Background: p53 is a tumor suppressor protein involved in regulating a wide array of signaling pathways. The role of p53 in the cell is determined by the type of imposed oxidative stress, its intensity and duration. The last decade of research has unravelled a dual nature in the function of p53 in mediating the oxidative stress burden. However, this is dependent on the specific properties of the applied stress and thus requires further analysis.Methods: A systematic review was performed following an electronic search of Pubmed, Google Scholar, and ScienceDirect databases. Articles published in the English language between January 1, 1990 and March 1, 2017 were identified and isolated based on the analysis of p53 in skeletal muscle in both animal and cell culture models.Results: Literature was categorized according to the modality of imposed oxidative stress including exercise, diet modification, exogenous oxidizing agents, tissue manipulation, irradiation, and hypoxia. With low to moderate levels of oxidative stress, p53 is involved in activating pathways that increase time for cell repair, such as cell cycle arrest and autophagy, to enhance cell survival. However, with greater levels of stress intensity and duration, such as with irradiation, hypoxia, and oxidizing agents, the role of p53 switches to facilitate increased cellular stress levels by initiating DNA fragmentation to induce apoptosis, thereby preventing aberrant cell proliferation.Conclusion: Current evidence confirms that p53 acts as a threshold regulator of cellular homeostasis. Therefore, within each modality, the intensity and duration are parameters of the oxidative stressor that must be analyzed to determine the role p53 plays in regulating signaling pathways to maintain cellular health and function in skeletal muscle.Abbreviations: Acadl: acyl-CoA dehydrogenase, long chain; Acadm: acyl-CoA dehydrogenase, C-4 to C-12 straight chain; AIF: apoptosis-inducing factor; Akt: protein kinase B (PKB); AMPK: AMP-activated protein kinase; ATF-4: activating transcription factor 4; ATM: ATM serine/threonine kinase; Bax: BCL2 associated X, apoptosis regulator; Bcl-2: B cell Leukemia/Lymphoma 2 apoptosis regulator; Bhlhe40: basic helix-loop-helix family member e40; BH3: Borane; Bim: bcl-2 interacting mediator of cell death; Bok: Bcl-2 related ovarian killer; COX-IV: cytochrome c oxidase IV; cGMP: Cyclic guanosine monophosphate; c-myc: proto-oncogene protein; Cpt1b: carnitine palmitoyltransferase 1B; Dr5: death receptor 5; eNOS: endothelial nitric oxide synthase; ERK: extracellular regulated MAP kinase; Fas: Fas Cell surface death receptor; FDXR: Ferredoxin Reductase; FOXO3a: forkhead box O3; Gadd45a: growth arrest and DNA damage-inducible 45 alpha; GLS2: glutaminase 2; GLUT 1 and 4: glucose transporter 1 and 4 ; GSH: Glutathione; Hes1: hes family bHLH transcription factor 1; Hey1: hes related family bHLH transcription factor with YRPW motif 1; HIFI-α: hypoxia-inducible factor 1, α-subunit; HK2: Hexokinase 2; HSP70: Heat Shock Protein 70; H2O2: Hydrogen Peroxide; Id2: inhibitor of DNA-binding 2; IGF-1-BP3: Insulin-like growth factor binding protein 3; IL-1β: Interleukin 1 beta; iNOS: inducible nitric oxide synthase; IRS-1: Insulin receptor substrate 1; JNK: c-Jun N-terminal kinases; LY-83583: 6-anilino-5,8-quinolinedione; inhibitor of soluble guanylate cyclase and of cGMP production; Mdm 2/ 4: Mouse double minute 2 homolog (mouse) Mdm4 (humans); mtDNA: mitochondrial DNA; MURF1: Muscle RING-finger protein-1; MyoD: Myogenic differentiation 1; MyoG: myogenin; Nanog: Nanog homeobox; NF-kB: Nuclear factor-κB; NO: nitric oxide; NoxA: phorbol-12-myristate-13-acetate-induced protein 1 (Pmaip1); NRF-1: nuclear respiratory factor 1; Nrf2: Nuclear factor erythroid 2-related factor 2; P21: Cdkn1a cyclin-dependent kinase inhibitor 1A (P21); P38 MAPK: mitogen-activated protein kinases; p53R2: p53 inducible ribonucleotide reductase gene; P66Shc: src homology 2 domain-containing transforming protein C1; PERP: p53 apoptosis effector related to PMP-22; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PGM: phosphoglucomutase; PI3K: Phosphatidylinositol-4,5-bisphosphate 3-kinase; PKCβ: protein kinase c beta; PTEN: phosphatase and tensin homolog; PTIO: 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) has been used as a nitric oxide (NO) scavenger; Puma: The p53 upregulated modulator of apoptosis; PW1: paternally expressed 3 (Peg3); RNS: Reactive nitrogen species; SIRT1: sirtuin 1; SCO2: cytochrome c oxidase assembly protein; SOD2: superoxide dismutase 2; Tfam: transcription factor A mitochondrial; TIGAR: Trp53 induced glycolysis repulatory phosphatase; TNF-a: tumor necrosis factor a; TRAF2: TNF receptor associated factor 2; TRAIL: type II transmembrane protein. Skeletal muscle is a highly malleable tissue, capable of conforming to numerous metabolic and physiological requirements imposed by various stressors. This adaptable property of skeletal muscle is a trait that is essential for regulating cellular homeostasis in response to various stressor intensities and durations. As skeletal muscle comprises approximately 40% of the total body mass, maintaining muscle health and function is imperative to sustaining whole body health and protection against chronic disease . DysfuncOxidative stress is a result of the imbalance between the induction of reactive oxygen species (ROS) and the cells ability to metabolize them ,6. Many p53 is a well-known tumor suppressor protein and is commonly referred to as the ‘Guardian of the Genome’ for its role as a major determinant of cell fate. p53 regulates the expression of an assortment of genes involved in maintaining homeostasis, including those involved in cell cycle regulation, redox homeostasis (i.e. antioxidant enzyme production), DNA replication and repair, apoptosis, and autophagy . VariousWith this purpose, we set out to analyze various modalities of imposed oxidative stress and the subsequent signaling pathways activated in skeletal muscle, with a specific focus on the role of p53 in regulating these pathways. This review will describe the signaling mechanisms controlled by various stressors and how, under these influences, p53 ultimately functions to maintain muscle health and function, while additionally focusing on the pathways that lead to pathological consequences.A systematic review of the experimental evidence was performed on the role of p53 in regulating oxidative stress in skeletal muscle. Articles published in the English language were evaluated through Pubmed, ScienceDirect, and Google Scholar search engines using the key terms ‘p53’ and ‘Skeletal Muscle’ and ‘Oxidative Stress’, while restricting against ‘Cancer’ and ‘Tumor’. Primary research studies included for comparison involve only animal and cell culture models. Important studies involving human subjects published in this area are discussed where applicable, but not compiled in the data tables for analysis in order to keep the review focused. However, a list of excluded human studies can be found in the appendix. All materials published between January 1, 1990 and March 1, 2017 were included for review.Studies were initially screened based on title; duplicate articles and articles not publishing original research were excluded. Studies were then screened by a review of abstracts fitting the appropriate inclusion criteria . If abstStudies were organized based on the oxidative stress intervention and categorized into one of six groups: exercise, diet modification, tissue manipulation, chemical and medicinal agents, irradiation, and oxygen deprivation. A limited number of studies fulfilled more than one category. Data extraction was performed to assess study methodology, the effect of upstream regulators and downstream targets of p53-regulated signaling pathways affected by the oxidative stress, and the application and relevance of the model. Qualitative analyses were performed to compare within and between interventions on the effectiveness of the model based on the induction of oxidative stress, and the role of p53 and other signaling mechanisms in regulating stress intensity and duration. A meta-analysis was not performed on this data.Acute and chronic contractile activity triggers a plethora of signals that induce beneficial metabolic and biochemical adaptations to enhance muscle health and performance. For example, the moderate increases in ROS produced by exercise are able to repair and strengthen the oxidative capacity of the cell by increasing mitochondrial content and fuel oxidation, and thus has dissimilar effects compared to the consequences of chronic, high levels of ROS . In this15 phosphorylation of p53, allowing for both nuclear and mitochondrial localization [α leading to the expression of nuclear genes encoding mitochondrial proteins (NUGEMPs) such as Tfam, COX-IV, SCO2, and AIF [One bout of acute exercise is sufficient to initiate transcriptional signaling towards mitochondrial biogenesis, and thus ultimately improves the oxidative capacity of skeletal muscle with the assistance of p53 . Increaslization ,22,24. I and AIF ,17,22–26 and AIF ,28. p53  and AIF .2 max and skeletal muscle performance [In contrast to acute exercise, training is classified as repeated bouts of endurance exercise interspersed with recovery sessions over a period of time. The result of chronic exercise is a heightened adaptive state in which the signaling response to each exercise bout is attenuated, including reduced ROS production . This adformance ,26,32,33formance ,18,33. Tformance .15 phosphorylation and PGC-1α nuclear localization [The subcellular localization of p53 is indispensable for exercise-mediated mtDNA repair and mitochondrial biogenesis, notably through the initiation of the Tfam-Polg1 complex in the mitochondria, independent of the nuclear regulation of TIGAR, SCO2, p53R2, and FDXR expression ,32. An ilization . Indeed,lization . Howeverlization . TherefoThere is ample evidence confirming that the modification of nutritional resources can alter physiological equilibria leading to specific muscle adaptations. As such, diet modification is considered a cellular stressor through its disruption of homeostasis. p53 is an essential upstream regulator of metabolism whereby it specifically transcribes TIGAR, SCO2, HK2, GLS2, and GLUT 1 and 4 genes ,37. UpreCaloric restriction extends longevity by reducing metabolic risk factors including blood pressure, serum fasting glucose, and total cholesterol . Further1987 phosphorylation [18 phosphorylation of p53 leading to increased nuclear localization [α in the nucleus, it can control Lipin-1 expression, a regulator of fatty acid metabolism, to increase fatty acid oxidation (FAO) and triglyceride synthesis, as well as upregulate TIGAR to reduce the emphasis on glycolytic energy production [α-ketoglutarate to enhance mitochondrial respiration and ATP production [α to enhance Nrf2 and SOD2 