between the regulatory pathways of abiotic stress and biotic stress responses.The individual effects of isolated abiotic and biotic stresses on plants and their physiological responses to each of them have been studied extensively under experimental conditions, along with the corresponding regulatory mechanisms at the genetic and molecular levels ,2,3,4. Hesponses . Besidesesponses . In this2+ and change their conformations and functions upon binding. Therefore, calcium signaling is considered to be the translator of different stimuli, and the first step in initiating downstream responses. Several calcium sensors have been shown to play roles in both abiotic and biotic stresses. Therefore, calcium sensors have also been regarded as hubs in plant responses to concurrent abiotic and biotic stresses [The fluctuations in cytosolic calcium level upon external stimuli are known as calcium waves, which are perceived by calcium sensors. Calcium sensors are proteins that bind to Castresses .Calcium sensors are categorized into calmodulins (CaMs) or calmodulin-like proteins (CMLs), calcineurin B-like proteins (CBLs), calcium-dependent protein kinases (CPKs), and calcium/calmodulin-dependent protein kinases (CCaMKs) ,12.2+-binding pocket. Within each pair, the two EF-hands are connected by an α-helix. Since each EF-hand pair forms a globular structure, the whole CaM protein consists of two globular domains. Upon the binding of Ca2+, the conformation of CaM changes to expose hydrophobic surfaces for binding to its targets [2+. However, CMLs have low sequence similarities with CaMs. The targets of CaMs/CMLs include promoters, enzymes such as kinases and phosphatases, transcription factors, and ion channels [2+ binding and their specific targets. Several CaMs were found to be involved in abiotic stress, while some were found to be involved in biotic stress (The CaM protein consists of two pairs of elongation factor (EF)-hands, each made up of a helix-loop-helix motif forming a Ca targets . CMLs archannels ,14. The c stress .Solanum lycopersicum), the expressions of SlCaM1, SlCaM2, SlCaM3, SlCaM4, SlCaM5, and SlCaM6 were reported to be induced by mechanical wounding and Botrytis cinerea infection [SlCaM2 had enhanced resistance to Botrytis cinerea infection, while transgenic tomato overexpressing the antisense SlCaM2 and thus having reduced expression of SlCaM2 was less resistant to the infection [SlCaM1, SlCaM2, SlCaM3, SlCaM4, SlCaM5, and SlCaM6 could also be induced by salicylic acid (SA) and jasmonic acid (JA) [Arabidopsis thaliana, the mutation of AtCaM3 led to a reduced tolerance to heat stress [SCaM-4 and SCaM-5 in soybean cell suspensions were induced by Fuasrium solani infection [SCaM-4 or SCaM-5 in tobacco led to enhanced resistance to infection by Phytophthora parasitica var. nicotianae [In tomato (nfection . Transgenfection . The expcid (JA) . In Arabt stress . In soybnfection . The concotianae .ShCML44 could be induced by cold, drought, mannitol, (osmotic stress), salt, ABA, methyl-jasmonate (MeJA), and ethephon (a plant growth regulator) [Arabidopsis thaliana, several CMLs were found to be involved in both abiotic and biotic stresses. The mutation of AtCML9 led to increased sensitivity to ABA, and enhanced tolerance to salt stress and water deficit [AtCML9 led to reduced resistance to Pseudomonas syringae pv. tomato (Pto) strain DC3000 infection [AtCML37 resulted in increased susceptibility to drought stress [AtCML42 led to enhanced resistance to herbivory but reduced tolerance of UV-B [For CMLs, in tomato, the expression of gulator) . In Arab deficit . On the nfection . The mutt stress  and herbt stress . On the  of UV-B . It was  of UV-B . Similar of UV-B ,22,23.Brassica napus), the expression of BnCBL1 was induced by both salt and osmotic stress [BnCBL1 in Arabidopsis conferred improved tolerance of low inorganic phosphate stress and salt stress [Arabidopsis thaliana, the overexpression of AtCBL5 resulted in improved tolerance of salt and drought stress [CBL10 in Arabidopsis thaliana led to a reduced tolerance of salt stress [CBL10 led to improved resistance to Pto DC3000 infection [All CBL proteins have a conserved four-EF-hand calcium-binding domain, and they specifically interact with a family of protein kinases, namely, CBL-interacting protein kinases (CIPKs) . In rapec stress . The ovet stress . In Arabt stress . The mutt stress . On the nfection .2+, CPKs can then phosphorylate their specific targets [Arabidopsis thaliana, the mutation of CPK10 led to an enhanced tolerance of drought [AtCPK3 in Arabidopsis thaliana protoplasts was induced by cold, salt, heat, hydrogen peroxide (H2O2), flagellin, and laminarin [AtCPK3 led to reduced tolerance of sodium chloride (NaCl) [AtCPK6 led to improved tolerance of salt and drought stress [AtCPK3 functioned together with AtCPK6 to regulate stomatal aperture when stimulated by ABA [ZmCPK4 conferred tolerance to drought stress by increasing the sensitivity to ABA [OsCPK9 was induced by ABA, NaCl, and polyethylene glycol (PEG) (osmotic stress). The overexpression of OsCPK9 conferred tolerance to drought stress [OsCPK12 is involved in both abiotic and biotic stress. The overexpression of OsCPK12 promoted tolerance of salt stress, increased sensitivity to ABA and increased susceptibility to blast fungus (Magnaporthe grisea) by reducing the accumulation of reactive oxygen species (ROS) [The typical CPK protein consists of four functional domains including an N-terminal variable domain (N-VD), a protein kinase (PK) domain, an autoinhibitory junction (AJ), and a calcium-binding domain (CBD) made up of one to five EF-hands . Upon bi targets . The tar targets . In Arab drought . The expaminarin . The mute (NaCl) . The ovet stress . It was d by ABA . In maizy to ABA . In ricet stress . OsCPK12es (ROS) .2+-binding domain consisting of three EF-hands. The binding of Ca2+ leads to the autophosphorylation of the protein and enhances its affinity to CaM, resulting in the activation of kinase activity [GsCBRLK was induced by cold, ABA, NaCl, and PEG [GsCBRLK in Arabidopsis thaliana led to improved tolerance of NaCl stress and reduced sensitivity to ABA [Sclerotinia sclerotiorum infection but repressed by Pto DC3000/Xanthomonas oryzae pv. oryzae (Xoo) infection [SlCCaMK reduced resistance to S. scelrotiorum and Pto DC3000 in tomato [TaCCaMK was reduced by ABA, NaCl, and PEG [TaCCaMK in Arabidopsis led to decreased sensitivity to ABA and improved tolerance of NaCl during seed germination, but increased sensitivity to NaCl, with heavier chlorosis in seedlings [CCaMKs have a serine-threonine kinase domain, an autoinhibitory domain, a CaM-binding domain, and a neutral visinin-like Caactivity . Severalactivity ,41,42. C and PEG . The ovey to ABA . In tomanfection . Knockinn tomato . In whea and PEG . The oveeedlings .A lot of calcium sensors were found to be involved in stress responses in plants. Among the characterized calcium sensors, some of them were found to be involved in both abiotic and biotic stresses. Examples are AtCML37, AtCML42, AtCML9, and OsCPK12.AtCML37 and AtCML42, AtCML9 is also involved in both abiotic and biotic stresses. It was reported that AtCML9 regulates ABA sensitivity [OsCPK12 was also reported to play a dual role in abiotic and biotic stresses [AtCML37 and AtCML42 are regulators of JA signaling. JA has been known for its dual role in both abiotic and biotic stress signaling, and the application of JA to plants protects them against both types of stresses . JA has sitivity . Besidesstresses . It is aABA is an important phytohormone that plays multiple roles in abiotic and biotic stresses. ABA closely interacts with several other stress-response hormones including JA, ethylene, and SA. In this section, the interactions between ABA and other stress hormones will be discussed.Pseudomonas syringae on the leaf, flg22, a 22-amino acid epitope on the flagellum of the bacterial cell and a typical pathogen-associated molecular pattern (PAMP), is recognized by the receptor, FLS2, on the leaf epidermal cells. The receptors then trigger SA- & ABA-mediated responses to close the stomata to prevent the pathogen from gaining entry into the plant [Pseudomonas syringae then injects the phytotoxin, coronatine (COR), which mimics the action of jasmonic acid–isoleucine (JA–ILeu), which is the active form of JA, into the host plant, in order to stimulate the re-opening of the stomata. COR, thus termed an effector, functions by stimulating the biosynthesis of JA via Jai1, which in turn induces JA–ILeu-inducible wound-responsive genes via the transcription factor, MYC2, while repressing PATHOGENESIS-RELATED (PR) genes [Upon contact with he plant . A virulR) genes ,49,50. TIn the absence of the virulence factor COR or actual wounding, induction of wounding-responsive genes is inhibited by the repression of MYC2 by jasmonate ZIM-domain (JAZ) proteins . Howevernog1-2 mutant of Nicotiana benthamiana is more susceptible to the pathogen Pseudomonas syringae, due to its inability to close its stomata [Pseudomonas syringae. NOG1-2-mediated stomatal closure is stimulated in the presence of COR, indicating that its function is dependent on JA–Ileu [A small GTPase protein, NOG1-2, stimulates stomatal closure during abiotic stresses, and the  stomata , proving JA–Ileu . In Arab JA–Ileu . JAZ 9 a JA–Ileu .nog1-2 and jaz mutants are often induced in response to ABA or drought, indicating the cooperative nature of these two proteins in inducing ABA-responsive genes [nog1-2 mutant of Nicotiana benthamiana has reduced accumulation of ROS  in guard cells, even after external application of ABA, indicating the role of NOG1-2 in ABA-mediated stomatal closure [The commonly downregulated genes in ve genes . Also, t closure . Thus NO closure . Though PDF1.2 defensin gene is induced upon fungal infection or MeJA challenge, but not by wounding or salicylic acid (SA) challenge, indicating that its induction is triggered only by pathogen attack but not by wounding [PDF1.2 requires both JA and ethylene (ET) for its activation [PDF1.2 occurs when ERF-1 is overexpressed both in the ethylene insensitive (ein2) and JA insensitive (coi1) mutants [The wounding . PDF1.2 tivation , or at ltivation . Constit mutants . At the  mutants . This fuDEHYDRATION RESPONSIVE ELEMENT (DRE) genes in response to drought stress [The plant ethylene receptor family consists of five members that function in conjunction with its negative regulator, CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) ,60. CTR1t stress ,70. Amont stress .The biotic stress-inducible Arabidopsis ERF proteins are also found to be associated with various abiotic stresses such as salt, drought, cold, heat, and light ,72,73. AOsEREBP1 in rice and Tsi1 in tobacco, and ectopic expression of ERF genes such as JERF3 from tomato and GmERF3 from soybean in tobacco, induce the expression of PATHOGENESIS-RELATED (PR) genes and enhance the resistance to various biotic and abiotic stresses [GmERF3 (from soybean) and JERF1 (from tomato) in tobacco, and overexpression of SlERF5 (in tomato), TaERF3 (in wheat), and OsEREBP1 (in rice), conferred resistance to drought, possibly by binding both the DRE promoter sequence along with the GCC box, which results in the upregulation of ABA biosynthesis genes and the accumulation of proline [OsWR1) of the Arabidopsis wax/cutin-synthesizing gene, WIN1/SHN1, is also an ERF gene. Overexpression of OsWR1 improves drought tolerance by inducing wax biosynthesis [ERF1-overexpression increases resistance to drought stress by upregulating the drought response-specific genes, GEA6 and LEA4-5, accumulating ABA and proline, and closing the stomata, whereby ERF1 binds only to the DRE promoter sequence, whereas ERF1 binds the GCC box exclusively during biotic stress responses [Overexpression of ERF related genes such as, stresses ,76,77,78 proline ,80,81. Tynthesis . In Arabesponses . AtERF4,esponses .ERF1-overexpression also causes upregulation of the heat-inducible genes, HSP101, HSP70, and HSP23.6, and the transcription factor, AtHsfA3, responsible for inducing heat-responsive genes, possibly by binding to the DRE promoter sequence [sequence .ETHYLENE AND SALT INDUCIBLE1 (ESE1), one of the salt- and ethylene-inducible genes of the ERF family in Arabidopsis. ESE1 confers salt tolerance by positively modulating the salt tolerance genes, COR15A and RD29A [AtERF1 caused the induction of salt-responsive genes, P5CS1, SRO5, GLP9, and ATOSM34, during salt stress by binding to the DRE sequence of the promoter [Though the overexpression of many ERF transcription factors, such as those found in soybean, tomato, wheat, and tobacco, has been shown to confer salt tolerance ,80,81,84nd RD29A . In a sepromoter .COR gene in an ABA-independent pathway [ERFs from various species, including Arabidopsis, tomato, and wheat ,86,87 ar pathway .Many ERFs that confer abiotic stress tolerance are induced not only by ethylene, but also by the other phytohormones involved in biotic stress response pathways, such as SA and JA, and hence these could be the facilitators of crosstalks between biotic and abiotic stress response pathways ,78,84,89Tamarix hispida was found to negatively regulate drought tolerance by inhibiting superoxide dismutase (SOD) and peroxidase (POD) activities in transgenic Arabidopsis, which are both downstream components of the ABA-mediated stress response pathway [A unique ERF (ThERF1) from  pathway . It also pathway .ACS4 and ACS8, in Arabidopsis, and represses ethylene production in the presence of ABA [ein2, caused higher ABA accumulation during germination, thus demonstrating the antagonistic relationship between ABA and ethylene [On the other hand, the ABA-INSENSITIVE4 (ABI4) protein binds to the promoters of the ethylene biosynthesis genes, e of ABA . One mutethylene .RD22 gene, which decreases lateral root formation [myb96-1d mutant, but it is reduced in the less-active myb96-1 mutant [SA-INDUCTION DEFICIENT 2 (SID2), the gene responsible for expressing isochorismate synthase, a key enzyme in the biosynthesis of SA and normally induced upon infection [myb96-1d mutant. The myb96-1d mutant is more responsive to ABA-mediated signaling, and it also produces higher levels of SA and is more resistant to pathogens, compared to the wild type [NahG plants [NahG encodes SA hydroxylase, which degrades SA [In response to drought stress, ABA promotes cuticular wax biosynthesis mediated by the transcription factor, MYB96, possibly by upregulating the enzyme, 3-ketoacyl-CoA synthase 2 (KCS2), involved in very-long-chain fatty acid biosynthesis in Arabidopsis . MYB96 aormation . This ph1 mutant . MYB96 anfection , is founild type . These mG plants , in whicrades SA . This shrades SA ,104,105.SID2, as indicated by the absence of SID2 induction upon abiotic stress in the aba3 mutant [The ABA3 protein promotes ABA biosynthesis by activating aldehyde oxidase through sulfuration. The enzyme then catalyzes the final step of ABA biosynthesis by converting abscisic aldehyde to ABA . ABA3 is3 mutant .ABI3, since the siz1 mutant accumulates a higher level of the ABI3 protein [siz1 mutant has a higher accumulation of SA, higher expression levels of PR genes, and it exhibits greater resistance to bacterial infections and drought, and these effects can be reversed by introducing the NahG gene [SUMO  E3 Ligase 1 (SIZ1) negatively regulates the ABA-responsive gene,  protein . At the ahG gene ,109. ThePLD gene family, such as PLDα, β, γ, δ, ε, and ζ, classified into subfamilies [pldα1 mutant. MPK6 in turn activates a Na+/H+ antiporter via phosphorylation to confer salt tolerance [pldδ mutants [PLDδ knockout mutants were found to be more sensitive to freezing stress, having less PA content, while overexpression increased the PA content, as well as conferring resistance [Phospholipid pathways are divided into those that activate phospholipase C (PLC), and those that activate phospholipase D (PLD). Here we will focus on the PLD pathway, which is known to be involved in both abiotic and biotic stresses. There are different genes in the families . PLDs mefamilies ,111,112.families  or bactefamilies  infectiofamilies ,116. Salolerance . PLDδ ac mutants . PLDδ knsistance . pldα1 mutants, but co-treatment with ABA and microtubule-disassembling drugs (orzyline and propyzamide) results in stomatal closure in these mutants [Disassembly of microtubules is responsible for stomatal closure, which is inhibited in mutants that overexpress microtubules . ABA, th mutants . PLDα1-d mutants .2+ mediated by ABA [2+ to the catalytic domain of PLDs [2+ concentration [2+ does not play any role downstream of PLDα1, as demonstrated by the fact that exogenous Ca2+ treatment could dis-assemble microtubules in guard cells, and this resulted in stomatal closure in wild-type Arabidopsis but not in the pldα1 mutant [2+ levels quickly in the wild type but slowly in the pldα1 mutant, demonstrating the role of PA produced by intact PLDα1. Treatment with PA increases Ca2+ concentrations more in the wild type than in the pldα1 mutant, showing the possibility that Ca2+ influx may occur both upstream and downstream of PLDα1 [2+ produced by PA in turn induces PLDα1 to produce more PA. Exogenous treatment with Ca2+ increases the PLDα1 enzyme activity without increasing the protein production because the cellular concentration of PLDα1 remains the same [The intracellular increase in Cad by ABA  helps to of PLDs . The downtration . Ca2+ do1 mutant . Treatmeof PLDα1 .. There the same .The PLDα1-PA system stimulates NADPH oxidase activity, resulting in ROS production . This isDuring biotic stress, pathogen-associated molecular patterns (PAMPs) such as flg22 are recognized by the cell surface receptors of plants, initiating PAMP-triggered immunity (PTI). To circumvent this plant defense, bacteria and fungi produce effector proteins that disrupt PTI. In turn, plants produce intracellular resistance (R) proteins that can bind to pathogen-produced effector molecules and initiate effector-triggered immunity (ETI) . Hence, G-proteins are cellular molecules for relaying signals. The involvement of G-proteins in JA signaling has been discussed in A heterotrimeric G-protein typically comprises three subunits . In plants, a heterotrimeric G-protein consists of one α-subunit (AGA1), one β-subunit (AGB1), and three γ-subunits . It transduces signals from a G-protein-coupled receptor (GPCR) in response to environmental stimuli.P. syringae pv. tomato DC3118 is significantly increased in gpa1 mutant plants [d1 mutant, which is deficient in the Gα subunit, exhibits more severe disease symptoms when inoculated with Xanthomonas oryzae pv. oryzae, and it has significantly lowered production of the probenazole-inducible protein, PBZ1, which belongs to PR10 proteins and exhibits RNase activity, and thus induces cell death against necrotrophic pathogens, compared to the wild type [The Arabidopsis Gα subunit, namely GPA1, plays an important role in stomatal defense against bacterial pathogens. For example, growth of the coronatine-deficient t plants . In addiild type .bir1-1 mutant of Arabidopsis, the G-protein β-subunit (AGB1) was found to function downstream of the leucine-rich repeat receptor-like kinase (LRRR-RLK), SOBIR1, to initiate cell death [agb1 null-mutant plants, the induction of PAMP-triggered defense responses as mediated by three other RLKs: FLS2, EFR, and CERK1, are severely compromised, suggesting that AGB1 is a signaling component for plant immunity in common among pathways mediated by different RLKs [On the other hand, using the ll death . In agb1ent RLKs .Fusarium oxysporum f. sp. conglutinans and Alternaria brassicicola (isolate UQ4273). Using the agb1 mutant against these pathogens, AGB1-mediated signaling was found to suppress the induction of PR genes by SA, JA, ethylene, or ABA during the initial phase of the infection. However, with the use of double mutants, leaf necrosis resulting from the agb1-2 mutation was shown to be rescued by simultaneous coi1-21 and jin1-9 mutations. Thus, AGB1 works upstream of COI1 and ATMYC2 in JA signaling during the late phase of infection by necrotrophic pathogens [Arabidopsis AGB1 also plays a role in the defense against the necrotrophic pathogens, athogens .For the Gγ subunits, AGG1 and AGG2 have redundant roles in Arabidopsis in response to elf18-induced oxidative bursts (a PTI response), although AGG1 seems to play the leading role. However, AGG3 is found to be involved in stomatal ion channel regulation and the development of reproductive organs, and it has no effect on the resistance against necrotrophic pathogens. Thus, AGG3 is probably not involved in PTI .agb1-2 mutant exhibited lower cotyledon greening rates, fresh weight, root length, seedling germination rates, and survival rates [Heterotrimeric G-proteins also participate in abiotic stress responses. The Gα subunit from carrot seedlings were shown to exhibit lowered expression under heat stress and prolonged salt treatment ,135 MeanThe unconventional G-proteins are unimolecular and are similar in size to the heterotrimeric Gα subunit. They are classified into extra-large G-protein, GPCR-type G-proteins (GTGs), and the Obg superfamily.agb1 mutants, suggesting the existence of functional similarities between XLGs and Gβ. Similar to the agb1 mutants, xlg2 mutants displayed increased susceptibility to Pseudomonas syringae pv. tomato DC3000. XLG2 interacts directly with AGB1 and acts as a positive regulator in the salicylic acid pathway [In the case of extra-large G-proteins, the Arabidopsis genome contains three genes encoding proteins with limited homology to Gα subunits, but more than twice the size of GPA1, and thus they were named extra-large G-proteins (XLGs) upon their discovery . XLGs co pathway gtg1gtg2 double mutant is defective in a variety of classic ABA responses, including inhibition of seed germination, prevention of cotyledon greening, retardation of primary root elongation, induction of ABA-responsive genes such as RAB18, RD29B, DREB2A, DREB2B, and ERD10, and promotion of stomatal closure.Another class of unconventional G-proteins, GPCR-type G-proteins (GTGs), has also been functionally characterized in Arabidopsis . GTGs arThe Obg superfamily is a TRAFAC class of P-loop GTPases that have a highly conserved amino acid sequence in various organisms from prokaryotes to eukaryotes, and are thus believed to be the descendants of ancient proteins. Some of them are found to play important roles in basic cellular processes, e.g., ribosome biogenesis, translation, and signal transduction. This superfamily includes the Obg, EngB, HflX, Era, TrmE, EngA, EngD/ YchF, Drg, and Nog1 G-protein families. Spo0B gene in Bacillus subtilis. It is found to be essential for the viability of nearly all bacteria. However, its functions in plant are least to be studied. Rice OsTSV3 encodes a Spo0B-associated GTP-binding (Obg) protein was reported to exhibit functions related to cold stress responses. The cold-sensitive virescent tsv3 mutant of rice exhibits an albino phenotype at 20 °C, but develops normal green leaves at 32 °C before the three-leaf stage, and has a normal green phenotype at 20 °C after the 4-leaf stage. The expressions of genes associated with the biogenesis of the chloroplast ribosome 50S subunit in the tsv3 mutant were severely decreased in the three-leaf stage seedlings at 20 °C. This observation implied that the nuclear-encoded Obg G-protein member, TSV3, plays critical roles in chloroplast development at early developmental stages [Spo0B-associated GTP-binding (Obg) proteins was named as such because it was found to be downstream to the l stages .AtDRG1-3 is induced during seed drying and heat stress [AtDRG1-3, which is stress inducible, is not yet elucidated [Developmentally regulated