Glucagon, a hormone secreted by pancreatic alpha cells, contributes to the maintenance of normal blood glucose concentration by inducing hepatic glucose production in response to declining blood glucose. However, glucagon hypersecretion contributes to the pathogenesis of type 2 diabetes. Moreover, diabetes is associated with relative glucagon undersecretion at low blood glucose and oversecretion at normal and high blood glucose. The mechanisms of such alpha cell dysfunctions are not well understood. This article reviews the genesis of alpha cell dysfunctions during the pathogenesis of type 2 diabetes and after the onset of type 1 and type 2 diabetes. It unravels a signaling pathway that contributes to glucose- or hydrogen peroxide-induced glucagon secretion, whose overstimulation contributes to glucagon dysregulation, partly through oxidative stress and reduced ATP synthesis. The signaling pathway involves phosphatidylinositol-3-kinase, protein kinase B, protein kinase C delta, non-receptor tyrosine kinase Src, and phospholipase C gamma-1. This knowledge will be useful in the design of new antidiabetic agents or regimens. The hormone glucagon, produced by pancreatic alpha cells, contributes to the regulation of blood glucose by promoting hepatic glucose production in response to declining blood glucose. However, its excessive secretion contributes to the development of type 2 diabetes , 2. MoreGcg) gene to produce proglucagon mRNA, which is translated to proglucagon, whose cleavage by prohormone convertase 2 produces glucagon [Glucagon synthesis involves transcription of the preproglucagon  [+ efflux to alter the membrane potential to a range that permits the opening of voltage-dependent Na+ channels; the resulting Na+ influx causes subsequent opening of P/Q type voltage-gated calcium channels (VDCCs); calcium influx through these VDCCs is coupled to fusion of glucagon vesicles with the plasma membrane, resulting in glucagon secretion; and too low or too high ATP levels induce excessive opening or closure of the K-ATP channels, respectively, leading to the inhibition of this pathway [+ channels activated by intracellular calcium  were recently found to contribute to glucagon secretion and were suggested to be useful in limiting voltage-dependent inhibition of P/Q type VDCCs during prolonged periods of low glucose [+ efflux was observed; instead, there was membrane depolarization due to Na+/K+ ATPase inhibition [As illustrated in hannels) , 8. Acco pathway –9. K+ ch glucose . Neverth glucose , 11. Forhibition , it has Other glucagon secretion pathways depend on the reduction of ER calcium content . For exa3) [3, which induces the release of calcium from the ER, thus raising the cytosolic calcium, which is amplified by SOCE and L-type VDCCs to promote glucagon exocytosis [+/K-ATPase activity for membrane repolarization, and preventing excessive opening of K-ATP channels [ER calcium release and glucagon secretion can be induced even at high glucose subsequent to the intracellular increase in cyclic adenosine monophosphate (cAMP) or inositol triphosphate (IP3) , 13, 14.ocytosis , 14–16. channels , 14, 17.+/K+ ATPase activity, inhibition of ER calcium release, and reduction of cAMP concentrations [Isolated alpha cells have a V-shaped glucagon secretion curve in response to increasing glucose concentrations from 0 mM, with maximal suppression at moderate glucose concentrations of 5-7 mM , 18. At trations –8, 13. ctrations , 13. Thetrations , 21.δ) [δ, Src, and EGFR should be important components of the suggested signaling pathway  and involving the generation of reactive oxygen species (ROS) can explain this phenomenon . This isδ) . Thus, P pathway .+) through SGLT-1 is responsible for initiating signaling, through PI3K activation that leads, via PKC-δ and Src, to NADPH oxidase (Nox) and the production of hydrogen peroxide (H2O2). This is based on the analogy that in cardiomyocytes exposed to high glucose, glucose transport through SGLT-1 induces Nox2 activation in a process dependent on sodium and glucose transport but not metabolism, and which is associated with PKC activation [+ entry through SGLT-1, can trigger activation of PI3K and Akt, upstream of Nox2 [According to tivation . SGLT1 wtivation . Membran of Nox2 . Akt pro of Nox2  and also of Nox2 , which p of Nox2 .γ1, even at high glucose such as 16.7 mM [γ1 through production of phosphatidylinositol 3-phosphate, but Akt can also activate this phospholipase, especially when EGFR is also activated [γ1 generates IP3, which causes ER calcium release and glucagon secretion as already described in Glucagon Secretion Pathways. PKC-δ promotes trafficking of glucagon secretory granules to sites close to L-type VDCCs that participate in ER-dependent glucagon secretion [Human alpha cells express the melatonin 1 receptor (MT1) . Melaton 16.7 mM . PI3K acctivated . PLC-γ1 ecretion . Thus, becretion , 38. Theecretion . Moreoveecretion , 39, 40.δ [γ1, and EGFR [γ and PI3-K [2O2). Hydrogen peroxide, via Src, activates PI3K [PI3K activates PKC-δ , which aδ –43. Src and EGFR , 44, 45.nd PI3-K . Nox protes PI3K , thus estes PI3K .+/K+ ATPase additionally promotes Src signaling by freeing the latter from an inhibitory interaction with the former, and this has been reported to be involved in the pathogenesis of obesity and cardiovascular dysfunctions [1O2) is a more selective ROS, which can be formed by the reaction of hydrogen peroxide with glucose [ε-(hexanoyl) lysine [+/K+ ATPase may be mediated by singlet oxygen according to Hydrogen peroxide-mediated Src activation depends on sulfenylation of two cysteine residues . ROS-medunctions , 49. Altunctions , this isunctions . Singlet glucose , oxidize glucose , and suc) lysine . Thus, iDysregulation of glucagon secretion starts before the development of type 2 diabetes , 55. TheInsulin inadequately inhibits glucagon secretion in insulin-resistant alpha cells , resultiPrediabetic individuals either revert to normoglycemia or progress to diabetes. Systemic oxidative stress is an important factor associated with progression to diabetes –73. Hypeδ, Src, and ROS generation in diabetic islets [In normal pancreatic islets, unlike isolated alpha cells, the rise in glucagon secretion at glucose concentrations above 7 mM is suppressed by paracrine action of somatostatin and insulin produced by beta cells and delta cells, respectively, and by gap junction coupling between these cells , 83; butc islets –27, 81. c islets , 84, 85.c islets , 85. On c islets , 84, 85.c islets . Oral adc islets . Apart fc islets . Notablyc islets . Althougc islets , it is lGlucagon-like peptide 1 (GLP-1) and GLP-1 receptor agonists such as exendin reduce hyperglycemia and are associated with lower risk of hypoglycemia , 90, 91.Exogenous insulin administration promotes somatostatin secretion, which aggravates hypoglycemia by reducing cAMP formation; while somatostatin receptor antagonists improve hypoglycemia by increased cAMP , and assAlthough according to δ inhibition [+/K+ ATPase-dependent Src activation and has been found to be beneficial against various other metabolic disorders [There has been increasing interest in the antidiabetic effects of the flavonoid quercetin, but this has mainly been limited to animal studies . Quercethibition , and it hibition . Furtherisorders .In healthy individuals, glucose increases ATP to promote glucagon secretion in hypoglycemia and to suppress glucagon secretion at higher glucose levels to prevent hyperglycemia. At high glucose, such as in the postprandial state, glucagon secretion is suppressed by paracrine action of somatostatin and insulin produced by beta cells and delta cells, respectively. Alpha cell dysfunctions such as insulin resistance, mitochondrial alterations, and oxidative stress contribute to the pathogenesis of type 2 diabetes and glucagon dysregulation in diabetes. A signaling pathway that can be initiated by glucose and sodium transport through SGLT-1 or by hydrogen peroxide promotes glucagon secretion and, if overactivated, may induce oxidative stress and ATP reduction as key contributors to glucagon dysregulation in diabetes. This pathway can be targeted in the search for new antidiabetic agents.


Lysine-specific demethylase 5B (KDM5B/PLU1/JARID1B) is found to be overexpressed in numerous malignancies, including breast, lung, skin, liver, and prostate cancer. Identification of molecules targeting the KDM5B enzyme could be a potential lead in cancer research. Although many KDM5B inhibitors with promising outcomes have been developed so far, its further application in clinical practice is limited due to toxicity and lack of target specificity. Here, we summarize the significance of targeting KDM5B in anticancer therapy and report the molecular docking studies of some known anti-viral agents, decitabine, entecavir, abacavir, penciclovir, and 3-deazaneplanocin A in the catalytic domain JmjC of KDM5B. These studies show the repurposing potential of identified anti-viral agents in cancer therapy. Studies over the past few decades and ongoing research reveal that, along with genetic alterations, epigenetic aberrations play a vital part in the evolution and progress of numerous diseases, including several types of cancers . EpigeneHistone, being the functional protein, not only actively takes part in gene expression, but also acts as a spool for DNA to wind around. Any modification on histone molecules via methylation, acetylation, phosphorylation, ubiquitylation, or sumoylation alters the compact chromatin architecture, leading to abnormal gene expression . HistoneLysine-specific demethylation is finely tuned by two enzyme categories, called lysine-specific demethylase (KDM1/LSD1) and the JumonjiC (JmjC) domain, encompassing histone demethylases (KDMs). JmjC, being the largest among two families, displays high substrate specificity, with demethylase activity targeting histones H3 and H4 . In mammThe KDM5/JARID1 subfamily consists of four interrelated histone demethylase enzymes—KDM5A, KDM5B, KDM5C and KDM5D—involved in several human biological processes, including hormonal response, stem cell regeneration, genomic stability, cellular proliferation, and differentiation ,9. KDM5BN-terminal Jumonji (JmjN) domain, a catalytic JmjC domain, an AT-rich domain (ARID), a C5HC2 zinc finger domain, a PLU1 motif and three plant homeobox domain . The domain architecture of KDM5B is highly conserved and homologous from yeast to humans. JmjN and JmjC are vital domains required for enzymatic activity, whereas AT-rich domain binds to GC rich DNA sequences. PHD domains contribute to substrate recognition in which it prevents reverse reaction ..2+ (20].These results also show that small molecules, which possess planar bicyclic heterocyclic rings, exhibit favorable binding toward the JmjC domain of human KDM5B. This is due to their ability to undergo interactions with the catalytic site metal ion, which is an important criterion in KDM5 inhibition and is consistent with previous studies ,77,88. OThe leading role of KDM5B oncogene in tumorigenesis and cancer progression is well established in various malignancies and is therefore, considered as a potential therapeutic target. Although many small molecule inhibitors of KDM5B have been identified so far, their possible clinical application in cancer treatment is a work in progress. This study provides a rationale on how repurposing antiviral drugs could contribute to the discovery and development of KDM5B inhibitors. We anticipate that these studies will stimulate further research in considering drug repurposing approaches to target KDM5B, and to discover novel therapies for various cancers.


