The investigated extract’s ultra-performance liquid chromatography and gas chromatography coupled with mass spectrometry (UPLC/MS and GC/MS) revealed several constituents with anticancer activity. Biochemical investigation displayed several favorable effects, including the inhibition of hypoxia-inducible factor1α (HIF1α), c-Myc, pyruvate kinase-M2 (PKM2), lactate dehydrogenase-A (LDH-A), glucose-6-phosphate dehydrogenase (G6PD), and glutaminase by asparagus and rue extracts. To less extent, HIF1α, c-Myc, PKM2, and LDH-A were partially inhibited by green tea extract, and HIF1α and glutaminase activity was inhibited by avocado oil. Undesirably, green tea extract increased glutaminase; avocado oil rose c-Myc, and both increased G6PD. In conclusion, our study confirms the potential cytotoxic effects of these plant extracts. It highlights a strong association between the ability of asparagus, green tea, rue, and avocado to sensitize HepG2 cells to doxorubicin and their power to amend cell metabolism, suggesting their use as add-on agents that might aid in clinically lowering the doxorubicin dose.Increasing cancer cell sensitivity to chemotherapy by amending aberrant metabolism using plant extracts represents a promising strategy to lower chemotherapy doses while retaining the same therapeutic outcome. Here, we incubated HepG2 cells with four plant extracts that were selected based on an earlier assessment of their cytotoxicity, viz asparagus, green tea, rue, and avocado, separately, before treatment with doxorubicin. MTT assays elucidated a significant decrease in doxorubicin-IC Hepatocellular carcinoma (HCC) ranks fourth among cancer-related deaths worldwide . Egypt rPlant-derived metabolites and phytochemical nutrients have long been investigated for their antiproliferative effect. More recently, some phytochemicals of different chemical classes have shown an ability to alter the microenvironment of cancer cells characterized by low pH, hypoxia, and oxidative stress . NaturalThe metabolism in cancer is reprogrammed to accommodate the high energy demands for the highly proliferating cells required for biomass buildup and tumor growth . DecadesPulsatilla and Anemone could alter cancer cell metabolism by regulating the MAPK pathway -, peak 8), coumaroyl feruloylglycerol , and diferuloylglycerol , which were previously reported in Asparagus officinalis  and asparanin D . Other potential saponins were detected at m/z 943, 1001, 1017, 1019, and 1105 but could not be identified due to the limited fragmentation of their molecular ion peaks. Other phenolic compounds identified in the extract included norlignans  and phenylacetylenes , Ultra-performance liquid chromatography coupled to mass spectrometry (UPLC/MS) analysis of asparagus extract showed early eluted peaks identified as organic acids and phenolic acid glycosides A, includicinalis . Severalm/z 457 for [M-H]-, peak 6) along with epi/catechin gallate  which were readily identifiable by the loss of gallic acid moiety (−170 amu) upon fragmentation. Six flavan-3-Ol were identified in peaks 2, 3, 4, 5, 6, 9, Meanwhile, green tea extract’s LC/MS chromatogram showed its enrichment in flavonoid compounds, especially catechin derivatives B. Gallicm/z 315 for [M + H]+, peak 11) and chalepensin , m/z corresponding to alkaloids . Several quinoline and acridone alkaloids were tentatively identified in The LC/MS chromatogram of rue methanolic extract showed fewer polar constituents than observed for both asparagus and green tea extracts, Besides the activity for the three-plant extract, fixed oil obtained from avocado pulp also showed appreciable activity. Gas chromatography analysis of fatty acids methyl esters of avocado oil identified unsaturated fatty acids as significant constituents of the FAMEs composition at 83.13% of total FAME, with oleic acid as the major fatty acid representing 53% of FAME content as outlined in Our data showed a significant decrease in doxorubicin IC50 in pretreated cells compared to untreated counterparts. Doxorubicin inhibited untreated HepG2 cell growth at IC50 value equal to 7.98 µg/mL. When cells were pretreated with a high and low dose of asparagus extract, doxorubicin showed much lower IC50 equal to 3.38 and 1.73 µg/mL, respectively. To less extent, rue-pretreated HepG2 cells were sensitized towards doxorubicin represented by IC50 at 5.57 and 4.32 µg/mL for cells treated with a low and a high dose of rue extract, respectively. Additionally, doxorubicin IC50 was observed at lower values for HepG2 cells pretreated with green tea extract or avocado oil as shown in .Hypoxia-inducible factor1α (HIF1α) was reported to regulate rate-limiting glycolytic enzymes such as lactate dehydrogenase-A (LDH-A) and pyruvate kinase-M2 (PK-M2) ,42,43. FMoreover, PKM2 levels in HepG2 cells treated with low and high doses of rue, asparagus, and green tea extracts were significantly lower than that shown in the untreated cells (high doses induced significantly higher effects as compared to low doses). Nevertheless, only the high dose of avocado oil produced a significant reduction in PKM2 levels compared to untreated cells C. LDH-A In cancer cells, glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the pentose-phosphate pathway (PPP) is up-regulated by hypoxia . Our resMeanwhile, rue and green tea extracts showed non-significant effects compared to untreated cells. Paradoxically, avocado fruit extract showed a significant increase in G6PD in HepG2 cells compared to untreated counterparts E.The glutamine consumption rate increases in many cancers, promoted by glutaminase, reflecting the crucial role of glutaminolysis in tumor biology . In our 50 and the metabolic proteins/enzymes level in each plant extract-pretreated HepG2 cells, a correlation study was performed between such parameters. As illustrated in 50 and HIF1α, C-Myc, PKM2, LDH-A, G6PDH, and glutaminase levels. Additionally, in rue-treated HepG2 cells, there was a significant positive correlation between doxorubicin IC50 and the HIF1α, C-Myc, PKM2, and glutaminase levels. In contrast, LDH-A and G6PDH levels have a non-significant correlation with doxorubicin IC50. Furthermore, in green tea-treated HepG2 cells, a significant positive correlation between doxorubicin IC50 and HIF1α, C-Myc, PKM2, and LDH-A was obtained, while G6PDH glutaminase levels have a significant negative correlation with doxorubicin IC50. Finally, in avocado-treated HepG2 cells, there was a significant positive correlation between doxorubicin IC50 and the HIF1α level, a significant negative correlation between doxorubicin IC50 and c-Myc, and G6PD levels. In contrast, correlations between doxorubicin IC50 and PKM2, LDH-A, and glutaminase levels were non-significant.To detect the association between the reduction (%) in doxorubicin ICNatural products have been widely reported for their cytotoxic effects by acting as antioxidants, anti-proliferative, and/or apoptotic agents . A recenThe four selected plant extracts showed very different phytochemical compositions, as revealed by LC/MS analysis, which spanned other secondary metabolites.Asparagus extract produced the most significant sensitization of Hep2G cells to doxorubicin. It showed various chemical constituents, including saponins, phenolic acids, lignans, and acetylenes. Phenolic acids such as coumaric, ferulic, and sinapic acids represent the significant constituents of the asparagus extract, and these compounds have been recognized as solid antioxidants . In addiCommon rue extract was the second primary substance in our study that showed an ability to sensitize Hep2G cells to the effect of doxorubicin. Rue is used as a condiment and herbal tea in many cultures. It has been reviewed for various biologically active constituents . At leasGreen tea extract and avocado oil showed an appreciable, but the less, ability to sensitize HepG2 to doxorubicin compared to asparagus and rue. Both functional foods are highly reputed for their antioxidant activities attributed to catechin and unsaturated fatty acids, respectively ,59, consWe demonstrated that treating HepG2 cells with either asparagus, rue, green tea extracts, or avocado oil amended the metabolism and augmented the doxorubicin cytotoxic effect on such cells. Pretreatment with asparagus was the most effective in sensitizing HepG2 cells to doxorubicin and, less extent, pretreatment with rue, green tea, and avocado oil, respectively. Asparagus and rue showed the most proficiency in correcting the aberrant metabolism as they inhibited HIF1α, c-Myc, PKM2, LDH-A, G6PD, and glutaminase in HepG2 cells. With a lower ability, green tea and avocado oil also corrected the aberrant metabolism. We concluded that the ability of the plant extracts to improve doxorubicin efficacy might be partially ascribed to the amended metabolism; that is, the more amended metabolism accomplished by the extract, the more its ability to sensitize HepG2 cells. Our study recommends that other chemotherapeutic drugs should be examined to confirm whether this approach can be generalized.