GTP-binding proteins (DRGs), being one of the family in the Obg superfamily, belongs to the Archaea-related type with Nog1. As suggested by the name, its functions are mainly related to development. However, current findings showed that it is also related to cold stress responses. Arabidopsis t stress . The expucidated .The YchFs are a group of ancient proteins, and their sequences are highly conserved among all kingdoms of life except Archaea . The ricOryza sativa GTPase-activating protein 1 (OsGAP1). OsGAP1 is a protein specific to higher plants, and it is induced during wounding in the bacterial blight-resistant rice line that expresses the R gene, Xa14, against bacterial blight. OsYchF1 is identified as its protein interacting partner through yeast two-hybrid experiments, in vitro pull-down and bimolecular fluorescence complementation (BiFC). OsGAP1 not only interacts with OsYchF1, but it also regulates OsYchF1 in several aspects. OsGAP1 alters the subcellular localization of OsYchF1, its RNA-binding affinity, and the substrate specificity of NTP hydrolysis [OsYchF1 is the interacting partner of a wounding-inducible protein, drolysis .Xanthomonas oryzae pv. oryzae (Xoo) and Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), respectively [PR genes involved in the SA, JA, and ethylene pathways [npr1-3 mutant failed to enhance resistance against Pto DC3000, and could not induce the above-mentioned PR genes, thus demonstrating that the function of OsGAP1 on biotic stress is upstream of NPR1, which is a well-known key regulator in biotic stress responses, and plays important roles as the convergence point of several signaling pathways, including the SA-dependent systemic acquired resistance, as well as the JA/ET-dependent induced systemic resistance [More importantly, OsGAP1 is able to enhance plant resistance towards bacterial pathogens. OsGAP1-transgenic rice and Arabidopsis exhibit phenotypes with fewer disease lesions after inoculations with ectively . Besidespathways . In addisistance .Pto DC3000 [However, the positive role of OsGAP1 in biotic stress responses function through its deactivation of OsYchF. Arabidopsis transgenic lines ectopically expressing OsYchF1 show increased susceptibility to o DC3000 . Thus, io DC3000 .AtYchF1-knockdown mutant exhibits higher tolerance towards salt treatment when compared to the wild type. Meanwhile, transgenic Arabidopsis lines over-expressing AtGAP1 demonstrated higher salt tolerance when compared to the wild type while AtGAP1-knockdown lines showed lower salt tolerance [AtYchF1-knockdown mutant, AtGAP1-over-expressing lines, and OsGAP1-transgenic Arabidopsis lines. In addition, OsYchF1-transgenic tobacco BY-2 cells show higher levels of ROS generation and percentage of cell death under salt treatment [YchF1 and GAP1 proteins not only play roles in biotic stress, they are also involved in abiotic stress responses. The ectopic or over-expression of rice and Arabidopsis YchF1 proteins in transgenic Arabidopsis enhances the sensitivity of the plants towards salt stress. On the other hand, olerance . Higher reatment .GAP1 is induced, and the GAP1 protein would inactivate YchF1 and then trigger defense responses.Therefore, YchF1 acts as a negative regulator to suppress both biotic and abiotic stress responses under favorable growing conditions. When there are stresses present, One of the special features of YchF1 is its lower nucleotide-binding specificity than typical GTPases; it can bind both GTP and ATP. By resolving the structure of OsYchF1 via crystallization, it is deduced that the G4 motif of OsYchF1 is responsible for the lowered specificity, and therefore its lower ability to use both ATP and GTP as a substrate. The canonical sequence of the G4 motif for GTP specificity is NKXD, while the sequence of the OsYchF1 G4 motif is NMSE. Mutation of the native noncanonical sequence of the G4 motif of OsYchF1 to the canonical sequence  precludes the binding/hydrolysis of ATP by OsYchF1. Transgenic Arabidopsis over-expressing the mutated versions of OsYchF1-G4 and AtYchF1-G4 could only bind GTP but not ATP, and could no longer function as a negative regulator of plant defense responses. On the other hand, being only able to bind/hydrolyze GTP and not ATP still allows YchF1 to function as a negative regulator of abiotic stress responses. This study showcases the specific nature of the ATP-binding/hydrolyzing ability of YchF1 in determining its function in disease resistance .In animals, there are two subfamilies within the Ras superfamily of small G-proteins: Ras and Rho. However, plants do not have Ras, but they contain Rho-like small G-proteins called RACs or ROPs.ROPs in the barley genome [The Arabidopsis genome encodes a family of 11 ROP small GTPases, while the rice genome comprises seven members and there are six y genome ,146.Magnaporthe grisea and elicitor (sphingolipid)-triggered responses. It positively regulates NADPH oxidase and cinnamoyl-CoA reductase, so as to increase ROS production and lignin biosynthesis, respectively [The rice Rac protein (OsRAC1) is involved in defense against ectively .Nicotiana tabacum results in higher production of SA and pathogenesis-related (PR) proteins, and increases resistance towards tobacco mosaic virus (TMV) [Overexpression of a Ras-related small G-protein, RGPL, in us (TMV) .In response to abiotic stress, Arabidopsis Rac1 (AtRac1) responds to ABA by disrupting the actin skeleton in guard cells, and this eventually leads to stomatal closure during drought conditions .OsRAN2 exhibit increased sensitivity towards salt (NaCl) and osmotic stress (PEG6000), as well as exogenous ABA (10 μM). Meanwhile, transgenic rice lines with knocked-down OsRAN2 expressions showed the reverse phenotypes. OsRAN2 gene expression declines to around 20% to 40% of that in the wild type during the first 10 h of treatments, including salt stress, osmotic stress, and ABA treatment. Besides, both rice and Arabidopsis transgenic lines with higher or ectopic OsRAN2 expressions develop shorter and fewer roots, and smaller leaves under the aforementioned stress conditions. To conclude, the OsRAN2 protein is a negative regulator in abiotic stress responses [Transgenic rice and Arabidopsis lines overexpressing esponses .OsRAN2 was induced during cold stress, and transgenic lines over-expressing OsRAN2 showed higher survival rates under cold treatment, where over 50% of seedlings were able to grow and stay green after a two-week recovery period in normal greenhouse conditions when compared to the wild type, where only around 20% survived after the recovery period. OsRAN2 was found to maintain cell division via promoting the export of intra-nuclear tubulin at the end of mitosis, and it was thus able to maintain the normal nuclear envelope under cold stress [On the other hand, OsRAN2 plays a positive role under cold stress. d stress .A list of the different classes of G-proteins and their roles in abiotic and biotic stress responses is presented in 2+ sensors are responsible for decoding the natures of the stimuli, and the signals are then transduced through the appropriate stress hormone pathways to bring forth a series of physiological responses. Some major plant hormones, such as ABA and JA, are closely coordinated with Ca2+ signaling. Therefore, it has been speculated that ABA and JA are the convergent points between abiotic stresses and biotic stresses. This speculation is supported by studies showing the dual roles of Ca2+ sensors as both the initial stress signal detectors and also the regulators of ABA and JA signaling. At the same time, G-proteins are also known to play dual roles in abiotic stress and biotic stress responses, and it has been reported that G-proteins are also involved in the signaling pathways of plant hormones, such as JA. However, as G-proteins consist of so many different sub-groups (Upon environmental challenges, a huge network of signaling events occur in the plant cell. Cab-groups , their d


Severe asthma (SA) is a chronic lung disease characterized by recurring symptoms of reversible airflow obstruction, airway hyper-responsiveness (AHR), and inflammation that is resistant to currently employed treatments. The nucleotide-binding oligomerization domain-like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome is an intracellular sensor that detects microbial motifs and endogenous danger signals and represents a key component of innate immune responses in the airways. Assembly of the NLRP3 inflammasome leads to caspase 1-dependent release of the pro-inflammatory cytokines IL-1β and IL-18 as well as pyroptosis. Accumulating evidence proposes that NLRP3 activation is critically involved in asthma pathogenesis. In fact, although NLRP3 facilitates the clearance of pathogens in the airways, persistent NLRP3 activation by inhaled irritants and/or innocuous environmental allergens can lead to overt pulmonary inflammation and exacerbation of asthma manifestations. Notably, administration of NLRP3 inhibitors in asthma models restrains AHR and pulmonary inflammation. Here, we provide an overview of the pathophysiology of SA, present molecular mechanisms underlying aberrant inflammatory responses in the airways, summarize recent studies pertinent to the biology and functions of NLRP3, and discuss the role of NLRP3 in the pathogenesis of asthma. Finally, we contemplate the potential of targeting NLRP3 as a novel therapeutic approach for the management of SA. Asthma represents a serious global health problem that affects 1%–18% of the population of all age groups. Its prevalence has increased in the last decades, especially among children ,3. A smaIn the lung, innate immune responses provide the first line of defense against environmental signals, including pathogens, allergens, and other irritants, and act through downstream signaling by numerous extracellular and intracellular receptors, termed pattern recognition receptors (PRRs) ,9,10. NOTo address SA complexity, the concept of asthma endotyping has emerged ,13,14. DUpon allergen exposure, dendritic cells (DCs) in the lung mucosa take up allergens, reach the mediastinal lymph nodes, and present allergen components to naive T cells in the context of major histocompatibility complex class II . AllergeApart from DCs, the asthmatic airway epithelium represents a major source of cytokines termed “alarmins”, such as, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), and chemokines, including RANTES , TARC (Thymus- and Activation-Regulated Chemokine or CCL17), eotaxins , and MCP-3 (Monocyte Chemotactic Protein-3 or CCL7) that trigger Th2 cell polarization upon exposure to allergens, pollutants, viral, fungal, and bacterial components . As mentType 2 asthma is characterized by any combination of the following processes: eosinophilia in the sputum or blood, atopy and a high level of fractional exhaled nitric oxide (FeNO) . SeveralIFNG expression was detected in BAL cells, accompanied by increased secretion in SA patients compared to mild-to-moderate asthmatics (MMA) [+IFN-γ+ T cells in the BAL [Chlamydia pneumoniae, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus) infections augment IFN-γ secretion in SA patients, highlighting a key role for respiratory tract infections in disease severity and asthma exacerbations [Clustering studies revealed that one of the major reasons SA patients remain unresponsive to corticosteroid treatment is that apart from Th2 inflammation, other mediators are also implicated in disease pathogenesis ,30. Indecs (MMA) . The sam the BAL . In line the BAL ,33. It mrbations .