Arabidopsis (TAA)/YUCCA) enzymes catalyze the key rate-limiting step of the tryptophan-dependent auxin biosynthesis pathway, from IPA (Indole-3-pyruvateacid) to IAA (Indole-3-acetic acid). Here, 13 YUCCA family genes were identified from Isatis indigotica, which were divided into four categories, distributing randomly on chromosomes (2n = 14). The typical and conservative motifs, including the flavin adenine dinucleotide (FAD)-binding motif and flavin-containing monooxygenases (FMO)-identifying sequence, existed in the gene structures. IiYUCCA genes were expressed differently in different organs  and developmental periods . Taking IiYUCCA6-1 as an example, the YUCCA genes functions were discussed. The results showed that IiYUCCA6-1 was sensitive to PEG (polyethylene glycol), cold, wounding, and NaCl treatments. The over-expressed tobacco plants exhibited high auxin performances, and some early auxin response genes  were upregulated with increased IAA content. In the dark, the contents of total chlorophyll and hydrogen peroxide in the transgenic lines were significantly lower than in the control group, with NbSAG12 downregulated and some delayed leaf senescence characteristics, which delayed the senescence process to a certain extent. The findings provide comprehensive insight into the phylogenetic relationships, chromosomal distributions, and expression patterns and functions of the YUCCA gene family in I. indigotica.Auxin is one of the most critical hormones in plants. YUCCA (Tryptophan aminotransferase of  ARF, Aux/IAA, GH3, SAUR, and LBD, which are involved in plant response to stress [OsIAA24 and OsIAA20 were up-regulated by high salt stress in Oryza sativa [GH3.13 increased under drought treatment, which resulted in IAA content decreasing, with enhanced resistance to drought [PIN2 were affected at a low temperature in Arabidopsis thaliana, which inhibited auxin transport in roots [IAA (Indole-3-acetic acid) is the main type of natural auxin in plants, which plays important roles in a series of growth and developmental processes, such as embryonic polarization, vascular bundle differentiation, apical dominance, shoot architecture, root formation, phototropic response, gravity response, and senescence ,3,4,5,6.o stress ,8. For ea sativa , and the drought . Furtherin roots .YUCCA genes play an important role in IAA biosynthesis process.IAA is generally synthesized using tryptophan as a substrate through the IPA (indole-3-pyruvate synthesis pathway), IAM (indole-3-acetamide), IAOx , and tryptamine pathways, of which the IPA synthesis pathway is the most important one . The IPAYUCCA genes were identified from A. thaliana, providing some evidence for the significant roles of AtYUCCA1, AtYUCCA2, AtYUCCA4, AtYUCCA5, AtYUCCA6, and AtYUCCA7 in the biosynthesis of auxin [YUCCA1, YUCCA2, YUCCA5, and YUCCA6 in A. thaliana could result in a high-auxin phenotype; on the contrary, interference of single genes did not produce an auxin-deficient phenotype [YUCCA6 activation mutant, yuc6-1D, presented an elevated IAA level and typical high-auxin phenotype with epinastic cotyledons, long hypocotyls, long narrow leaves, elongated petioles, and strong apical dominance [YUCCA genes caused developmental defects, while the over-expression of any one of YUCCA genes could promote auxin biosynthesis. When YUCCA6 was over-expressed in A. thaliana, dark-induced senescence was dramatically delayed. Consequently, it was confirmed that auxin delayed senescence by directly or indirectly regulating the expression of senescence-associated genes  [AtYUCCA6 could increase the tolerance to 5 μM methyl viologen-mediated oxidative stress in Ipomoea batatas (L.) Lam [YUCCA1 apparently delayed the senescence of strawberry fruit (Fragaria × ananassa Duch.) [YUCCA genes from Populus trichocarpa [Glycine max L. [Citrullus lanatus [O. sativa [Zea mays [Triticum aestivum [Cucumis sativus [Brassica rapa [Fragaria vesca [YUCCA genes. Furthermore, some evidence suggested that the FAD- and NADPH-binding motif GxGxxG is central to YUCCA activity [There were several research works reported on IAA accumulation and the regulation process. So far, 11 of auxin ,17,18,19henotype . The YUCominance . It was nd NAC6) . Over-ex(L.) Lam , and thea Duch.) . As a rechocarpa , 22 frome max L. , 10 from lanatus , 14 from. sativa , 14 fromZea mays , three faestivum , 10 from sativus , 18 fromica rapa , and eigia vesca  were ideia vesca , which mactivity .Isatis indigotica Fort. is a biennial herb, just like A. thaliana, belonging to Cruciferae. Its roots and leaves have great medicinal and economic value. Clinically, its secondary metabolites have significant effects on influenza, epidemic encephalitis, hepatitis, mumps, and other diseases [I. indigotica are not as clear as for A. thaliana and other plants [YUCCA genes, the phylogenetic, gene expression, and functional characterization of IiYUCCA genes could be similar or different, which needs to be further explored. Furthermore, it is extremely necessary to understand the molecular mechanism of auxin synthesis and regulation in I. indigotica. Based on the previous references of YUCCA genes in other species and the whole I. indigotica genome data , we colYUCCA genes in total were identified in I. indigotica , referring to the homologous YUCCA sequences in A. thaliana. Among them, IiYUCCA6-2 was identified as a pseudogene, because there was a 2-bp deletion at the 450-bp position, which caused its translation to be terminated prematurely at the position of the 456-bp site when compared to IiYUCCA6-1. The CDS (Coding sequence) lengths of IiYUCCAs ranged from 1044 bp (IiYUCCA10) to 1335 bp (IiYUCCA6-1). Most IiYUCCA genes included three exons, while three genes  had four exons, and two genes (IiYUCCA1 and IiYUCCA4) had five exons. The theoretical isoelectric point (pI) and molecular weight (Mw) ranged from 8.36 to 9.58 (average pI = 9.03) and from 38.64 kDa to 49.77 kDa (average Mw = 46.28 kDa), respectively. The detailed information of each IiYUCCA gene is listed in 13 digotica  and the IiYUCCA genes on the chromosomes (2n = 14) was investigated (IiYUCCA6-1 and IiYUCCA6-2 (which were not mapped to any chromosomes), the other 11 IiYUCCA genes were randomly distributed on five chromosomes: Chr01, Chr02, Chr03, Chr06, and Chr07. The distributions of IiYUCCA genes on the different chromosomes were not uniform. Chr06 had four IiYUCCA genes, the highest number, while Chr02 and Chr07 both had only one. Furthermore, there were three IiYUCCA genes on Chr03. It should be noted that IiYUCCA5-1 and IiYUCCA5-2 (located on Chr01) and IiYUCCA3 and IiYUCCA9 (located on Chr06) were very close to each other, and whether they might have similar biologic functions or not still needs to be confirmed.Based on the genome data, the genetic mapping of stigated . Except IiYUCCA genes, an exon–intron composition map was drawn as shown in in IiYUCCA10 and IiYUCCA11 had three exons and similar coding sequence lengths, which indicated that they might have a close evolutionary relationship. Otherwise, 12 IiYUCCA genes (except for IiYUCCA6-2) were subjected to analysis with the MEME program to identify the conserved protein motifs. A total of 11 distinct motifs were found; among them, six motifs were identified. These were the FAD-binding motif, GC motif, ATG-containing motif 1, ATG-containing motif 2, FMO-identifying sequence, and NADPH-binding motif. It was evident that the closely related proteins shared the same motif profiles, which suggested that the IiYUCCA proteins within one branch might share conservative functions. Moreover, it was not difficult to find that the conserved motifs were nearly the same, both in terms of number and type. However, there were some differences between IiYUCCA10 and IiYUCCA11. IiYUCCA10 lacked ATG-containing motif 2 and motif 8  [I. indigotica.In order to uncover the evolutionary relationships of YUCCA proteins among different species, we constructed the phylogenetic tree implemented in MEGA7.0, including digotica . It was lection) , which iIiYUCCAs in different organs and different developmental periods (except IiYUCCA5-2 and IiYUCCA6-2) were carried out. Here, YUCCA5-2 and YUCCA6-2 were more likely to be regarded as pseudogenes; thus, we did not determine their expression levels, but this does not mean that they have no biological function. As shown in IiYUCCA gene expressions varied significantly in nine organs. It was notable that IiYUCCA7 was specifically expressed in buds, which could play a key role in the bud development process. Interestingly, IiYUCCA6-1 was expressed at relatively high levels in most organs, indicating its importance in I. indigotica. IiYUCCA1, IiYUCCA9, and IiYUCCA11 were found to be predominantly expressed in the main roots, implying their specific functions in the root developmental process. IiYUCCA4 and IiYUCCA5-1 were mainly expressed in young leaves. IiYUCCA2 and IiYUCCA8 were expressed more highly in flowers than in other organs. Moreover, IiYUCCA3 and IiYUCCA10 were preferentially expressed in green siliques, reveling that they might participate in the reproductive development process. In summary, IiYUCCA members had various expression patterns in different organs, and they could play different roles in the accumulation and regulations of auxin in different organs of I. indigotica.The expression patterns of IiYUCCA2, IiYUCCA4, and IiYUCCA10 increased gradually, whereas the levels of IiYUCCA3, IiYUCCA5-1, IiYUCCA8, and IiYUCCA9 decreased with the developmental process. Moreover, IiYUCCA11 showed a trend of decreasing firstly and then rising. IiYUCCA1 expressions were detected only 21 days and 60 days after germination, whereas IiYUCCA6-1 was expressed highly compared to other developmental periods. Therefore, these YUCCA genes had distinct spatiotemporal expression patterns in I. indigotica, laying a foundation for further function research on IiYUCCA genes.In addition to differences for different organs, the expression patterns in different developmental periods were also observed, and the four developmental periods  were detected. As shown in YUCCA genes and how they were regulated by different environmental signals. Due to the higher expression level in different organs, IiYUCCA6-1 was taken as an example to examine expression patterns in response to various stress treatments. As shown in IiYUCCA6-1 showed a strong response toward PEG treatment, and the expression changes firstly showed a sharp increase and then a rapid decrease. After 1 h of PEG treatment, the transcript level of IiYUCCA6-1 was 1.13-fold higher than the control group. Additionally, the expression levels stimulated by NaCl showed no obvious changes, with a slight increase (0.21-fold) after 6 h of treatment. Furthermore, IiYUCCA6-1 showed a completely opposite expression pattern when exposed to the wounding stimulation, and the expression level was 41% of the control group level after 1 h of treatment, before rising to 142%, demonstrating its sensibility to wounding stress. In addition, IiYUCCA6-1 exhibited time-dependent and inconspicuous responses to the low-temperature treatment, but only demonstrated a 0.58-fold change after 12 h of treatment. In summary, IiYUCCA6-1 exhibited the most obvious response to PEG treatment, but showed tiny variations when induced by low temperature. Therefore, the functions of the IiYUCCA6-1 gene were correlated with the kinds of stress resistance to some extent.Plants usually implement various signal regulatory networks to respond and adapt to different natural environments. Thus, it was important to understand what kinds of factors controlled the specific expressions of YUCCA gene functions, taking IiYUCCA6-1 as an example. IiYUCCA6-1 was identified and cloned from I. indigotica (GenBank Accession Number: KY318462), encoding a protein highly homologous to AtYUCCA6 (91%). The genome DNA of IiYUCCA6-1 was 2835 bp, containing four exons and three introns. Its ORF (Open Reading Frame) length was 1335 bp, encoding 444 amino-acid residues. Detailed sequences and protein structure predictions are shown in the IiYUCCA6-1, the gene was transformed into N. benthamiana. After screening with Hygromycin B, 10 transgenic lines (OE1–OE10) were tested at DNA and RNA levels. The detection results at the transcriptional levels demonstrated that IiYUCCA6-1 was significantly expressed in 10 transgenic