CD3G, CD3D, CD8B, LAT, LCK, ZAP70, CCR5, CXCR3, CXCL9, CXCL10, and CXCL11, were highly expressed in VLS, while NR4A family genes , whose coding products inhibit T cell activity, were significantly downregulated, suggesting heightened T cell response in VLS. Neutrophil chemoattractant genes CXCL1, CXCL2, CXCL3, CXCL8, and their cognate receptor CXCR2 were downregulated, suggesting dampened neutrophil activity. We also found the downregulation of genes involved in cell cycle progression, including cyclins  and centrosome factors , while microRNA-203a and let-7, microRNAs known to inhibit cell growth, were found to be upregulated. These data collectively indicate that cell proliferation in VLS is compromised. In sum, these findings comprehensively deciphered key regulatory genes and networks in VLS, which could further our understanding of disease mechanisms and point toward therapeutic strategies.Vulvar lichen sclerosus (VLS) is a chronic inflammatory dermatosis that affects female anogenital skin. Although VLS is considered a T cell-mediated autoimmune disease, the diagnosis criteria, molecular mechanism, and universally accepted therapies for this disease remain largely unresolved. To explore disease pathogenesis and potential biomarkers, we performed an RNA-Seq-based transcriptome analysis to profile the gene expression of VLS lesions. Differentially expressed gene (DEG) analysis revealed profound changes in expressions of coding genes, microRNAs, and long non-coding RNAs. Pathway and network analysis suggested that T cell activation-associated genes, including  Lichen sclerosus (LS) is a chronic progressive dermatosis that most commonly affects the genitalia . The disDespite being clinically defined for more than a century, much about LS remains unclear, and debate over its etiology, histological diagnostic criteria, and treatment persists. Immune dysregulation is one of the most widely recognized explanations for LS. According to reports, 82% of the overall cohort had at least one form of autoimmune illness  . AccumulIn the current study, we performed an RNA-seq-based gene expression profiling of skin biopsy samples from VLS patients to systematically interrogate disease pathogenesis at the molecular level. Differentially expressed genes were identified and further analyzed with functional bioinformatics tools. We further depicted molecular signatures and key regulatory networks of VLS, providing insights into VLS pathogenesis and potential biomarkers for disease management.Six female patients with VLS and four healthy individuals were included. All patients recruited had classical signs of lichen sclerosus with histopathology confirmation. This study was approved by the Medical Ethics Committee of Beijing Jishuitan Hospital (202109-04).After receipt of written informed consent signed by the patients and healthy individuals, vulval skin samples were obtained by punch biopsy so that it contained the layers of epidermis and dermis. Control samples were collected from healthy women who underwent cosmetic vulvar surgery. There is no statistically significant difference between the control and patient groups in terms of age. The biopsies obtained from each sample was put into sterile tubes and immediately frozen in liquid nitrogen until further analysis.® UltraTM RNA Library Prep Kit , sequencing libraries with different index labels were created for each sample according to the manufacturer’s instructions. The library construction procedures were as follows. First, ribosomal RNA was removed using an Illumina Ribo-Zero™ Gold rRNA Removal Kit. Following that, RNA fragmentation was performed. The first and second cDNA strands were then generated consecutively. Purification of the library fragments was then performed, followed by terminal repair, dA-tailing, and adapter ligation. The library fragments were purified and digested using UNG enzyme. Finally, polymerase chain reaction amplification was used to finish the library construction.Frozen skin tissues were ground in liquid nitrogen and extracted using Trizol  according to the manufacturer’s instructions. For library construction, total RNA that met the following criteria was used: the RNA integrity number (RIN) ≥ 7; 28S/18S rRNA ratio ≥ 1.5. The initial material for library construction was 1 μg of total RNA per sample. Using a NEBNextThe index-coded samples were clustered using a TruSeq PE Cluster Kit v4-cBot-HS  according to the manufacturer’s instructions using a cBot cluster generation system. Following cluster generation, the libraries were sequenced on an Illumina Hiseq X10 platform , and CapitalBioTech produced 125 bp paired-end reads .The FastQC program was used to acquire quality control metrics for raw sequencing reads . TopHat2The Limma package of R language (v. 3.22.7) was used to conduct differential gene expression analysis . Paired http://metascape.org) was employed to perform the gene enrichment and functional annotation analyses (https://string-db.org) was used to construct a protein-protein interaction network.Metascape  value for each sample was used for data analysis. The 2−ΔΔCt method was used for determining the fold-change in the expression level, and GAPDH was used for normalization. All data were presented as the mean ± SD after normalization.To confirm the RNA-Seq results, RT-qPCR was performed on 12 genes. RNA was extracted using Trizol . The sequences of the qPCR primers used in this study are shown in All patients fulfilled the diagnostic criteria for VLS. The demographic characteristics of the patients are detailed in p-value (FDR < 0.05) and fold change (FC) ratio (|log2FC| ≥ 1) were used to determine the DEGs. A total of 1641 differentially expressed coding genes were identified. Of those, 972 were upregulated and 669 were downregulated  were labeled  were then identified by comparing VLS transcriptomes with those of the healthy donors. An adjusted egulated . Moreoveegulated . A heatmegulated . Hierarc labeled ; the top factors . NR4A1,  process . Among tagocytes    in VLS by using Metascape (ape.org) , a web-bape.org) , supportape.org) . Besidesape.org) .CD8B, CD3G, CD3D, LAT, LCK, and ZAP70   software, we performed Canonical Pathway Analysis and Upstream Regulator Analysis to unravel signaling pathways and key regulators implicated in VLS pathogenesis. Sixteen pathways were identified using an absolute z-score value greater than 1 as a threshold  . Among tp-value) . RNA-seqnd CCL20  were signd CCL20 . Glycosand CCL20 , which cp-value < 0.05, |z-score| ≥ 3), and 15 top-ranked activated upstream regulators were demonstrated   , transglutaminases (TGM1 and TGM5), desmogleins (DSG1 and DSG3), and desmocollin (DSC3), were downregulated, while late cornified envelope family genes  were upregulated  , we buildb.org/) . We foundb.org/) . Heatmapd AURKB)   . These dd AURKB) . In thisegulated . The CXCegulated ; thus, tegulated . In contor CXCR2  were dowor CXCR2 , suggestor CXCR2 . We alsoor CXCR2 , in whicegulated . This obegulated . Of noteegulated . In addiegulated . Genes eegulated , such asegulated . The coregulated . Thus, tCXCR3, CXCL10, and CXCL11 were chemokine-chemokine receptor pairs involved in Th1 cell activation, and LCE2A, LCE2C, LCE2D are cornified envelope family genes specifically upregulated in VLS. Of down-regulated genes, CXCL8 is the critical chemotactic factor for neutrophils, and HBEGF, LDLR, NR4A1, NR4A2, and NR4A3 are randomly selected. Transcript levels for each gene were measured in comparison with GAPDH and then normalized to healthy control by using the 2−ΔΔCt method. We found that all selected upregulated and downregulated genes in RNA-Seq were successfully validated by qPCR analysis (We next verified the RNA-seq results by quantitative polymerase chain reactions (qPCR) analysis. Six upregulated and six downregulated genes identified by the RNA-seq analysis were tested. Of selected upregulated genes, analysis , indicatComplications of VLS mainly include pruritus, pain, sexual and/or urinary dysfunction, posing detrimental effects on quality of life. If not appropriately treated, VLS is associated with a greater degree of the lesion and an increased risk of genital cancer . To dateCXCR3 and CCR5, both of which are markers for Th-1 type reactions, as well as the TCR activation-associated molecules CD3G, CD3D, ZAP70, LAT, LCK, and CD8B, were significantly upregulated, suggesting a dominant CD8+ T cell response in VLS. Of note, the orphan nuclear receptor NR4A family proteins, including NR4A1, NR4A2, and NR4A3, are among top downregulated genes. This downregulation was further verified by our quantitative rt-PCR analysis. NR4A genes were recently suggested to be associated with T cell hyporesponsiveness, as evidenced by the fact that NR4A-depleted T cells exerted potent effector functions in the context of cancer and infection (VLS is considered lymphocyte-mediated dermatosis because one of the classic histological features is a lymphatic infiltrate belt underneath a condensed region of sclerosis . Corrobonfection . As a reCXCR2 gene, the major chemokine receptors on neutrophils, and its ligand genes CXCL1, CXCL2, CXCL3, and CXCL8, were significantly downregulated, suggesting the depletion of neutrophils in VLS. Neutrophils are the most abundant leukocytes in the circulation and play critical roles in immune and inflammatory responses (Intriguingly, we found that esponses . Infiltresponses , anotheresponses . Becauseesponses , the absCCNB1, CCNB2, CCNL1, CCNE1, CCNK. In contrast, only one cyclin gene, CCNG2, was upregulated in VLS. These data strongly suggested that cell cycle progression in LS lesions has been impeded. As a result, cell proliferation may be inhibited, resulting in skin atrophy and basal cell degeneration observed in VLS. Intriguingly, let-7, a microRNA known to repress cell proliferation (CCNB1, CCNE2, CCNF, CCNJ in HepG2 cells (CENPA, CENPE, CENPF, CENPN, BUB1B, AURKB, and SKA1, were significantly downregulated. As centromere proteins play crucial roles in maintaining chromosome stability (One of the noticeable features observed in the VLS transcriptome is the decreased expression of genes involved in cell cycle progression. These include cyclin genes feration , was sigG2 cells , it can G2 cells . Besidestability , downregIn sum, our study provides a thorough gene expression profile and molecular interaction network for VLS, a disease with an elusive etiology. We revealed that coding genes, microRNAs, and long non-coding RNAs are profoundly dysregulated in VLS lesions. Furthermore, gene networks that regulate immune cell chemotaxis, mitotic nuclear division, antigen processing and presentation, or epidermal barrier construction were perturbed. These alterations may play crucial roles in the pathophysiology of VLS. It should be noted that the current study included a small number of cases, and a larger sample size need be used to generalize the findings. Future research with advanced methodologies, such as combining single-cell level transcriptomics with in-depth bioinformatics analysis, will shed further light on VLS pathogenesis and therapy.