+ T cells isolated from bronchial biopsies of SA patients produce copious amounts of IL-17 and IL-22 upon ex vivo polyclonal stimulation [+ T cells from asthmatics bearing the IL4RR576 mutation exhibited defective induction of Treg cells and were skewed towards a Th17-like phenotype, exemplified by increased secretion of IL-17 [The pathophysiology of nontype 2 asthma remains less well characterized. Nontype 2 asthma is denoted by the absence of type 2 biomarkers, such as eosinophils and IgE, and the predominance of Th17 cells and neutrophils in the airways . In factmulation ,37. Anotmulation . Moreovemulation . Increasmulation . In addimulation . A very mulation . In brieof IL-17 . Still, of IL-17 ,43.To date, biomarkers of type 2-low or neutrophilic asthma have not been identified. Several studies have shown that there is no inverse correlation between sputum eosinophil and neutrophil numbers in SA patients and that eosinophils are present in excess, additionally to neutrophilic accumulation, in the airways . MoreoveApart from type 2 and nontype 2, asthma can be categorized into four endotypes  based on the type of airway infiltrating immune cells . Among tIn sharp contrast to adult asthma, wherein the type of airway inflammation has been extensively investigated due to the use of invasive and semi-invasive techniques, such as bronchial biopsies, BAL, and induced sputum, the majority of analyses in childhood asthma have been performed only in severe forms of disease ,65,66,67In-depth investigation of the molecular and cellular mechanisms underlying SA pathophysiology has significantly contributed to the development of novel therapeutic strategies for disease management. Indeed, antibodies targeting factors involved in SA pathology are already being used as a first line medication. An excellent example is omalizumab, a monoclonal antibody directed against IgE that has become an established add-on therapy for patients with uncontrolled allergic asthma . In addiTaken together, it becomes evident that as the airway lumen is continually exposed to external and endogenous stimuli, its ability to distinguish between innocuous environmental allergens and pathogenic agents is crucial for the maintenance of immune tolerance and lung homeostasis. In fact, the complex interactions between innate and adaptive immune responses in the lung micromilieu represent a major determinant of the development of tolerance or allergic inflammation. Innate immune responses have considerable bearing on ensuing adaptive responses, and if left uncontrolled, can lead to detrimental pathological consequences. Hence, delineation of the precise mechanisms involved in the regulation of innate immune reponses in the airways is essential for the design of more efficient treatment modalities for SA patients.The innate immune system acts as the first line of defense during exposure to environmental pathogens. In the lung, innate immune responses act through downstream signaling by numerous extracellular and intracellular receptors termed pattern recognition receptors (PRRs). PRRs recognize pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), bacterial and viral RNA, danger-associated molecular patterns (DAMPs) in damaged and/or dying cells, including reactive oxygen species (ROS), ATP and mitochondrial DNA, and homeostasis-altering molecular processes (HAMPs) that detect alterations in cell homeostasis and elicit inflammatory responses in the host ,9. PRRs NLRs consist of a conserved nucleotide binding and oligomerization domain (NACHT), a carboxy-terminal ligand-binding region, composed of leucine-rich repeats (LRRs), involved in ligand binding or activator sensing, and an amino-terminal effector domain required for protein–protein interactions ,86,87. TNLRP3 is expressed in granulocytes, monocytes, DCs, T cells, and epithelial cells . The NLP+ and CD8+ T cells, natural killer cells, and macrophages, and is essential for antiviral immunity. IL-18 also plays a role in Th2 cell differentiation and is involved in AHR in experimental models, as well as in asthmatic patients [IL-1β secretion upon NLRP3 inflammasome activation initiates acute phase reactions, including the recruitment of inflammatory cells at the site of infection and expression of proinflammatory cytokines, such as, IL-6, TNF-α, and chemokines . Brieflypatients ,102.Apart from proinflammatory cytokine release, a key outcome of NLRP3 inflammasome activation is pyroptosis, a form of lytic cell death characterized by cell swelling, membrane rupture, and release of proinflammatory cellular contents ,104. TheNLRP3, CASP1, and the IL1B and IL18 genes. The priming signal is usually a PAMP, such as, LPS, lipoproteins, carbohydrates, and flagellin [Activation of the NLRP3 inflammasome through the canonical pathway requires 2 steps: a priming signal and a second activating signal . The prilagellin . IL-1β alagellin ,109. Intlagellin .+, Ca+2, and Cl-, b) lysosomal destabilization, c) ROS production, and d) mitochondrial dysfunction [A wide variety of secondary stimuli activate the NLRP3 inflammasome, including bacteria, viruses, environmental nanoparticles such as alum and silica, and endogenous molecules, including ATP, monosodium urate (MSU), and cholesterol crystals. The main mechanisms through which these PAMPs and DAMPs trigger NLRP3 inflammasome activation are associated with: a) changes in cytosolic levels of ions, such as, Kfunction ,111. The+ levels trigger NLRP3 activation, while high extracellular K+ concentrations block NLRP3 signaling [+ efflux and initiates NLRP3 assembly and downstream signaling [+ efflux in a pannexin-1-dependent manner [+ efflux. Moreover, the complement cascade component membrane attack complex (MAC) activates NLRP3 [+ efflux, mobilization of Ca+2 in the cytosol through the opening of plasma membrane channels or the release of endoplasmic reticulum (ER)-linked Ca+2 stores represents another upstream event in NLRP3 activation [+ efflux regulates Ca+2 flux and these two channels act cooperatively to activate NLRP3. In fact, NLRP3 activation induced by nigericin, alum, MSU crystals, and the MAC depends on Ca+2 flux along with K+ efflux [- efflux through chloride intracellular channel proteins (CLICs) enhances NLRP3 activation via the polymerization of ASC [- efflux occurs downstream of K+ efflux [+2 in the cytosol, which, in turn, drive NLRP3 assembly and activation in a K+ efflux-dependent manner [Decreased extracellular Kignaling . For exaignaling ,114. Thet manner . In addies NLRP3 . Apart ftivation . It shou+ efflux . Cl- effn of ASC . Translo+ efflux . Lysosom+ efflux ,121. Brit manner ,121  enhance NLRP3-driven responses ,128,129 Altogether, these secondary activating signals lead to IL-1β and IL-18 secretion downstream of NLRP3 activation and also to pyroptosis . Still, Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis [+ efflux which activates the canonical NLRP3 pathway and the release of IL-1β and IL-18. Hence, caspases 4, 5, and 11 do not cleave IL-1β and IL-18, but only lead to pyroptosis, and the subsequent canonical NLRP3 inflammasome activation pathway is responsible for caspase-1 activation and cytokine secretion. Activated caspase-11 also cleaves pannexin-1, a membrane ATP channel, which induces K+ efflux and activates the canonical NLRP3 pathway [The noncanonical pathway of NLRP3 activation is associated with the detection of intracellular LPS generated following infection by Gram-negative bacteria, such as andensis ,132,133.andensis . Pyropto pathway ,132,133.The cytosolic accessibility of LPS is driven by Guanylate-Binding Proteins (GBPs) and the Immunity Related GTPase family member 10 (IRGB10) which lyse the Gram-negative bacterium-containing vacuoles, releasing bacterial LPS into the cytoplasm ,135. Bac+ efflux and ASC speck formation. An additional unique feature is that it does not trigger pyroptosis, and the secretion of IL-1β is independent of GSDM [An alternative NLRP3 inflammasome pathway was recently discovered in human and porcine monocytes and does not require a secondary signal . LPS rec of GSDM . Notably of GSDM . This tyNLRP3 gene are associated with the dominantly inherited autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS), including familial cold autoinflammatory syndrome, Muckle–Wells syndrome, and chronic infantile neurological cutaneous and articular syndrome [The activation of the NLRP3 inflammasome is associated with a diverse range of human diseases. Mutations in the syndrome ,140,141.syndrome ,144,145.syndrome . Hence, NLRP3 regulation takes place both at the transcriptional and the post-transcriptional levels. Type I-IFNs repress the expression of pro-IL-1β through the secretion of IL-10  Figure . Type-I Beclin or Lc3b, leads to increased secretion of mtDNA and ROS, and increased activation of caspase-1 and release of IL-1β and IL-18 [Nod2 and Ripk2 results in defective mitophagy, leading to excessive activation of NLRP3 and increased IL-18 production [One of the most important mechanisms that restrain NLRP3 functions is autophagy . Autophand IL-18 ,153. Furnd IL-18 . In addind IL-18 . Moreoveoduction .+) activates sirtuins (Sirt2) which inhibit NLRP3 inflammasome activation and decrease pro-IL-1β production [+ efflux and the oligomerization of ASC, and by reducing ROS levels [Signaling through metabolic regulators can also inhibit NLRP3 activation. Dimethyl fumarate (DMF) activates the transcription factor NF-E2-related factor 2 (NRF2) that represses IL-1β and NLRP3 gene expression in LPS-treated microglia and in the human acute monocytic leukemia cell line (THP-1) . NRF2 aloduction  , as well as in HDM-instilled mice, increased ROS and mtROS production in BAL cells and in primary tracheal epithelial cells was detected, inducing augmented NLRP3, caspase-1, and IL-1β protein expression in the airways [NLRP3 inflammasome activation is involved in the initiation and the propagation of allergen-driven inflammatory responses in the airways. Studies using mouse models of allergic asthma induced by adjuvant-free ovalbumin (OVA) and adjuvant -coupled OVA, demonstrate enhanced protein levels of NLRP3 and caspase-1, along with elevated IL-1β and TNF-α release by epithelial cells and macrophages in the airways, compared to Phosphate-buffered saline (PBS)-treated mice . Moreove airways . Interes airways . Moreove airways . Notably airways .NLRP3 and ASC in a model of OVA-induced AAI demonstrated decreased eosinophil influx, dampened AHR and reduced airway inflammation, and goblet cell accumulation, accompanied by decreased IL-1β expression in the airway, compared to wild type (WT) littermates [-/-NLRP3 mice exhibit decreased pulmonary inflammation with suppressed mucus secretion, Th2 cytokine and chemokine production, and IgE levels [NLRP3 and caspase-1, the authors demonstrated a reduction in infiltrating neutrophils in the lung and decreased inflammatory cytokine/chemokine release  in the BAL compared to WT controls [Il4 promoter, enhances Th2 cell differentiation, and exacerbates asthma symptoms in a mouse model [Pertinent to the role of NLRP3 activation in allergic responses, studies using mice deficient in termates . Other sE levels . InteresE levels . These sE levels . Other rE levels . In factcontrols . Anothercontrols . Interesse model . Moreovese model . These sAspergillus fumigates followed by challenge with OVA or A. fumigates, respectively, increased ROS production in lung homogenates mediated by mitochondrial Ca2+/calmodium-dependent protein kinase II (CaMK II), which induced NLRP3, active caspase-1 and mature IL-18 at the mRNA, and protein levels in allergic lungs, as well as exacerbated OVA-AAI [Chlamydia muridarum or Haemofilus influenza, demonstrated an increased expression of NLRP3 in airway epithelial and infiltrating immune cells, as well as enhanced IL-1β and caspase-1 mRNA levels in the lungs of infected mice [NLRP3 and IL-1β, another study documented reduced airway inflammation and cytokine release following rhinovirus (RV) infection in HDM-challenged mice, highlighting the implication of NLRP3 activation in RV-induced disease exacerbations [-/-NLRP3 mice, the authors showed that the protective effects of a high-fibre diet against colitis were