lines, and the expression levels of OE3 and OE9 were 200 times higher than in the control groups  were isolated and treated with dark stress. The contents of chlorophyll and hydrogen peroxide and the expression of reatment C. These YUCCA genes in plants are gene families with multiple members, except for Petunia, which only has one YUCCA gene [A. thaliana, rice, maize, tomato, and so on [I. indigotica for the first time. There are 13 YUCCA genes in I. indigotica, which are distributed on five chromosomes, but IiYUCCA6-1 and IiYUCCA6-2 were not located. Although IiYUCCA6-1 and IiYUCCA6-2 are very similar, the latter is a pseudogene. Therefore, it was speculated that IiYUCCA6-1 might have vital functions because of the functional reduction of IiYUCCA6-2. Moreover, due to the similarities in both gene structures and conservative motifs, the IiYUCCA genes were deservedly clustered in one branch, showing the differences between I. indigotica and other plants  based on the phylogenetic tree analysis. Furthermore, the IiYUCCA genes were divided into four subfamilies according to the conservative motifs. Meanwhile, the results showed that the YUCCA family members of several dicotyledons of Cruciferae were closely related and highly conserved in evolution, far from monocotyledon rice, consistent with the clustering analysis results of OsYUCCA gene family members in rice [As far as we know, most CCA gene . The YUCnd so on ,38,39,40 in rice .IiYUCCA gene expressions in nine organs and four developmental periods, which was conducive to assessing their possible biological functions. However, IiYUCCA5-2 was not quantified by qRT-PCR, because its prediction exon sequence showed a base deletion at the 960-bp position, suggesting an early translation termination, hinting that IiYUCCA5-2 might be a pseudogene. In addition, our transcriptome dataset of I. indigotica under different stress conditions (unpublished) showed no information for IiYUCCA5-2  and the reproductive organs . Furthermore, IiYUCCA9 and IiYUCCA11 showed similar expression patterns, implying that there could be a certain degree of functional redundancy, which also contributed to stabilizing the genetic networks [YUCCA genes in P. trichocarpa [Lycopersicon esculentum [Zea mays [YUCCA genes of A. thaliana [Fragaria vesca [I. indigotica need to be discussed. Some IiYUCCA gene expressions were upregulated along with the developmental process, such as IiYUCCA2, IiYUCCA4, IiYUCCA10, and IiYUCCA11. In contrast, the expression levels of IiYUCCA2, IiYUCCA3, IiYUCCA5, IiYUCCA8, and IiYUCCA9 were downregulated. The results implied that these genes could play important roles in the developmental process. Therefore, the analyses of expression patterns in IiYUCCA genes provided some information to explore the functions of YUCCA genes in different organs and during different developmental processes, as well as contribute to speculating how spatio-temporal patterns of auxin biosynthesis are regulated in I. indigotica.Gene expression patterns are usually consistent with their functions. Here, qRT-PCR analysis was conducted to investigate networks . It was chocarpa , Lycoperculentum , and ZeaZea mays  displayethaliana  and Fragia vesca  were maiYUCCA genes performed potential regulatory functions under different kinds of stress conditions [IiYUCCA6-1 was sensitive to NaCl, PEG, cold, and wounding treatments. Its changes in expression pattern reflected that this gene might be involved in complex regulatory processes, with a function in defense responses. Similar results were found in G. max, whereas C. sativus CsYUCCA8 and CsYUCCA9 responded significantly to high temperature, and CsYUCCA10b was sensitive to low temperature and salt stress [GmYUCCA5 and GmYUCCA8 were proven to be sensitive to different stress stimuli, including drought, copper, zinc, low temperature, high temperature, and salinity [YUCCA genes in improving the stress-resistance capacities in plants. It is necessary to confirm the expression patterns of YUCCA genes in I. indigotica, which contribute to enriching the YUCCA gene family characteristics and functional researches in plants.There was some evidence exhibiting that nditions ,43. IiYUt stress . GmYUCCAsalinity . These fIiYUCCA6-1 in the auxin biosynthesis process in I. indigotica were also discussed. Its functions in the IAA synthesis pathway were relatively consistent with those of AtYUCCA6 reported in A. thaliana [AtYUCCA6 increased not only IAA content, but also the expression levels of some auxin-responsive genes [NbIAA8, NbIAA16, NbGH3.1, and NbGH3.6 were significantly higher than those in the control group. The results demonstrated that the over-expressed IiYUCCA6-1 gene might upregulate the auxin content, whereas the expression of early responsive genes was activated. Moreover, it was reported that YUCCA family genes could participate in plant resistance to stress [2O2 content. Furthermore, the expression level of NbSAG12 decreased in the transgenic lines, which could be explained by the senescence process being delayed to a certain extent. Hence, the over-expression of IiYUCCA6-1 in tobacco could promote IAA content, as well as upregulate the expression of auxin biosynthesis genes, which caused the typical hyperauxin phenotype. Furthermore, with a decrease in the contents of total chlorophyll and H2O2, as well as a downregulation of NbSAG12, the leaf senescence process was delayed, and stress resistance could also be improved. However, there are more stress conditions that still need further research.Auxin plays important roles in cell division, lateral root formation, flowering time, and leaf senescence in plants . The funthaliana  and strathaliana . It was ve genes . Here, so stress , which wI. indigotica, the nucleotide sequences of YUCCA genes from A. thaliana, B. rapa, B. oleracea, R. sativus, and O. sativa were downloaded from TAIR (https://www.arabidopsis.org/), brassicaDB (http://brassicadb.org), and RGAP (http://rice.plantbiology.msu.edu/). The BLASTn program  with a threshold of 1 × 10−20 was used to obtain the predicted sequences based on I. indigotica genome data. Then, Pfam (http://pfam.sanger.ac.uk/) and CDD (Conserved Domain Database) analyses were performed to confirm the encoded sequences of IiYUCCA proteins. GeneWise  was used to predict the pseudogenes. Furthermore, the physical and chemical properties  of IiYUCCA proteins were calculated using the ExPASy website (http://web.expasy.org/protparam/).To identify YUCCA family genes in http://gsds.cbi.pku.edu.cn/, Peking University, Beijing, China). MEME software  was used to find the common motifs, and the maximum number of motifs was set to 12 with the width of motifs set from six to 20. The multiple sequence alignments of YUCCA gene family proteins from six species were performed using the MUSCLE program  with default parameters. The full-length amino-acid sequences of 96 YUCCA proteins from six species were downloaded from their genome database, while the phylogenetic tree with aligned amino-acid sequences was constructed using MEGA 7.0 software with the maximum-likelihood method and verified with 1000 bootstraps. In addition, PAML 4.9d software was used to calculate the synonymous substitution rates (Ks) and non-synonymous substitution rates (Ka) of YUCCA genes in I. indigotica.The chromosome locations of IiYUCCA members were investigated using MapChart software. The gene structures were analyzed using the Gene Structure Display Server (I. indigotica seeds were sown in pots (three plants each pot) in the greenhouse of the National Engineering Laboratory for Resources Development of Endangered Crude Drugs in Northwest China. The plants were cultivated at 23 ± 2 °C under a 16-h light/8-h dark photoperiod. Leaf materials at different developmental periods were taken from seedlings at seven days (cotyledon unfolding), 21 days (the second pair of euphylla unfolding), 60 days, and 150 days. Moreover, 60-day-old seedlings were treated with different abiotic stresses, namely, 5% PEG600, 100 mM NaCl, 4 °C, and 40% mechanical wounding, and the control group was irrigated with the same amount of deionized water. Then, all the leaf materials were collected after 0, 1, 3, 6, 12, and 24 h of treatment. The different organs, including roots, stems, leaves, buds, flowers, and siliques were taken from the plants in the field. All samples were quickly frozen in liquid nitrogen and stored at −80 °C for further research.® reagent  according to the manufacturer’s protocol. The quality of RNA was determined using a Nanodrop instrument  and agarose gel electrophoresis. The first-strand complementary DNA (cDNA) was synthesized using the PrimeScript TM II First Strand cDNA Synthesis Kit , with 1.0 µg of total RNA in 20 µL of water, stored at −20 °C. The RT-PCR products were diluted (1:20) for qRT-PCR analysis, which was performed using the SYBR® Premix Ex Taq™ II  on a Roche LightCycler®96 System . All primer sequences for qRT-PCR are listed in the I. indigotica, IieIF2, IiPP2A-4, and IiRPL15, were used as the internal controls [−∆∆CT method. In each experiment, three biological and technical repeats were conducted.Total RNA of each sample was extracted with TRIzolcontrols . The relIiYUCCA6-1 was cloned by the specific primers (forward (F): 5′–ATGGATTTCTGCT GGAGGA–3′, reverse (R): 5′–TCAACCGTCTAGTAATCTACTAT–3′), with the following PCR conditions: 94 °C, 3 min; 30 cycles of 94 °C, 30 s, 57 °C, 30 s, 72 °C, 2 min; 72 °C, 10 min. Subsequently, the product was cut with the restriction enzymes, Bgl II and Spe I, then inserted into the pCAMBIA1302 vector with the CaMV35S promoter. Furthermore, the vector was transformed into Agrobacterium tumefaciens strain GV3101, and the transformation of N. benthamiana was conducted using Agrobacterium-mediated leaf disc method. After screening by Hygromycin B, the over-expressed IiYUCCA6-1 plants were sampled separately. One part of the plants was used to extract the genomic DNA, and the other part was used to extract RNA and reverse-transcribe to cDNA. Then, the transgenic lines were detected at the DNA and RNA level.NbIAA8, NbIAA16, NbGH3.1, NbGH3.6), and free IAA contents were measured with the help of a Plant Indole-3-acetic acid ELISA Kit , following the manufacturer’s instructions. Next, some leaves were soaked in 3 mM MES (2-(N-morpholine) ethanesulfonic acid) medium (pH 5.7), and then placed in the dark. The total contents of chlorophyll and H2O2 , and the expression level of senescence related gene NbSAG12  were detected after dark treatments (zero and seven days), with NbActin  as the reference gene.The fresh leaves from control and transgenic lines were selected for the gene function analysis. The cDNA was used to detect the expressions of early responsive genes . All experimental data were assessed using one-way analysis of variance (ANOVA), and results were presented as the means ± standard error (SE) at the levels of * I. indigotica. One of YUCCA gene family members, IiYUCCA6-1, could promote IAA accumulation and the upregulation of some auxin-responsive genes, which triggered a typical hyperauxin performance. Moreover, the contents of total chlorophyll and H2O2 decreased and the expression of NbSAG12 was downregulated, which could delay the senescence process while improving the stress resistance (I. indigotica YUCCA gene family using phylogenetic analysis, gene expression analysis, and functional characterization by heterologous expression in tobacco. All the findings demonstrated the experimental basis for the identification of the YUCCA genes in I. indigotica, with an exploration of their function.In brief, this study systematically analyzed the YUCCA gene family structures, phylogenetic characteristics, and expression patterns in sistance . This st