Thiosulfate: cyanide sulfurtransferase (TST), also named rhodanese, is an enzyme widely distributed in both prokaryotes and eukaryotes, where it plays a relevant role in mitochondrial function. TST enzyme is involved in several biochemical processes such as: cyanide detoxification, the transport of sulfur and selenium in biologically available forms, the restoration of iron–sulfur clusters, redox system maintenance and the mitochondrial import of 5S rRNA. Recently, the relevance of TST in metabolic diseases, such as diabetes, has been highlighted, opening the way for research on important aspects of sulfur metabolism in diabetes. This review underlines the structural and functional characteristics of TST, describing the physiological role and biomedical and biotechnological applications of this essential enzyme. The tst gene encoding the human TST, also named rhodanese, is located on chromosome 22q12.3 [m of the recombinant human TST are 39.5 ± 2.5 mM and 29 ± 4 mM for thiosulfate and cyanide, respectively [The enzyme thiosulfate sulfurtransferase (TST) EC: 2.8.1.1), originally identified in 1933 by Lang [.1, origi 22q12.3 . In mamm 22q12.3  and its  22q12.3 . In part 22q12.3 ,8. Accor 22q12.3 ,11. The m = 2.6 mM), a substrate processed also by mercaptopyruvate sulfurtransferase (MST) in cyanide detoxification. There is strong evidence that TST and MST are evolutionarily related because, in addition to being able to interact with the same substrate  although with distinct kinetics and different affinities, they show a striking similarity in amino acid sequences around the active site (66% of sequence homology between TST and MST in rat liver) [Among the sulfur donors of TST, there is mercaptopyruvate (Kt liver) . In mammt liver) . In factt liver) ), herbivt liver) ,17. TST t liver) .In the last twenty years, several studies have highlighted the involvement of TST in metabolic processes, as well as its relevance in various metabolic and non-metabolic diseases. This review underlines the structural aspects of this enzyme, which has represented and still represents an enzymatic structural model. Moreover, it highlights the involvement of this mitochondrial enzyme in different and relevant biochemical processes, its involvement in the onset of some diseases, as well as its possible application in biotechnology.Bos taurus (Rhobov) and Azotobacter vinelandii (RhdA) [35Cl NMR relaxation studies [15N NMR relaxation studies together with essential dynamics studies on the prokaryotic TST from Azotobacter vinelandii (RhdA) [m value for thiosulfate and decrease the kcat for rhodanese 6-fold [The TST superfamily members are characterized by an alpha-beta topology with a structural module in which alpha-helices surround a central five-stranded beta-sheet core . The suli (RhdA) ,25,26, ti (RhdA) ,27. TST i (RhdA) ,29,30, ai (RhdA) ,32, whili (RhdA) . Figure i (RhdA)  or in thi (RhdA) ,35, thati (RhdA)  and has i (RhdA) ,39,40,41i (RhdA) . The N-ti (RhdA) ,44. The i (RhdA)  that havi (RhdA) . These di (RhdA) , Cys-247i (RhdA) . Proteini (RhdA) . The obsi (RhdA) ,51,52. Wi (RhdA)  and 35Cl studies  on eukar studies . Howeveri (RhdA) ,56 did ni (RhdA) . The sube 6-fold .http://sanger.ac.uk/cgi-bin/Pfam) suggests that the rhodanese domain can be involved in distinct physiological roles [The high number of members of the homology superfamily of rhodanese (accession number: PF00581 (accessed on 1 April 2021); al roles .TST/Rhodanese, in addition to cyanide detoxification, either alone or in association with other proteins, can perform a variety of biological roles, ranging from the transport of sulfur and selenium in biologically available forms ,60,61 an2+, Zn2+ [2+ [2− [2−, oxaloacetate, pyruvate [2O2 [The E form of TST can be phosphorylated as a result of cellular signaling , at seri2+, Zn2+ , Cu2+ [8Zn2+ [2+  ions, SO [2+ [2− , SO42−, pyruvate , H2O2 [8ate [2O2  and in gate [2O2  and L-cyate [2O2 , and redate [2O2 , where tate [2O2 ,88.3), which is an active selenium donor required for SeCys-tRNA synthesis, a relevant precursor of selenocysteine. Therefore, the TST enzyme can have a relevant role in the synthesis of selenoenzymes and so indirectly facilitate the removal of hydrogen peroxide by GSH and provide reducing equivalents to thioredoxin (TRX). All members of the sulfurtransferase family can oxidize and reduce, respectively, the thiol and persulfidated forms of TRX [2S) has been recognized. However, the detailed molecular role of TST is still debated. TST/Rhodanese is a crucial enzyme in the H2S oxidation route that leads to the formation of hydropersulfides (-SSH), thiosulfate and sulfate. TST/Rhodanese catalyzes the transfer of sulfane sulfur from glutathione persulfide (GSSH) to sulfite, which is produced in a reaction catalyzed by persulfide dioxygenase (SDO), i.e., ETHE1, to form thiosulfate [Another role of TST is in selenium metabolism, as demonstrated in vitro that the E form of TST binds selenium at a 1:1 molar ratio, leading to the formation of the stable perselenide form of TST . Accordis of TRX ,73,89. Is of TRX ,91,92,93s of TRX . In agres of TRX . During osulfate ,95,96.2S using dihydrolipoic acid (DHLA) [2S production from L- and D- cysteine [2S [2S in vivo. However, although the reaction is reversible, the efficiency for thiosulfate production is much greater than that for thiosulfate utilization in this reaction [TST is also able to catalyze the formation of Hd (DHLA) ,98 whichcysteine ,98. The eine [2S ,76 has sreaction .2S [2S from thiosulfate in the presence of GSH is only ~1% of the ability of cystathionine -synthase and -cystathionine lyase to produce H2S from cysteine and homocysteine. This suggests a primary role of rhodanese in H2S catabolism rather than in its production. The involvement of TST in the metabolism expands the relevance of this enzyme in several biochemical pathways and diseases, considering the remarkable properties of the endogenous H2S as a gasotransmitter, cytoprotective agent, mediator of vascular response and platelet adhesion, and regulator of glucose metabolism, redox balance and the detoxification of intra-mitochondrial oxygen free radicals [This is suggested due to the activity of the sulfide quinone oxidoreductase (SQR), which is the first enzyme in the mitochondrial sulfide oxidation pathway, and predominantly catalyzes the synthesis of GSSH. The amount of rhodanese is about 4-fold lower than of CSE, but is 14-fold higher than of cystathionine-synthase (CBS) , the sul2S ,101. Theradicals ,105,106.Historically, the first disease related to an alteration in the expression of TST/rhodanese is Lebers hereditary optic neuropathy (LHON). LHON is a rare neurodegenerative disease that results in significant visual loss or even total blindness. In most cases, this disease occurs in young men and leads to a selective degeneration of retinal ganglion cells (RGCs) with optic atrophy within a year of disease onset. LHON is caused by the presence of mitochondrial DNA point mutations  that aff2S metabolism, such as colonic diseases and, according to recent studies, also obesity-associated type 2 diabetes mellitus  H2S concentration is increased [2S in the colon [The effects of alteration in TST expression and activity are detected in several other diseases with mitochondrial dysfunction and/or unbalance in redox state or in Hncreased , with a he colon . A reduche colon . Howeverhe colon . It is dhe colon ,123,124.tst gene has been identified as a candidate obesity resistance gene involved in low adiposity and metabolic health. Morton et al., using a polygenic lean mouse model, showed a connection between the up-regulation of the tst gene expression and reduced adiposity and insulin sensitization in white adipose tissue [2S, both involved in adipocyte function, has been mentioned [2S effects on adipocyte function. Notably, since several recent studies highlight a link between insulin resistance and an altered electron transport chain [In recent years, a dramatic increase in obesity and in diabetes mellitus type 2 (DMT2) was observed worldwide, due mainly to the globalization of a diet with a high content of animal fat and protein, refined grains and added sugar. Obesity is related not only to an obesogenic environment, but also to a genetic susceptibility ,126 and e tissue . TST enze tissue . Furthere tissue . Among tentioned ,132, leaentioned  and leadentioned ,135. Thientioned , which rentioned , determirt chain ,139 due rt chain ,140.Deregulation of tst/rhodanese gene expression has also been related to other diseases, such as Friedreich’s ataxia (FRDA). FRDA is an autosomal-recessive neuro- and cardio-degenerative disease characterized by cardiomyopathy and progressive ataxia is related to the inhibition of frataxin expression , a proteAll these findings that show the association between the expression of rhodanese and degenerative states can make rhodanese a potential tumor/disease biomarker and treatment target.In the last few decades, TST, due to the capability of detoxifying cyanide, has been investigated for its use in many environmental and biomedical biotechnologies see . Among tCyanide is a nitrile which is extensively employed in many industry fields such as pharmaceuticals, food processing, coal cooking, jewelry, mining, electroplating, plastics, dyes and paints . It is pEscherichia coli expressing recombinant TST from Pseudomonas aeruginosa [Azotobacter vinelandii [Bacillus pumilus [Rhizopus oryzae [Trametes sanguinea [Aureobasidium pullulans [Aureobasidium pullulans and performed an immobilization via cross-linking with glutaraldehyde prior to enzyme entrapment in alginate supports, with the aim to develop a new bio-device for the biodegradation of cyanide in cassava mill effluents [Instead, the biological methods, which involve the use of biological agents such as microorganisms (bacteria/fungi) and enzymes, represent an inexpensive, eco-friendly, highly efficient approach with no release of toxic by-products ,152. Henruginosa , Azotobanelandii ,159, Bac pumilus  and the  pumilus , have bes oryzae , Trameteanguinea  and Aureullulans  was alsoullulans  and sephullulans  has beenffluents .−) by means of the exogenously administered sulfane sulfur and sulfurtransferase enzymes [5(CH2)17 chain modified surface in combination with the classic inorganic sodium thiosulfate (TS) and a chemical component of garlic, diallyldisulfide (DADS), a sulfur donor, to prevent cyanide lethality in a prophylactic mice model. They showed that DADS is not a more efficient sulfur donor than TS; however, the use of external TST significantly enhanced the in vivo efficacy of both sulfur donor-nitrite combinations, indicating the potential usefulness of enzyme nano-delivery systems in developing antidotal therapeutic agents [Trichormus variabilis VRUC 168, with the addition of polyethylene glycol diacrylated (PEGDa) [Azotobacter vinelandii [Other applications of TST are more related to civilian and military research, trying to develop effective catalytic bio-scavengers for a therapeutic and prophylactic defense against cyanide as a chemical toxic agent. The treatment of cyanide intoxication requires the use of scavengers  and/or the transformation of cyanide into the less toxic thiocyanate  . Bellininelandii ,30,56,89nelandii . They denelandii .4 ppm KCN significantly increased the rate of ruminal HCN degradation. Moreover, they found that, though the in vitro dry matter digestibility (IVDMD) was suppressed when increasing doses of KCN were administered at 600 ppm, the supplementation of rhodanese enzymes at 1.0–1.35 mg/104 ppm KCN enhanced IVDMD [Another potential application of TST concerns the use of this enzyme to reduce ruminant animals’ mortality due to cyanide-containing feed ingestion . In facted IVDMD .Finally, the cyanide-detoxifying ability of rhodanese has also been exploited to develop biosensors for cyanide detection ,184,185.Azotobacter vinelandii and incorporated the TSTMBs into photo-polymerizable polyethylene glycol-fibrinogen hydrogel (PFHy), with the aim to realize H2S-releasing scaffolds to promote the human Lin− Sca-1pos cardiac progenitor cells (hCPCs) proliferation and differentiation [2S release over time which exerts an antioxidant protective effect on cells, therefore stimulating cell growth [More recently, the application field of TST is represented by the optimization of hydrogel-based cellular scaffolds for tissue engineering and regenerative medicine ,188,189.Certainly, this property of TST together with the recent discovery of its relevance in metabolic diseases, such as diabetes, could favor the development of new bio-medical applications of this enzyme in the future.