mediated through NLRP3 activation in colonic epithelial cells. Altogether, these studies suggest that epithelial cells at distinct sites respond differently to inflammasome activation, not only in terms of cytokine secretion but also in the inflammasome sensor utilized, and propose a key role of the tissue micromilieu in governing epithelial cell-induced inflammatory responses. Notably, sensitization with OVA-alum or  OVA-AAI . In cont OVA-AAI . Furtherted mice . Notablyted mice . Furtherrbations . Notablyrbations . Notably-/-NLRP3, -/-,Casp or -/-PYCARD mice [-/-NLRP3 mice did not exhibit significant differences in airway eosinophilia, mucus production, AHR, and Th2 cell responses upon exposure to uric acid crystals compared to their WT counterparts [-/-NLRP3, -/-,Casp or -/-PYCARD exhibited enhanced lung inflammation and pathology, including eosinophilic inflitration and Th2 cytokine release, upon exposure to HDM, supporting the notion that NLRP3 activation exerts protective functions against HDM-induced allergic lung inflammation [Contradictory data have been generated by studies showing that NLRP3 activation is not essential for allergic disease development in OVA and HDM induced AAI. Allen et al. showed that the adjuvant effects of aluminum hydroxide in the OVA-AAI model were not affected in -/- mice . In anotterparts . Similarterparts . Finallyammation .10. In fact, they demonstrated that NLRP3 silencing using short hairpin (sh) NLRP3 attenuated PM10-induced release of IL-1β by human airway epithelial cells [NLRP3 and pro-IL-1β expression, resulting in increased IL-1β secretion in an APOE-dependent manner [A series of recent studies suggest that NLRP3 activation is involved in human asthma pathogenesis. Hirota et al. was among the first to describe NLRP3 and caspase-1 protein expression in human lung sections and in primary airway epithelial cells from healthy volunteers following exposure to PMal cells . In otheal cells . Knock dal cells  (Figure al cells . Notablyal cells ,194 (Figal cells . In anotal cells . NLRP3 gal cells  (Figure al cells  (Figure t manner . Lui et t manner .Pertinent to inflammasome related-cytokines, most studies have shown that IL-1β is increased in the sputum and BAL of patients with neutrophilic asthma , and in In summary, a growing body of evidence suggests that NLRP3-induced inflammasome responses are implicated in AAI both in experimental models and human asthma . Still, The past decade has witnessed a burgeoning appreciation of the existence of a wide range of SA endotypes. Still, and particularly in the type 2 low asthma endotypes, there is a considerable gap in our understanding of the cellular and molecular mechanisms involved and a remarkable scarcity of relevant biomarkers. More importantly, no effective treatments targeted at these endotypes have emerged. Growing evidence has illuminated a key role for NLRP3 inflammasome activation in the development and exacerbation of allergic responses. Hence, targeting NLRP3 inflammasome pathways in the airways of allergen-challenged mice, particularly in the context of SA, will improve our understanding of how NLRP3 signaling contributes to the development of specific aspects of disease severity. In fact, the direct comparison of the expression and activation of NLRP3 in mouse models and patients with distinct asthma severities is essential for the identification of novel biomarkers pertinent to the diverse asthma endotypes.Current treatment modalities of NLRP3-related inflammatory human diseases target IL-1β with IL-1β antibodies or recombinant IL-1βR antagonists, such as canakinumab and anakinra, respectively. Nevertheless, most of these inhibitors are relatively nonspecific and have low efficacy. Thus, the development of targeted NLRP3 inflammasome site-specific therapeutics may be more beneficial in suppressing inflammasome-associated disease whilst not predisposing to infection. However, a deeper understanding of the NLRP3 inflammasome assembly and activation is needed before translation of these findings into therapies in clinical practice, especially in the context of SA. To achieve this, we need the development and use of better in vivo models of SA, along with complementary human studies using physiologically relevant in vitro models. Ultimately, this will facilitate the development of personalized medicine for the growing numbers of patients with SA.


Liver fibrosis is a common consequence of chronic liver disease. Over time, liver fibrosis can develop into liver cirrhosis. Current therapies for liver fibrosis are limited, and liver transplant is the only curative therapy for patients who progress to end-stage disease. A potential approach to treat chronic liver disease with increasing interest is cell-based therapy. Among the multiple cell types which have been proposed for therapeutic uses, human amnion epithelial cells and amniotic fluid-derived mesenchymal cells are promising. These cells are highly abundant, and their use poses no ethical concern. Furthermore, they exert potent anti-inflammatory and antifibrotic effects in animal models of liver injury. This review highlights the therapeutic characteristics and discusses how human amnion epithelial cells can be utilised as a therapeutic tool for chronic liver disease. Chronic liver disease (CLD) results in the development of chronic hepatic wound healing, characterised by persistent liver inflammation and the accumulation of extracellular matrix proteins (ECM), collectively described as fibrosis , 2. CrucCirrhosis is associated with reduced liver function and an increased risk of developing liver cancers. Currently, a liver transplant is the only cure for patients who progress to end-stage liver cirrhosis. However, transplantation is complicated by low organ availability, high cost, and long-term immunosuppression . ConsequResearch has highlighted the potential of stem cell-based therapies for CLD , 9. For Perinatal stem cells are derived from extraembryonic tissues, such as the foetal membrane and umbilical cord , 11. TheOver the decades, multiple cell types have been isolated from extraembryonic material, including the foetal membrane, umbilical cord, and amniotic fluid. Of these sources, one of the most extensively investigated is the amniotic component of the foetal membrane , 16. TheGenerally, chronic wound healing involves a highly complex and dynamic interplay between injured parenchymal cells, myofibroblasts, inflammatory cells, and tissue-specific stem/progenitor cells  32, 33], 3332, 3A hallmark of tissue fibrosis is the accumulation of ECM-producing myofibroblasts , 46. MyoMyofibroblasts can be derived from a variety of precursor cells type; however, liver myofibroblasts are almost exclusively derived from hepatic stellate cells (HSCs) . These n4-) induced liver injury and bleomycin-induced lung injury, hAEC therapies are reported to reduce the levels of profibrotic factors, namely, transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF) [β stimulates HSC activation, maintains myofibroblast survival, and promotes ECM synthesis. Furthermore, TGFβ reduces the activity of matrix-degrading matrix metalloproteinases (MMPs) by upregulating the expression of tissue inhibitor of metalloproteinases (TIMPs) by myofibroblasts [Preclinical studies in animal models of the liver, lung, and skin fibrosis report that hAEC therapy reduces the number of myofibroblasts within injured tissue , 40, 48.r (PDGF) , 36, 40.roblasts . PDGF isroblasts . Thus, bβ and PDGF activity, hAECs directly suppress the fibrotic activity of HSCs and myofibroblasts through paracrine signalling. For instance, Hodge et al. demonstrated that when cultured in hAEC-conditioned medium, HSCs adopt an antifibrotic phenotype characterised by a reduction in proliferation, activation, and ECM production [β signalling in myofibroblasts by secreting soluble human leukocyte antigen G5 [In addition to reducing TGFoduction . Furthertigen G5 . Other atigen G5 . Overallβ, PDGF, interleukin- (IL-) 1, IL-6, tumor necrosis factor alpha (TNFα), and TNF-related weak inducer of apoptosis (TWEAK) [CLD is closely associated with the enrichment of liver macrophages . Macroph (TWEAK) , 53. The (TWEAK) –57. ConsThe liver houses a specialised macrophage population known as Kupffer cells; however, following injury, the liver's macrophage population expands dramatically through monocyte recruitment. These monocytes differentiate into monocyte-derived macrophages (MDMs) upon infiltration into the injured liver . Researc4 or a high fat diet show a reduction in macrophage numbers when hAECs are infused systemically [β, PDGF, IL-1, IL-6, and TNFα are reduced [The benefits of hAEC therapy are associated with reduced macrophage recruitment. Studies using bleomycin-induced lung injury and liver injury caused by CClemically –40, 45.  reduced , 38–40.  reduced , 39. CCL reduced , 64. In  reduced , 65. Fur reduced . Hence, Animal models of macrophage depletion have shown that macrophages can both promote and resolve tissue fibrosis. For example, Duffield et al., using the CD11b-DTR transgenic mouse, established that macrophage depletion during liver injury and inflammation prevents the development of liver fibrosis. Conversely, depletion during recovery attenuates the degradation of matrix proteins . These cα, IL-1β, and IL-6 [β, and MMPs [M1 macrophages are predominantly proinflammatory, releasing cytokines such as TNFand IL-6 , 69. In and IL-6 , 69. M2 and MMPs , 70  are important regulators of inflammation and fibrosis. This subset of CD4 subsets . An imba−/− mice, only when an adoptive transfer of either Tregs or naive T cells is coadministered [The induction and expansion of Tregs is a crucial component of hAEC therapy. hAECs increase the number of Tregs in the lung following bleomycin-induced injury . Additionistered . Importanistered . Conversnistered . TherefoMost studies investigating hAEC-based therapies for liver pathologies focus on the inflammatory and fibrotic aspects. Currently, it is unknown whether hAEC therapy enhances hepatocyte regeneration. Regardless, as hAEC therapy improves markers of tissue function in models of chronic liver and lung injury, it is likely that tissue regeneration is occurring , 36, 37.4-indcued liver injury [α, IL-1β, and TGFβ are known to stimulate hepatocyte apoptosis [hAEC therapy may support liver regeneration through multiple mechanisms. Firstly, hAECs secrete growth factors such as EFG and IGF2 which are known to induce hepatocyte proliferation , 85, 86.r injury , 36. Thipoptosis , 89. Excpoptosis , 89. ThehAEC therapy may also promote hepatocyte regeneration by stimulating the proliferation and differentiation of liver progenitor cells (LPCs). LPCs are a population of bipotential epithelial cells that reside in the canals of Hering, located within the biliary tree , 91. LPCIt is difficult to discern the exact impact hAEC therapy will have on LPCs. On the one hand, several of the factors such as IGF2 and galectin 3 which are secreted by hAECs may stimulate LPC proliferation –97. ConvPreclinical research over the last decade has illustrated the potential of hAEC-based therapies as a treatment for CLD . These s