The term “ovarian carcinoma” encompasses at least five different malignant neoplasms: high-grade serous carcinoma, low-grade serous carcinoma, endometrioid carcinoma, mucinous carcinoma, and clear cell carcinoma. These five histotypes demonstrated distinctive histological, molecular, and clinical features. The rise of novel target therapies and of a tailored oncological approach has demanded an integrated multidisciplinary approach in the setting of ovarian carcinoma. The need to implement a molecular-based classification in the worldwide diagnostic and therapeutic setting of ovarian cancer demanded a search for easy-to-use and cost-effective molecular-surrogate biomarkers, relying particularly on immunohistochemical analysis. The present review focuses on the role of immunohistochemistry as a surrogate of molecular analysis in the everyday diagnostic approach to ovarian carcinomas.  TP53-mutated, were characterized by aggressive clinical behavior and poor outcomes [Ovarian epithelial cancer (OC) is the eighth most common malignant neoplasm in female patients and the seventh greatest cause of cancer death worldwide ,4,5,6,7.outcomes ,9. From outcomes ,11. AlthClassic histotype: characterized by a papillary, micropapillary, and/or solid architecture, marked nuclear pleomorphism and high mitotic index a.BRCA1/2 mutation and SET morphology [“SET” variant : characterized by an admixture of solid, glandular/endometrioid-like, and transitional/malignant Brenner-like growth patterns), higher mitotic index compared to the classic histotype, and a high number of tumor-infiltrating lymphocytes (TILs) b. In 201rphology HGSC is the most common epithelial malignancy of the tubo-ovarian district, responsible for almost 70% of OCs . The majTP53 mutation has been observed in almost all tubo-ovarian HGSCs (96%), followed by alteration of homologous recombination repair-related genes such as BRCA1/2. The TCGA project revealed a surprisingly simple spectrum of mutations in HGSC . TP53 muTP53 mutations was investigated by Köbel et al., 2016 [TP53 mutation was obtained when a binary p53 IHC scoring system was adopted: p53 intense nuclear positivity in >80% tumor cells (overexpression pattern), complete absence of expression (null pattern) or cytoplasmic expression without nuclear staining were scored as p53 “abnormal” (p53abn) and correlated with TP53 mutation a,b.TP53-wildtype gene (heterogeneous p53 expression was scored as “wild-type” (p53wt) and correlated with a ype gene c.The use of an optimized p53 IHC staining as a molecular surrogate for l., 2016 ,14. An aTP53-wildtype HGSC, albeit rarely encountered, can represent a potential diagnostic pitfall, as grade 3 endometrioid carcinoma, often TP53-wildtype, can mimic a SET variant of HGSC. The following criteria could help in the differential diagnosis:CCNE1, BRCA1/2, and MDM2 amplification support the diagnosis of TP53-wildtype HGSC [Association with serous tubal intraepithelial carcinoma (STIC), low-grade serous-like areas, WT1 IHC positivity, mutation of ype HGSC .Association with endometriosis, endometrioid cystadenofibroma, and borderline endometrioid tumor, as well as WT1 IHC negativity, support the diagnosis of p53abn grade 3 ENOC .Though p53 mutational pattern is now considered a reliable marker for a diagnosis of HGSC, two important caveats have to be considered: (1) 4% of HGSCs are p53 wild-type; (2) almost 20% of ovarian endometrioid grade 3 carcinomas  and a minor subset of CCC are p53abn ,16. TP53BRCA mutation and extensive adnexal sampling via the so-called SEE-Fim protocol (sectioning and extensively examining the FIMbriated end) provided a basis to identify potential early lesions in the tubo-ovarian district [TP53 mutation [WHO classification of the Tumours of the Female Genital Tract and the AJCC Cancer Staging Manual encouraged thorough examination of the fimbria to search for STIC [The adoption of prophylactic salpingo-oophorectomy in patients with district . This remutation ,21,22,23mutation . Though for STIC . Albeit BRCA1 and BRCA2 are tumor suppressor genes involved in the so-called homologous recombination repair system (HR). In the event of DNA double-strand breaks or DNA damage at a replication fork, the cells undergo a repair via HR, recruiting the sister chromatid as a template for DNA replication [BRCA1 together with BARD1 and BRIP1 recruit the so-called MRN complex  at the site of the damage. The MRN complex then resects the 5′ strand, while BRCA2 is responsible for loading RAD51 protein at the site of the single-strand DNA stretch to initiate the homologous DNA invasion [BRCA1/2 germinal loss of function is the driver mutation of the so-called hereditary breast and ovarian carcinoma syndrome (HBOC). HGSCs are the most commonly found ovarian histotype in HBOC patients.lication . In partinvasion . In the BRCA1 and BRCA2 pathogenic variants (8% and 9% of the cases respectively), BRCA1 and BRCA2 somatic mutations , and other alterations such as hypermethylation of BRCA1 promoter and mutations in other HR-related genes such as RAD51C, ATM, PALB2, CDK12, CHEK2, etc., almost 50% of HGSCs could be classified as homologous recombination deficient (HRD) [Considering germline nt (HRD) ,29,30,31BRCA1/2 as a surrogate biomarker for BRCA mutation showed high sensitivity (86.2%) and negative predictive value (95.4%). However, it demonstrated poor specificity (78.3%) and positive predictive value (52.1%), therefore IHC-only testing should not be considered sufficient for the detection of BRCA genes status [In a study by Meisel et al., immunohistochemistry for s status ,33. Homos status ,35,36,37BRCA1/2 mutations has been highlighted with the introduction of poly(ADP-ribose) polymerase inhibitors (PARPi) as maintenance therapy in platinum-sensitive OCs [BRCA1/2 loss, tumor cells are unable to undergo DNA repair via HR and single-strand break repair, causing irreparable damage and, ultimately, cell death. Several clinical trials, after the milestone SOLO1 trial, demonstrated a statistically significant better outcome in BRCA germline-mutated patients [BRCA somatic tumors and/or HRD non-BRCA patients [The adoption of a routine workflow to identify tive OCs ,39. PARPpatients . Howeverpatients ,43,44,45BRCA-reflex testing seems to be particularly useful to:BRCA germline mutations eligible for PARPi therapy, prophylactic surgery, and genetic counseling;identify patients with BRCA somatic mutations potentially eligible for PARPi therapy;identify patients with BRCA-negative tumors, in order to be cost-effective and reduce patients’ psychological distress.exclude unnecessary germline testing for somatic Thus a pathology-based, BRCA status, but demonstrated a statistically significant correlation with better survival in late-stage HGSCs  ,2,51. LGTP53 and HR-related gene mutations, while LGSC is thought to be the result of a neoplastic transformation of cortical inclusion cysts into serous cystadenomas/adenofibromas and serous borderline tumors [Despite their similar pathological denomination, LGSCs share little in common with HGSCs. HGSCs are thought to arise from the surface epithelium of the distal fimbria via e tumors . FurtherKRAS, NRAS, BRAFV600E, BRAFnon-V600E, and ERBB2 were found in almost 50% of LGSCs, while TP53 was wild-type in all reported cases [KRAS p.Gly12Val (KRASG12V) mutation has been particularly associated with worse prognosis and higher recurrence rate, while BRAFV600E demonstrated a statistically significant correlation with better prognosis [LGSCs are characterized by a completely different mutational spectrum compared to HGSCs: mutations in ed cases ,55,56. KTo further complicate the landscape of serous carcinomas, Zarei et al. recently published a case series of serous carcinomas with mixed low-grade and high-grade features . This evBRAF inhibitors, with promising results: this evidence is particularly noteworthy as LGSC is a stage-dependent disease, poorly responding to conventional chemotherapy in advanced stages [BRAFV600E demonstrated 96% sensitivity, 96% specificity, 93% positive predictive value, and 98% negative predictive value for BRAFV600E mutation [BRAFV600E is particularly helpful and relatively cost-effective for the identification of patients eligible for target therapy, especially in advanced-stage LGSC. Hormonal replacement therapy is currently included in the National Comprehensive Cancer Network (NCCN) guidelines in advanced-stage LGSC with expression of estrogen receptor (ER) and progesterone receptor (PGR) . Some std stages ,59,60,61d stages . In a stERBB2-mutated LGSCs  represent a heterogeneous group of neoplasms regarding morphological and molecular features. ENOCs typically exhibit cribriform, maze-like or glandular architecture with frequent squamous differentiation ,2. TumorPOLE) ultramutated, microsatellite instable (MSI-H), carcinomas with high somatic copy number alterations (CN), and carcinomas with low CN [POLE is a gene involved in DNA repair and replication. The exonuclease proofreading activity replaces erroneously incorporated nucleotides during DNA replication. Mutations in its exonuclease domain lead to proofreading errors, with increase in mutation rates during DNA replications by about 100-fold, and, ultimately, neoplastic transformation [MLH1, PMS2, MSH2, and MSH6) are nuclear enzymes involved in repair of base–base mismatch during DNA replication in proliferating cells: these proteins form heterodimers binding to areas of abnormal DNA and initiating its removal. Loss of MMR proteins leads to accumulated errors during DNA replication, especially in short repetitive nucleotide sequences (“microsatellite instability”). Mutation of MMR-genes and hypermethilation of MLH1 promoter has been associated with the so-called MSI-H group and, for germline mutations, with Lynch Syndrome (LS) [high ECs exhibited frequent mutation in TP53. CNlow ECs were characterized by an heterogeneous spectrum of mutated genes such as PTEN, ARID1A, CTNNB1, and PIK3CA [The 2013 TCGA analysis of endometrial carcinomas (ECs) identified four main molecular subgroups: DNA polymerase ε  ,66,67. Cd PIK3CA . low and MSI subgroups while high-grade EECs were almost equally distributed in all four molecular subgroups [POLE ultramutated carcinomas demonstrated an excellent prognosis, MSI-H and CNlow ECs showed intermediate prognosis, while the CNhigh group was associated with worse prognosis [WHO classification of the Tumours of the Female Genital Tract tried to implement the usual histological classification with a new molecular approach in pathologists’ routine practice: a diagnostic algorithm  was proposed to identify molecular EC-subgroups  were represented in all four subgroups. In particular, low-grade EECs were most frequently encountered in the CNubgroups ,70. Thisrognosis . As to tubgroups .high tumors. No immunohistochemical marker is available for POLE, thus Sanger sequencing is the gold standard to evaluate POLE status. Finally, the assignment to the CNlow/no special molecular profile (NSMP) group could be made once POLE/MMR/p53 mutations are excluded. Immunohistochemistry for MMR proteins and p53 are excellent surrogates for MSI-H and CNPOLE sequencing, with subsequent MMR and p53 immunohistochemical evaluation of POLE-wt cases  ENOCs were also rarer than MMRd EECs, while NSMP ENOCs were more frequently represented than NSMP EECs: both MMRd ENOCs and NSMP ENOCs were associated with an intermediate prognosis. Finally, p53abn tumors, rarely encountered, were significantly correlated with poor prognosis. POLE-mutant ENOCs [POLE ENOCs were associated with an excellent prognosis. Thus, the role of molecular classification in ENOCs appears promising as in EECs, especially to guide neoadjuvant treatment.In another study by Parra-Hernan et al., MMRd ENOCs demonstrated excellent prognosis, overlapping with nt ENOCs . These mPOLE mutations by Van Gool et al. [POLE-mutant ECs were statistically associated with a high number of TILs, tumor giant-cells, p53wt IHC pattern, and intact MMR IHC expression. The same morphological features were also observed in POLE ENOCs [POLE mutation, albeit Sanger sequencing represents the gold standard assay to establish POLE mutational status. Histopathological and IHC analysis, blinded of the molecular status, was carried on a series of ECs to identify morphological features associated with l et al. . POLE-muLE ENOCs . TherefoMLH1 promoter hypermethylation was the most commonly found alteration (86% of the cases), but germline loss of function of MLH1, MSH2, and MSH6 (highly suspicious for LS) was observed in 35% of the cases. Furthermore, MMRd OCs were associated with younger age at presentation and increased TILs. A two-antibody approach with PMS2 and MSH6 is considered sufficient and more cost-effective than an upfront four-antibody approach, due to the binding heterodimeric nature of the MMR complexes: a loss of expression of MLH1 or MSH2 would inevitably lead to degradation of PMS2 and MSH6, respectively [MLH1 promoter