antioxidant expression, concomitant with sestrin activation, to buffer the harmful effects of ROS/RNS to protect the cell during moderate to prolonged fasting states [Glucose withdrawal upregulates nitric oxide production and activates ATM kinase through Serrylation . This inlization ,49. Whenoduction ,49–52. pnges ROS . In addig states ,49. Furtg states . Therefoα and its downstream targets NRF-1 and Tfam, which are involved in vascular angiogenesis and mitochondrial biogenesis, respectively [α-target genes involved in FAO including Acadl, Acadm, and Cpt1b can lead to fat accumulation [On the other hand, the effect of chronic supplementation of metabolic fuels in excess to a normal diet enhances vascular oxidative stress leading to the development of pathological conditions including obesity, diabetes, and coronary artery disease ,56. Withectively . Reductimulation . p53 uprmulation . Interesmulation . Howevermulation ,59.Another dietary modification includes the addition of toxic substances into the diet, such as alcohol in excessive quantities, which can lead to reduced contractile proteins  in the muscle itself causing alcoholic myopathy, defined by cramps, impaired muscle strength, difficulties in gait, and reduced lean tissue mass . InteresIn contrast to the effect of healthy physical activity on skeletal muscle, this tissue is also subject to reduced activity levels brought about by trauma, denervation, or immobilization. With the knowledge that p53 plays a role in regulating overall cellular homeostasis, the intensity of the various physical manipulations of this kind may lead to alternate functions required of p53 to mediate or attenuate this stress. A total of five studies have been selected to assess the potential variations in p53-mediated regulation of muscle adaptations under these conditions .Table 4α and IL-1β expression, while concomitantly increasing insulin resistance and apoptosis respectively, to induce muscle wasting [The most common manipulations of muscle include crush injury, tissue autografting, disease, and burn injury which can lead to direct skeletal muscle atrophy, or indirect atrophy through immobilization . Tissue  wasting ,74. Anot wasting . Additio wasting . Interes wasting ,74.α-mediated inhibition of myogenin and MyoD, and activation of p53-dependent cell death proteins, leads to the inhibition of myogenic differentiation via upregulation of atrogin-1 to enhance apoptosis and atrophy [Additional roles for p53 can be observed in disease settings that induce pathological muscle degeneration, such as seen with diabetes and cancer cachexia. A precursor to many diseases, diabetes leads to cellular stress by reducing proteasomal degradation of p53 through the inhibition of the negative Mdm2/Akt upstream regulatory pathway, leading to an abnormal accumulation of p53 and the induction of apoptosis . Interes atrophy ,77,78. T atrophy ,79.Cellular respiration, an essential metabolic process for energy generation, requires oxygen as the terminal electron acceptor in oxidative phosphorylation to produce ATP energy. Low oxygen tension denotes a hypoxic microenvironment which may be induced by mechanisms such as ischemia or high-altitude exposure, causing detrimental physiological consequences . p53, a α and glycolysis), and downregulated 224 genes involved in protein catabolism and muscle organ development [Hypoxia, defined as ∼1% of physiological oxygen volume in skeletal muscle following an ischemic insult, represses myogenesis through numerous transcriptome changes ,83. One elopment . Of the elopment ,84.37, Ser46, and Ser392 phosphorylation and decreased Lys373 acetylation occur at 5-h of anoxic exposure (complete oxygen deprivation), with additional phosphorylation at Ser6, Ser9, Ser15, and Ser20 at 20 h [It is well known that hypoxia inhibits myogenin and myogenic differentiation ,85. Rece at 20 h . These p at 20 h . Further at 20 h . Under h at 20 h . This re at 20 h . Repress at 20 h ,88,89. TIonizing radiation energy is an extreme stress signal which, depending on the length of exposure, can lead to cell death via two mechanisms. The first is through p53-dependent and -independent initiation of apoptosis which serves as a protective mechanism to eliminate heavily damaged or mutated cells . The secα-driven apoptotic augmentation, leading to muscle atrophy [The role of p53 on skeletal muscle differentiation following DNA damage is a field of interest because of its implications for the regenerative capacity of muscle. Previous work has revealed that p53 maintains the genetic stability of mouse embryonic stem (MES) cells following DNA damage by repressing Nanog, a cancer-stem cell marker, to then regulate and promote differentiation ,93. Regu atrophy ,92,94. TCoordinated commitment to cell division is clearly monitored by p53-mediated intrinsic and extrinsic stress signals, such as DNA damage, hypoxia, low levels of ribosomal biogenesis, or oncogene activation, which introduces infidelity to cellular division through the inhibition of specific signals. Repression of such signals including the IGF-1-Akt and mTOR pathways allows for the coordinated activation of autophagy and apoptosis in a tissue-specific manner . UltraviTo further understand the activation of p53 and its downstream signaling pathways, various naturally occurring and synthetically manufactured drugs have been examined. These chemical agents, depending on whether they are direct or indirect oxidizing agents, trigger specific mechanisms to combat oxidative stress. Therefore, to determine how these oxidizing agents lead to variable signaling mechanisms, particularly stress signals activated by p53, a total of 11 studies (5 direct oxidizing agents and 6 indirect oxidizing agents) were analyzed .Table 72O2, increase p53 expression which can activate kinases PKCβ and JNK to phosphorylate p66Shc on its Ser36 residue, allowing for its translocation to the mitochondria [2O2 generation and the release of cytochrome c[2O2, also inhibits the ERK signaling pathway, leading to inappropriate actin cytoskeleton polymerization and irregular glucose transport into skeletal muscle [2O2 include the JNK-mediated Thr447 and Thr452 phosphorylation of FOXO3a and FOXO4, respectively, which can then upregulate pro-apoptotic proteins Fas, TRAIL, and Bim [2O2 oxidative stress additionally leads to the transcription of pro-apoptotic proteins, including Bax, BH3-only family members, and PERP [33 phosphorylation of p53 [α-B-crystallin, E2 (17β-estradiol), and testosterone can protect against the H2O2-mediated p53 induction of apoptosis by enhancing the de-acetylation of p53 by SIRT1 or by binding certain pro-apoptotic family members [Direct oxidizing agents crucially impact metabolism and cellular homeostasis by acting as the final electron acceptor for many reactions. Numerous pharmacologic agents have been naturally discovered or synthetically constructed that fit the description of a direct oxidizing agent and consequentially play an important role in inducing or mediating oxidative stress through the activation of p53. Harmful oxidizing agents, such as Hchondria . Once inochrome c,100. p66l muscle ,102,103.l muscle ,104. Spe and Bim . Phosphoand PERP ,108. Antand PERP . This acand PERP ,109. Furand PERP ,109. Snan of p53 ,111. Thin of p53 ,110,111. members ,112. p53 members ,112. ThiIndirect oxidizing agents function through additional and alternate pathways to ultimately lead to similar results as direct oxidizing agents. Synthetic agents, in particular, have been created to mediate ROS production and regulate p53 signaling pathways depending on the pathology. For example, in diabetic hyperglycemia, p53 accumulates and activates downstream genes involved in myocyte death ,114. Des124 S-nitrosylation of p53 due to NO flux, allowing it to localize to the nucleus [α promoter to enhance its transcription [α then act synergistically to co-activate the Nrf2-mediated antioxidant response to buffer harmful ROS/RNS [α–NF-kB pathway under chemically induced oxidative stress [α, p53-mediated regulation of damage associated molecular patterns (DAMPs), or increased binding between p53 and MURF1 to trigger ubiquitination [α)pyrene and C60 fullerene pollutants act to initially increase antioxidant defences by increasing glutathione levels [Specific indirect oxidizing agents, such as PTIO, LY-83583, and buthionine sulfoximine, induce oxidative stress through the upregulation of NO/iNOS/nNOS and/or GSH depletion pathways ,73,116.  nucleus ,73,116. cription ,116,117. ROS/RNS ,117. Howe stress ,118. Thie stress ,78,119. tination ,73. Thestination . For exae levels ,122. Howe levels ,122. TheOxidative stress in skeletal muscle is a result of impaired redox homeostasis due to either the overproduction of ROS, or a compromised antioxidant defence system. ROS overproduction, caused by either endogenous or exogenous sources, can lead to damaged proteins, lipids, and DNA, inevitably causing pathologies such as metabolic (type II diabetes and cancer), neuromuscular (dystrophies), neural (Alzheimer’s disease) or cardiovascular diseases, as well as promoting muscle aging ,124. EndDepending on the type, duration, and intensity of the imposed stress, various signaling pathways are activated within specific organelles, such as the mitochondria or nucleus, to mitigate the cellular damage caused by these non-physiological levels of ROS. Specifically, p53 is deemed to be of key importance in regulating these cellular stress responses. The studies outlined in this review confirm a dual ability for p53 activation of specific signaling mechanisms, dependent on the intensity and length of the oxidative stress. With low levels of oxidative stress induced by dietary restriction, non-ionizing radiation, hypoxia, and oxidizing agents, there is an upregulation of p53-mediated activation of the antioxidant enzymes Nrf2, SOD2, sestrins, and catalase. Furthermore, cell cycle arrest and cellular senescent proteins p21 and Gadd45a are upregulated, concomitant with increased autophagy to maintain genomic integrity as well as a healthy organelle pool, respectively. Interestingly, metabolic modifications additionally occur exemplified by increased TIGAR, PGM, and GLS2 expression which reduce glycolysis and pyruvate levels, while increasing Lipin-1 to upregulate FAO. Surprisingly, with exercise, p53 enhances cellular function by increasing mitochondrial biogenesis and maintaining mtDNA utility.α, IL-1β, Bhlhe4, and Notch. Immobilization of muscle on the other hand, a mechanism solely dependent on duration, is characterized by increased ATF4, p21, and atrogin-1 expression to cause tissue atrophy. With even greater levels of stress induced by radiation and chronic exposure to oxidizing agents, there is an increase in numerous signaling pathways including TNF-α-PWI-caspase-Bax, PTEN, FOXOs, p66Shc, MURF1, and IGF-BP3, which initiate the accumulation of harmful free radical species leading to cell death. In these cases, p53 acts as a pro-oxidant to further exacerbate the stress load. This culminates in the inhibition of preventative oxidative signaling pathways and enhances the caspase cascade to initiate apoptosis. Interestingly, this duality function of p53 is observed with specific oxidative stress sources such as radiation, nutrient regulation, hypoxia, and oxidizing/non-oxidizing agents, but not with exercise and tissue manipulation.Yet with greater and prolonged levels of stress, signaling pathways that exacerbate oxidative dysfunction, such as those involved in inflammatory cascades and cell death, are intensified. As observed with chronic nutrient abundance, anoxia/hypoxia, and burn injuries, there is increased expression of inflammatory proteins and cytokines including c-myc, TNF-This review outlines for the first time the various modalities of imposed oxidative stress in skeletal muscle that analyze the regulation of p53 and its numerous downstream signaling pathways involved in maintaining cellular homeostasis . Clearly