Moreover, a small amount of Ti17P10, CaTiO3, Ca3(PO4)2 were also detected. The reaction that may occur during the preparation process is suppressed to some extent, which is because that the addition of second phases can prevent the direct contact of titanium with HA and reduce the contact areas. Transmission electron microscope (TEM) analysis proved the existence of elemental diffusion and chemical reactions in sintered composites. Compared with results of composites prepared by hot-pressed sintering before, mechanical properties  of 0.5-GNFs composites prepared by SPS were increased by about 2.8, 4.8, and 4.1 times, respectively. The better mechanical properties of 0.5-GNFs composite in nano-carbon reinforced composites are mainly due to the lower degree of agglomeration of tubular carbon nanotubes (CNTs) compared to lamellar graphene nanoflakes (GNFs). Moreover, the strengthening and toughening mechanisms of nano-carbon reinforced titanium alloy/HA biocomposite prepared by spark plasma sintering (SPS) mainly included second phase strengthening, grain refinement strengthening, solution strengthening, graphene extraction, carbon nanotubes bridging, crack tail stripping, etc. In addition, in vitro bioactivity test revealed that the addition of nano-carbon was beneficial to promote the adhesion and proliferation of cells on the surface of titanium alloy/HA composite, because nano-carbon can enhance the formation of mineralized necks in the composites after transplantation, stimulate biomineralization and promote bone regeneration.Nano-carbon reinforced titanium matrix/hydroxyapatite (HA) biocomposites were successfully prepared by spark plasma sintering (SPS). The microstructure, mechanical properties, biocompatibility, and the relationship between microstructure and properties of biocomposites were systematically investigated. Results showed there are some new phases in sintered composites, such as β-Ti, TiO Therefore it is easy to have poor bonding with bones and may even fall off after being implanted into the body ,2. Hydroreatment . This wareatment . Neverthreatment ,7,10; . The microstructure and compositions of milled powders and sintered samples were analyzed by SEM  and Electron Probe Microanalyzer . Transmission electron microscopy  was used to further analyze the microstructure of sintered composites. In terms of mechanical properties, the density, microhardness were measured by the Archimedes method and Vickers microhardness tester  with the load of 500 g and holding time of 10 s. Compressive and shear strength of composites were tested using a microcomputer control electron universal testing machine  with a movement rate of 0.1 mm/min. The morphology and characteristics of fracture surface were analyzed by SEM equipped with energy dispersive X-ray spectroscopy (EDS). The bending strength were analyzed by a three point bending test  with the samples of 20 mm × 3 mm × 2.5 mm, load of 0.5 kN load with 0.5 mm min4/well. After the cells were cultured in an incubator for 24 h, the culture solution was removed. Then the cells were washed three times with phosphate buffer salin (PBS), fixed with 4% paraformaldehyde for 15 min, and dehydrated with alcohol gradient . After the process of critical point drying and gold spraying, the cell adhesion and proliferation morphology were analyzed by SEM. On the other hand, each group of composites was immersed in 75% alcohol for 1 h, then the alcohol was removed by soaking in α-MEM. Next, each set of materials were placed into a 24-well plate, and MC3T3-E1 cells were seeded on the surface of materials in the 24-well plate (5 × 104 cells per well). After adding α-MEM + 10%FBS  + 1%P/S (Penicillin-Streptomycin), the cells were cultured in an incubator for 24 h. Finally, the cells were stained with Dapi and phalloidin  and observed by fluorescence microscopy .In vitro biocompatibility of nano-carbon reinforced titanium matrix/HA composites for MC3T3-E1 cells was evaluated by phalloidin staining and SEM ,34,35. F4 [4 . There w17P10, CaTiO3, Ca3(PO4)2, TiO3, ZrO2, which showed that chemical reactions occur in the composite during SPS sintering [17P10 and CaTiO3 at a position of about 33° on the XRD spectra of nano-carbon reinforced titanium matrix/HA composite. Therefore, it was considered that there is no significant difference in the XRD spectra of different sintered samples, and the existence of La, GNFs, and CNTs phases added in the sintered samples are not detected by XRD due to their low content. There were only high-intensity peaks of Ta and Nb detected in sintered composites, and no new phases formed by Ta or Nb were found in the XRD results, which indicated that no chemical reactions occur concerning Ta and Nb during the sintering process. In addition, the XRD did not detect the reactions between titanium and nano-carbon, which indicated that the vacuum environment effectively protects the purity of sintering process. Moreover, researchers also found that the pyrolysis of oxygen-containing functional groups occurs during the preparation of graphene-reinforced titanium-based composites by laser sintering [It can be seen from intering ,16. Moreintering . But no intering .40Zr10Cu36Pd14-HA composites prepared SPS, and there is almost no obvious interface [nterface . A smallnterface . This wanterface . It was seen from 3, CaTiO3, Ca3(PO4)2, etc., and matrix phases contained Ti, Ta, Nb, Zr, TixPy and so on [TEM was used to further analyze the microstructure and composition of the composite, and nd so on ,17,41. Tnd so on . Figure nd so on . This innd so on . Moreove3, Ca3(PO4)2 and TixPy are formed at the boundary of titanium particles [On the other hand, the analysis found that shear fracture surface of composites has a cleavage step (indicated by the red arrows). The shear fracture belonged to the cleavage fracture, and researchers also found that there are some cleavage steps and river patterns on the compression fracture surface of Ti-5/10%HA composite . Zhang earticles . There warticles . There warticles ,43.In vitro biocompatibility of nano-carbon reinforced titanium matrix/HA composites was evaluated by culturing MC3T3-E1 cells on the surface of composites. 17P10, CaTiO3, Ca3(PO4)2, TiO3, ZrO2, TiO3, etc. were detected in the experiment. TiO3 and ZrO2 were formed by the reaction of titanium and zirconium with oxygen in the environment, respectively. Although the experiment was carried out in the vacuum environment, the oxygen in the sintering furnace was not completely removed. The researchers also found that titanium atoms tend to oxidize to form amorphous or crystalline TiO2 at the HA/Ti interface during high temperatures sintering [2 was continuously formed [Studies have shown that HA will decompose at about 800 °C in titanium matrix/HA composites, the reaction process is shown as Equations (1) and (2) ,16. Thenintering ,47. The y formed . Ye and y formed ,50.(1)CIt can be clearly seen from 5(PO4)3(OH), Ca4O(PO4)2, CaTi4(PO4)6, etc., which were mentioned in other literatures [17P10, CaTiO3, Ca3(PO4)2 were detected. On the one hand, this was due to adjustment of the sintering method and reduction of HA content. In fact, SPS sintering is a pressure-assisted sintering method based on spark discharge sintering between particles. It is heated by the spark plasma discharge mechanism and heat transfer mechanism during sintering [In addition, the existence of Caeratures ,51,52,53intering . On the intering . In the intering . Therefointering . Due to intering  was not 17P10, CaTiO3, Ca3(PO4)2, TiO3, ZrO2, etc., but the reactions that may occur during the preparation process are suppressed to some extent. TEM analysis proved the existence of elemental diffusion and chemical reactions in sintered composites. The shear fracture belonged to cleavage fracture, and there were obvious cleavage steps, pores, and some cracks on the surface of the composites.Nano-carbon reinforced titanium matrix/HA biocomposites were successfully prepared by spark plasma sintering. Results showed that there are some new phases detected in sintered composites, such as β-Ti, TiCompared with the traditional hot press sintering, the mechanical properties of the composites prepared by SPS, such as density (>98%), compressive strength (847–1134 MPa), shear strength (178–228 MPa) and bending strength (190–220.15 MPa), have been significantly improved. The compressive strength and shear strength of 0.5-GNFs composite in all Nano-C reinforced Titanium matrix/HA composites were slightly higher. Because entanglement and agglomeration is more prone to occur in tubular CNTs than lamellar GNFs.The strengthening and toughening mechanisms existing in nano-carbon reinforced titanium matrix/HA composites mainly included second phase strengthening, grain refinement strengthening, solution strengthening, graphene extraction, carbon nanotubes bridging, crack tail stripping, etc. In vitro cells proliferation test showed that nano-carbon reinforced titanium matrix/HA composites have good biocompatibility. Moreover, the addition of nano-carbon in the titanium matrix/HA composites was beneficial to promote cell adhesion and proliferation.


Punica granatum) has also gained considerable recognition as a functional food in the modern era. A large body of literature has linked pomegranate polyphenols, particularly anthocyanins (ATs) and hydrolyzable tannins (HTs), to the health-promoting activities of pomegranate juice and fruit extracts. However, it remains unclear as to how, and to what extent, the numerous phytochemicals in pomegranate may interact and exert cooperative activities in humans. In this review, we examine the structural and analytical information of the diverse phytochemicals that have been identified in different pomegranate tissues, to establish a knowledge base for characterization of metabolite profiles, discovery of novel phytochemicals, and investigation of phytochemical interactions in pomegranate. We also assess recent findings on the function and molecular mechanism of ATs as well as urolithins, the intestinal microbial derivatives of pomegranate HTs, on human nutrition and health. A better understanding of the structural diversity of pomegranate phytochemicals as well as their bioconversions and bioactivities in humans will facilitate the interrogation of their synergistic/antagonistic interactions and accelerate their applications in dietary-based cancer chemoprevention and treatment in the future.Having served as a symbolic fruit since ancient times, pomegranate ( Punica granatum) can be dated back to at least 3000 BC. Historically, pomegranate has served as a symbol of fertility and prosperity. In addition, various parts of the pomegranate have been used in traditional medicine for treating a wide variety of illness. Pomegranate fruits have purported use for expelling parasites [Cultivation and consumption of pomegranate , and tissues of identification . Knowledf values of pomegranate phytochemicals to those of the authentic standards)  identified in different pomegranate tissues using diode array detection (DAD), electron spin resonance (ESR), fluorescence detection (FD), flame ionization detection (FID), infrared spectroscopy (IR), mass spectrometry (MS), nuclear magnetic resonance (NMR), and thin layer chromatography  . It shouw/w) of total tannins extracted from pomegranate fruit peel [HTs are among the most studied phytochemicals in pomegranate; they can be further grouped into ETs and gallotannins (GTs) based on the different phenolic acids that are esterified to the core cyclic polyol molecule (it is often a glucose molecule). Overall, more than 60 HTs have been identified from pomegranate using MS and/or NMR . Pomegrauit peel . EA, metuit peel .C-glycosides, punicacorteins A–D , and punigluconin (an ET with a gluconic acid core) [Besides fruit peel, pomegranate stem barks are also abundant in HTs and have been used historically in tanneries for making leather. In addition to the HTs identified in fruit peel, stem barks also contain ET id core) . The denid core) ,16,17.The composition of HTs in pomegranate leaves is largely different from that in fruit peel. In leaves, the major HTs are granatins A and B, whereas punicalagins and punicalins are present at negligible levels . AdditioO-β-d--glucopyranoside showed measurable accumulations in pomegranate flowers [In pomegranate flowers, EA and two oxidized derivatives of EA, pomegranatate and phyllanthusiin E, were discovered . Punicat flowers ,23,24.Pomegranate fruit peel, aril, and juice are abundant in flavonoids of diverse structures, including the aglycones and glycosides of