methylation test should also be carried out in all cases showing MLH1 IHC loss.A large cohort of 502 early-stage ovarian carcinomas, representative of all five main histotypes was tested for MMRd by Leskela et al. MMRd was significantly associated with endometrioid and clear cell histotypes . As for ectively . Conversectively ,75,76. HPOLE-mutant tumors, because of their high immunogenic characteristics [PD1/PD-L1 expression has been tested in OC with particular attention to MMRd and eristics ,48,77,78eristics .Hormone receptor expression has been linked to better prognosis supporting the possibility of hormonal therapy in ENOCs. In a large multicentric study by Sieh et al., ER and PGR expression, scored as negative (<1% positive nuclei), weak (1–50%), or strong (≥50%), were correlated with better disease-specific survival in ENOCs . AnotherPTEN and ARID1A genes. PTEN loss could represent a target for PI3K/AKT inhibitors, while ARID1A loss for EZH2 and HDAC inhibition, but further studies are needed to clarify their potential role [IHC evaluation of PTEN and ARID1A proteins are good surrogates of loss-of-function mutations in ial role ,82,83.CTNNB1-mutated ENOCs, examined with surrogate IHC staining for nuclear β-catenin, were associated with a favorable outcome in two independent studies, while abnormal expression of p16 in ENOCs has been associated with a worse outcome: however, these data still need further validation  [The mutational spectrum of CCCs is characterized by frequent mutation of SWI/SNF chromatin remodeling complex protein ion (5%) ,93,94,95ion (5%) . This ision (5%) ,97.Primary ovarian mucinous carcinoma is extremely rare as the vast majority of mucinous malignant tumors represents metastatic localization from the cervical or gastrointestinal tract . CommonlCDKN2A and KRAS mutations are the most commonly encountered followed by TP53 and ERBB2 mutations [ERBB2 amplified tumors demonstrated a significantly better prognosis when Trastuzumab was added to the conventional chemotherapy treatment, suggesting a potential actionable target in the treatment of ovarian primary MCs [Molecularly, utations ,104. MSIutations . ERBB2 aSeromucinous carcinomas (SCs), previously considered an independent entity, are now considered a subtype of endometrioid carcinoma due to similar pathogenic background (endometriosis-associated) and mutational spectrum: thus, the last WHO Classification of Female Genital Tract Tumors considers SCs as a morphologic variant of ENOCs ,106. KRAS, NRAS, and PIK3CA mutations [A new entity introduced in the 2020 WHO Classification of Female Genital Tract Tumors is the mesonephric-like adenocarcinoma (MLA). This rare tumor may arise from a malignant transformation of mesonephric ovarian remnants or transdifferentiation of Müllerian epithelium. The latter event could explain the frequent association of MLA with endometriosis or other carcinomatous histotypes, most commonly ENOCs. MLAs usually exhibit an admixture of glomeruloid, tubular, sieve-like, solid, and glandular architectural patterns and a distinctive GATA3+/TTF1+/CD10+/PAX8+ immunophenotype, while ERs and PGRs are typically negative . Though utations ,109. No ARID1A, ARID1B, SMARCB1/INI1, and SMARCA4 genes [POLE EDM. All these molecular alterations can be also easily identified via IHC and sequencing with good reliability and can be of help in the differential diagnosis with other undifferentiated metastatic neoplasms. This can also be helpful to identify a subset of patients potentially eligible for immunotherapy, as demonstrated for their endometrial counterparts. In a case series published by Espinosa et al., a neuroendocrine differentiation was encountered in four ovarian DED/UCs, one of which, characterized by POLE EDM, demonstrated excellent prognosis [Dedifferentiated/undifferentiated carcinomas (DED/UCs) are uncommon ovarian tumors with poor prognosis, thought to represent the latter stage of neoplastic transformation of endometrioid tumors. Frequent mutations in the SWI/SNF chromatin remodeling complex have been found, in particular involving rognosis .TP53, PIK3CA, BRCA1/2, PTEN, MLH1, MSH6, PPP2R1A, ARID1A, KRAS, CDH4, and BCOR [BRCA1/2: these tumors can be potentially treated with PARPi. In fact, as for HGSCs, CSs with PTEN loss could be eligible for PI3K/AKT inhibitors and, although rarely encountered, microsatellite instable-CSs could be eligible for anti-PD1 therapy [Carcinosarcomas (CSs) are rare biphasic ovarian malignant tumors with both carcinomatous and sarcomatous elements . The twoand BCOR ,115,116. therapy .BRCAmut HGSCs show better prognosis compared to BRCAwt tumors, particularly after the introduction of PARPi in maintenance treatment [BRAFV600E LGSC are characterized by better outcomes compared to KRAS-mutated LGSCs [POLE-ultramutated, MSI hypermutated, CNhigh and CNlow tumors with different histologic and clinical features [POLE-ultramutated tumors have demonstrated an excellent prognosis, MSI and CNlow intermediate outcomes and CNhigh tumors poor survival rates. Almost 25% of ENOCs, 20% of MCs, and 14% of CCCs could be related to Lynch Syndrome with notable genetic and clinical implications, such as the adoption of preventive familiar strategies and, in the event of adjuvant therapy, of potential checkpoint inhibition [OCs are a heterogeneous group of diseases with specific histologic, molecular, and clinicopathologic features. Ten years after the first attempt at an OC molecular classification, the introduction of new technologies allowed us to expand our knowledge of the complex molecular landscape of this disease. To date, at least five main independent histotypes are recognized: HGSCs, LGSCs, CCCs, ENOCs, and MCs ,8. A molreatment . BRAFV60ed LGSCs . ENOCs efeatures . POLE-ulhibition . The nee


CsATGs—autophagy-related genes (ATGs) of Citrus sinensis, Cs) in a genome-wide manner from sweet orange (Citrus sinensis). Bioinformatic analysis showed that these CsATGs were highly similar to Arabidopsis ATGs in both sequence and phylogeny. All the CsATGs were randomly distributed on nine known (28 genes) and one unknown (7 genes) chromosomes. Ten CsATGs were predicted to be segmental duplications. Expression patterns suggested that most of the CsATG were significantly up- or down-regulated in response to drought; cold; heat; salt; mannitol; and excess manganese, copper, and cadmium stresses. In addition, two ATG18 members, CsATG18a and CsATG18b, were cloned from sweet orange and ectopically expressed in Arabidopsis. The CsATG18a and CsATG18b transgenic plants showed enhanced tolerance to osmotic stress, salt, as well as drought (CsATG18a) or cold (CsATG18b), compared to wild-type plants. These results highlight the essential roles of CsATG genes in abiotic stresses.Autophagy is a highly conserved intracellular degradation pathway that breaks down damaged macromolecules and/or organelles. It is involved in plant development and senescence, as well as in biotic and abiotic stresses. However, the autophagy process and related genes are largely unknown in citrus. In this study, we identified 35 autophagy-related genes ( ATGs) are responsible for the regulation and control of the autophagy process. The first ATG gene was identified from yeast, and at least 32 ATGs have been shown to participate in yeast autophagy [ATGs have been identified from various plant species, including 40 AtATGs in Arabidopsis [Arabidopsis thaliana, At), 33 OsATGs in rice [Oryza sativa, Os), 30 NtATGs in tobacco [Nicotiana tabacum, Nt), 45 ZmATGs in maize [Zea mays, Zm), 29 CaATGs in pepper [Capsicum annuum, Ca), 37 SiATGs in foxtail millet [Setaria italic, Si), 32 MaATGs in banana [Musa acuminate, Ma), and 35 VvATGs in grapevine [Vitis vinifera, Vv). According to the reported characterizations of ATGs in yeast and Arabidopsis, these ATGs can be divided into the following functional groups: (1) the ATG1/13 kinase complex consisting of ATG1, ATG13, ATG20, and TOR (target of rapamycin kinase), mainly functioning on autophagy induction and initiation; (2) the PI3K (phosphatidylinositol 3 kinase) complex consisting of ATG6, VPS15 (vacuolar protein sorting-associated protein), and VPS34 that is involved in vesicle nucleation and autophagosome formation; (3) the ATG9/2/18 complex consisting of ATG9, ATG2, and ATG18 that is responsible for the delivery of membranes for autophagosome formation; (4) the ubiquitin-like ATG8-PE (phosphatidylethanolamine) conjugation pathway  and ATG12–ATG5 conjugation pathway  that are involved in the elongation of autophagic vesicles; and (5) the VTI12  protein) family belonging to the SNARE group, which contributes to the fusion of autophagosomes with vacuoles [Autophagy is a highly conserved intracellular degradation process that occurs in eukaryotes including yeasts, animals, and plants ,2. Durinutophagy ,6. To dabidopsis ,3  was significantly up-regulated in response to nitrogen depletion and oxidative stress, and overexpression of MdATG8i in apple callus conferred enhanced tolerance to nutrient-limited conditions [MdATG3a, MdATG3b, MdATG7b, and MdATG18a of apple were shown to confer tolerance to nitrogen/carbon starvation, drought, salt, or osmotic stresses by ectopic-expressing in Arabidopsis and apple [MaATG8f improved drought stress tolerance in transgenic Arabidopsis through modulating reactive oxygen species (ROS) metabolism, abscisic acid biosynthesis, and autophagy activity [ATG8 in Arabidopsis that increased autophagic activity and improved nitrogen remobilization efficiency [SiATG8a of foxtail millet conferred tolerance to both nitrogen starvation and drought in transgenic Arabidopsis and rice [AtATG18a in Arabidopsis reduced drought and salt tolerance [ATG6 of wheat inhibited autophagy and resulted in accelerated programmed cell death (PCD) under drought stress [ATG10 and ATG18f significantly reduced HsfA1a-induced drought tolerance and autophagosome formation in tomato [Arabidopsis showed hypersensitive phenotype [Arabidopsis displayed visibly impaired pollen development  [atg2-1, atg5-1, atg12ab, and atg18a-2) [Arabidopsis [The autophagy process maintains a basal level under normal conditions, but it can be quickly induced by external stimuli such as nutrition starvation, drought, salt, heat, and oxidative and osmotic stresses ,14,15,16nditions . MdATG3and apple ,20,21. Lactivity . Chen etficiency . Moreoveand rice ,24. In colerance . Knockdot stress . Loss ofn tomato . Under shenotype . Under hatg10-1)  or enhantg18a-2) . Recentlbidopsis . Moreovebidopsis ,32,33.http://faostat.fao.org/default.aspx). However, citrus production is limited by abiotic stresses including drought, heat, cold, and nutrient disorders. Although the underlying physiological and molecular mechanisms triggered in response to these stresses have been well studied, the autophagy process and ATG genes in citrus are largely unknown. In this study, we systematically identified 35 ATGs from citrus by using the genome of sweet orange [ATGs were conducted and discussed. Moreover, two ATGs of sweet orange, CsATG18a and CsATG18b , were cloned and overexpressed in Arabidopsis. This further supported their functions in improving tolerance to drought, salt, cold, and osmotic stresses.As the largest fruit crop, citrus is widely grown around the world. In 2017, the global citrus growing area and yield were 14.1 million hectares and 146.6 million tons, respectively  to 7095 bp (CsTOR), encoding 104 to 2364 amino acids  contained the most CsATGs (seven genes), followed by chr2 (four genes) and chr4 (four genes). Gene duplication analysis showed that 10 CsATGs were predicted to be segmental duplicated genes, accounting for about 29% of all CsATGs. In these segmental duplicated genes, five pairs—CsATG8a and CsATG8d, CsATG8c and CsATG8d, CsATG18a and CsATG8e, CsATG8f and CsATG8g, and CsVTI12b and CsVTI12c—were predicted to have collinear correlations.The chromosomal distribution of 35 CsATGs had 4 to 17 exons, but 1 gene (CsTOR) had 54 exons. All the CsATG genes contained introns, but three members  had no untranslated region (UTR). The main conserved domains of CsATGs were predicted to be protein kinase superfamily (PKc)-like, ATG, phox homology (PX) domain, Bin/amphiphysin/Rvs (BAR), PI3K, WD40, breast carcinoma amplified sequence 3 (BCAS3), chorein N, peptidase C54, ubiquitin homologues (UBQ), E1 enzyme family, and vesicle transport v-SNARE (V-SNARE). In general, the CsATGs in the same functional group or subfamily had similar domains  during at least one time-point of drought, heat, cold, mannitol, or salt treatments, respectively. In particular, CsATG2, CsATG8g, CsATG13, and CsATG18h under drought; CsATG2, CsATG18e, CsATG18g, and CsVTI12b under heat; CsATG4 under cold; and CsATG8a, CsATG18e, and CsVTI12c under mannitol and salt showed high expression levels, suggesting their potential key roles in response to these stresses. Apart from CsATG1d, CsATG5, CsATG7, CsATG8d, CsATG8g, CsATG16, CsATG18e, CsTOR, and CsVTI12d, the other 26 tested genes were significantly up-regulated during both drought and heat stresses. Twenty CsATGs showed significant up-regulation under both mannitol and salt stress. Remarkably, CsATG2, CsATG4, CsATG8i, CsATG9, CsATG10, CsATG18a, CsATG18b, and CsVTI12c were significantly up-regulated under all tested stresses simultaneously, suggesting their important roles in response to these stresses. In addition, some genes were significantly up-regulated at all tested time points of treatment, such as CsATG9 and CsATG13 under drought. Apart from these up-regulated CsATGs, we also noticed that CsATG1a was down-regulated under cold, mannitol, and salt treatments, which differed from the expression of the other two members of the ATG1 subfamily, CsATG1b and CsATG1d. The expression of CsATG5 was not obvious  under most of the stresses, except for a significant down-regulation under 12 h of heat treatment. Similarly, the expression of CsATG8d was also steady, except for a significant up-regulation under mannitol and salt treatments.As shown in CsATGs were down-regulated or not obvious in roots and leaves, and only a few CsATGs were significantly up-regulated at one or two time-points of treatment  in both roots and leaves, CsATG18g, CsATG18h, and CsATG20 were significantly down-regulated in leaves, and CsATG13 was significantly down-regulated in roots under +Cu, +Mn, and +Cd stresses simultaneously. In addition, the expression of CsATG1a in +Cd roots and +Cu leaves, as well as CsATG8g and CsATG18c in +Cu leaves showed extreme down-regulation. Expression of CsATG8g and CsATG9 in +Cd roots, CsATG8c in +Cu leaves, and CsATG12 in both +Cu and +Mn leaves showed obvious up-regulation (Under excess Cu (+Cu), Mn (+Mn), and Cd (+Cd) treatments, the expression of most reatment . Generalgulation . These gCsATGs in abiotic stresses, CsATG18a and CsATG18b were cloned and overexpressed in Arabidopsis. Three homozygous overexpression (OE) lines (T4 generation) and wild-type  of Arabidopsis were selected to perform germination experiments. The results showed that the OE lines of CsATG18a  and CsATG18b  had significantly higher germination rates than WT on 200 mM NaCl and 400 mM mannitol . However, after recovery for 2 days, the three OE lines had less leaf damage. In addition, the relative electrolyte leakage of OE lines was significantly lower than that of WT after cold treatment for 5 min  [MaATGs) [OsATGs) [SiATGs) [CsATGs, we found that CsATG1, CsATG8, and CsATG18 contained multiple members, which is similar to that in Arabidopsis and other plants [ATG4, ATG12, and ATG13 genes and one VTI12 in Arabidopsis, sweet orange has one member of CsATG4, CsATG12, and CsATG13 and four members of CsVTI12. This indicates a citrus crop-specificity in the number of these ATGs [CsATGs, especially for CsATG8. This suggests that expansion of these CsATG subfamilies was possibly derived from gene duplication during evolution [ process . Most ATin yeast . More th VvATGs) , banana [SiATGs) , suggestr plants . HoweverCsATG genes showed higher than 60% sequence identity to homologues of Arabidopsis. Most of these high identities of CsATG and AtATG were also closely clustered together in phylogenetic trees. This result indicates that some CsATG genes may have functions similar to their homologues in Arabidopsis. On the basis of analysis of conserved domains, three CsATG1 members have similar serine/threonine-protein kinase domains that function in forming a complex with ATG13 and regulating the initiation of autophagy [CsATG8 and CsVTI12 members, respectively. In general, the similar domain containing in the family members indicates the possible functional redundancy of this family [CsATG1, CsATG8, and CsVTI12 were obviously clustered in two different branches, indicating that functional divergences are also existing in these subfamilies. A functional divergence had been reported for ATG8b and ATG8i during the hydrotropic response in Arabidopsis roots [CsATG18 subfamily, all members contain the WD40 domain, except CsATG18f, CsATG18g, and CsATG18h with an extra BCAS3 domain. The phylogenetic analysis also showed that the CsATG18f, CsATG18g, and CsATG18h were clustered in an independent branch, indicating functional divergence of these three CsATGs with the other members in this family [Before functions were identified via transgenic strategy, the potential functions of unknown genes were often predicted according to their orthologous genes, conserved domains, and expression patterns. In this study, 29 of the utophagy . The UBQis roots , which as family .ATG genes have played key roles in plant responses to different abiotic stresses. We found that the expression of most CsATG genes were induced under drought, heat, cold, mannitol, or salt stresses. Our results support the previous findings regarding the involvement of autophagy in abiotic stresses [CsATGs were induced by these stresses, some CsATGs showed different expression patterns to different stresses. For example, CsATG1a was significantly up-regulated under drought and heat but down-regulated under cold. In contrast, CsATG1d was significantly up-regulated under cold. In the CsATG8 subfamily, CsATG8a, CsATG8c, CsATG8g, and CsATG8i were obviously induced by drought and heat, but this was not true for CsATG8d and CsATG8f. Similar results have been reported for ATGs of other plant species, such as VvATGs of grape [NtATGs of tobacco [CaATGs of pepper [CsATGs were analyzed under high levels of Cu, Mn, and Cd. Unlike the predominant up-regulation of CsATGs under drought, heat, cold, and salt stress, more down-regulated CsATGs were found upon exposure to excess Cu, Mn, and Cd. In addition, the expression patterns were different between heavy metal-treated roots and leaves. Although we could not find similar reports to support our result at present, these data provide a new reference for understanding potential roles of ATGs in heavy metal stress.stresses ,25,28. Aof grape , NtATGs f pepper . These rf pepper ,15,25. CRNAi-AtATG18a transgenic Arabidopsis plants were autophagy-defective and more sensitive to oxidative, salt, and drought stresses than WT [MdATG18a of apple showed significant up-regulation in response to leaf senescence, drought, heat, oxidative stress, and nitrogen starvation, and overexpression of MdATG18a in tomato and apple enhanced drought tolerance [CsATG18a in Arabidopsis also improved tolerance to osmotic stress, salt, and drought, which is consistent with previous results. Another member, CsATG18b, was shown to confer enhanced tolerance to osmotic stress, salt, and cold. This result provides new evidence regarding the functions of ATG18b in plants. Under abiotic stresses, a common response in cells is ROS accumulation and oxidative damage, including membrane breakage and protein oxidation and aggregation [CsATG18a and CsATG18b transgenic plants might be because the damaged proteins and components are more efficiently degraded and recycled by the autophagy pathway, thereby reducing ROS accumulation and maintaining membrane integrity under stress [CsATG18a and CsATG18b was noticed under drought and cold. Overexpression of CsATG18a conferred drought but not cold tolerance, whereas overexpression of CsATG18b conferred cold but not drought tolerance. This result appears reasonable and explainable. First, CsATG18a and CsATG18b have very different gene structures and predicted subcellular localization, which indicates their functional divergence (CsATG18a has a higher expression level under drought but a lower expression level under cold than CsATG18b (CsATG18a and CsATG18b may be regulated by different transcription factors (TFs) under drought and cold. There is evidence that ATG18a interacts with the WRKY33 transcription factor to mediate autophagosome formation and necrotrophic pathogen resistance in Arabidopsis [HsfA1a transcription factor binds to the ATG18f promoter to regulate drought tolerance in tomato [ATG18a and ATG18f are regulated by two different TFs, indicating that CsATG18a and CsATG18b may be also regulated by different TFs, thereby resulting in their functional differentiations. ATG18 is a WD40 domain containing protein family with multiple members. They can function in vesicle formation in autophagy by binding to the phosphatidylinositol 3-phosphate sites in plants . Among t than WT ,37. MdATolerance ,38. In tregation ,37. Thusr stress . Interesvergence . Second,CsATG18b . Third, n tomato . ATG18a Citrus sinensis (L.) Osbeck) was used as the plant material in this study. The seeds of “Hamlin” sweet orange were collected from the Citrus Research Institute, Southwest University, Chongqing, China. The seeds were sterilized and germinated, and the seedlings were hydroponically cultured at 25 °C and 16 h photoperiod (50 µmol m−2 s−1), as described by Fu et al. [“Hamlin” sweet orange  [Arabidopsis ATG proteins (AtATGs) as query sequences [e-value ≤ e−10 [ATG genes were submitted to the PFAM database (http://pfam.xfam.org/) to confirm the existence of the ATG domains. All of the identified ATG genes of sweet orange were named as CsATGs.To identify all putative ATG proteins of the sweet orange, a BLASTP search of the sweet orange genome database (orange/)  was perfequences ,3. The re ≤ e−10 . After rhttp://www.softberry.com/berry.phtml?topic=protcomppl&group=programs&subgroup=proloc). To construct phylogenetic trees, all putative CsATGs and the ATG proteins from Arabidopsis thaliana [Nicotiana tabacum) [Oryza sativa) [Vitis vinifera) [CsATG genes were provided by the sweet orange genome database, and MapInspect software (http://mapinspect.software.informer.com) was used to draw the location images. For the gene structure analysis, the generic feature format (GFF) file of each CsATG was downloaded from the sweet orange genome database and then submitted to the Gene Structure Display Server  to display intron-exon structures [https://www.ncbi.nlm.nih.gov/cdd/).The subcellular locations of the CsATG proteins were predicted online by using the ProtComp v. 9.0  , rice (O sativa) , and grainifera)  were firinifera) . The geninifera) . The booructures . The GFFructures . The conCsATG genes were designed using the online primer-blast program in the NCBI website, whereas Actin (Cs1g05000.1) was used as a reference gene to normalize the relative expression levels of the tested genes. Three biological replicates and three technical replicates were performed for each treatment.Total RNA was extracted from the CK and stress-treated leaves or roots of “Hamlin” sweet orange seedlings using a RNAprep pure plant kit , and the RNA concentration and quality were determined with a Nanodrop 2000 spectrophotometer . Then, 1 μg of high-quality RNA was used for cDNA synthesis with an iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. qRT-PCR was performed on the Bio-Rad CFX Connect RealTime system using ChamQ Universal SYBR qPCR Master Mix . Each PCR reaction contained 5.0 μL SYBR mix, 0.2 μM primers, and 1.0 μL diluted cDNA in a final volume of 10 μL. Specific primers of CsATG18a and CsATG18b were first isolated from “Hamlin” sweet orange. After being confirmed by sequencing, the full CDS fragments of CsATG18a and CsATG18b were recombined into a pGBO vector to produce 35S-CsATG18a and 35S-CsATG18b constructs, respectively. The two constructs were then transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. The Arabidopsis thaliana wild-type  plants were used to perform Agrobacterium-mediated transformation by the floral dipping method [−1 kanamycin until a homozygous T4 generation was obtained. The expression levels of transgenes were measured by qRT-PCR.The CDS (coding sequence) fragments of g method . The traCsATG18a and CsATG18b transgenic Arabidopsis plants to different abiotic stresses, three independent T4 homozygous lines with relatively high expression levels of CsATG18a and CsATG18b were selected for NaCl, mannitol, drought, and cold treatments, and WT (Col-0) plants were used as controls. For the NaCl and mannitol treatments, the seeds of transgenic lines and WT were sown on 1/2 MS with 200 mM NaCl or 400 mM mannitol, and the germination rate was calculated at 3 to 7 days. For the drought treatment, 30 day old transgenic and WT plants were dewatered for 15 days and then watered for recovery. For the cold treatment, 30 day old transgenic and WT plants were transferred to a −20 °C freezer for 5 min and then back to room temperature for recovery. After drought and cold treatments, the relative electrolytic leakage was measured as described by Shi et al. [To evaluate the tolerance of i et al. .CsATG genes were identified from the genome of sweet orange. These CsATGs showed high sequence and phylogeny similarities with the AtATGs of Arabidopsis. Most CsATG genes were significantly up- or down-regulated in response to drought, heat, cold, salt, and osmotic stresses, as well as to excess Cu, Mn, and Cd stresses. Overexpression of CsATG18a and CsATG18b enhanced tolerance to both salt and osmotic stresses in Arabidopsis. Overexpression of CsATG18a in Arabidopsis conferred drought but not cold tolerance, whereas overexpression of CsATG18b conferred cold but not drought tolerance. This study analyzed CsATG genes in citrus and preliminarily revealed their important roles in response to abiotic stresses. In future, their functions and the underlying mechanisms will be further uncovered by overexpressing in citrus plants.A total of 35