N-phenmethyl-N,N-dimethyl chitosan (PDCS), double quaternized chitosan N-(1-pyridylmethyl-2-ylmethyl)-N,N-dimethyl chitosan (MP2MDCS), N-(1-pyridylmethyl-3-ylmethyl)-N,N-dimethyl chitosan (MP3MDCS), and N-(1-pyridylmethyl-4-ylmethyl)-N,N-dimethyl chitosan (MP4MDCS) were designed and synthesized successfully through chemical modification of chitosan. Besides, three kinds of antioxidant activities, including hydroxyl radicals, superoxide radicals, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals were tested in vitro. As shown in this paper, the scavenging ability was ranking in order of MP3MDC > MP4MDCS > MP2MDCS > PDCS > chitosan at 1.6 mg/mL in all assays. All double quaternary ammonium salts were better than chitosan or the single quaternary ammonium salt. In addition, MP3MDCS could scavenge hydroxyl radicals totally at 1.6 mg/mL. MP2MDCS and MP4MDCS with more than 90% scavenging indices both had great scavenging ability on hydroxyl radicals or DPPH radicals. Furthermore, these data demonstrated that the increasing number of the positive charge would improve the antioxidant property of chitosan derivatives, and the N-pyridinium position would influence the scavenging radical ability.With the specialty of improving the water solubility of chitosan, quaternary ammonium salts have broadened the application of this polysaccharide in food, medicine and pesticides. To identify the effect of quaternary ammonium salts’ quantity, single quaternized chitosan  Free radicals, especially oxygen free radicals in one’s body, may damage the chemical structure of the organized cell and cause symptoms, such as ruptures of the main chain of the nucleic acid and protein peptide bond, membrane lipid peroxidation, enzyme inactivation, and cell apoptosis in certain pathological conditions ,2,3,4,5.N-acetylglucosamine, is usually obtained from the exoskeletons of the shellfish and the insects. As a natural renewable resource, chitosan has advantages in unique physicochemical characteristics and bioactivities expressed as its antifungal, antioxidant and antitumor properties  × 100, where Acontrol 560nm is the absorbance of the control (distilled water instead of NADH for each concentration) and Ablank 560nm is the absorbance of the blank (distilled water instead of the samples).The superoxide radical ability was assessed by the method of Nishikimi et al. . Superoxsample 517nm − Acontrol 517nm)/Ablank 517nm] × 100, where Acontrol 517nm is the absorbance of the control (ethanol instead of DPPH for each concentration) and Ablank 517nm is the absorbance of the blank (distilled water instead of the samples).According to HU , the DPPN-pyridylmethyl chitosan, a series of derivatives of chitosan with single or double quaternary ammonium salts were synthesized successfully. In addition, antioxidant activities of chitosan and quaternized chitosan derivatives against hydroxyl radicals, DPPH radicals, and superoxide radicals were tested in vitro. It was found that all quaternized chitosan derivatives had good water solubility and stronger antioxidant ability compared with chitosan, especially double quaternized chitosan derivatives that might be further developed into more effective antioxidant biomaterials. These data demonstrated that the higher positive charge density of quaternized chitosan derivatives might contribute to antioxidant activities. Furthermore, MP3MDCS was more effective than MP2MDCS and MP4MDCS in all assays especially at 1.6 mg/mL. It was reasonable to presume that the N-pyridinium position of double quaternized chitosan derivatives could influence the antioxidant property. Besides, it was reported that pyridinium derivatives were showed to be non-toxic for genen delivery in vitro among the quaternary ammonium chitosans [Via hitosans , so our


In this study, a library of biologically active small molecular weight calpain inhibitors was used for model validation and inhibition site recognition. Subsequently, two natural neuroactive polyphenols, curcumin and quercetin, were tested for their sensitivity and activity towards calpain’s proteolytic sequence and compared with the known calpain inhibitors via detailed molecular mechanics (MM), molecular dynamics (MD), and docking simulations. The MM and MD energy profiles  and the docking analysis  demonstrated that polyphenols conferred comparable calpain inhibition profiling. The modeling paradigm used in this study provides the first detailed account of corroboration of enzyme inhibition efficacy of calpain inhibitors and the respective calpain–calpain inhibitor molecular complexes’ energetic landscape and in addition stimulates the polyphenol bioactive paradigm for post-SCI intervention with implications reaching to experimental  In broader terms, the secondary injury mediating tissue damage and destruction prevails over a primary injury’s cellular and molecular pathogenic events, eventually leading to neurological dysfunction and functional paralysis -4-methyl-N-[(2S)-1-oxohexan-2-yl]pentanamide] and calpain Inhibitor II (ALLM) [(2R)-2-[(2S)-2-acetamido-4-methylpentanamido]-4-methyl-N-[(2S)-4-(methylsulfanyl)-1-oxobutan-2-yl] pentanamide] have been reported to prevent proteolysis and neuronal apoptosis when administered intravenously after spinal cord injury  and ALLN   than calpain–ALLN  with both component kinetic and potential energies contributing to final geometrical and energetic equilibrium   , Table 1R-2- carbamoyl}-3-methylbutyl]carbamate) and AK295 (benzyl N-[(1S)-3-methyl-1-{[(1S)-1-[(morpholin-4-ylmethyl)carbamoyl]propyl]carbamoyl}butyl]carbamate) are potent, highly specific, and permeable neuroentities differing in their aqueous solubility characteristics, with AK295 being more soluble than AK275, and are known to inhibit calpain-induced degeneration in vivo as well as inhibition of in vitro proteolysis of cytoskeletal proteins  as compared to AK275 [calpain I (Ki = 0.25 μM) and calpain II (Ki = 0.21 μM)]  , Table 1ilibrium .binding = −3.05 kcal/mol) demonstrated better inhibition of calpain, with a Ki value of 5.81 mM as compared to AK275 , which showed a Ki value of 6.48 mM. All the intermolecular energy components—van der Waals + H-bond + desolvation + electrostatic—were more stabilized in the case of the calpain–AK295 complex, and the larger interaction surface produced a better geometrical fit. The binding pocket of calpain–AK275 was formed by GLU72, LYS79, GLN109, CYS115, THR210, SER206, SER251, ILE254, ARG258, ALA262, VAL269, ARG270, GLY271, HIS272, ALA273, TRP298, 347LYS, and 349GLU, with hydrogen bonding interactions , polar bonds , and hydrophobic involvements  contributing to the binding energy -3-methyl-1-{[(2R)-1-oxohexan-2-yl]carbamoyl}butyl]carbamate) and leupeptin ((2S)-N-[-1-{[(2R)-5-Carbamimidamido-1-hydroxypentan-2-yl]amino}-1-hydroxy-4-methylpentan-2-yl]-2-acetamido -4-methylpentanamide)—differ in terms of chemical structure, permeability, charge, and selectivity, with calpeptin containing benzyl functionality and being cell permeable, less aqueous soluble, and higher in selectivity, while leupeptin is positively charged due to the presence of guanidinium group, water soluble, poorly cell permeable, and less selective towards calpain inhibition. Calpeptin and leupeptin are among the first calpain inhibitors to be employed to demonstrate “the involvement of calpain in apoptotic death of rat glial and neuronal cells”  and leupeptin   than calpain–leupeptin , with both component kinetic and potential energies contributing to final geometrical and energetic equilibrium ] , Table 1S-3-methyilibrium .binding = −4.88 kcal/mol) demonstrated better inhibition of calpain with a Ki value of 263.03 μM as compared to leupeptin , which showed a Ki value of 842.98 μM. The major intermolecular energy components—van der Waals + H-bond + desolvation—were more stabilized in the case of the calpain–calpeptin complex. However, the calpain–leupeptin complex demonstrated higher interaction surface, which may be due to a larger molecular size and hence may not be able to provide a preferable geometrical fit. The binding pocket of calpain–calpeptin was lined by GLU72, GLN109, CYS115, TRP116, THR210, SER251, ASN253, ILE254, ARG258, ALA262, VAL269, GLY271, HIS272, ALA273, TRP298, and GLU349, with hydrogen bonding interactions (GLY271 and CYS115), polar bonds , hydrophobic involvements , and cation–π interaction (TRP298) contributing to the binding energy  conferred better interaction and complexation with calpain than PD151746 , which supported the reported experimental inhibition capability of PD150606 [calpain I (Ki = 0.21 μM) and calpain II (Ki = 0.37 μM)] as compared to PD151746 [calpain I (Ki = 0.26 μM) and calpain II (Ki = 5.3 μM)]  than calpain–PD151746  with both component kinetic and potential energies contributing to final geometrical and energetic equilibrium (PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) and PD151746 (sulfanide) represent the most potent α-mercapto acrylic acids, characterized by higher cell permeability, small molecular weight, and the presence of a halogen such as fluorine or iodine ,23,24. Eilibrium .binding = −3.97 kcal/mol) demonstrated better inhibition of calpain, with a Ki value of 1.23 mM as compared to PD151746 , which showed a Ki value of 2.53 mM. All intermolecular energy components—van der Waals + H-bond + desolvation + electrostatic—were more stabilized in the case of the calpain–PD150606 complex, which also had a larger interaction surface, producing a better geometrical fit. The binding pocket of calpain–PD150606 was lined by GLN109, GLY113, CYS115, TRP116, SER251, ILE254, ALA262, VAL269, ARG270, GLY271, HIS272, TRP298, LYS347, and 349GLU, with hydrogen bonding interactions (SER251 and TRP298), halogen bonds , polar bonds , hydrophobic involvements , and π–π bonds (HIS272 and TRP298) contributing to the binding