chalcones, flavanones, flavones, flavonols, ATs, flavan-3-ols, and procyanidins . Two flaO-α-l-arabinofuranosyl(1-6)-β-d-glucoside, naringenin-4′methyl ether 7-O-α-l-arabinofuranosyl(1-6)-β-d-glucoside, and quercetin-3,4′-dimethyl ether 7-O-α-l-arabinofuranosyl(1-6)-β-d-glucoside, respectively, by NMR analysis [Similar to other plants, leaves of pomegranate also accumulate high levels of flavone  glycosides . Two flaanalysis ,27. Highanalysis ,28.n studies -MS studies , while i30) are the biosynthetic precursors of steroids in plants (phytosterols) and animals (steroid hormones). Triterpenoids and phytosterols have been found in pomegranate seed, leaf, flower, fruit peel, and bark tissues -∆1piperideine, norpseudopelletierine, and the pyrrolidine alkaloids (with a five-membered N-containing ring) hygrine and norhygrine, were also found in root barks at low quantities [N-pyridinium chloride was identified in pomegranate leaves [N-methylpyrrolidine) was recently characterized in pomegranate fruit peel [Pelletierine, pseudopelletierine, and ot barks . Sedridiantities . In addie leaves , and a puit peel . Besidesuit peel .6, C8, C10, and C12), long , and very long (C22 and C24) chain length have been identified from pomegranate seeds, juice, and fruit using GC-FID, MS, or NMR analysis  represents the most abundant FA in pomegranate seeds, accounting for over 60% of seed oil. Triacylglycerols (TAGs) containing 9E, 11Z, 13E-octadecatrienoic acid, 3-O-octadec-2-enoic acid, 9Z, 11E, 13Z-octadecatrienoic acid, and 8Z, 11Z, 13E-octadecatrienoic acid are produced in pomegranate seeds and their structures were determined by NMR [N-palmitoyl cerebroside was identified from pomegranate (cv. Nana) seed oil by TLC and GC-FID analyses [Fatty acids (FAs) of medium  have suggested synergistic interactions among pomegranate phytochemicals, this is a promising, but currently under-explored topic.Fresh arils and juice products of pomegranate fruit, as well as seeds of the soft-seeded cultivars, are mostly consumed. Phytochemicals in pomegranate fruit peel extracts, fermented pomegranate juice, and pomegranate seed oil exhibited cooperative interactions toward limiting the proliferation, metastasis, and invasiveness of human prostate cancer cells in vitro . A subseWhen the commercial pomegranate-nectarine juice was separated into predominately sugar, organic acid, neutral phenol, and AT fractions, complex antagonistic or synergistic effects were observed among different fractions on the total phenol or total antioxidant content . The antATs are colorful, water-soluble polyphenol pigments that are found in many plant foods, such as berries and pomegranate fruits. Plant ATs are often investigated collectively as a group of phytochemicals for their bioactivities, and have been linked to many aspects of human disease prevention and treatment ,54,55,56Aside from cardioprotection, the anti-inflammatory property of ATs also contributes to their anti-obesity effect and action in adipose tissue . StudiesIn addition to their antioxidant and anti-inflammatory activities, ATs act in cancer chemoprevention by inducing terminal differentiation of tumor cells and thus impeding tumorigenesis . PreventSimilar to other AT-rich fruits, the pomegranate also has a cardioprotective effect by targeting two major causal factors of atherosclerotic lesion and cardiovascular disease, namely the accumulation of cholesterol and oxidized lipids, and arterial macrophage foam cell formation . PomegraThe bioactivities of pomegranate ATs have mostly been studied in the context of pomegranate juice and fruit extracts. However, these AT-rich sources are abundant in vitamin C, carotenoids, and HTs, which can also contribute to the ascribed bioactivities. To this end, “white” (AT-less) pomegranate cultivars are available, which lack the accumulation of ATs in leaves, fruits (including peels and arils) and flowers ,72. CompETs are abundant in pomegranate fruit peel, as well as juice and extracts that are produced commercially from the whole fruit. Besides pomegranate, ETs are also present in a wide range of medicinal and food plants, such as many berries, nuts, and herbs ,74. As sEA, a hydrolysis product of ETs A, can beUrolithins contain a common core structure of dibenzopyranone and are more hydrophobic than EA B. The miAromatase that converts androgens to estrogens has been considered to be a therapeutic target for treating the hormone-sensitive type of breast cancer . AlthougBesides breast cancer, urolithins can also reduce the risks of another sex steroid hormone-related cancer, endometrial cancer. UA and UB (at 10 μM for 48 h) caused over 50% reduction in the proliferation of human endometrial cancer cells . UA (at 50 = 35.2 ± 3.7 μM), UB (IC50 > 40 μM), and urolithin C  impaired the proliferation of the “androgen-sensitive” prostate cancer cells (LNCaP) [UA (IC (LNCaP) . UA and  (LNCaP) . Further (LNCaP) . Urolith (LNCaP) ,85.50 = 1.15 ± 0.65 μM), UB (IC50 = 1.55 ± 0.49 μM), and methyl-UA  effectively inhibited the CYP1-mediated ethoxy resorufin-O-deethylase (EROD) activities and were selective toward CYP1-B1 over CYP1-A1 (CYP1-B1 and CYP1-A1 are two members of the CYP1 family) in in vitro assays [50 = 32 ± 8.9 μM) and UB (IC50 = 38 ± 3.9 μM) also led to inhibition of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1 activities and decreased CYP1-B1 protein accumulation in the “androgen-responsive” 22Rv1 prostate cancer cells. However, the anti-proliferative effects of UC, dimethyl-UC, and mUA toward 22Rv1 cells were due to their cytotoxic activities rather than inhibition of the TCDD-induced CYP1 expression [Cytochrome P450 1 (CYP1) family members activate pro-carcinogens and often exhibit enhanced expression in tumor and cancer cells . UA  reduced specifically the EphA2-Ephrin-A1 interaction (this receptor-ligand pair is known to be involved in cancer development and progression) of the Eph-Ephrin system . UD actsOverall, these studies collectively suggest that urolithins may find applications as chemo-preventive agents against prostate cancers, through different mechanisms in androgen-sensitive, -responsive, and -independent cells.Fragaria ananassa), and Jamun berry (Eugenia jambolana), as well as pure EA and UA compounds, were tested in the human embryonic kidney 293T cells that express a reconstructed canonical Wnt signaling pathway [50 value (at 39 μM) that was physiologically relevant in the lumen of colons when taking enterohepatic circulation of urolithins into consideration [The canonical Wnt/β-catenin signaling pathway has been shown to activate T-cell factor transcription and function in colon carcinogenesis . To dete pathway . Althougderation .−1 for 48 h) impaired the proliferation of HT-29 cells and led to cell cycle arrest at the G2/M phase, as well as activation of CDKN1A expression. In addition, treatment with urolithins resulted in activation of caspases 3, 8, and 9, suggesting that urolithins induced both the extrinsic  and intrinsic  apoptotic pathways in HT-29 cells [During the initiation stage of the HT-29 colon cancer cells, urolithins A–D inhibited TCDD-induced, CYP1-mediated EROD activities . At the 29 cells .50 values of 43.9, 35.2, and 46.3 μM, respectively [50 of 39 μM [2O2-induced oxidative stress and induced apoptosis through activation of caspase 3 and PPAR-γ protein expression [UA, UB, and mUA inhibited the proliferation of the T24 human bladder cancer cells in vitro, with ICectively , comparaof 39 μM . The trapression .Inflammatory responses involving the activation of neutrophils and monocytes play a central role in the development of cardiovascular diseases . IntriguIn addition to recruitment of neutrophils, monocyte adhesion to endothelial cells represents another key event in inflammatory responses . A mixtuNO plays multifaceted roles in combatting cardiovascular diseases . AlthougGordonibacter urolithinfaciens correlated positively with the in vivo production of UA, but inversely with that of UB and isoUA [G. urolithinfaciens levels were higher in the metabotype A than the metabotype B individuals [Based on the urinary excretion of urolithins by healthy volunteers after ingesting ET-rich foods or fruit extracts, three urolithin metabolic types (metabotypes) have been defined, including A (excretes only UA), B , and 0 (does not excrete urolithins) . Interesnd isoUA . A recennd isoUA . A relatividuals . FurtherLarge inter-individual variations in the cardiovascular risk biomarkers were observed in healthy overweight-obese individuals after consuming pomegranate supplement . HoweverCaenorhabditis elegans  was also achieved by mUB treatment [Of the various pomegranate phenolic metabolites exhibiting anti-Alzheimer activities in in vitro assays, only urolithins, including UA, UB, mUA, and methyl-urolithin B (mUB), were predicted to be capable of crossing the blood-brain barrier . Methylareatment . HoweverAdvanced protein glycation (non-enzymatic glycosylation) products have been implicated in the development of chronic diseases, such as diabetes and Alzheimer’s disease. The inhibitory effects of pomegranate fruit extract, punicalagin, EA, gallic acid (GA), UA, and UB toward the early, middle, and late stage of protein glycation were compared using in vitro assays . AlthougC. elegans, mammalian cells, and rodent models [C. elegans with UA led to a dosage-dependent extension of the worm’s lifespan. It was shown that UA could activate mitophagy (degradation of mitochondria by autophagy) and eliminate damaged mitochondria in C. elegans, mammalian cells derived from muscle or intestinal tissues, and muscles of young and old rodents. UA was also found to be bioavailable in the skeletal muscle and could improve muscle function in mice and rats [The mechanistic basis underlying UA’s anti-aging activities was explored recently using t models . Feedingand rats . The rolPomegranate fruits, leaves, flowers, and seeds have all been used in traditional herbal medicine for treating various illness. However, bioactive metabolites present in pomegranate fruit peel and juice have received considerable more attention than those found in other tissues. Bioactivities of the phytochemicals accumulating in tissues other than fruit should be investigated more rigorously in the future. In addition, detailed metabolite profiling and characterization of different pomegranate cultivars, grown under non-standard conditions , can be carried out to explore further the phytochemical diversity in pomegranate. Metabolic enzymes and pathways that confer the structural diversity of pomegranate phytochemicals also warrant further investigation. For example, the production of diverse HT monomers and polymers has been suggested to involve the activity of multiple laccase enzymes. However, the HT-forming laccases have not been cloned and characterized from pomegranate or any other plant species. Elucidation of the laccase family enzymes in pomegranate that bring about the great diversity of HT structures will have broad implications in dissecting the biological functions of HTs.In vitro assays and animal model studies have demonstrated various health benefits of urolithins, the ET derivatives. It is critical to obtain clinically relevant data on their efficacies prior to widespread applications in human disease interventions. Overall, exploration of the diversity and interactions of pomegranate phytochemicals, as well as preclinical and clinical investigations of their bioactivities, holds great promise of fully realizing the potential of this ancient fruit and modern functional food. Since a majority of the phytochemicals identified in pomegranate have also been found in other plants, examining the interactions and health-promoting functions of pomegranate phytochemicals will also have a far-reaching impact on exploiting the bioactive components of various edible medicinal plants and functional plant food.