Botrytis cinerea. This deepening understanding of the function of ERFs in fruit ripening and pathogen responses may identify novel approaches for engineering transcriptional regulation to improve fruit quality and pathogen resistance.Fleshy fruits are generally hard and unpalatable when unripe; however, as they mature, their quality is transformed by the complex and dynamic genetic and biochemical process of ripening, which affects all cell compartments. Ripening fruits are enriched with nutrients such as acids, sugars, vitamins, attractive volatiles and pigments and develop a pleasant taste and texture and become attractive to eat. Ripening also increases sensitivity to pathogens, and this presents a crucial problem for fruit postharvest transport and storage: how to enhance pathogen resistance while maintaining ripening quality. Fruit development and ripening involve many changes in gene expression regulated by transcription factors (TFs), some of which respond to hormones such as auxin, abscisic acid (ABA) and ethylene. Ethylene response factor (ERF) TFs regulate both fruit ripening and resistance to pathogen stresses. Different ERFs regulate fruit ripening and/or pathogen responses in both fleshy climacteric and non-climacteric fruits and function cooperatively or independently of other TFs. In this review, we summarize the current status of studies on ERFs that regulate fruit ripening and responses to infection by several fungal pathogens, including a systematic ERF transcriptome analysis of fungal grey mould infection of tomato caused by  Solanum lycorpersicum) involved include MADS, particularly RIN (MADS-RIN), several different NACs including NOR and NOR-like1, CNR and other TFs responsive to auxin, ABA and ethylene [Botrytis cinerea (B. cinerea), such as the KNOX TFs (SlTKN4 and SlTKN2), Golden2-like (SlGLK2) and TFs and SlAPRR2-like, which regulates chlorophyll accumulation and other aspects of chloroplast development before ripening onset [B. cinerea because of their increased sugar content [The changes that occur to fleshy fruits during ripening make them more sensitive than unripe fruit to infection by bacteria and fungi ,2. The rethylene . Some TFethylene . These iethylene , SlMYB75ethylene , bHLH TFethylene ,14 and aethylene . Other Tng onset ,17,18,19 content . Tomatoe content ,21,22, a content . This isB. cinerea and Colletotrichum gloeosporioides, are the most widespread cause of fruit rotting [B. cinerea is a well-researched airborne necrotrophic plant pathogen, which can infect more than 200 crop hosts throughout the world [C. gloeosporioides causes anthracnose, a major disease of many tropical fruits such as avocado (Persea americana Mill.), mango (Mangifera indica L.), papaya (Carica papaya L.) and strawberry (Fragaria ananassa Duch.). While the host fruit are still unripe, C. gloeosporioides spores produce an infection peg, which penetrates the cuticle of unripe fruit and then becomes quiescent. Growth is only resumed when the fruit begin to ripen and the quiescent fungus is stimulated to produce hyphae and initiate necrotrophic growth, causing the anthracnose fruit-rot symptoms. It is possible that the fungus perceives ethylene or monitors other signals produced by the fruit during ripening or wounding [C. gloeosporioides contrasts with the behaviour of B. cinerea, which can infect both unripe and ripe fruits through wounds, causing grey mould. Alkan et al. [C. gloeosporioides pathogen and fruit during different stages of infection in tomato and showed that over 3000 transcripts were significantly up-regulated, compared to control fruit. The upregulated genes were involved in processes such as the hypersensitive response (HR), phenylalanine biosynthesis, flavonoid biosynthesis and cell wall modification (xyloglucan endotransglucosylases and expansin-like proteins). Several responses were involved in promotion of cell death, including enhanced salicylic acid (SA) signaling, which promotes the respiratory burst, involving nicotinamide adenine dinucleotide phosphate (NADPH) oxidase transcripts, and repression of the jasmonic acid (JA) and ethylene response pathways. Hong et al. [C. gloeosporioides and identified transcripts for defense-related pathways and other proteins involved in signaling pathways. Genes encoding 373 TFs including MYBs (10.7%-), WRKYs (8.0%-), and ERFs  showed increased expression and 13 ERF unigenes were up-regulated 1.5- to 85-fold in infected fruits.Fungal diseases such as those caused by  rotting . B. cinehe world ,24, leadhe world . C. gloewounding , but thin et al.  carried g et al.  characteThe challenge for fruit production is to balance fruit ripening and pathogen resistance to maintain postharvest fruit quality. Understanding the underlying processes of ripening control and the pathogen response, both of which involve ERFs, may provide novel approaches for engineering improvements to fruit quality and pathogen resistance. Ethylene response factors (ERFs) belong to the APETALA2/ethylene response factors (AP2/ERFs) TF family, one of the largest of the 58 plant TF families, defined by their AP2/ERF domains that consist of approximately 60 to 70 amino acids . ERFs acIn this article, we review and summarize the current status of studies on ERFs that regulate fleshy fruit ripening and fruit resistance to pathogens, as part of an investigation into novel approaches for altering transcriptional regulation to improve fruit quality and pathogen resistance.Malus pumila Mill.), pears (Pyrus spp.), bananas (Musa spp.), melons (Cucumis sativus L.) and tomato) or non-climacteric , strawberry, citrus (Citrus reticulata Blanco.)) types. Tomato is a typical climacteric fruit which shows a characteristic rise in respiration (the respiratory climacteric) and ethylene production at the onset of ripening. Ethylene is required for ripening initiation in climacteric fruits and is a major cue that initiates most aspects of ripening [Fleshy fruits are classified as climacteric  ethylene biosynthesis [Increases in ethylene production in plants involve changes in expression of multiple ACC synthase (ACS) and ACC oxidase (ACO) enzymes   display an increase in their expression at the ripening initiation stage (Breaker) and reach a peak at the pink or fully ripe stage; one gene (ERF.F2) displays an increase in transcripts at the mature green (MG) stage and remains relatively highly expressed during ripening. Six genes  show an increase at a specific ripening stage (pink or fully ripe) but show no significant changes when ripening is initiated. The other genes  show obvious increases and peak transcript levels at the breaker stage, which supports their potential functions in ripening initiation  . Twelve itiation . It shoumulation .ERF.F5 , a repressor class ERF gene that does not respond to abiotic stresses or wounding, accumulate before ripening onset and decline sharply thereafter [ERF.F4  transcripts also increase significantly late in tomato fruit developmental and their transcript accumulation is negatively correlated with flavonoid biosynthesis [ERF genes appear to be involved in tomato fruit ripening, such as SlPti4  [ERF.F12 [Previous studies recognized that ERF.E4   and ERF.[ERF.F12 .ACC oxidase (ACO), ACC synthase (ACS), polygalacturonase (PG) and phytosynthase (PSY), etc., which are involved in multiple ripening pathways. In peach (Prunus persica L.), ABA is also involved in ripening regulation, and PpERF3 activates and PpeERF2 suppresses ABA biosynthesis by, respectively, positively and negatively regulating transcription of 9-cis-epoxycarotenoid dehydrogenase (NCED) genes PpeNCED2/3, involved in ABA biosynthesis [PpPG and activate its expression in peach fruit [Durio zibethinus L.), DzERF6 and DzERF9 have been recognized as the repressor and activator, respectively, of ethylene biosynthesis [Musa acuminata AAA group, cv. Cavendish) MaERF9, apple  MdERF1, and apple  MdERF3 are positive activators [ACO1 and ACS1 by binding to their promoters, and they have also been reported to physically interact with ACO1 protein [MaACO1 and expansins, in a process involving histone deacetylation [Malus domestica), MdERF2 is involved in three interactions regulating MdACS expression. The repressor MdERF2 and activator MdERF3 regulate the transcript level by binding to the promoters of MdACS genes. MdERF2 also inhibits MdERF3 activity by binding to the dehydration-responsive (DRE) element (a core sequence of CCGAC) in the promoter, indirectly suppressing MdACS expression. Additionally, a direct interaction between MdERF2 and MdERF3 modulates the binding of MdERF3 to the MdACS promoter, suppressing its expression in fruit flesh [MdPSY1 and MdPSY2 are involved in carotenoid accumulation in apple  fruits and are activated by AP2/ERF TFs such as AP2D15, AP2D21, AP2D26 and MdAP2-34 [Other climacteric fruits, in addition to tomato, also undergo ethylene synthesis and evolution at the onset of ripening and multiple members of the AP2/ERFs family appear to have ripening-related functions in other fruits (discussed later). Ethylene signaling via ERFs is transmitted to the promoters of genes such as ynthesis ,54. PpERch fruit . In duriynthesis . The bantivators ,58,59, wtivators . MaERF9  protein . MaERF11tylation . In applMdAP2-34 ,62.CitSWEET11d expression in citrus (Citrus unshiu), which promotes sucrose accumulation in both citrus and tomato fruit [CitAP2.10 is involved in regulating the synthesis of (+)-valencene in Newhall orange (Citrus sinensis), by upregulating the transcription of terpene synthase 1 (CsTPS1) [CsTPS16 promoter and contributes to E-geraniol synthesis in sweet orange (C. sinensis Osbeck) fruit [FaQR) promoter. FaQR catalyses the last step in the 4-hydroxy-2,5-dimethyl-3(2H)-furanone biosynthesis pathway, which makes a major contribution to strawberry fruit aroma [Citrullus lanatus), the accumulation of ClERF069 transcripts is negatively correlated with the sugar and lycopene content, and its overexpression in tomato delayed the ripening process, reducing ethylene production and the accumulation of lycopene and β-carotene, confirming that ClERF069 negatively regulates fruit ripening [ERFs have also been reported to function in non-climacteric fruit ripening and development of quality attributes. This offers some support for the suggestion that ethylene signaling is required for the process, although another interpretation is that some ERF-like genes are actually regulated by other hormones. CitERF16 activates to fruit . CitAP2.