energy -2-methyl-1-{[(2S)-1-oxo-3-phenylpropan-2-yl]carbamoyl}propyl]amino 2-phenylacetate) and SJA6017 ((2S)-2-[(4-fluorobenzene)sulfonamido]-3-methyl-N-[(2R)-4-methyl-1-oxopentan-2-yl]butanamide) have been reported to have calpain inhibition capability. Yu and Geddes, 2007, concluded that the combined intravenous and daily intraperitoneal administration of MDL-28170 resulted in “significant improvement in both functional and pathological outcome measures” in a rat SCI model  and SJA6017  . The electrostatic component—the major contributor to the calpain–calpain inhibitor geometrical stabilization—of the calpain–MDL-28170  was significantly more stabilized than that of calpain–SJA6017 . The 3D-mapped isosurface plots of calpain–MDL-28170  and calpain–SJA6017  depicted major electrostatic surface with very strong positive and negative potentials, respectively  demonstrated better inhibition of calpain, with a Ki value of 204.69 μM as compared to SJA6017 , which showed a Ki value of 254.11 μM. All the intermolecular energy components—van der Waals + H-bond + desolvation + electrostatic—was more stabilized in the case of the calpain–MDL-28170 complex, which also had a larger interaction surface, producing a better geometrical fit. The binding pocket of calpain–MDL-28170 was lined by GLU72, GLN109, CYS115, TRP116, SER206, GLY208, SER251, ASN253, ILE254, ARG258, ALA262, VAL269, GLY271, HIS272, ALA273, and TRP298, with hydrogen bonding interactions , polar bonds , hydrophobic involvements , and π–π interactions (HIS272 and TRP298) contributing to the binding energy  produced a geometrically and energetically stable complex with calpain -7-(3-methoxy-4-oxidophenyl)-5-oxido-3-oxohepta-1,4,6-trien-1-yl]benzen-1-olate)), a polyphenolic phytochemical extracted from the rhizome of apeutics . The pre calpain . The cal calpain .binding = −4.11 kcal/mol) demonstrated inhibition of calpain with a Ki value of 978.21 μM. The intermolecular energy component was stabilized by van der Waals forces, H-bonding, and desolvation energy. The docking studies established two interesting results: (1) the electrostatic energy destabilized the total intermolecular energy and was +ve in magnitude  as compared to –ve magnitude in case of the reference compounds tested, and (2) the interaction surface was narrower than all the reference compounds tested in this study. The binding pocket of calpain–curcumin was lined by GLU72, LYS79, GLN109, CYS115, TRP116, SER206, ASN253, ILE254, ARG258, GLU261, ALA262, VAL269, HIS272, and TRP298, with hydrogen bonding interactions (GLN109 and CYS115), polar bonds , hydrophobic involvements , π-–π interaction (TRP298), and cation–π interaction (HIS272) contributing to the binding energy  produced a geometrically and energetically stable complex with calpain -3,4-dioxo-3,4-dihydro-2H-1-benzopyran-5,7-bis(olate)), a naturally occurring polyphenolic flavonoid, has shown immense potential in SCI therapeutics as an antioxidant and anti-inflammatory agent. This modeling study was inspired by the fact that quercetin consists of a very active chromenone functionality as well as five –OH groups capable of forming H-bonds with biomacromolecules such as calpain . The abo calpain . The cal calpain .binding = −4.69 kcal/mol) demonstrated inhibition of calpain with a Ki value of 365.57 μM ≈ three times more potent than curcumin. The intermolecular energy component was stabilized by van der Waals forces, H-bonding, and desolvation energy, as well as the electrostatic energy. The docking studies established two interesting results: (1) the electrostatic energy destabilized the total intermolecular energy and was +ve in magnitude  as compared to –ve magnitude in the case of the reference compounds tested, and (2) the interaction surface was narrower than all the reference compounds tested in this study. In the case of calpain–quercetin, the ligand–protein binding included GLU72, GLN109, CYS115, TRP116, SER206, THR210, SER251, ASN253, ILE254, ARG258, GLU261, ALA262, VAL269, HIS272, ALA273, TRP298, LYS347, and GLU349, with interaction arising from hydrogen bonding , polar bonds , hydrophobic deliberations , π–π interactions (HIS272 and TRP298) and cation–π interactions   . The hydin silico simulation can replicate the in vitro/in vivo inhibition profile of various calpain inhibitors against calpain and (2) ascertain if polyphenols such as curcumin and quercetin can act as calpain inhibitors with efficacy comparable to that of 10 well-known calpain inhibitors. The MM and MD energy stabilization profile of the tested calpain inhibitors can be plotted as follows: SJA6017 < AK275 < AK295 < PD151746 < quercetin < leupeptin < PD150606 < curcumin < ALLN < ALLM < MDL-28170 < calpeptin (The present modeling simulation study was planned to (1) provide a molecular modeling algorithm, thereby testing if alpeptin . The aboalpeptin . The mutin silico results revealed a strong correlation with previously reported in vitro results, the estimated inhibition constants of the compounds examined in this study displayed an important deviation from the literature [in vitro and in vivo calpain inhibition potential of curcumin and quercetin alone and in combination.Spinal Cord Injury (SCI) has been a challenge to treat for several decades, even more so today with the host of experimental treatments available that have many inherent risks for patients. The ideal combination of drugs to be used is not optimized and many clinicians have failed to achieve a therapeutic response due to ignorance of the multiple confounding formulation factors and side effects when dosing a particular steroid. Apart from steroidal interventions, small molecule calpain inhibitors have been employed alone as well as in combination with steroids. However, the non-specificity and low potency of known calpain inhibitors jeopardize their administration after SCI. Polyphenols, if administered in the correct dose, may offer multifaceted therapeutic benefits countering inflammation, free radical-mediated oxidation, and neurodegeneration. In addition, polyphenols such as curcumin and quercetin may offer distinctive advantages via the inhibition of calpain, as described in this study. Individually, these compounds have shown promise but have an unfavorable toxicity profile due to the high doses required and hence there is significant debate over the use of natural compounds such as curcumin and quercetin as alternatives to conventional high-dose steroidal drug therapy. However, a synergistic combination of lower doses of each may prove to be the best option. This paper adds value by hypothetically exploring the magnitude of interactions that exist between polyphenols and calpain. The varied binding pocket of curcumin and quercetin with calpain in addition to wide-ranging energetic modification of calpain’s proteolytic site further confirmed the synergistic potential of curcumin and quercetin. Although the terature ,29. Thesterature . TherefoTM 8.0.8 Molecular Modeling software  and ChemBio3D Ultra 11.0 . The calpain 20-mer peptide (PQFKIRLEEVDDADDYDSRE) was constructed using the Sequence Builder Module on HyperChemTM 8.0.8. The structures of the ligands calpain inhibitor I (ALLN), calpain inhibitor II (ALLM), AK275, AK295, calpeptin, leupeptin, PD150606, PD151746, MDL-28170, SJA6017, curcumin, and quercetin were built with pre-defined natural bond angles and charges , θV denotes the bond angle contributions , φV represents the torsional contribution arising from deviations from optimum dihedral angles, ijV incorporates van der Waals interactions due to non-bonded interatomic distances, hbV symbolizes the hydrogen-bond energy function, and elV stands for electrostatic energy.Molecular mechanics energy relationship (MMER), a method for analytico-mathematical representation of potential energy surfaces, was used to provide information about the contributions of valence terms, noncovalent Coulombic terms, and noncovalent van der Waals interactions for ligand–peptide interactions. The MMER model for the potential energy factor in various molecular complexes can be written as:Etotal, was calculated as the difference between the total potential energy of the complex system and the sum of the potential energies of isolated individual molecules, as follows:In addition, the total potential energy deviation, ΔThe molecular stability can then be estimated by comparing the total potential energies of the isolated and complexed systems. If the total potential energy of the complex is smaller than the sum of the potential energies of isolated individual molecules in the same conformation, the complexed form is more stable and its formation is favored .The ligand–peptide complexes initially minimized by molecular mechanics were then minimized by molecular dynamics for 1.0 ps (time step = 0.001 ps) at 300 K with the Nose−Hoover thermostat. For evaluation of the stability of a simulation and the extent of equilibration and for identification of the interesting low energy conformations, molecular dynamics calculations were averaged and saved as kinetic energy (EKIN), potential energy (EPOT), total energy (ETOT), and temperature (TEMP) Equilibrium was established before recording the measurements, and the instantaneous potential and kinetic energy were monitored to determine when the system reaches equilibrium. Thereafter, the simulation was allowed to run for 1000 time-steps before taking measurements .Docking computations were performed using DockingServer. The MMFF94 force field was used for energy minimization of the ligand molecule. Gasteiger partial charges were added to the ligand atoms and nonpolar H-atoms were merged, with rotatable bonds defined. The docking computations were performed using the inhibitor protein model. Essential H-atoms, Kollman united-atom type charges, and solvation parameters were added with the aid of AutoDock tools. Affinity (grid) maps of 20 × 20 × 20 °A grid points and 0.375 °A spacing were generated using the Autogrid program. AutoDock parameter set- and distance-dependent dielectric functions were used for computation of the van der Waals and electrostatic terms, respectively. The docking simulations were performed using a Lamarckian Genetic Algorithm as well as the Solis and Wets local search method. The initial position, orientation, and torsions of the ligand molecules were set randomly. All rotatable torsions were released during docking and each docking experiment was derived from 10 different runs that were set to terminate after a maximum of 25,000 energy evaluations. The population size was set to a value of 150. During the search, a translational step of 0.2 °A and quaternion as well as torsion steps of to the value of 5 were applied ,35,36.in silico evidence across pharmaceutical and medicinal interventions for the potential inhibition of calpain-induced apoptosis. The library of biologically active small molecules such as