GJB2, SLC26A4, and MT-RNR1, are much more prevalent than those of other genes worldwide. However, mutation spectra of common deafness genes differ remarkably across different populations. Here, we performed comprehensive genetic examination and haplotype analyses in 188 unrelated Mongolian families with idiopathic SNHI, and compared their mutation spectra and haplotypes to those of other European and Asian cohorts. We confirmed genetic diagnoses in 18 (9.6%) of the 188 families, including 13 with bi-allelic GJB2 mutations, three with bi-allelic SLC26A4 mutations, and two with homoplasmic MT-RNR1 m.1555A>G mutation. Moreover, mono-allelic mutations were identified in 17 families (9.0%), including 14 with mono-allelic GJB2 mutations and three with mono-allelic SLC26A4 mutations. Interestingly, three GJB2 mutations prevalent in other populations, including c.35delG in Caucasians, c.235delC in East Asians, and c.-23+1G>A in Southwest and South Asians, were simultaneously detected in Mongolian patients. Haplotype analyses further confirmed founder effects for each of the three mutations, indicating that each mutation derived from its ancestral origin independently. By demonstrating the unique spectra of deafness-associated mutations, our findings may have important clinical and scientific implications for refining the molecular diagnostics of SNHI in Mongolian patients, and for elucidating the genetic relationships among Eurasian populations.Genetic factors are an important cause of idiopathic sensorineural hearing impairment (SNHI). From the epidemiological perspective, mutations of three deafness genes:  It is estimated that permanent sensorineural hearing impairment (SNHI) occurs in approximately 1.9 per 1000 live births , 3. MoreGJB2 (MIM *121011), SLC26A4 (MIM *605646), and the mitochondrial 12S rRNA gene , are particularly prevalent in deaf patients across different populations [GJB2 mutations in Caucasians [GJB2 c.-23+1G>A mutation was identified uniquely in Southwest [SLC26A4 mutations differ among populations, including p.T416P and c.1001G>A in Caucasians [Among the plethora of HHI genes, mutations in three: 4 MIM *60646, and ucasians , Ashkenaucasians , and Eas2 MIM *1211, SLC26outhwest , 14 and outhwest , 16. Simucasians , 18, p.Hucasians  and Koreucasians , and c.9ucasians . These fGJB2 mutations, and the contribution of other deafness genes to SNHI in these patients was not addressed [The genetics of SNHI in the Mongolian population have been documented in several previous studies –26. HoweThe scientific value of investigating genetic diseases in Mongolian patients also lies in the geographic location of Mongolia. As an intersection between the European, Middle Eastern, and East Asian civilizations, dissecting the genetic underpinnings of Mongolians may offer insights into the genetic diversity and genetic relationships among the Eurasian populations –30. In tFrom November 2016 to January 2018, a total of 188 unrelated Mongolian families with idiopathic bilateral SNHI were recruited from the EMJJ Otolaryngology Hospital and the Department of Otolaryngology, National Center for Maternal and Child Health, Ulaanbaatar, Mongolia. Patients were excluded if they (1) were aged more than 40 years, (2) had conductive or mixed-type hearing impairment, (3) had previous noise or ototoxic medical exposure, (4) had a history of perinatal insults, such as prematurity or kernicterus, or (5) had no complete records of their medical history available.For the proband of each family, comprehensive family history, personal medical history, physical examination, audiological results, and imaging results were ascertained. The audiological results were evaluated with pure tone audiograms or auditory brainstem response, depending on age or neurological status . For imaGJB2, SLC26A4, and MT-RNR1 [GJB2. Real-time PCR was performed on two mutation hotspots of SLC26A4 (c.919-2A>G and p.H723R), and on the m.1555A>G mutation of MT-RNR1. Patients with enlarged vestibular aqueduct (EVA), a common inner ear malformation caused by recessive SLC26A4 mutations [SLC26A4. The NGS data were filtered and analyzed as previously described [http://deafnessvariationdatabase.org/) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) were used to identify known causative variants. All subjects and/or their parents provided informed consent before genetic testing, and all procedures were approved by the Research Ethics Committees of National Taiwan University Hospital, National Center for Maternal and Child Health of Mongolia, and the EMJJ Otolaryngology Hospital of Mongolia.Dried blood spot specimens were collected from the patients and their family members, and genomic DNA was extracted using a MagCore HF16 Automatic DNA/RNA Purification system  with a MagCore Genomic DNA Tissue Kit  according to the manufacturer's instructions , 36. We  MT-RNR1 , 38 in autations , 40, werescribed . The DeaGJB2, namely rs747931, rs3751385, rs11147592, rs9509086, and rs9552102, were selected and genotyped by Sanger sequencing  within or in the vicinity of quencing . Haplotyr cohort , 37, andr cohort .p values < 0.05 were interpreted as being statistically significant.Differences between groups were tested with Fisher’s exact test . Corresponding GJB2 alleles and three mutant SLC26A4 alleles were identified  was higher than that of SLC26A4 mutations  and that of the mitochondrial m.1555A>G mutation . More prevalent GJB2 mutations included c.-23+1G>A , c.235delC , and c.35delG ; whereas the most prevalent SLC26A4 mutation was c.919-2A>G (2.1%). The SLC26A4 c.2168A>G mutation, common in the East Asian populations [GJB2 c.235delC, none of these variants was identified in the 47 normal-hearing controls. Because of the limited number of the normal-hearing controls, there was no difference in the allele frequencies of these variants between the patient and control groups . However, as all these variants have been previously shown to be disease-causing in other populations, they were interpreted as causative pathogenic mutations in this study.In the 188 unrelated Mongolian families with SNHI, nine different mutant entified . The allGJB2 mutations, three with bi-allelic SLC26A4 mutations, and two with homoplasmic m.1555A>G mutations , including 13 with bi-allelic utations  & Fig 2. reports –46, a seGJB2 c.35delG mutation segregated exclusively with the A-A-C-G-T haplotype , and all eight chromosomes with the GJB2 c.235delC mutation segregated exclusively with the A-G-T-T-A haplotype . On the other hand, the GJB2 c.-23+1G>A mutation was associated with a major haplotype G-G-C-T-A (9/12 chromosomes) and a minor haplotype A-G-C-T-A (3/12 chromosomes), which also differed in haplotype distribution as compared to the control chromosomes . These results indicated founder effects for all the three prevalent GJB2 mutations in the Mongolian patients.To investigate whether prevalent mutations in the Mongolian patients derived from common origins, we performed haplotype analyses by genotyping five SNPs, in the patients and in 47 normal-hearing Mongolian controls . Our resGJB2 haplotypes, namely A-A-C-G-T, A-G-C-T-A, and A-G-T-T-A, were the same in the Mongolian and East Asian populations; whereas a common GJB2 haplotype in the European and South Asian populations, G-G-C-T-A, was relatively rare in the Mongolians and East Asians.Population-specific haplotype structures, generated from the data of the 1000 Genomes Project, revealed that the Mongolian population shares a closer genetic background with East Asians than with the Europeans and South Asians . The thrGJB2 c.235delC mutation in Mongolians originated from a common ancestor with other East Asians, whereas the GJB2 c.35delG originated from a common ancestor with the Europeans  patients carried bi-allelic SLC26A4 mutations. However, only 31 of the 135 patients were Mongolians, and the authors did not classify their genetic results by ethnicity [GJB2, SLC26A4, and MT-RNR1 in 189 deaf patients from northwest China, of whom only 19 were Mongolians. The authors did not identify any GJB2 mutations, and reported the SLC26A4 c.919-2A>G mutation as the most common deafness mutation in the Mongolian patients [GJB2, SLC26A4, MT-RNR1, and GJB3 in 738 deaf children recruited from the Inner Mongolia region of China, including 216 Mongolians. The authors also reported a higher prevalence of SLC26A4 mutations than that of GJB2 mutations [GJB2 mutations are more prevalent than SLC26A4 mutations in Mongolian patients with SNHI. This difference between our study and previous reports might result from the more thorough sequencing strategy we adopted, since the GJB2 c.-23+1G>A mutation, which is prevalent in Mongolians but rare in other East Asian populations, was not targeted in previous reports. It is also notable that the genetic structure of the Mongolian people who live in China could have been influenced by that of other races through inter-population marriage.Prior to the current study, several studies have investigated the genetics of SNHI in Mongolian patients . Most ofthnicity . Yang etpatients . Liu et utations . In contGJB2 mutations, three (1.6%) with bi-allelic SLC26A4 mutations, and two (1.1%) with homoplasmic m.1555A>G mutations, by screening the three common deafness genes. We additionally identified mono-allelic GJB2 or SLC26A4 mutations in 17 families (9.0%); however, given the recessive inheritance pattern of mutations in these two genes, the genetic results in these families were regarded as unconfirmed. Accordingly, the rate of confirmed results in our study is lower than that documented in other studies on European [GJB2 mutations in Mongolian patients from a deaf school in Ulaanbaatar [GJB2 mutations in 36 (16.6%) of their 216 Mongolian patients from the Inner Mongolia region of China [GJB2 mutations might be considerably lower than 16.6% [In the present study, definite genetic diagnoses could be achieved in 18 (9.6%) of the 188 families, comprising 13 (6.9%) with bi-allelic European , 54 and European , 55–57 pof China . Howeveran 16.6% .GJB2 deafness in many western populations [GJB2 mutations has been reported in several populations with lower socioeconomic status, such as patients in Sudan [The relatively low mutation rates of deafness genes in Mongolians have been ascribed to lower assortative mating rate and decreased genetic fitness of the deaf in Mongolia as compared to other populations . It has ulations . Indeed,in Sudan , Kenya [in Sudan , Indonesin Sudan , and Camin Sudan .http://www.grandviewresearch.com/industry-analysis/aminoglycoside-market). To prevent aminoglycoside-induced hearing loss, it might be reasonable to perform mutational screening of the MT-RNR1 gene before the initiation of antibiotic therapy, especially in regions where aminoglycosides are frequently used [Notably, two hearing-impaired patients in this study were identified to have the mitochondrial m.1555A>G mutation. Their medical history revealed that both patients were exposed to aminoglycosides. According to a recent report, the Asia-Pacific region is the largest market for the aminoglycoside industry, probably owing to a high incidence rate of tuberculosis  and 14 (7.4%) of the 188 Mongolian families, respectively. This finding is also consistent with Tekin et al., who reported mono-allelic GJB2 mutations in 5.4% of their Mongolian patients [GJB2 mutation might have occult mutations in the non-coding regions of GJB2, such as untranslated exon 1, intron 1, promoter, enhancer, or other regulatory elements, leading to compound heterozygosity, which has been observed previously for GJB2 mutations [GJB2 mutations and contribute to hearing impairment via digenic or polygenic inheritance [GJB2 mutations, hearing impairment is in fact caused by mutations in other deafness genes, and these “mono-allelic” patients are incidental carriers of certain GJB2 variants that are prevalent in the population [By comprehensively sequencing both patients . Patientutations , 65. Alteritance –68. The pulation .MYO15A mutations in a multiplex family. The employment of NGS-based genetic examination and cCMV screening may help improve the evaluation, diagnosis, and management of Mongolian patients with SNHI.The major strength of this study lies in the demonstration of the mutation spectra of all the three common HHI genes in a single large cohort of pure Mongolian ethnicity. However, some limitations of this study merit further discussion. Genetic mutations, with confirmed and unconfirmed results counted together, were only identified in 35 (18.6%) of the 188 families; for the other families, the etiology remained unclear. It is conceivable that in a certain portion of these families SNHI may be caused by mutations in other deafness genes, especially in families with multiple affected members. In addition, SNHI of acquired causes, such as perinatal insults or congenital cytomegalovirus (cCMV) infection, may be more prevalent in developing countries like Mongolia than in industrialized countries . RecentlIn conclusion, by performing comprehensive genetic examination and haplotype analyses in a large Mongolian cohort with SNHI, this study throws light on the genetic epidemiology of HHI in Mongolians. It also provides insights into the development of the unique mutation spectra observed in Mongolians. These findings may have clinical implications for the refinement of molecular diagnostics in Mongolian patients, as well as scientific implications for the delineation of genetic relationships among the Eurasian populations.S1 Table(DOCX)Click here for additional data file.