(CsTPS1) ; CitERF7k) fruit . In strait aroma . In riperipening . Thus, tripening .rin, nor, Nr, are unable to ripen and do not rot for many months. The senescence process that normally begins after ripening greatly increases the susceptibility of fruits to pathogenesis and rotting. Alterations in fruit texture, increases in the accumulation of excess reactive oxygen species (ROS), increases in cell membrane permeability and metabolic disorder lead to irreversible damage, cell disintegration and increased sensitivity to fungal infection [Ripe fruit are much more sensitive to pathogen infection then unripe ones, as shown by taking the climacteric fruit tomato as an example. First, tomato pleiotropic ripening mutants, e.g., nfection ,69,70. Tnfection  and, tognfection ,27. For nfection ,73. Thirnfection . ROS is nfection .B. cinerea, Rhizopus stolonifera, Fusarium acuminatum, C. gloeosporioides, and plant immune responses against these fungi involve pathogen-associated molecular pattern (PAMP) recognition and mitogen-activated protein kinase (MAPK) cascades. Phytohormones such as ethylene, JA and SA also coordinate and regulate the production of multiple complex stress-related secondary metabolites [Fruit pathogens include pathogenic fungi, bacteria, viruses, viroids, etc. . Compareabolites .pathogenesis-related (PR) genes, and the accumulation of antimicrobial compounds [PR genes are regulated by several plant hormones, such as SA and JA [PR genes by binding to the W-box in their promoters [PR-1 during infection by Piriformospora indica, and ERF9 negatively regulates defense gene expression mediated by ET/JA signaling in response to B. cinerea infection [Active defenses switched on when plants are infected by pathogens include an oxidative burst, expression of defense-related genes, such as ompounds ,76,77. PA and JA . TFs areA and JA . WRKY62 A and JA ,80, and romoters . In addiromoters  by bindiromoters  element,romoters ), and thromoters . In contnfection .B. cinerea causes one of the biggest problems, and the tomato-B. cinerea pathosystem has become a model for studying quantitative host–pathogen interactions [B. cinerea infection, we carried out comprehensive transcriptomic profiling of tomato ERF genes in WT fruits among AC_healthy, AC_wounded (wounded but not inoculated), AC_B. cinerea (wounded with B. cinerea inoculation) at two different ripening stages mature green (MG) and red ripe (RR)) using information from the NCBI database. A heatmap representing their expression pattern shows 33 genes, excluding those with low levels of transcripts (selected by FPKM > 10)  a.ERF.A1, A4, B2, B12, B13, C1, C3, C4, C6, D2, D6, D7, F4, F5, G2, H9) display an obvious increase (fold change above 2.0) in their expression after B. cinerea inoculation compared to wounding alone at either or both ripening stages (MG and RR), and seven of them  have strongly increased transcript levels (fold change above 10). Transcripts of ERF.A1 and C6 were strongly induced at both MG and RR ripening stages; the other genes  showed no obvious increased transcript levels after wounding or B. cinerea inoculation.These 33 genes have different pathogen-response-related patterns; 16 genes (B. cinera (ERF.A2 (ERF1) and ERF.F5 (ERF3) were upregulated in fruit after B. cinerea infection at the MG and RR stage, respectively [Pathogenesis-Related (PR1-5), Plant Defensin (PDF1.2), the pathogenesis response marker gene PR-STH2 (formerly pathogen-activated gene of potato and the corresponding protein) and Thionin (Thi2.1) involved in B. cinerea infection [Related-to-ABI3/VP1 (RAV1), MYC2, cytokininresponsefactor1 (SlCRF1), tomato stress-responsive factor 1 (TSRF1), Pti5 and Pti6 involved in B.cinerea resistance [Multiple ERF TFs are involved in tomato fruit resistance to . cinera . Expressectively . ERF.A3,nfection ,88,89,90sistance ,90,91,92SlERF2 (ERF.E1) gene participates in the disease resistance response and ERF2 overexpression enhanced tomato fruit resistance against B. cinerea. Methyl jasmonate (MeJA) is involved in this process and supplying it externally increased ethylene production, chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia-lyase activities, and increased the PR proteins and phenolic content. Furthermore, MeJA enhanced the disease response in ERF2-overexpressing tomato fruit, whereas the effect was reduced in antisense SlERF2 tomato fruit [ERF.B2 (SlERF5) and ERF.F5 (SlERF3b) plays an important role in the immune response to B. cinerea by upregulating the JA/ET signaling pathways [B. cinerea infection leads to the upregulation of SlERF.A1, SlERF.A3, SlERF.B4, and SlERF.C3 defense signaling hormones such as SA, MeJA, and 1-aminocyclopropane-1-carboxylic acid . The inhibition of either SlERF.B1 or SlERF.C2 by virus-induced gene silencing (VIGS)-proved lethal and silencing of SlERF.A3 (Pit4) significantly reduced vegetative growth of tomato plants. Silencing of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 increased the susceptibility to B. cinerea and reduced accumulation of jasmonic acid and ethylene-responsive defense genes and H2O2 accumulation, which is involved in the plant hypersensitivity response. In response to B. cinerea, SlERF.A3 silencing also reduced the resistance to Pseudomonas syringae pv. tomato (Pst) DC3000, whereas silencing of SlERF.A1, SlERF.B4 or SlERF.C3 had no effect on resistance to this bacterium [The tomato to fruit . The ovepathways . B. cineacterium .ripening inhibitor (rin) [non-ripening (nor) [colorless non-ripening (cnr) [Never-ripe (Nr) [A typical sign of tomato fruit ripening is the burst of ethylene production, which is essential for ripening initiation and progression ,37. Compor (rin) , non-ripng (nor) , colorleng (cnr)  and Neveipe (Nr) , make itnor and rin fruit have similar disrupted ripening phenotypes and severely inhibited ethylene production [Nr fruit have disrupted ethylene signaling, which inhibits the ripening phenotype, and Cnr fruit remain partially unripe because of genome DNA methylation [B. cinerea infection is determined by disease incidence and disease severity  after inoculation. It was reported that MG-like fruit of Cnr was the only unripe fruit susceptible to B. cinerea infection, and Cnr RR-like showed greater susceptibility than wildtype (WT) Ailsa Craig (AC) fruit. Both MG-like and RR-like stages of rin fruit showed similar or slightly reduced susceptibility compared to WT AC fruit [Nr fruit were fully resistant at the MG-like stage but only partially resistant at the RR-like stage [It has been reported that mutant oduction ,103, whihylation . Tomato AC fruit , and Nr B. cinerea infection in WT, rin and Nr tomato fruit, which was shown by the fold change of ethylene production comparing inoculation to wounded fruits at 3 days post-inoculation (dpi), at the MG stage of AC (1.3-fold), rin (15.5-fold), nor (4.4-fold), Nr (22.5-fold) fruit and RR-like stage of AC (2.6-fold), rin (75.0-fold), nor (3.0-fold), Nr (3.2-fold) fruit [Cnr fruit after inoculation was not checked). If ethylene was involved in disease susceptibility or response, in WT AC and rin fruit, treatment with 1-MCP might be expected to reduce their susceptibility to infection by pathogens compared to controls, but 1-MCP-treated WT RR fruit were not more resistant [However, ethylene production was induced by ge of AC .3-fold, ge of AC .3-fold, cnr, medium sensitive WT AC, slightly resistant rin and resistant nor ripening mutants. Nr was not considered due to its altered ethylene signaling mechanism. In interpreting the results of these experiments, it is important to recognize that it has recently been demonstrated that the nor and rin mutations produce aberrant TF proteins (either truncated NOR in nor or fused RIN-MC in rin) that have, through mutation, acquired the ability to negatively regulate some of their gene targets [CNR knock-out fruit displayed only slightly delayed ripening, which is puzzling since it was previously recognized as a master ripening regulator [B. cinerea has been re-examined using knocking out (KO) technology, where RIN is absent [rin mutant, where MADS-RIN is a repressor [rin (cnr (nor (B. cinerea infected (wounded plus B. cinerea) fruits.To understand more about the role of ERFs in fruit ripening, we compared the accumulation of their transcripts at the MG and RR ripening stages in the extremely sensitive  targets ,110,111.egulator . Functios absent , which cepressor . The expsor [rin b, cnr  show obvious increases (fold change above 2.0) in response to B. cinerea infection and ERF.A1, A4, C1, C3, C4, C6 and ERF.A5, B12, H9 display much higher transcript levels (fold change above 10.0) at one or both ripening stages  show obvious increases (fold change above 2.0) in response to B. cinerea infection, and 2  and 11  of them display strongly induced transcript levels (fold change above 10.0) at one or both ripening stages  showed an obvious increase (fold change above 2.0) in response to B. cinerea infection, and 1 (ERF.C6) and 3  were strongly induced (fold change above 10.0) at both or one ripening stage  showed obvious increases in all of the four tomato types; transcripts of six genes  were obviously increased in AC, rin and cnr, but not in the B. cinerea-resistant nor mutant fruit, and only ERF.E1 expression was significantly induced in three ripening mutant but not in WT AC fruit. The susceptibility to B. cinerea infection of AC and rin mutant fruit was similar, and they share the expression of fifteen genes in common, which displayed obvious upregulated expression patterns in both fruit during the response to infection  and Rhizopus stolonifer (R. stolonifer) have also been reported to infect various plant tissues, especially fleshy fruit [F. acuminatum is one of the most toxic species of Fusarium and induces host cell death and tissue necrosis and by producing strong mycotoxins, such as trichothecene and fumonisins [R. stolonifer is able to grow rapidly and is a very destructive postharvest pathogen [Gray mold, caused by ractions . Both Fuhy fruit . F. acummonisins . R. stolpathogen , causingERF genes in WT fruits of healthy, uninoculated wounded F. acuminatum (wounded with F. acuminatum inoculation), R. stolonifer (wounded with R. stolonifer inoculation) at two different ripening stages (MG and RR) was carried out using information from the NCBI database. A heatmap representing their expression pattern shows 37 ERF genes excluding those with a low level of transcripts (selected by FPKM > 10) (Comprehensive transcriptomic profiling of tomato KM > 10) .ERF.A1, A4, B2, C1, C6) showed an obvious increase (fold change above 2.0) compared to wounded fruits after F. acuminatum inoculation, but there were no obvious changes in levels of transcripts after R. stolonifer inoculation  showed an obvious increase (fold change above 2.0) in their expression after F. acuminatum inoculation compared to wounded fruit, and three  showed strong induction of transcript levels (fold change above 10). At the RR stage, after R. stolonifer inoculation, 22 genes  displayed an obvious increase (fold change above 2.0) in their expression and nine of them  were strongly induced (fold change above 10). The others  showed no obvious increased transcript levels after F. acuminatum or R. stolonifer inoculation  in tomato-fruit-enhanced resistance to Rhizopus nigricans, and this was associated with a substantial accumulation of transcripts of PR1a, PR5, Chi1 and PAL. It was concluded that although changes in ethylene production can occur, it does not play a pivotal role in fruit resistance to R. nigricans [ERF.C1  is thought to be involved in the chitosan (CHT)-induced systemic acquired resistance (SAR) response [ERF.B3  and ERF.D2 are also induced by infection by Trichoderma harzianum (strain T22) and aphid Macrosiphum euphorbiae infestation [ERF.D2  gene is required for N. benthamiana non-host resistance to Xanthomonas oryzae pv. Oryzae [ERF68  silencing has been shown to increase susceptibility of tomatoes to two incompatible Xanthomonas spp. and is associated with the altered expression of genes involved in ethylene production and SA, JA and hypersensitive response pathways. Target genes assay by chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) indicated that ERF68 is involved in promoting response to infection, including hypersensitive cell death, by modulating multiple signaling pathways [Except for above expression patteren related to fungal pathogen response, multiple ERFs also function in resistance to other pathogens, as listed in igricans . Tomato response . ERF.B3 estation  and the . Oryzae . ERF68 -silenced tomato plants showed susceptibility after inoculation with S. lycopersici, which might be due to a decreased hypersensitive response involving reduced catalase, peroxidase and superoxide dismutase leading to a reduction in ROS production. Furthermore, the results indicated that ERF2 may directly or indirectly regulate Pto, PR1b1 and PR-P2 