calpain inhibitor I (ALLN), calpain inhibitor II (ALLM), AK275, AK295, calpeptin, leupeptin, PD150606, PD151746, MDL-28170, and SJA6017 successfully validated the modeling algorithm. The MM, MD, and protein–ligand docking studies effectively quantified the molecular attributes of the protein–ligand(s) interactions in the terms of various pertinent energy attributes and generated preliminary data for protein–ligand sensitivity analysis and interaction studies. The MM and MD energy stabilization profiles displayed the following interaction profiling: SJA6017 < AK275 < AK295 < PD151746 < quercetin < leupeptin < PD150606 < curcumin < ALLN < ALLM < MDL-28170 < calpeptin. The docking analysis demonstrated a calpain inhibition profile in the following order: AK275 < AK295 < PD151746 < ALLN < PD150606 < curcumin < leupeptin < quercetin < calpeptin < SJA6017 < MDL-28170 < ALLM, wherein curcumin interacted with GLU72, LYS79, GLN109, CYS115, TRP116, SER206, ASN253, ILE254, ARG258, GLU261, ALA262, VAL269, HIS272, and TRP298, whereas quercetin exhibited a binding pocket formed by GLU72, GLN109, CYS115, TRP116, SER206, THR210, SER251, ASN253, ILE254, ARG258, GLU261, ALA262, VAL269, HIS272, ALA273, TRP298, LYS347, and GLU349. The modeling paradigm used in this study provided the first ever detailed account of the enzyme inhibition efficacy of calpain inhibitors and the respective calpain–calpain inhibitor molecular complexes’ energetic landscape along with a detailed polyphenol–calpain interaction profile with implications reaching to in vivo studies. The whole study can be concluded under the following points:Calpain inhibitors may significantly improve biochemical, functional, and behavioral outcomes after SCI by reducing the calpain-mediated proteolysis of cytoskeletal and neurofilament proteins.The unique symmetrical chemical structure of curcumin, with two aromatic rings on either side of a diketone, conferred the best van der Waals interaction profile among the calpain inhibitors tested, with an energy stabilization of ≈−32 kcal/mol .The presence of hydroxyl groups on the entire chemical structure accounts for the calpain–quercetin complexation having the best H-bonding profile among the calpain inhibitors tested.The experimental inhibitory potential of calpain inhibitors is directly related to the molecular modeling energy relationship (MMER) developed in this study—a first in the field of computational elucidation of inhibitory potential of enzyme inhibitors.The varied binding pocket of curcumin and quercetin with calpain, in addition to the wide-ranging energetic modification of calpain’s proteolytic site, confirmed the synergistic potential of curcumin and quercetin.This study provided the foremost comprehensive


MTP family in tobacco remain unclear. In the present study, 26, 13, and 12 MTPs in three main Nicotiana species  were identified and designated, respectively. The phylogenetic relationships, gene structures, chromosome distributions, conserved motifs, and domains of NtMTPs were systematic analyzed. According to the phylogenetic features, 26 NtMTPs were classified into three major substrate-specific groups that were Zn-cation diffusion facilitators (CDFs), Zn/Fe-CDFs, and Mn-CDFs, and seven primary groups . All of the NtMTPs contained a modified signature sequence and the cation_efflux domain, whereas some of them also harbored the ZT_dimer. Evolutionary analysis showed that NtMTP family of N. tabacum originated from its parental genome of N. sylvestris and N. tomentosiformis, and further underwent gene loss and expanded via one segmental duplication event. Moreover, the prediction of cis-acting elements (CREs) and the microRNA target sites of NtMTP genes suggested the diverse and complex regulatory mechanisms that control NtMTPs gene expression. Expression profile analysis derived from transcriptome data and quantitative real-time reverse transcription-PCR (qRT-PCR) analysis showed that the tissue expression patterns of NtMTPs in the same group were similar but varied among groups. Besides, under heavy metal toxicity, NtMTP genes exhibited various responses in either tobacco leaves or roots. 19 and 15 NtMTPs were found to response to at least one metal ion treatment in leaves and roots, respectively. In addition, NtMTP8.1, NtMTP8.4, and NtMTP11.1 exhibited Mn transport abilities in yeast cells. These results provided a perspective on the evolution of MTP genes in tobacco and were helpful for further functional characterization of NtMTP genes.Plant metal tolerance proteins (MTPs) comprise a family of membrane divalent cation transporters that play essential roles in plant mineral nutrition maintenance and heavy metal stresses resistance. However, the evolutionary relationships and biological functions of  Metal ions such as Zinc (Zn), Cobalt (Co), Iron (Fe), Manganese (Mn), and Copper (Cu), which constitute essential trace elements in plants, have vital biological functions at low levels but can cause toxic effects at excessive amounts . MeanwhiMembers of the cation diffusion facilitator (CDF) family are integral membrane divalent cation transporters that are involved in metal ions efflux from the cytoplasm either to the outside of the cell or into subcellular compartments . Since tArabidopsis MTPs , also called AtMTP1, was the first identified MTP protein , and form seven groups  according to the results of the phylogenetic analysis and annotation of sis MTPs . There w protein . Previou vacuole . OsMTP1, vacuole . AtMTP5  vacuole . There w vacuole . Rice ha vacuole . Moreove vacuole . In addi vacuole . CsMTP7  vacuole . CsMTP8  vacuole . CsMTP9 tiporter .Vitis vinifera, Brachypodium diastychon, Zea mays, Sorghum bicolor, Populus trichocarpa, Brassica rapa, Triticum aestivum, and Citrus sinensis (Nicotiana tabacum) is one of the most widely cultivated non-food crops worldwide and is also an important model plant organism for molecular plant biological research  with a large genome of approximately 4.5 Gb, which originated through the hybridization of the ancestral parents N. sylvestris (2n = 24) and N. tomentosiformis (2n = 24)  and comprehensively analyzed their sequence and structural characteristics, as well as the evolutionary relationships. Besides, the cis-acting regulatory element distributions, and the potential microRNA target sites in NtMTP genes were further predicted. In addition, the expression profiles of NtMTP genes in different tobacco tissues and in response to heavy metal toxicity were also investigated. In the end, the metal transport abilities of six representative NtMTPs in yeast mutant cells were investigated. Results in this study would provide a basis for the isolation and functional characterization of NtMTP genes in future studies.As the genome sequences become available for more species, a number of MTP proteins have been genome-widely identified in several plants species, including sinensis . Tobaccoresearch . Like ma2n = 24) . However2n = 24) , and theMTP genes in tobacco, the protein sequences of 12 MTPs in Arabidopsis which were obtained from TAIR101 were used as queries in TBLASTN search against the genomes of N. tabacum , and the candidates containing any of the typical domains of MTP proteins were recognized as MTP proteins.To identify the rProScan 3, and thN. tabacum and A. thaliana were analyzed in blastp suite – 2 sequences program at National Center for Biotechnology Information (NCBI4). Each protein sequence of MTPs in Arabidopsis was used as the query sequence, and all 26 NtMTP protein sequences were used as the subject sequence.The sequence similarity of MTPs proteins between Vitis vinifera, Brachypodium diastychon, Zea mays, Oryza sativa, Sorghum bicolor, and Populus trichocarpa were downloaded from the corresponding database as described by Cucumis sativus were retrieved from the GenBank database with the accession numbers of NP_001295856.1 (CsMTP1), AFJ24701.1 (CsMTP4), APM86800.1 (CsMTP5), APM86801.1 (CsMPT6), APM86799.1 (CsMPT7), AFJ24703.1 (CsMTP8), AFJ24702.1 (CsMTP9), XP_004147705.1 (CsMTP11), and APM86802.1 (CsMTP12).For phylogenetic analysis, multiple sequence alignments at amino acid level were performed by ClustalW, and MEGA 6.0 software was used for phylogenetic tree construction by the Maximum Likelihood method based on the Jones-Taylor-Thornton (JTT) matrix-base model with bootstrap of 1000 replicates . The seq5). Sub-cellular localizations were predicted by Plant-mPLoc server (6). The putative transmembrane regions in NtMTP proteins were predicted by using the TMHMM Server V. 2.0 (7). Conserved motifs and domains in NtMTP sequences were predicted by the MEME program and the Pfam tool, respectively .Molecular weight (MW), theoretical isoelectric pint (pI) and grand average of hydropathicity (GRAVY), were calculated using ProParam tool 5. Sub-cec server 6. The pur V. 2.0 7. ConserNtMTP genes were determined according to the genome annotation files at Sol Genomics Network10. Gene duplication events were analyzed by using Multiple Collinearity Scan toolkit (MCScanX) with the default parameters (11). The number of synonymous (Ks) and non-synonymous (Ka) substitutions per site of duplicated gene pair were calculated by DnaSP v6 . The coding sequences of NtMTP genes were analyzed by psRNATarget server for miRNA target sites prediction (14).The promoter sequences (up-stream 1000 bp) of analysis 13. The cediction 14.NtMTP genes, the Illumina RNA-sequencing data of N. tabacum TN90 were downloaded from GenBank Sequence Read Archive (SRA) with the accession code SRP029183 and analyzed. The fragments per kilobase per million reads (FPKM) were calculated and log2 transformed to estimate the expression levels of NtMTP genes in eight different tobacco tissues. The resulting values were used to generate a heat map by TBtools software (15).To investigate the tissue expression patterns of software 15.4, 1 M MnSO4, 0.1 M CoCl2, 0.1 M CdCl2, 0.5 M FeSO4-EDTA, and 1 M MgSO4, respectively, and those grown in nutrient solution without any heavy metal supplied were regarded as control (CK). Twelve tobacco plants were used for each treatment. After 24 h of treatments, the leaves and roots of the plants were harvested separately and were immediately frozen in liquid nitrogen, and stored at -70°C for RNA extraction.Tobacco plants (variety K326) were grown hydroponically with half-strength Hoagland solution (pH 6.0) in the greenhouse with a 16:8 h light:dark cycle under a temperature of 24°C at day, 18°C at night. The nutrient solutions were renewed every 4 days and were continuously aerated and exchanged every 4 h per day. Four weeks old plants were transferred in nutrient solutions containing 0.5 M ZnSOTotal RNAs were extracted and treated with DNase I to degrade any residual genomic DNA contamination using the RNAprep pure Plant Kit  according to the manufacturer’s instructions. The purity and concentration of total RNA was estimated by micro volume