Protein folding is a substantively error prone process, especially when it occurs in the endoplasmic reticulum (ER). The highly exquisite machinery in the ER controls secretory protein folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol; these misfolded proteins are then degraded by the ubiquitin–proteasome system termed as the ER-associated degradation (ERAD). The 26S proteasome is a multisubunit protease complex that recognizes and degrades ubiquitinated proteins in an ATP-dependent manner. The complex structure of the 26S proteasome requires exquisite regulation at the transcription, translation, and molecular assembly levels. Nuclear factor erythroid-derived 2-related factor 1 , an ER-resident transcription factor, has recently been shown to be responsible for the coordinated expression of all the proteasome subunit genes upon proteasome impairment in mammalian cells. In this review, we summarize the current knowledge regarding the transcriptional regulation of the proteasome, as well as recent findings concerning the regulation of Nrf1 transcription activity in ER homeostasis and metabolic processes. Proteins are essential to the vast majority of cellular and organismal functions. Cellular protein levels are defined by the balance among protein synthesis, folding, and degradation, and the failure to accurately coordinate these processes leads to disease . Under nThe ubiquitin–proteasome system (UPS) is the main machinery responsible for protein degradation in all eukaryotes. Substrate proteins are covalently modified by a small protein called ubiquitin that is activated by the coordinated actions of E1, E2, and E3 enzymes . The 26SApproximately one-third of all cellular proteins enter the endoplasmic reticulum (ER) and are exported to their final location. Proteins retrotranslocated from the ER to the cytosol are mainly degraded by the UPS in a process known as ER-associated degradation (ERAD) ,10. The Proteasomal degradation is vital in all cells and organisms, and dysfunction or failure of which is associated with a diverse range of human diseases, including autoinflammatory syndromes and neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS); on the other hand, an increase in proteasome activity is observed in cancer cells, emphasizing the importance of proper functional maintenance of the proteasome ,14,15. T+ ATPase p97/valosin-containing protein (VCP) for degradation by the proteasome. Hrd1, a major ER-resident E3, forms a complex with the ER-resident Sel1L  and plays a central role in the degradation of a subset of ER-resident misfolded proteins [Proteins newly synthesized by ER-bound ribosomes are inserted into the ER ,19,20. Mproteins . It is kproteins ,23. In aproteins . Recent proteins ,25,26,27In addition to misfolded proteins, ERAD degrades properly folded proteins in the ER. Human 3-hydroxyl-3-methylglutaryl-coenzyme A reductase (HMGR), an ER-resident integral membrane, limits the rate of mevalonic acid production and undergoes feedback-regulated degradation. When sterol levels are high, HMGR associates with insulin-induced gene (INSIG), which brings an ER-resident E3 gp78 close to HMGR, causing its ubiquitination and degradation by the proteasome . Ligand-Chlamydomonas reinhardtii recently revealed that the cytosolic ERAD machinery is concentrated within 200 nm foci that contact specialized patches of the ER membrane [The ERAD plays an essential role in maintaining protein homeostasis by alleviating the protein burden on the ER. Although a fraction of proteasomes are known to associate with the ER membrane ,31, in smembrane . These nSaccharomyces cerevisiae (budding yeast) to mammals, and is composed of a 20S core particle (CP) and a 19S regulatory particle (RP) that binds to either one or both ends of the CP [+ ATPase subunits (Rpt1-Rpt6) and four non-ATPase subunits . The six AAA+ ATPases form a ring and participate in the unfolding and translocation of the substrates towards the interior cavity of CP in an ATP-dependent manner. Rpn10 and Rpn13 function as ubiquitin receptors [The 26S proteasome is highly conserved in eukaryotes from f the CP ,34. In tf the CP . Recognif the CP ,37. The eceptors ,39. Rpn1eceptors ,41. CP aCaenorhabditis elegans (C. elegans), Drosophila melanogaster (D. melanogaster), and mammals, which is similar to yeast even though Rpn4 is not conserved [D. melanogaster), SKN-1 (C. elegans), and Nrf1  are responsible for the expression of proteasome subunit genes, especially in the compensatory response (termed as the bounce-back response) [C. elegans and fruit flies, although its exact mechanism remains elusive [Increasing the proteasome abundance when needed is important for sustaining protein degradation and cell viability, as the proteasome is essential for a broad range of housekeeping functions. As each proteasome subunit exhibits a distinct structure and specific function that cannot be fully substituted by the other subunits, the expression of all proteasome subunits is coordinately regulated at the transcriptional level . In yeasonserved . In thesesponse) ,17,41,45 elusive ,49,50,511/2 ≈ 2 min) as it is continually degraded by the proteasome [Rpn4 is essential for stress-induced proteasome expression in a negative feedback loop, as well as for constitutive proteasome expression in yeast . Rpn4 bioteasome . Therefooteasome . In addioteasome .C. elegans [Three protein isoforms  that show different expression patterns are generated by differential splicing and transcription start site utilization in  elegans . All thr elegans . Of the  elegans . SKN-1B  elegans . SKN-1C  elegans .The fruit fly transcription factor CncC regulates the expression of phase I and II enzymes, antioxidant proteins, and proteasome subunits . AlthougVertebrates have four Nrf genes, namely, Nrf1, Nrf2, Nrf3, and nuclear factor erythroid 2 (NF-E2), implying that a diversification of the protein family occurred during vertebrate evolution. A recent study has revealed that Nrf1, Nrf2, and Nrf3 contribute to proteasome subunit gene expression with varying significance ,64. Nrf1In mammals, several transcription factors such as nuclear transcription factor Y (NF-Y), forkhead box protein O4 (FOXO4), and signal transducer and activator of transcription 3 (STAT3) are known to regulate the expression of proteasome subunit genes ,68,69. ND. melanogaster CnC gene and is a 43-amino acid region that flanks the N-terminal of the bZIP domain. In mammals, the CNC-bZIP family includes six members, namely, Nrf1, Nrf2, Nrf3, NF-E2 p45 subunit, BTB, and CNC homolog 1 (Bach1), and Bach2 [SKN-1, CncC, and Nrf1 are among the CNC basic leucine zipper (CNC-bZIP) family of transcription factors that require the Maf dimerization partner to stably bind DNA ,71. The nd Bach2 . The bZInd Bach2 . Each SKnd Bach2 ,75,76. And Bach2 . NotablyIt is generally known that Nrf1 is co-translationally inserted into the ER membrane and that only a small portion of its N terminus is protruded into the cytosol, whereas the bulk of its polypeptide, including the transcriptional activation and DNA-binding domains, is embedded in the ER lumen. ER-resident proteins are generally degraded by the ERAD when they are misfolded. Interestingly, Nrf1 is constantly subjected to ERAD under normal conditions . In factC. elegans, the E3 substrate adaptor WDR-23 binds simultaneously to DDB-1, the cullin4 (CUL4) ortholog, and SKN-1, and is thought to trigger ubiquitylation and degradation of SKN-1 [Moreover, Nrf1 stability is regulated in the nucleus. Nuclear Nrf1 interacts with beta-transducin repeat-containing E3 (β-TrCP). β-TrCP forms a Skp1–Cul1–F-box (SCF) E3 complex that recognizes the DSGLS sequence in Nrf1, and ubiquitinates Nrf1 for degradation . Recentlof SKN-1 . Thus, NThe nascent Nrf1 is tethered to the ER membrane at its N-terminal transmembrane domain, and a large proportion of its polypeptide is N-glycosylated in the ER lumen . The lum2+-regulatory calpains act as a class of limited proteases that may contribute to the selective proteolytic processing of Nrf1 [It has also been shown that Nrf1 is proteolytically processed into a soluble, active C-terminal fragment that enters the nucleus and activates transcription upon partial proteasome inhibition. If proteasome activity is inhibited to a fuller extent, Nrf1 becomes incorporated into protein aggregates and becomes insoluble. These results imply that some proteasome functionality is necessary for Nrf1 processing; however, the exact mechanism is still unknown . In addi of Nrf1 . However of Nrf1 ,88. Alth of Nrf1 ,90. Howe of Nrf1 . TherefoC. elegans, ddi1 (C. elegans ortholog of DDI2) mutants were identified as having suppressed SKN-1-dependent induction of proteasome gene expression [C. elegans and mammals. This indicates that the activation mechanism of Nrf1/SKN-1 by the DDI proteins is widely conserved in multicellular animals.DDI2 was identified as a Nrf1 nuclear translocation regulator by an image-based screening of mammalian cells . By genepression . DDI famStudies on yeast Ddi1 (yeast ortholog of DDI2) provided evidence for its proteolytic activity, which was found to be required for sufficient checkpoint regulation, and it also contributes to DNA replication stress response and to DNA-protein crosslink repair; however, very little is known regarding the physiological roles of DDI proteins in mammals ,94,95. YThe UBL domain of human DDI2 is involved in Nrf1 processing , probablNrf1 transcription activity is regulated both positively and negatively by phosphorylation and glycosylation.Drosophila [png-1; C. elegans ortholog of human NGLY1) which is involved in the bounce-back response of proteasome gene expression. Recently, it was reported that NGLY1 edits the sequence of SKN-1A by converting its glycosylated Asn (-N-S/T-) into acidic Asp (-D-S/T-) residues. This editing is required for DDI-1-mediated processing of SKN-1A and the expression of proteasome genes in defending against proteotoxic stress in C. elegans [N-linked glycosylation and subsequent deglycosylation steps have critical roles in Nrf1 transcription activity. Glycosylation occurs in its Asn/Ser/Thr-rich (NST) domain that consists of seven Asn-X-Ser/Thr consensus motifs for glycosylation. NGLY1 is thought to be responsible for deglycosylating misfolded proteins prior to cytosolic degradation ,100. NGLosophila . These r elegans . Accordi elegans . Glycosy elegans . These dIn addition to the effects of N-glycosylation on Nrf1, O-GlcNAcylation of Nrf1 by OGT is also essential for Nrf1 stabilization and for the bounce-back response ,104. OGTNrf1 activity is increased by treatment with okadaic acid, which is a phosphatase inhibitor, and is suppressed by the protein kinase C (PKC) inhibitor