expression, believed to be involved in the defense response, thereby also enhancing tomato resistance [Tomato SlERF01  activates the expression of nfection . ERF2  in tomato and tobacco activates the expression of PR genes, enhancing plant resistance to R. solanacearum, the causative agent of bacterial wilt disease [TSRF1 is enhanced by ethylene-induced salicylic acid accumulation and activates the expression of PR1, PR2 and PR3 [The transcriptional activator TSRF1 enhances plant resistance to the bacterial pathogen  disease . Express and PR3 .SlERF84 (ERF.D6) overexpression in Arabidopsis plants, on the other hand, led to more severe symptoms with extensive chlorotic lesions after inoculation with Pst DC3000. Several other well-documented genes, such as AtPR1 and AtPR3, have been suggested to play important roles in resistance to pathogens, and their transcript levels were much lower in ERF.D6-overexpressign lines compared to controls, after inoculation with Pst DC3000 [AP2d in Solanum pimpinellifolium L3708 conferred greater resistance to Phytophthora infestans (P. infestans) infection [t DC3000 . The miRnfection .Begomovirus in the Geminiviridae family induces serious damage to tomato production and quality due to the serious symptoms, which include leaf yellowing and curling as well as the occurrence of shrunken new leaves and cessation of plant growth [ERFs were identified in response to tomato TYLCV using transcriptome data; Two of them (Soly19 (ERF.C6), Soly36 (ERF.B2), Soly66 (ERF.B3), Soly67 (ERF.B1) and Soly106 (ERF.C4)) were selected for further assay and showed different response to TYLCV virus between these cultivars [The monopartite tomato yellow leaf curl virus (TYLCV) of the genus t growth . Five diultivars .ERF genes play critical roles not only in tomato fruit resistance to pathogens but also in other fruit of both climacteric and non-climacteric  types, listed in MaERF1 [CpERF1, 2, 3, 4 from papaya that appear to play different roles during fruit development, ripening and responses to stress. CpERF2 and CpERF3 are closely associated with fruit ripening, and the accumulation of CpERF1, CpERF3 and CpERF4 transcripts were induced by the ethylene perception inhibitor 1-MCP. Conversely, CpERF2 was repressed by 1-MCP and its transcripts accumulated in response to ethylene. CpERF2 and CpERF4 are involved in the response of papaya to C. gloeosporioides stress, and the accumulation of CpERF2, CpERF3 and CpERF4 transcripts is rapidly activated by low temperatures, whereas CpERF1 was induced by high temperature.Interestingly, heat (52 °C for 3 min) reduced lesion sizes in infected banana, and it has been suggested that heat activates defense responses by the up-regulation of MaERF1 . Li et aMaERF1  characteFragaria vesca) fruit infected with B. cinerea, the expression of WRI1, ERF061, ERF053 were significantly upregulated in unripe (white stage) fruit [Malus domestica) ‘Jonagold’ fruit, accumulation of transcripts for all four MdERFs (MdERF3–6) was ethylene-dependent and induced by wounding or by B. cinerea infection [MdACS genes and represses its expression by recruiting repressor MdERF2 [MdERF11 in apple  callus cells significantly increased their resistance to B. dothidea infection, whereas silencing MdERF11 resulted in reduced resistance. MdERF11 increases plant defense against B. dothidea by stimulating the SA biosynthesis pathway [Malus domestica Gala) interacts directly with MdbHLH92 to mediate resistance to powdery mildew by upregulating the JA and SA signaling pathways [VvERF1 in grape (Vitis vinifera Kyoho) fruits reduced the susceptibility to B. cinerea infection [Vitis quinquangularis VqERF112, VqERF114 and VqERF072 transcripts increased in response to the powdery mildew pathogen Pseudomonas syringae pv. Tomato (Pst) DC3000, B. cinerea and to treatments with ET, SA, MeJA or ABA hormones. When VqERF112, VqERF114 and VqERF072 were overexpressed in Arabidopsis, resistance to Pst DC3000 and B. cinerea increased and the accumulation of transcripts for SA signaling-related genes AtNPR1 and AtPR1 and JA/ethylene signaling-related genes PLANT DEFENSIN 1.2(PDF1.2), LIPOXYGENASE 3 (LOX3), BASIC CHITINASE (PR3) and HEVEIN-LIKE (PR4) also increased [In strawberry (e) fruit . Transcre) fruit . In applnfection . Noticear MdERF2 ,59, whic pathway , and MdEpathways . Overexpnfection . In Chinncreased .VvERF041 and VvERF069, VvERF071, VvERF072 and VvERF099 showed they were induced after infection of a B. cinerea-resistant variety, Shuangyou (Vitis amurensis) and a susceptible variety, Red Globe (Vitis vinifera), indicating that they have a potential role in responding to attack by pathogens [Vitis pseudoreticulata were associated with disease resistance, and it was demonstrated that VpERF2 and VpERF3 over-expression in transgenic tobacco and VpERF1 overexpression in transgenic Arabidopsis led to enhanced resistance to the fungal pathogen Phytophtora parasitica var. nicotianae Tucker and also the bacterial pathogen Ralstonia solanacearum [Expression profiling of genes such as athogens . ERF memnacearum .Citrus sinensis), the role of CsAP2-09 was characterized using over-expression and RNAi silencing strategies. The diseased lesions and disease index in transgenic citrus overexpression lines infected with Xanthomonas citri subsp (Xcc) were significantly decreased while they were significantly enhanced in RNAi lines. Comparison of the transcriptomes of WT and overexpression lines revealed that some of the genes associated with increased expression were involved in phenylpropanoid biosynthesis, pathogen responses, and transcriptional regulation [In citrus  are not very high. Twenty three ERF genes  respond to B. cinerea infection, and eight ERF genes also respond to infection by three fungi investigated; five of these ERF genes  were not involved in ripening initiation or progression, and the other three  showed a peak expression at a specific ripening stage; twenty-eight ERF genes could respond to one or more fungi; five of them  also showed ripening-related expression patterns, and could be recognized as both tomato fruit ripening and fungal resistance regulators.Of the 81 identified tomato ERF genes, eighteen are recognized as the best candidates for being involved in ripening initiation and progression, although the transcript levels of one  and non-climacteric fruits . Different ERFs regulate aspects of ripening and responses to infection and are involved in the stress-related synthesis of PR proteins, phytohormones ethylene and JA signaling pathways. In tomato, key ERFs regulating ripening, responses to infection, or both have been identified . Further


High-grade serous ovarian carcinoma (HGSOC) represents the most common form of epithelial ovarian carcinoma. The absence of specific symptoms leads to late-stage diagnosis, making HGSOC one of the gynecological cancers with the worst prognosis. The cellular origin of HGSOC and the role of reproductive hormones, genetic traits , chromosomal instability, or dysregulation of crucial signaling pathways have been considered when evaluating prognosis and response to therapy in HGSOC patients. However, the detection of HGSOC is still based on traditional methods such as carbohydrate antigen 125 (CA125) detection and ultrasound, and the combined use of these methods has yet to support significant reductions in overall mortality rates. The current paradigm for HGSOC management has moved towards early diagnosis via the non-invasive detection of molecular markers through liquid biopsies. This review presents an integrated view of the relevant cellular and molecular aspects involved in the etiopathogenesis of HGSOC and brings together studies that consider new horizons for the possible early detection of this gynecological cancer. Ovarian cancer (OC), situated among the most aggressive and deadly gynecological malignancies, is the fifth leading cause of cancer-related death in females in developed countries, with a total of 313,959 new cases diagnosed in 2020 worldwide  and 12,810 predicted annual deaths and 19,880 predicted diagnoses in 2022 in the US ,5,6. TheCurrent clinical approaches that guide physicians in managing EOC remain primarily based on imaging, histological evaluation, serum markers such as CA125, or predictive models, which do not display sufficient sensitivity and sensibility ,19; howeDifficulties in the early detection of HGSOC, before the disease develops to advanced stages, can be attributed to the lack of specific symptoms, which are usually missed or attributed to other pathologies . In clinMUC16 gene—in HGSOC compared to healthy ovarian tissue [Initial studies found elevated levels of CA125—a transmembrane cell-surface protein encoded by the n tissue ,27. CA12n tissue ,30,31,32n tissue ,42,43,44n tissue .The Risk of Malignancy Index (RMI) algorithm combines CA125 levels, ultrasound results, and menopausal status ; howeverOther strategies based on the Risk of Ovarian Cancer Algorithm (ROCA)  aimed toAlternative strategies such as the Risk of Ovarian Malignancy Algorithm (ROMA), OVA1™, or OVERA™ deserve special consideration given their U.S. Food and Drug Administration (FDA) approval and their relevance to routine clinical practice. ROMA, which displays high performance in menopausal patients, evaluates HE4 and CA125 levels and stratifies patients with adnexal masses into low or high risk of malignancy ,64. HE4 WT1, TP53, and P16 expression profile that distinguishes HGSCO from LGSOC [While ongoing gene expression profiling studies have suggested that HGSOC represents a highly heterogeneous pathology , expressom LGSOC , while Lom LGSOC .HOX, FAP (myofibroblast markers), and ANGPTL1/2 (markers of microvascular pericytes) or high expression of HMGA2, SOX11 (transcription factors), MCM2, and PCNA (proliferation markers) is correlated with worse prognosis [MUC gene (MUC16) expression or CXCL11, CXCL10 (chemokine ligands), and CXCR3 (receptor) expression [Different gene expression profiles reported by studies such as the Cancer Genome Atlas (TCGA) project ,83 can brognosis ,86,87. Apression ,86,87.NR5A1, GATA4, FOXL2, TP53, and BMP7 possess different profiles when comparing primary OC tumors and their metastases, and could eventually be used to predict patient survival [BRCA1, ATR, FANCD2, BRIP1, BARD1, and RAD51) is associated with a better prognosis in HGSOC cases, whereas the expression of epithelial-to-mesenchymal genes  is associated with a worse prognosis [Gene expression panels such as survival . Additiorognosis .HIF1α and TP53 in therapy-unresponsive patients as an indicator of a worse overall prognosis [CTNNBL1, CKB, GNG11, IGFBP7, and PLCG2) for classifying wild-type BRCA HGSOC patients into sensitive- and resistant-to-therapy groups [Studies have also assessed differential gene expression in HGSOC to guide the optimal therapeutic choice and measure expected responses . A meta-rognosis . Lee et rognosis . Most rey groups .UBE2Q1 as a prognostic biomarker associated with poor relapse-free overall survival in HGSOC patients [Novel bioinformatic approaches are currently attempting to refine gene expression signatures that predict therapeutic responses  and diffpatients .Overall, using DEG analysis in early diagnosis has the potential to improve the management and survival of HGSOC patients.Scientists and physicians have attempted to elucidate the possible cellular origin of HGSOC for over a century; unfortunately, a clear origin has yet to be established, since no specific precancerous lesion has been recognized, which represents a significant obstacle to early diagnosis approaches ,96,97.The first theory for HGSOC’s cell of origin points to the ovarian surface epithelium (OSE) . FathallBRCA1 or BRCA2 