spectrophotometer Q6000 , the quality and integrity of which was assessed by 1% (w/v) agarose gel analysis. After that, 2 μg of total RNA was reverse transcribed into cDNA using ReverTra Ace qPCR RT Kit .NtL25 (GenBank accession L18908.1), Ntubc2 (GenBank accession AB026056.1), NtEF-1α (GenBank accession AF120093.1), NtRL2 (GenBank accession X62500.1), and NtCYP1 (GenBank accession AY368274.1), were chosen as internal reference gene candidates for qRT-PCR. The geNorm v. 3.5  using the CFX96 Real-Time System . All the primers used for qRT-PCR analysis are presented in m v. 3.5  was usedt method .NtMTP genes by PCR using specific primers list in KpnI and XbaI or KpnI and EcoRI sites of pYES2 to yield recombinant plasmids pYES2-NtMTP1.2, pYES2-NtMTP5.2, pYES2-NtMTP7.2, pYES2-NtMTP8.1, pYES2-NtMTP8.4, and pYES2-NtMTP11.1, respectively.To generate the yeast expression constructs, the cDNA of the leaves of control plants obtained as above was used as the template to amplify the full coding regions of six Saccharomyces cerevisiae strain BY4741 and five deletion mutants Y00829 (zrc1△), Y04534 (pmr1△), Y01613 (cot1△), Y04069 (ycf1△), and Y04169 (ccc1△) were obtained from the Euroscarf16. The plasmids were introduced into yeast by using the LiOAc/PEG method  and five dicots , were comprehensively analyzed and a phylogenetic tree was constructed. According to the classification of previous studies  to 4653 bp (NtMTP6.2), while the length of their encoded protein ranged from 296 to 1550 amino acid, and the relative MW ranged from 32.808 to 171.021 kDa, respectively , except for group 5 which possessed 9–10 exons, while Zn/Fe-CDFs comprised the highest number of exons   . Two gro3 exons) . In addi group 7 .N. tabacum genome, the chromosomal localizations of NtMTP genes were determined. 14 of the 26 NtMTP genes were located in 10 out of the 24 tobacco chromosomes  found as segmental duplication event despite of their same chromosome location, however, no tandem duplication event was identified (NtMTP7.1/NtMTP7.2 was 0.5349 (<1), suggesting that this duplicated pair might undergo negative selection (To better understand the impact of gene duplication on the expansion of the entified . Based oentified . The reselection .cis-acting elements (CREs) are regions of non-coding DNA which regulate the transcription of neighboring genes through binding by transcription factors and/or other regulatory molecules  responsiveness, P-box, GARE-motif, TATC-box in gibberellin responsiveness, TCA-element and SARE in salicylic acid responsiveness, CGTCA-motif and TGACG-motif in jasmonic acid (MeJA) responsiveness, TGA-element and AuxRR-core in auxin responsiveness and ERE in ethylene responsiveness. Additionally, abiotic stress elements comprised LTR for low temperature responsiveness, MBS for drought inducibility, TC-rich repeat for defense/stress responsiveness, HSE for heat stress responsiveness, WUN-motif for wound responsiveness, ARE and GC-motif for anaerobic induction. Moreover, elements associated with tissue expression included CAT-box and CCGTCC-box for meristem expression and meristem specific activation, respectively, RY-element for seed-specific regulation, GCN4_motif and Skn-1_motif for endosperm expression, HD-Zip 1 for palisade mesophyll cells differentiation, HD-Zip 2 for leaf morphology development control and as-2-box for shoot-specific expression and light responsiveness. Notably, circadian involved in circadian control was distributed in the promoter regions of over half of the NtMTP genes. Whereas, elements involved in biotic stress responsiveness, secondary metabolism and site-binding were less abundant than others. Taken together, the presence of these elements indicated that NtMTP genes could be transcriptionally regulated by multiple stimuli, and participate in various plant metabolic processes.Among these elements, CAAT-box and TATA-box, which were are common CREs, appeared to be the most abundance elements  and were commonly shared by all NtMTP genes, their potential miRNA target sites were searched using plant small RNA target analysis server (psRNATarget). With the expectation score lower than 4.0, in total eight NtmiRNAs comprising target sites in ten NtMTP genes were identified  to 22.218 (nta-miR6020a-5p/NtMTP6.2).MicroRNAs (miRNAs) are small non-coding RNA molecules that can play important regulatory roles in gene expression by targeting mRNAs for cleavage or translational repression . To giveentified . All thrognition . The eneognition . The resNtMTP genes in eight different tobacco tissues, including mature flower, young flower, dry capsule, young leaf, mature leaf, senescent leaf, root, and stem, were investigated using the Illumina RNA sequencing data from GenBank SRA  and one representative macroelement Mg. The relative expression levels of NtMTP genes under normal conditions (CK) were analyzed first. 14 NtMTP genes showed relative higher expression levels in leaves than in roots. The expressions of NtMTP5.1, NtMTP8.3, NtMTP9.1, NtMTP10.2, NtMTP10.3, and NtMTP10.4 genes in the tobacco roots were higher than those in the leaves, whereas NtMTP1.1, NtMTP1.2, NtMTP5.2, NtMTP6.1, NtMTP7.1, and NtMTP10.1 genes exhibited similar expression levels between these two tissues  were up-regulated by Cd treatment. In contrast to Cd, other metal ions had diverse effects on the change of expression levels of NtMTPs. NtMTP6.1, NtMTP8.4, and NtMTP11.1 were up-regulated by Zn, whereas NtMTP10.2 was down-regulated; NtMTP11.1 was increased by Mn, whereas NtMTP10.2 and NtMTP12.2 were repressed; NtMTP8.4, NtMTP8.5, and NtMTP11.1 were up-regulated by Co, whereas NtMTP10.1 were down-regulated; NtMTP4.1, NtMTP4.2, NtMTP6.2, NtMTP7.3, NtMTP8.2, and NtMTP10.2 were repressed by Fe, whereas NtMTP10.3 was increased; NtMTP8.4 was up-regulated by Mg, whereas NtMTP10.2 was down-regulated  in wild type yeast strain BY4741 and five deletion mutants which were deficient in various metal transporters. As shown in zrc1△ to Zn and cot1△ to Co. Also, the growth of pmr1△ in toxic Mn was restored by NtMTP8.1, NtMTP8.4, and NtMTP11.1, respectively. In contrast, the expression of neither NtMTP5.2 nor NtMTP7.2 complemented the sensitive phenotypes of any tested mutant strains grown in excess of different metals. These results suggested that NtMTP1.2 was a transporter of both Zn2+ and Co2+. On the other hand, NtMTP8.1, NtMTP8.4 and NtMTP11.1 could transport Mn2+ in yeast cell.Previous studies found that NtMTP1a and NtMTP1b from ast cell . In ordeMTP genes encode membrane divalent cation transporters that participated in tolerating and transporting various heavy metals, and may play essential roles in plant mineral nutrition maintenance and resistance to stresses caused by metals , respectively, and named these MTPs based on the sequence similarities and orthologous relationships between them and AtMTPs.y metals . In the N. tabacum and Arabidopsis, and other eight representative plants species were assessed at first. According to previous studies, A. thalianan contained 12 MTPs (AtMTP1-12). Compared with Arabidopsis, N. tabacum genome carried multiple MTP homologs for each AtMTP, but the homologs for AtMTP2 and AtMTP3 were absent. This result indicated that the NtMTP gene family might have undergone gene expansion and/or gene loss in the evolutionary history, probably due to the polyploidization events. In addition, N. tabacum was found to have the largest number of MTP family members among all plant species studied here, which is probably due to the large size of the Nicotiana genomes. There were eight, seven, and thirteen NtMTP genes belonging to Zn-CDFs, Zn/Fe-CDFs, and Mn-CDFs, respectively. Considering the implications of phylogenetic distributions in inferring structure and functional roles across species , and might also respond to Cd stress through negatively regulating their target genes  was little affected by Mn2+ supplies that ranged from basal to severely toxic  were identified, respectively, in the present study, and a comprehensive analysis of NtMTP genes was further carried out. The 26 NtMTPs were divided into three major substrate-specific groups  and seven primary groups , and appeared to have underwent gene loss and expanded through segmental duplication after polyploidization. All the NtMTPs contained modified signature sequences and the cation_efflux domain, whereas some of them also harbored the ZT_dimer. The expression patterns of NtMTP genes in different tissues and in response to various heavy metal toxicity indicated the conserved and essential roles of NtMTP genes in tobacco growth and development, especially in heavy metal transport and tolerance. NtMTP8.1, NtMTP8.4, and NtMTP11.1 were found to function as Mn transporters in yeast cell. These results shed some light on the evolution of MTPs in tobacco as well as the regulatory mechanism controlling NtMTPs gene expression, and provided a valuable resource for better understanding the biological roles of NtMTP genes in tobacco.Twenty six, thirteen, and twelve JL and YfG conceived and designed the experiments. JL and YlG performed the experiments. JL analyzed the data. YT, DW, and XC contributed to reagents and equipments. JL wrote the manuscript. YfG and YY provided guidance on the whole manuscript. All authors reviewed and approved the final submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