staurosporine. This suggests that phosphorylation has a positive effect on Nrf1 activity ; howeverIn addition to controlling the expression of proteasome subunit genes, Nrf1 also controls the expression of Herpud1 and p97/VCP, which are components of the ERAD pathway ,109. NrfC. elegans revealed that activation of proteasome subunit expression by SKN-1A is triggered by misfolded endogenous proteins and the human amyloid beta peptide, even though proteasome function is not impaired, suggesting that SKN-1A activation is a general protective mechanism against defective proteostasis [Because studies on the activation of Nrf1 have been largely restricted to its response to proteasome inhibitors, the precise molecular mechanism behind the nuclear transport and activation of Nrf1 is not entirely clear. A recent study in eostasis . Indeed,eostasis .Recently, the TIP60 chromatin regulatory complex was shown to be necessary for the Nrf1-dependent transcription of proteasome subunit genes; however, the exact nature of this requirement remains to be uncovered .C. elegans [The proteasome actively contributes to ER quality control through the ERAD . Thus, p elegans .C. elegans, the ablation of germ-like stem cells causes SKN-1 activation, thereby increasing proteasome activity, stress resistance, and lifespan [D. melanogaster [Gst genes [HK1 mRNA expression, and HK1 protein was found to be highly increased in the mitochondrial fraction [In lifespan . Loss-oflifespan . Overexpnogaster ,129. Nrfnogaster ,17. In cst genes ,131,132.st genes . It was fraction . Transgefraction , implyinNfe2l1 gene, which encodes Nrf1 [In addition to stress-related stimuli, mTORC1-mediated SREBP activity is known to activate Nrf1 expression . mTORC1 des Nrf1 . Treatmedes Nrf1 ,140. ThumTORC1 also plays a role in the prevention of ER stress through the activation of SREBP and de novo lipid synthesis. Liver-specific deletion of Nrf1 in mice results in hepatic lipid accumulation by disruption of lipid homeostasis, suggesting that Nrf1 controls the lipid metabolism by expression of a subset of enzymes ,141. Of To date, Nrf2 has been assumed to act as a proteasome subunit gene transcription factor until further details on the molecular function of Nrf1 are revealed. In addition, a recent study suggests that the ER-resident Nrf3 has the potential to act with an activity overlapping that of Nrf1, even though the function of Nrf3 is tissue-specific.Nrf2, which is widely accepted to be a master regulator of adaptive responses to oxidative stress and electrophiles, contains the same consensus binding sequence as Nrf1 ,144. IntChop, IRE1, and ATF6 genes against tunicamycin, although Nrf2 is also partially involved in this response [-/- (but not Nrf2-/-) revealed that Nrf1 is necessary for cellular homeostasis and organ integrity, and that it cannot be replaced by Nrf2 [Pharmacologically, tunicamycin only increases Nrf1 protein levels, and sulforaphane only increases Nrf2 protein levels. Moreover, the Nrf1 activity of proteasome induction was shown to be repressed by tunicamycin ,151. Nrfresponse . In contresponse . Collect by Nrf2 ,155,156.CPEB3 gene expression, and induced CPEB3 represses Nrf1 translation through a cytoplasmic polyadenylation element in the 3′-UTR of the Nrf1 gene. Recent ChIP-seq data demonstrated that Nrf1 and Nrf3 peaks show relatively similar distributions, suggesting that Nrf1 and Nrf3 act complementarily [Nrf3 is controlled in part by a PEST degron sequence and has three isoforms with varying subcellular localizations, including isoform “A” in the ER, isoform “B” in the cytosol, and isoform “C” in the nucleus . Similarentarily . In contPSMB5 (a target subunit of the proteasome inhibitor bortezomib), aberrant expression of UPS pathway components, and induction of drug efflux from cells [Multiple pieces of evidence have unveiled the association between proteasome function and various diseases such as cancer, neurodegeneration, autoinflammation, and autoimmunity. Proteasome inhibitors have been demonstrated to exhibit remarkable effects in the treatment of multiple myeloma and other malignant neoplasms, while the emergence of drug resistance against proteasome inhibitors is a major concern during cancer treatment. Combating these resistance mechanisms can serve as a potentially useful strategy for making proteasome inhibitors more effective for treating cancers and solid tumors. However, there have been multiple observed mechanisms of resistance to proteasome inhibitors, such as mutations in om cells . The bouom cells . In thisom cells . It has om cells ,165. DDIom cells . Conversom cells . Pharmacom cells ,167. Rotom cells . Hrd1/syom cells . Anotherom cells . ASC-JM1om cells . Inorganom cells ,170. TheRecent studies have unveiled the regulatory mechanisms of Nrf1 activation; however, many questions still remain unsolved. Thus far, the mechanism of DDI2 activation is unclear because the protease activity of purified recombinant DDI2 has not yet been confirmed in vitro. A recent study has shown that yeast Ddi1 cleaves substrate proteins in vitro only when they are tagged with long ubiquitin chains (approximately longer than eight ubiquitins) . It has


Cancer is one of the highest prevalent diseases in humans. The chances of surviving cancer and its prognosis are very dependent on the affected tissue, body location, and stage at which the disease is diagnosed. Researchers and pharmaceutical companies worldwide are pursuing many attempts to look for compounds to treat this malignancy. Most of the current strategies to fight cancer implicate the use of compounds acting on DNA damage checkpoints, non-receptor tyrosine kinases activities, regulators of the hedgehog signaling pathways, and metabolic adaptations placed in cancer. In the last decade, the finding of a lipid peroxidation increase linked to 15-lipoxygenases isoform 1 (15-LOX-1) activity stimulation has been found in specific successful treatments against cancer. This discovery contrasts with the production of other lipid oxidation signatures generated by stimulation of other lipoxygenases such as 5-LOX and 12-LOX, and cyclooxygenase (COX-2) activities, which have been suggested as cancer biomarkers and which inhibitors present anti-tumoral and antiproliferative activities. These findings support the previously proposed role of lipid hydroperoxides and their metabolites as cancer cell mediators. Depletion or promotion of lipid peroxidation is generally related to a specific production source associated with a cancer stage or tissue in which cancer originates. This review highlights the potential therapeutical use of chemical derivatives to stimulate or block specific cellular routes to generate lipid hydroperoxides to treat this disease. Oxidative stress and inflammation are linked to cancer development ,2. Mutat•), which is a key radical that participates in peroxidation reactions.Cellular lipid peroxidation can occur through different reactions, but they can be categorized into enzyme and non-enzyme dependent reactions. The primary substrates in lipid peroxidation reactions are polyunsaturated lipids since carbon-carbon double bonds are susceptible to reactive oxygen species, such as the hydroxyl radical  and the phospholipids containing this fatty acid are essential molecules since they are precursors of pro- and anti-inflammatory mediators, sometimes enriched at cellular locations identified as signaling platforms, such as the plasma membrane lipid rafts -benzenesulfonamide (SC-236) and [2-acetoxy-(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS)  A, which Lipid hydroperoxides generated via enzyme or non-enzyme-dependent reactions can be further oxidized to form highly reactive species and lipid autoxidation products. Acrolein, malonaldehyde, and 4-hydroxynonenal can covalently modify proteins leading to functional and structural changes in proteins ,124,125.Antioxidants play a central role to counteract lipid peroxidation. In the non-enzyme-dependent reactions, the radical chain reactions can terminate when antioxidants react with the alkyl peroxyl or the alkoxyl radicals. Tocopherol is the main membrane antioxidant in charge of reacting with these radicals and one of the primary membrane antioxidants against reactions generating lipid peroxides, in general . Consequ2 (PGH2) is a PG generated by both COX-1 and -2 using AA as a substrate, which acts as a precursor for the generation of other PGs such as the thromboxane A2 (TxA2) and the PGs named PGI2, PGD2, PGE2, and PGF2α [2 and cancer development has been observed [The main AA oxidation products of COXs activity are PGs. Prostaglandin Hnd PGF2α . PGs arend PGF2α . In addind PGF2α ,144,145.nd PGF2α ,147. COXobserved ,35.2 by COXs, a peroxidation and cyclooxygenation reaction of the substrate is required. On the distal side from the heme moiety, an array of amino acids provides the site for hydroperoxides to bind and assists the heme-dependent reduction of hydroperoxides to alcohols [2 requires several steps for its production [2 (PGG2) through COX activity using AA as a substrate. This PG can be further metabolized to a second unstable endoperoxide intermediate named PGH2, which can be enzymatically broken down to generate the PGE2 [For the generation of PGEalcohols . Therefooduction . First, the PGE2 ,26,27,282 acts binding to any of the specific G-coupled receptors belonging to four subclasses, EP1-EP4, which have specific signal transduction activities, tissue localization, and regulation [2 activity highly dependent on the specific docking receptor [PGEgulation . The actreceptor . The rolreceptor .2 are compounds, such as 15-keto-PGE2, generated by 15-hydroxyprostaglandin dehydrogenase [2 and thiazolidinediones, have been proposed as potential pharmacological therapeutical drugs in cancer [2 (15d-PGJ2), are also known as PPARγ agonists. Besides, PGD2, PGJ2, Δ12-PGJ2 are also known to induce the apoptosis of tumoral cells [Moreover, downstream metabolites derived from PGErogenase ,153. Thirogenase ,155. Somn cancer ,158,159.n cancer . Other Aal cells ,161.1  have been identified in humans . HistoriS)-hydroperoxyeicosatetraenoic acid (12(S)-HpETE), which is converted to the end-product named 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) implicated in the promotion of tumorigenesis, proliferation, and metastasis by stimulation of the vascular endothelial growth factor (VEGF), and some integrins expression and controlling the cell cycle [S)-HETE, metabolites generated from 5-LOX such as 5-hydroxyeicosatetraenoic acid (5(S)-HETE), which precursor is 5(S)-HpETE, have been implicated in the stimulation of prostate cancer cell growth [S)-HpODE) [S-hydroxyoctadecadienoic acid (13-S-HODE), which are reported in human colorectal and esophageal cancers [The 12-LOX utilizes AA to synthesize 12-HpODE)  and 13-S cancers . These e cancers ,184,185, cancers . Other d cancers , support cancers . Notably cancers . These rMoreover, 15-LOX-1 activity is elevated when ferroptosis is induced in cancer cells . Some ofThe nonsteroidal anti-inflammatory drugs acting as inhibitors of COX/LOX named tepoxalin , and lic2O2 production dependent upon iron can not only be attributed to Fenton’s chemistry [In cancer, lipid peroxidation became interesting for researchers since its involvement, and implication in cancer cell ferroptosis was discovered . Many wehemistry . All 15-hemistry ,209, plahemistry ,211. Thehemistry ,212,213.S,3R)-RSL-3 (RSL3) was the first described irreversible inhibitor of GPX4 [Ferroptosis can be triggered by inhibition of GPX4 , a perox of GPX4 ,199,209. of GPX4 . Due to  of GPX4 ), can al of GPX4 . Moreove of GPX4 .Km value for quinones in the same range reported for FSP1 [Km values for quinones, are also putative targets for cancer treatment combined with GPX4 inhibitors. We proposed a list of described mammalian QRs with reported Km values, the name and structure of the specific inhibitors available for these enzymes, and the IC50 value reported for cancer cells as potential targets to inhibit to promote ferroptosis in conjunction with GPX4 inhibitors  nanoparticles co-encapsulating a COX-2 inhibitor (celecoxib) and a herbal compound (naringenin) has shown promising results for lung cancer treatment in in vitro studies [Regarding COX inhibitors, the use of nebulized colloidal poly  studies . A simil studies . As pert