mutations undergoing salpingectomies [BRCA gene mutations [P53 gene mutations, and a strong correlation in CCNE1 copy number amplification [The observation of genetically and phenotypically altered cells  in the ciliated end of the fallopian tube in female patients carrying ectomies ,106 provectomies ,110,111 ectomies ,112,113.ectomies , includiutations ,115, idefication ,117. STIfication ,118,119.fication  and telofication ,121; thefication ,123. Latfication , which wAlthough recent evidence suggests the likelihood of a tubular origin of HGSOC precursor lesions, a possible dual origin (OSE or FTE) for this tumor is also currently accepted ,127,128.Gonadotropin hypothesis: The risk of serous EOC increases due to the excessive ovarian tissue uptake of FSH and LH [(a) H and LH ,137. TheH and LH  and the H and LH ,140 or EH and LH  support Androgen/progestin hypothesis: This dual hypothesis acknowledges high androgen levels  or obesity) as an EOC risk factor [(b) k factor ,143,144 k factor ,133. Higk factor ,146 in Hk factor ,148 and k factor . Conversk factor ,150.The heterogeneous expression of estrogen and the estrogen receptor (ER) alters depending on tumor stage and subcellular location and even changes with different prognoses in HGSOC patients ,129,130;In line with these two hypotheses, differing physiological conditions could be considered to be risk factors  or protective factors . During ovulation and menopause, elevated FSH and LH flux from the pituitary gland to the ovarian epithelium increases the risk of tumor development . Indeed,Based on this knowledge, strategies that consider the expression of AR, ER, and PR have been explored to predict survival rates and responses to therapy in HGSOC ,162.The complex genomic landscape of HGSOC, which arises from a combination of inherited and/or somatic mutations and chromosomal abnormalities, epigenetic alterations, and signaling pathway dysregulation , has beeBRCA1 and BRCA2 genes, present in 13–22% of HGSOC cases [RAD51, BRIP1, PALB2, CHEK2, MRE11A, RAD50, and ATM, which belong to the Fanconi anemia–BRCA pathway and play crucial roles in the HRR system for double-stranded DNA breaks [RAD51 [BRCA genes [HGSOC possesses a robust heritable component involving germline mutations that contribute to the development of disease in 25% of cases . GermlinOC cases ,185,186,OC cases . Other rA breaks ,189,190.A breaks ,191,192.A breaks ,194. News [RAD51  or calcuCA genes .Genome-wide association studies (GWASs) have revealed associations between single-nucleotide polymorphisms (SNPs) and pathogenic phenotypes within the population  or the iHOXD9, CDC42, and CDA8 [CCNE1, CPNE1, HEATR3, and STRCP1 [As most SNPs occur in non-coding regulatory regions, transcriptome-wide association studies (TWASs) and expression quantitative trait locus (eQTL) analyses have sought to overcome this drawback when studying disease predisposition. Lawrenson et al. described three statistically significant eQTL associations for and CDA8 , while Gand CDA8 , indicatd STRCP1 . Novel ad STRCP1 . These dTP53, the most frequently mutated gene associated with HGSOC, functions as “the guardian of the genome” by regulating upstream or downstream genes to control tumor suppression and maintain cellular homeostasis [TP53 mutations in around 93–96% of HGSOC patients [TP53 mutation rates of 100% in late-stage cases and 82% in early-stage cases [TP53 mutational load in HGSOC patients [TP53 mutations are mostly missense (60.52%) but can also be frameshift (15.24%), splice-site (10.53%), nonsense (10.73%), and in-frame (3.22%) mutations [BRCA1/2 and RAD51D may also undergo somatic mutations [CSMD3 and FAT3 (tumor-suppressor genes), MLH3 (with known roles in DNA mismatch repair), and CDK12 (RNA splicing regulation) [eostasis ,202. Thepatients ,201,203.ge cases , consistpatients . TP53 muutations . R273, Rutations ,206,207 utations ,211,212.utations ,213,214.ulation) ,171.Corona et al. described several point mutations that converge on TEAD4/PAX8-binding sites during OC’s progression , highligTP53 mutations [CNNE1, MYC, and MECOM) and focal deletion areas  [CNNE1 amplification [PTEN loss [Chromosomal instability (CIN) leads to aberrant gene expression patterns and protein functions related to primary tumor growth/development and metastatic burden in several cancers . Specifiutations , cell-cyutations . CIN affand NF1) ,172; confication  or PTEN TEN loss . InteresTEN loss .MLF1, BRCA1, CCNE1, TP53, ARID1A, MYC, and PIK3CA [TP53 mutations, CNNE1 amplification, or RB1 loss [Chromothripsis, a well-reported form of CIN with a critical role in cancer , is corrd PIK3CA . FinallyRB1 loss ,225.Epigenetic mechanisms involving DNA methylation , histone modifications, or non-coding RNAs (ncRNAs) can regulate gene expression, participate in the onset of HGSOC, and influence therapeutic outcomes ,226,227.BRCA1 promoter hypermethylation in families affected by HGSOC without BRCA1/2 germline mutations is correlated with allelic BRCA1 loss [DNA methylation is generally associated with decreased gene expression and can affect the promoters of tumor-suppressor genes. For instance, CA1 loss . HypometCA1 loss ,177. MorCA1 loss .NOXA [Histone modifications carried out by methyltransferases (HMTs) or deacetylases (HDACs) have also been implicated in the development of HGSOC . SpecifiNOXA . FurtherNOXA ,231.CTBP1-DT lncRNA and its link to the malignant behavior of HGSOC cells and disease progression, suggesting this ncRNA as a possible biomarker for early diagnosis or a therapeutic target [The expression of ncRNAs—specifically, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)—represents an additional epigenetic mechanism with importance in the pathophysiology of cancer ,232. A sc target .Relevant signaling pathways involved in HGSOC include RB (67% of cases), PI3K/RAS (45% of cases), NOTCH (22% of cases), and FOXM1 (85% of cases) ,172,237 RB1 loss combined with HRD are associated with unusually long survival rates after chemotherapy, providing opportunities for more precise stratification of HGSOC [The RB pathway controls the G1-to-S phase transition in mammalian cells ,239. Oveof HGSOC .PIK3CA (20% of cases) or AKT isoforms (15–20% of cases) [KRAS oncogene amplifications (11% of cases), MAPK amplification (20% of cases), NF1 loss (8% of cases), and other less frequent changes in NRAS or BRAF [Alterations in the PI3K/Akt/mTOR pathway in HGSOC mainly derive from genomic amplification of f cases) ,243. Nonf cases) ,245. Ras or BRAF .BRCA1/2 promoters represent downstream targets of the RB, PI3K, and RAS signaling pathways, offering opportunities for therapeutic interventions using CDK, RTK, and MAPK inhibitors such as cediranib, nindetanib or pazopanib  [Interestingly, survival ,247), or tumors) ,248. Oth tumors) ,250.E2F copy number gain and amplification or the overexpression/inactivation of FOXM1 upstream transcription factors such as ERBB2 (tyrosine kinase receptor) or TP53, might cause overexpression of FOXM1 in HGSOC. Abnormal FOXM1 overactivation, in addition to copy number gains on this gene, coincides with abnormal cell-cycle processes that promote cell proliferation, cancer stemness, genomic instability, and poor prognosis [Different molecular traits, such as rognosis ,252.CCND, p21CIP1, NF-κβ, and c-MYC [CXCR4/SDF1α signaling dysregulation [DK1 overexpression  [Notch family members are transmembrane receptors that, upon binding and cleavage, migrate to the nucleus and target the promoters of genes such as nd c-MYC . Other fgulation  or DK1 o ligand) , with boThe upward trend in the number of clinical trials focused on OC—and on HGSOC in particular—has translated into an increase in survival and positive outcomes when comparing the last five years with the early 2000s .Up to 949 clinical trials have been registered in the clinicaltrials.gov portal of the U.S. National Institutes of Health (NIH) within the last five years , with 19It should be noted that while much existing research has focused on early and non-invasive detection, the need for a very large study population for general screening represents an obstacle to the design of such studies ,258,259.Liquid biopsies, based on the non-invasive detection of molecular biomarkers (mainly through NGS) released from tumor cells into the bloodstream, represent an alternative approach to the early detection of HGSOC . Here, wCTCs are released from a primary or metastatic tumor and shed into the peripheral blood, where they can be isolated by size selection  or by tactDNA is the fraction of total cfDNA released from tumor cells, mainly through necrosis and/or apoptosis . ctDNA lOPCML, RASSF1A, RUNX3, and APC [COL23A1, C2CD4D, and WNT6 [HOXA9 and HIC1 [TP53 mutations and other somatic variants have also been detected in ctDNA from stage I and II HGSOC cases [Early detection strategies using ctDNA rely on evaluating methylation patterns and detecting somatic mutations or aberrant fragmentation patterns ,279,280. and APC ,282,283,and WNT6 , or HOXAand HIC1  have beeOC cases ,287,288.OC cases ,290, as OC cases . FinallyOC cases .miR-1246, miR-595, and miR-2278) [CASC11 [Detection of ctRNA, as a fraction of the total cfRNA released from tumor cells, represents a promising approach in liquid biopsies, since this strategy offers a snapshot of the transcriptional landscape and mirrors potential pathogenic processes . ctRNA hiR-2278) ,297 overiR-2278)  or lncRNiR-2278)  and CASC [CASC11 ) upregul [CASC11 .Exosomes are a subtype of EVs that are released from tumors and extravasate into the bloodstream, where they can avoid the immune system , promoteRanked as one of the most aggressive and deadly forms of gynecological cancer, HGSOC is currently considered to be a public healthcare issue that significantly impacts female patients’ quality of life. Current challenges arise from the difficulty in establishing an early and effective diagnosis in patients with adnexal masses. HGSOC is mostly diagnosed in advanced FIGO stages (III-IV) due to the lack of symptoms and the use of traditional diagnostic methods. The performance of CA125—the only available molecular serum biomarker—alone or in combination with other biomarkers or TVS, has yet to support significant reductions in mortality rates.TP53 gene, genes belonging to the HRR system, and CIN. Furthermore, alterations in multiple signaling pathways and epigenomic mechanisms have also been described.Other strategies have focused on the possible dual origin of HGSOC (the FTE or OSE) and the influence of hormones in promoting or protecting against the development of disease. Furthermore, HGSOC is characterized by extensive genomic instability, promoted by almost universal mutations in the Research in this field is rapidly moving forward, allowing the molecular management of HGSOC by encompassing multiple disease-associated features in high-throughput, personalized approaches. Proof of such advances includes the putative gene panels and expression analyses intended for the stratification of HGSOC, the establishment of treatment-guided decisions, and the monitoring of disease progression, which can be extended to early detection.Most recently, liquid biopsies have gained momentum as an emerging strategy in the field of gynecological oncology, providing a non-invasive approach with promising applications from several clinical standpoints, including early diagnosis of adnexal masses or monitoring of HGSOC patients’ prognosis and treatment effectiveness.This review sought to provide an update of those studies concerning the molecular and cellular characterization of HGSOC tumors, which may serve as a starting point for the design of new strategies aimed at improving management of this subtype of cancer.