H-indole. The performance of the gold catalyst was strongly dependent on the Au NP size, with the system containing the smallest Au NPs being the more effective. Moreover, a slight drop of their catalytic efficiency was observed after three consecutive reaction runs, which was attributed to morphological changes as a consequence of fiber merging.Electrospun nanocomposite fibers consisting of crosslinked polyvinylpyrrolidone (PVP) chains and gold nanoparticles (Au NPs) were fabricated, starting from highly stable PVP/Au NP colloidal solutions with different NP loadings, followed by thermal treatment. Information on the morphological characteristics of the fibers and of the embedded Au NPs was obtained by electron microscopy. Cylindrical, bead-free fibers were visualized by Scanning Electron Microscopy (SEM) while Transmission Electron Microscopy (TEM) and Energy Diffraction X-ray (EDX) analysis supported the presence of Au NPs within the fibers and gave information on their morphologies and average diameters. These materials were briefly evaluated as heterogeneous catalytic supports for the gold-catalyzed intramolecular cyclisation of 2‑(phenylethynyl)aniline to form 2-phenyl-1 Significant advancements in the field of nanotechnology in terms of synthesis and materials characterization and processing, have led to new nanostructured materials with enhanced catalytic performance. The combination of inorganic catalytic nanoparticles with polymeric materials has led to cost-effective polymer-based nanocomposites that exhibit superior catalytic activity and selectivity in both homo- and heterogeneous catalysis, including photocatalysis, electrocatalysis and catalytic procedures adopted in organic synthesis .2, NO reduction and CO2 hydrogenation processes. Since then, nano-sized Au NPs have attracted significant interest in catalytic industrial and environmental processes [In 1989, Haruta et al. , reporterocesses ,4.There are several examples of Au NP-containing colloidal systems that were prepared and employed in homogeneous catalysis; however, these could not be readily recovered from the reaction products . MoreoveTo overcome these problems, catalytic Au NPs have been immobilized onto high surface-area insoluble matrices including hydrogels ,8,9,10, Recently, electrospinning has evolved into a versatile and powerful technique for the production of polymeric, carbon-based, ceramic and composite nano- and microfibers ,18,19. TAlthough several reports deal with the production of inorganic electrospun fibers employed as insoluble matrices for hosting Au NPs ,21,22,23To obtain electrospun polymer fibrous mats exhibiting structural and compositional stability in organic or aqueous solvents thus enabling their use as heterogeneous catalytic supports in organic synthesis, fiber crosslinking is required, which usually involves chemical treatment of the fibers ,48,49,502O NPs, that were successfully evaluated as heterogeneous catalytic supports in Heck, Suzuki and click chemistry reactions [1) to form 2-phenyl-1H-indole (2).Very recently, we fabricated PVP-based electrospun fibrous nanocomposite membranes with embedded Pd and Cueactions . Giving The gold activation of alkynes towards intramolecular cyclisation has been studied thoroughly and occurs mainly with homogeneous gold complexes . RecentlThe PVP/Au nanohybrids were prepared in the form of highly stable colloidal solutions in methanol. The reaction process involved the reduction of the Au(III) ions into metallic Au(0) nanoparticles (NPs) upon introducing hydrazine monohydrate as a reducing agent .The optical characterization of the PVP and the PVP/Au methanol solutions was performed by UV-vis spectroscopy .As seen in the spectra of the PVP/Au systems, a characteristic absorption band appeared at around 520 nm, which corresponded to the surface plasmon resonance (SPR) of the Au NPs ,62. SpecThe PVP/Au NPs solutions were used as precursors for the fabrication of Au NP-containing nanocomposite fibrous mats based on PVP, by means of the electrospinning technique .w/v was the optimum for the fabrication of continuous, cylindrical fibers without beads, which agreed with our earlier work [Systematic parametric studies helped determine the optimum experimental conditions for the production of fibrous, bead-free PVP/Au nanocomposite fibers. Variable parameters included the polymer solution concentration, the applied voltage, the delivery rate of the solution, the diameter of the needle and the needle-to-collector distance. In ier work .The thermal stability of the as-prepared PVP electrospun fibrous membranes was investigated in the presence and absence of Au NPs by means of thermal gravimetric analysis (TGA). 2O-containing electrospun heterogeneous catalysts [Thermal crosslinking of the fibrous mats was carried out to prevent membrane dissolution and consequently NP leaching of the heterogeneous catalytic supports in the organic reaction media. As demonstrated in atalysts , the theSEM images of the as-prepared (non-crosslinked) PVP membrane a and of To verify the success of the crosslinking process, the thermally-treated membranes were immersed in water at ambient temperature for several days and subsequently, the UV-vis spectrum of the supernatant was recorded. As seen in The resulting materials were also visualized by TEM to obtain information on the size and morphological characteristics of the Au NPs embedded within the polymer fibers. 1). To compare our catalyst to the literature we used similar reaction conditions to those of Perea-Buceta et al. [H-indole (2) in 78% yield , we investigated the gold-catalyzed intramolecular cyclisation of 2-(phenylethynyl)aniline . Tentatively, this indicated that higher gold loadings led to larger particle size and, therefore, a reduction of the surface area of the metal available for catalysis. The results were in agreement with the TEM investigations, which confirmed the particle size differences between the two systems: the one with the highest NP loading displayed larger particle diameters compared to the lowest NP loading analogue. Considering the exponential inverse relationship between the Au NP size and the catalytic activity, our findings on the catalytic efficacy differences existing between the two systems can be rationalized based on NP size differences. Interestingly, when our PVP-supported gold catalyst with a higher loading of Au (9% wt corresponding to 0.5 mol% Au) was used to perform the same reaction, the consumption of aniline 4·3H2O > 99.9%), and hydrazine monohydrate (N2H4·H2O) were purchased from Sigma-Aldrich . Methanol  was purchased from Scharlau . The above-mentioned reagents were used as provided by the supplier without further purification. Toluene was dried by azeotropic removal of water using a Dean-Stark apparatus . All volatiles were removed under reduced pressure. All reaction mixtures and column eluents were monitored by TLC using commercial glass backed thin layer chromatography (TLC) plates ). The plates were observed under UV light at 254 and 365 nm. The technique of dry flash chromatography was used throughout for all non-TLC scale chromatographic separations using Merck Silica Gel 60 (less than 0.063 mm)  [1) was prepared according to the literature [Polyvinylpyrrolidone , gold(III) chloride trihydrate (HAuClGermany) . 2-(Phenterature .w/v with respect to the polymer mass), with different Au % loading,  were prepared as follows: In the case of the system corresponding to the 171:1 molar ratio, in a round-bottomed flask (50 mL), PVP  was dissolved in dry MeOH (8 mL) and was stirred at room temperature. To the solution, HAuCl4·3H2O  dissolved in dry MeOH (2 mL) was added with the aid of the syringe. The mixture was left to stir at room temperature until complete solubilization of the HAuCl4·3H2O salt. Subsequently, hydrazine monohydrate  was added using a micropipette. The yellow solution turned immediately dark purple and it was left to stir under ambient conditions for another 2 h. The final polymer-Au nanohybrid solution was highly stable and no agglomeration or destabilization phenomena were observed even after several months.The PVP/Au nanohybrid colloidal systems , syringes with specially connected spinneret needle electrodes, a high-voltage power source (10–50 kV) and a custom-designed, grounded target collector, inside an interlocked Faraday enclosure safety cabinet. Systematic parametric studies were performed by varying the applied voltage, the needle-to-collector distance, the needle diameter and the flow rate so as to determine the optimum experimental conditions for obtaining bead-free fibrous materials.Insoluble PVP/Au electrospun nanocomposite fibrous mats were obtained via thermal crosslinking of the as prepared PVP/Au electrospun fibers . Thermal1) to 2-phenyl-1H-indole (2): To a stirred solution of 2-(phenylethynyl)aniline (1)  in dry PhMe (1 mL) at ca. 20 °C, was added the PVP/Au electrospun membrane . The mixture was heated to ca. 120 °C in a sealed tube until complete consumption of the starting material . The mixture was then cooled to ca. 20 °C and the polymer filtered and washed with t-BuOMe (10 mL). The membrane was reused in subsequent reactions without further treatment. The organic washings were combined, adsorbed onto silica and chromatographed  to give 2-phenyl-1H-indole (2)  as colorless plates, mp (hotstage) 183–185 °C ; νmax/cm−1 3443m (N-H), 3050w (C-H), 1605w, 1541w, 1493w, 1481m, 1456m, 1447m, 1404m, 1352m, 1339m, 1298m, 1242m, 1231m, 1190m, 1115m, 1074w, 1049w, 1028w, 1009w, 932w, 907w, 797m, 762s, 743s; δH 10.66 , 7.86 , 7.57 , 7.45 , 7.44 , 7.31 , 7,10 , 7.02 , 6.90 ; δC 138.8 (s), 138.4 (s), 133.6 (s), 130.2 (s), 129.8 (d), 128.3 (d), 125.9 (d), 122.7 (d), 121.1 (d), 120.5 (d), 112.0 (d), 99.9 (d); m/z (MALDI-TOF) 193 , 165 (100), identical to that reported [Gold catalyzed cyclisation of 2-(phenylethynyl)aniline (6–187 °C ); Rf 0.5reported .The UV-vis spectra of the PVP/Au nanohybrids stabilized in MeOH were recorded on a Jasco V-630 UV-vis spectrophotometer operating at ca. 20 °C, after appropriate dilution of the as prepared colloidal solutions. High Resolution Transmission Electron Microscopy (HRTEM) investigations of the membranes were performed by using a TECNAI F30 G2 S-TWIN microscope operated at 300 kV equipped with energy dispersive X-ray spectrometer (EDX). Samples were placed into a double copper grid (oyster) to be visualized by TEM. The morphological characteristics of the nanocomposite membranes were also determined by scanning electron microscopy (SEM) . The samples were gold-sputtered (~15 nm)  prior to SEM inspection. Thermal Gravimetric Analysis (TGA) measurements were performed on a Q500 TA instrument  under argon flow at a heating rate of 10 °C/min.1H and 13C-NMR spectra were recorded on a Bruker Avance 500 machine ). Deuterated solvents were used for homonuclear lock and the signals are referenced to the deuterated solvent peaks. CH assignments are made based on DEPT 135 spectroscopy. MALDI-TOF mass spectra were recorded on a Bruker Autoflex III Smartbeam instrument (Bruker).For the characterisation of the final product obtained from the catalytic reaction: Melting points were determined using a PolyTherm-A, Wagner & Munz, Koefler-Hotstage Microscope apparatus . IR spectra were recorded on a Shimadzu FTIR-NIR Prestige-21 spectrometer  with Pike Miracle Ge ATR accessory  and strong, medium and weak peaks are represented by s, m and w, respectively. H-indole. Based on the experimental findings, the catalytic efficiency of these materials depended strongly on the size of the embedded Au NPs. More precisely, the system containing the smallest Au NPs exhibited good catalytic efficiency, i.e., complete consumption of the starting material and product isolation at 78% yield, whereas the corresponding material enclosing the largest Au NPs led to an incomplete reaction and a lower product yield.Cost-effective, polymer-based heterogeneous catalytic supports comprised of insoluble PVP electrospun fibers and embedded catalytic Au NP were successfully prepared and evaluated in a typical gold-catalyzed intramolecular cyclisation reaction involving the transformation of 2-(phenylethynyl)aniline to 2-phenyl-1