Traditional theory based on physical constraints has assumed that b is 2/3 or 3/4, following natural law, but hundreds of studies have documented extensive, systematic variation in b. This overwhelming, law-breaking, empirical evidence is causing a paradigm shift in metabolic scaling theory and methodology from ‘Newtonian’ to ‘Darwinian’ approaches. A new wave of studies focuses on the adaptable regulation and evolution of metabolic scaling, as influenced by diverse intrinsic and extrinsic factors, according to multiple context-dependent mechanisms, and within boundary limits set by physical constraints.Life's size and tempo are intimately linked. The rate of metabolism varies with body mass in remarkably regular ways that can often be described by a simple power function, where the scaling exponent ( For over a century, these two key features of life have also attracted much attention because they are usually inversely related in remarkably regular law-like ways that can be described by simple mathematical formulae. In particular, the relationship between the rate of metabolism (ar plot) –8. Accorar plot) –7,9,10. b have attracted much interest for over the past 150 years. During the late 1800s and early 1900s, many biologists claimed that b was universally 2/3 or nearly so, the so-called ‘surface law’, based on simple Euclidean geometry of organismal surfaces across which metabolic resources, wastes and heat are exchanged [The value and significance of the metabolic scaling exponent xchanged –13. Thisxchanged ,15. Belixchanged , Williamxchanged  in threexchanged ,16–19. Hxchanged –31, have. 2The surface law and 3/4-power law, and theory supporting them, have so captivated the minds of many scientists that from the early 1900s to early 2000s, numerous studies showing significant diversity of metabolic scaling have been largely ignored or regarded  as being the result of factors having secondary importance to those causing a presumed primary universal scaling pattern. Ironically, some proponents of the 3/4-power law have suggested that the immense diversity of life has so distracted many biologists that it has inhibited their proclivity to develop general, coarse-grained theory based on universal natural laws ,33, thusb value (i.e. 3/4)  . Despiteb values  that have been reported for several taxa of unicellular and multi-cellular organisms  relates systematically to various intrinsic  and extrinsic  factors (b between approximately 0.5 (or 2/3) and 1.0, as predicted by context-dependent, multi-mechanistic theory [b observed between endothermic versus ectothermic vertebrates [b values have been reported for grazing versus folivorous and vertebrate- versus invertebrate-eating mammals [b. For example, temperature effects have been shown to interact with salinity [Variation in the metabolic scaling exponent  relatesalinity , predatisalinity ,88, actisalinity , and modsalinity ,78,79,82. 4b) includes dramatic, remarkably regular shifts observed between the allometric scaling (b < 1) of resting animals and the isometric or near-isometric scaling (b ∼ 1) of torpid or maximally active animals [b in response to individual changes in reproductive state [b derives from artificial selection experiments [b [Effects of various intrinsic and extrinsic factors on metabolic scaling may occur via phenotypic plasticity, at least partially driven by biological regulation at the organismal level, or via genotypic evolution driven by natural selection or genetic drift at the population level. Evidence for regulated phenotypic plasticity of the metabolic scaling exponent  stages of grief after losing a loved one, including ‘denial’, ‘anger’, ‘bargaining’, ‘depression’ and ‘acceptance’ , and a r (a)b) in 1916 [Many scientists, especially theoreticians with backgrounds in the physical sciences, continue to believe in a universal or nearly universal 3/4-power law, despite enormous evidence that metabolic scaling is highly diverse across the tree of life figures  and 2, w in 1916 . This de in 1916 ,105,106. in 1916 ,116,117. in 1916 ,116–121. (b)During the last two decades, I have witnessed rancorous debates about the existence of the 3/4-power law and theory supporting it at several international scientific conferences. This rancor seems to be driven by conflict between investigators who have different worldviews about how science should be carried out, particularly in biology ,122,123, (c)Some investigators continue to support the existence of a 3/4-power law by modifying its range of applicability in four major ways. First, some argue that the 3/4-power law applies best to large-scale metabolic scaling relationships encompassing diverse species and taxa with a very broad range of body masses, rather than small-scale relationships within specific species or taxa ,19,32,54b is not fixed at 3/4 in either vertebrates or vascular plants, but varies considerably between 0.4 or less to 1.2 or more for both intra- and interspecific scaling relationships  for intraspecific metabolic scaling relationships is significantly more or less than 3/4 for many major taxa or ecological groups of organisms, such as angiosperms  [n = 28 [n = 58 [b varies substantially, not only for individual species values, but also for means  of many large groups of species (approx. 2/3 to 1), often differing significantly from 3/4, which is clearly not a ‘magic’ number [Third, some posit that a  scaling ,126–129  scaling , but see scaling ). This bearly so ,19,127. early so . Second,6, n = 9 ,131), ga [n = 29 ), arachn [n = 23 ), insect [n = 89 ; 0.94 ± species) ), reptil [n = 28 ) and pel [n = 58 ), as wel [n = 58 ,25,124. ’ number .b is related to statistical or methodological error [b tends to converge toward 3/4 for the most rigorous datasets that have sufficiently large sample sizes and broad body mass ranges, especially over two orders of magnitude [b values do not become centred on 0.75, but rather become increasingly confined within the broad boundaries of 0.5 and 1.0. In fact, a greater proportion of b values are significantly different from 0.75 for scaling relationships involving larger body mass ranges. For example, in an extensive survey of 642 scaling relationships for 218 animal species, 50.2% were significantly different from 0.75, which increased to 72.7% and 88.5%, respectively, for relationships with body mass ranges at or above 2 or 2.5 orders of magnitude [Fourth, some investigators claim that much of the variation in al error ,55,128, agnitude ,134) useagnitude . Increas (d)I have talked to colleagues who have given up studying metabolic scaling because they feel that the field has become too acrimonious and divided among largely self-isolated working groups, has failed to make substantive progress during the past few decades and (or) no longer shows promise for developing a general theory. However, as I point out later, general theory need not be monolithic and completely deterministic, but may be multi-faceted and context dependent ,30. An o (e)Since the 1950s, several investigators have accepted the demise (inadequacy) of the 3/4-power law ,125,127, (f)Many biologists are now embracing a fundamentally new worldview of metabolic scaling as being highly variable and adaptable, which is revolutionizing the theory, empirical study and practical application of metabolic scaling relationships, as discussed further next.. 6Biological scaling relationships have been traditionally regarded as physically or developmentally constrained ,135,136,The ongoing paradigm shift in the theory of metabolic scaling involves five fundamental overlapping changes in focus. Essentially, the primary emphases are shifting from (i) a single universal law to the diversity of metabolic scaling, (ii) a single primary deterministic mechanism to multiple, context-dependent mechanisms, (iii) rigid internal physical constraints to adaptable phenotypic plasticity and genotypic evolution, highly responsive to multiple internal  and external  causal factors, (iv) centralized to bounded physical constraints and (v) restrictive effects of physically constrained resource supply (RS) and metabolic waste removal across body surfaces and through anatomical transport networks to flexibly regulated and evolvable effects of multiple kinds of resource-demand (RD) that support various vital fitness-related activities.The shift in focus from an adherence to a single universal law (in particular the 3/4-power law) to embracing the exuberant diversity of metabolic scaling has already been described. Many investigators no longer regard this diversity as being random or secondary to a single primary, physically constrained law, but in itself of primary interest and importance.b) depends on the overall metabolic level of an organism , which in turn depends on activity level, temperature and other biological and ecological factors in diverse taxa for both intra- and interspecific relationships [Consequently, many investigators have recently formulated several kinds of multi-mechanistic models to explain the diversity of metabolic scaling . Theionships ,73,78,87ionships ,78,144.b, but clearly not all of the systematic effects of various intrinsic and extrinsic factors that have been observed [b [b.Each of several possible mechanisms may, by itself, potentially explain some variation in observed ,30. For erved [b ,126,145.erved [b ,30, mosterved [b ,20,36, eerved [b ,146, anderved [b ,72,73,95erved [b ,90,96,97b, rather than as the cause of any central tendency in b [b often varies between 2/3 and 1 in isomorphic organisms with isometric resource-demanding processes, and over even larger ranges in organisms with variable body shapes or allometric scaling of specific resource-demanding processes [b values in the middle of a frequency distribution for multiple diverse species do not necessarily follow from a single predominant mechanism, as sometimes thought [In addition, several investigators now view physical or geometric constraints as acting as boundary limits on the variation of ncy in b ,106,147.rocesses ,56,69,87rocesses ,82). Acc thought ,32,126,  thought ,82).b to be 2/3 or 3/4 , recently many investigators have identified body size-dependent variation in RD by various vital biological structures and processes  as a major cause of variation in b .Furthermore, although traditional theory focusing on the surface law or 3/4-power law has emphasized how physical and geometric constraints on RS and (or) waste removal may cause 3 or 3/4 b,c, receigure 5d ,144,146)igure 5d ,95,146. igure 5d ). Secondnditions ,13,30,98redators ,150. Thiredators ,150. In redators ,98,151. redators . FollowiLastly, although ‘Newtonian’ law-based approaches to metabolic scaling theory focus primarily on how organisms physically uptake, transport and use energy and other resources to support metabolism, ‘Darwinian’ context-based approaches additionally focus explicitly on how biological information, as embodied in various genetic and regulatory systems, is used to control the uptake, transport and use of resources . Consequ. 7Based on the extensive evidence discussed in this review, I do not believe that it is too outlandish to say that a scientific revolution in our understanding about metabolic scaling is occurring. In a scientific sense, investigators studying metabolic scaling are increasingly appreciating ‘diversity and inclusion’ by showing more awareness of the empirical diversity of metabolic scaling patterns, as well as more inclusiveness about the kinds of theory used to explain this diversity. For 25 years, RTN theory that has focused primarily on an ideal, non-existent 3/4-power law has dominated the metabolic scaling field, but many investigators are now invoking multiple mechanisms to explain the diversity of metabolic scaling that actually exists ,98,144. b [b in relation to diverse intrinsic and extrinsic factors [b within conspecific populations, and macroevolution of b across species, including, in particular, multi-variate selection on metabolic rate, body mass and other related traits [b [New multi-faceted Darwinian approaches focused on adaptable phenotypic plasticity and evolvability show much promise for increasing an understanding of the diversity of metabolic scaling. Recommendations for further research on little-understood topics include studies examining (i) how biological regulatory systems at the molecular, cellular and organismal levels control the phenotypic plasticity of metabolic scaling ,98,148; b ; (iii) rb ,129,152; factors ,104–106;d traits ,103,144;raits [b ,82,147; raits [b –88,148. raits [b ,78,144. raits [b ,102. An b [b values for rates of metabolizing drugs based on age and drug type [An increased appreciation of the extensive, law-breaking evidence for variable scaling of metabolism and other associated biological processes has many important practical scientific, medical, agricultural, forestry and conservation implications. For example, comparative studies of variation in metabolic rate or other related traits can no longer control for effects of body size by simply assuming that metabolic rate scales with body mass according to a universal 3/4-power law, but must now consider complex interactions between body size and various other factors of interest, as revealed by significant effects of these factors on b . In addirug type ,153,154.rug type ,127,155.The big picture is no longer a physically constrained, deterministically caused 2/3- or 3/4-power scaling law applying to most or all of life, but rather the pervasive occurrence of extensive variability in metabolic scaling at multiple taxonomic levels, owing to adaptable phenotypic plasticity and genotypic evolution that are highly sensitive to a variety of biological and ecological influences that are best understood with multi-mechanistic, context-dependent theory. In short, metabolic scaling is a ‘many-splendoured thing’ , p. 1633