ASAH1, ASAH2, ACER1, ACER2, and ACER3, respectively). Notably, the neutral ceramidase N-acylsphingosine amidohydrolase 2 (ASAH2) shows considerable differences between humans and animals in terms of tissue expression levels. Besides, the subcellular localization of ASAH2 remains controversial. In this review, we sum up the results obtained for identifying gene divergence, structure, subcellular localization, and manipulating factors and address the role of ASAH2 along with other ceramidases in human diseases. Human pathologies such as Alzheimer’s disease, type 2 diabetes-induced insulin resistance, cancer, and cardiovascular diseases have altered lipid homeostasis. Among these imbalanced lipids, the bioactive sphingolipids ceramide and sphingosine-1 phosphate (S1P) are pivotal in the pathophysiology of these diseases. Several enzymes within the sphingolipid pathway contribute to the homeostasis of ceramide and S1P. Ceramidase is key in the degradation of ceramide into sphingosine and free fatty acids. In humans, five different ceramidases are known—acid ceramidase, neutral ceramidase, and alkaline ceramidase 1, 2, and 3—which are encoded by five different genes ( Ceramide and sphingosine-1 phosphate (S1P) are bioactive lipids of the sphingolipid pathway and play essential roles in cell signaling. In physical conditions, they play contrasting roles within cellular metabolism. Ceramide has been shown to be involved in stress-related cellular responses and apoptosis, whereas S1P stimulates cell survival, proliferation, and tissue regeneration ,3,4. HenCeramide is hydrolyzed by three types of ceramidases, which are differentially localized and are categorized according to their catalytic pH optimum: (i) acid ceramidase A, (ii) nN-acyl linkage of ceramide to produce sphingosine and free fatty acid (ASAH1 encodes for acid ceramidase N-acylsphingosine amidohydrolase 1 (ASAH1), which localizes to the p22 arm of chromosome 8. Neutral ceramidase N-acylsphingosine amidohydrolase 2 (ASAH2) is encoded by a gene located at q11.23 of chromosome 10. Finally, the genes for alkaline ceramidases ACER1, ACER2, and ACER3 are located at p13.3 of chromosome 19, p22.1 of chromosome 9, and q13.5 of chromosome 11, respectively (Ceramidases (EC3.5.1.23) cleave the tty acid D. In humectively  18]..N-acyl lKM value of ASAH1 is about 389 to 413 µM, as is shown for 14C-labeled and BODIPY-conjugated substrate N-lauroylsphingosine  is abundantly expressed in brain tissue. Most interestingly, its expression has been recognized to decrease in correlation with age and late-onset of Alzheimer’s disease [Human ASAH2 was first identified in the small intestine   galactose in a synthetic-complete medium (SC-Ura−) [+, Ca2+, Mg2+, and Mn2+ increased the enzyme activity of purified yeast microsomal ASAH2. In contrast, Zn2+, Cu2+, and Fe2+ inhibited ASAH2 activity [Using a genome-wide study, Maltesen el al. predicted that the hepatocyte nuclear factor 4α (HNF-4α) binding site is at position −207 to −221 of n (ASAH2 . In a yeSC-Ura−) . The dowactivity .Homo sapiens) is 780 amino acids in length, while ASAH2 isoform 2  comprises only 745 amino acids. Alternative splicing leads to the truncation of isoform 2, which lacks 35 amino acids, from position 410 to 444 , dogs (Canis lupus familiaris XP_005636694), hamsters , mice , rats , frogs , and zebrafish  revealed that the most variable region ranges from position 34 to 103, which includes hypothetical O-glycosylation sites ranging from amino acids 62 to 98. Besides, in hamsters, mice, rats, frogs, and zebrafish, some of the nucleotide sequences of ASAH2 are missing  and the amidase motif NLGDVSPNXLGPXC (a.a.353 to 366) are relatively conserved, and their functions have been well studied. However, there are multiple putative start codons in the N-terminal of ASAH2 mRNA. These might lead to various ASAH2 isoforms that differ in the length of their N-terminus and might significantly influence intracellular localization of the ASAH2 polypeptide. Two isoforms of human neutral ceramidases have been identified. ASAH2 isoform 1 . In the structure, a narrow hydrophobic substrate-binding pocket was identified. They suggested that His196, Arg257, Tyr579, and Tyr591 in the binding pocket of human ASAH2 play critical roles in catalyzing the hydrolysis of ceramide , and potentially Alzheimer’s disease ,75,76. AObesity cases show a compromised insulin sensitivity due to lipid oversupply and lipid-induced oxidative stress ,78,79. TAdiponectin is the most abundant hormone secreted by adipocytes. It is involved in regulating glucose metabolism and fatty acid breakdown. Its reduction plays a very significant role in obesity-linked diseases, including insulin resistance, T2DM, and cardiovascular diseases ,90. AdipAlzheimer’s disease is a neurodegenerative disorder characterized by amyloid-beta (Aβ) accumulation and phosphorylated-tau protein deposition . Many faTraumatic brain injury (TBI) is defined as a sudden mechanical injury to the brain that progresses in terms of neuronal degeneration, followed by the disruption of synaptic circuits . IncreasASAH2, which is highly expressed in the small intestine along the brush border, plays an essential role in the pathogenesis of colorectal cancer. Garcia-Barros et al. demonstrated that the inhibition of ASAH2 elevates the ceramide level (with an increase in apoptosis and autophagy) and also leads to the suppression of the components responsible for colon cancer development, β-catenin, and ERK. Additionally, ASAH2 inhibition induced delayed tumor growth in a xenograft model with an increased ceramide level and decreased proliferation, which confirmed the role of neutral ceramidase in the regulation and development of colon cancer . In a stInflammatory bowel disease (IBD) is defined as chronic inflammation of the intestinal tract due to abnormal mucosal immune responses. Affected individuals experience abdominal symptoms, including diarrhea, abdominal pain, vomiting, and bloody stool . In IBD,e-MAPP, and NOE [In diseases such as cancer, the overexpression of ceramidase results in the excessive generation of sphingosine-1 phosphate, which leads to larger tumor growth and more resistance to chemotherapy. Recently, targeting ASAH1 for cancer treatment has been shown to enhance the efficacy of chemotherapy, which identifies ASAH1 as a therapeutic target. Thus, several ASAH1 inhibitors have been developed for use in cancer therapies ,110. The and NOE . Among t and NOE . Further and NOE . The inh and NOE . For exa and NOE . Recentl and NOE . Carmofu and NOE . TherefoCeramide plays a crucial role in metabolic and disease states, and therefore, the degrading enzyme ceramidase is the critical regulator that maintains ceramide homeostasis inside cells. A disturbance in the balance between ceramide and sphingosine-1 phosphate interferes with healthy metabolism. Consumption of a high-fat diet leads to the development of obesity-related diseases, where ceramide is involved. An accumulation of ceramide has been observed in NAFLD, T2DM, cancer, IBD, and AD. Although ASAH2 may improve insulin sensitivity and hepatic steatosis and inhibits TNF-α-induced vascular inflammation, only a few studies have targeted the manipulation of ASAH2 expression as a therapeutic approach, as ASAH2 distribution and its functions remain unclear in many respects. In summary, finding a suitable animal model and gaining a better understanding of ASAH2 will be of critical importance for clarifying its role in human diseases and in terms of its potential use in the treatment of metabolic disorders and neurodegenerative diseases.


Caveolae, flask-shaped cholesterol-, and glycosphingolipid-rich membrane microdomains, contain caveolin 1, 2, 3 and several structural proteins, in particular Cavin 1–4, EHD2, pacsin2, and dynamin 2. Caveolae participate in several physiological processes like lipid uptake, mechanosensitivity, or signaling events and are involved in pathophysiological changes in the cardiovascular system. They serve as a specific membrane platform for a diverse set of signaling molecules like endothelial nitric oxide synthase (eNOS), and further maintain vascular homeostasis. Lack of caveolins causes the complete loss of caveolae; induces vascular disorders, endothelial dysfunction, and impaired myogenic tone; and alters numerous cellular processes, which all contribute to an increased risk for hypertension. This brief review describes our current knowledge on caveolae in vasculature, with special focus on their pathophysiological role in hypertension. CAV1, CAV2, and CAV3. Cav1 is expressed in most of the cell types and is essential for caveolae biogenesis; Cav3 is predominantly expressed in muscle cells  and is required for caveolar morphogenesis; Cav2 is generally expressed together with Cav1 in adipocytes, endothelial cells, pneumocytes, and fibroblasts, but appears to be dispensable for caveolar formation ; pa; paCAV1,ormation , 15. Dynormation . Cav1 isormation . DynaminIn accordance with major knowledge and understanding of caveolae structure, increasing interest has been focused on caveolae functions in physiology and pathophysiology. The physiological roles of caveolae vary depending on the organ systems and cell types examined . In the ex vivo and lower blood pressure in mice .–43.41–43produced  48, 49), 492+ thproduced . Furthermembrane , 50, 51.is of NO , 52, 53.is of NO , associais of NO . Cav1 knis of NO , 57. Conis of NO  reportedis of NO . This diis of NO , 61.2+ absorption and increases K+ excretion in renal distal convoluted tubule . However, there are several studies reporting no difference in systolic and diastolic blood pressure between Cav1 knockout and wild-type mice –64. Of nd tubule . The inv2+ entry  channels, where the NO-dependent vasodilation in arteries is triggered by Ca2+ entry , colocal2+ entry . These cchannels . Cav1 knal cells .Caveolae are also abundant in SMCs, which are known to express Cav1–3 . Similar2+ mobilization  (Angiotensin II (Ang II) working through AT1R is a well-known signaling pathway in vascular SMCs, which plays a great role in renal hypertension. Dysfunction of this pathway shows a predominant role in the pathophysiology of renal hypertension and several renal diseases , 72. In sponses) , epidermsponses) , 76, andsponses) . Cav1 shsponses) . Ang II-sponses) .1-integrins/Fyn/Shc, which mediates stretch-induced extracellular signal-regulated kinase (ERK) activation  (+-Ca2+ exchanger (NCX1)  , Na+-Ca2r (NCX1) , T-type channels , and trachannels  are showchannels ; the exa elusive .2+ via voltage-operated calcium channels  activation through sarcoplasmic reticulum Ca2+ content  channels and are essential for the restoration of internal Ca2+ , which directly expresses and secretes Cav1 , 114. NuAn increasing number of caveolae-associated diseases are explored in order to achieve a more precise assessment of caveolae function in various pathophysiological conditions in vasculature. With the convinced and diversified roles of caveolae in the vasculature, studies on future therapeutic targeting of caveolae in hypertension are necessary.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.