Since outbreak in December 2019, the highly infectious and pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over a million deaths globally. With increasing burden, the novel coronavirus has posed a dire threat to public health, social interaction, and global economy. Mutations in the SARS-CoV-2 genome are moderately evolving which might have contributed to its genome variability, transmission, replication efficiency, and virulence in different regions of the world.The present study elucidated the mutational landscape in the SARS-CoV-2 genome among the African populace, which may have contributed to the virulence, spread, and pathogenicity observed in the region. A total of 3045 SARS-CoV-2 complete protein sequences with the reference viral sequence (EPI_ISL_402124) were mined and analyzed. SARS-CoV-2 ORF1ab, spike, ORF3, ORF8, and nucleocapsid proteins were observed as mutational hotspots in the African population and may be of keen interest in understanding the viral host relationship, while there is conservation in the ORF6, ORF7a, ORF7b, ORF10, envelope, and membrane proteins.The accumulation of moderate mutations (though slowly), in the SARS-CoV-2 genome as seen in this present study, could be a promising strategy to develop antiviral drugs or vaccines. These antiviral interventions should target viral conserved domains and host cellular proteins and/or receptors involved in viral invasion and replication to avoid a new viral wave due to drug resistance and vaccine evasion.The online version contains supplementary material available at 10.1186/s43088-021-00102-1. The novel coronavirus infection was first reported in December 2019 in Wuhan, China. The viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially referred to as the Wuhan seafood market pneumonia virus before it was officially named by World Health Organization (WHO) on February 12, 2020, as coronavirus disease 2019 (COVID-19). As of January 2, 2021, 05:07 GMT, COVID-19 has spread in over 216 countries with 84,382,536 confirmed cases and 1,835,389 deaths, out of which 2,800,709 confirmed cases and 66,224 deaths were reported in Africa [ORF1ab, ORF3a, ORF6, ORF7a/b, ORF8, spike (S), envelope (E), membrane (M), and nucleocapsid (N) gene  among all RNA viruses. It encodes  to 32 kbComprehensive genomic analysis of epidemiologic viral sequences can be a promising approach to understanding SARS-CoV-2 transmission and pathogenesis. Thus, genomic variability studies of SARS-CoV-2 can help in gaining insight into its genomic diversity in order to adopt measures in curtailing the menace of COVID-19. The efforts of researchers, WHO, National Center for Biotechnology Information (NCBI), and GISAID in making scientific reports and SARS-CoV-2 genome sequences publicly available has aided rapid understanding of viral transmission and the host-viral mechanism of action , 11. VacPrevious studies , 13, 14 https://epicov.org/epi3) on January 2, 2021. A total of 3045 SARS-CoV-2 complete protein sequences from 27 African countries  were mined, after filtering as all lineage and clade, complete, Homo sapiens as host, high coverage only, collection and submission date of the viral sample , and originating from Africa, while low coverage sequences (> 5% NNNs) which might be the product of sequencing errors were excluded. Comprehensive geographic information, the laboratory where the viral samples were sequenced, and accession numbers of SARS-CoV-2 genomic sequences used in the present study are provided as We used a slightly modified methodology . A totalhttps://epicov.org/epi3). Viral sequences with incomplete genomic sequence, low coverage, undetermined residues (labeled as X), and genomic sites with few or single mutations were excluded. While recurrent mutations were focused on as they are likely candidates for SARS-CoV-2 transmissibility, adaptability to the human host, and possible target for drug and vaccine development. The viral sequences were classified into clades  based on the mutational distribution. Mutation frequencies were presented numerically and as percentages using basic arithmetic operations.In the present study, the mined 3045 SARS-CoV-2 datasets were analyzed with respect to the reference sequence WIV04 (EPI_ISL_402124) using the EpiCoV web interface (pro); G3278S , nsp6; L3606F , and RNA dependent RNA polymerase (RdRp) region; P4715L. Mutations at RdRp P4715L occurred in 2787 viral sequences (91.5%) flagging the RdRp region as a SARS-CoV-2 mutational hotspot. The nsp2 I739V and P765S variants were simultaneously observed in 12 viral sequences, predominantly from Nigeria (66.67%). More gene variants were observed in the ORF1ab non-structural proteins; nsp2, 3CLpro, and RdRp.Our analysis of 3045 SARS-CoV-2 sequences from 27 African countries studied highlights high rate recurrent mutations observed at the following genomic sites in the SARS-CoV-2 ORF1ab polyprotein site: nsp2 region; T265I , 3 chymotrypsin-like proteinase region  carry the N501Y mutation. The spike D614G mutation was observed in 2881 viral sequences (94.6%), flagging this position as a SARS-CoV-2 mutational hotspot in Africa. Highly recurrent mutations were also observed in the SARS-CoV-2 ORF3 protein: Q57H and G251V mutations in 383 and 22 viral sequences, respectively. The prevalence of the ORF3 Q57H missense mutation in South African samples is relatively low (6.4%) with a relatively high incidence in Egypt (62.7%).The ORF8 L84S variant was observed in 169 viral sequences with very few/no observance in viral sequences from Southern and Central Africa. Recurrent mutations in the viral genome are presented in Table The genome, being the molecular architecture of life encodes its phenotypic and genotypic expression. Evolving SARS-CoV-2 gene variants play a significant role in its replication, spread, and pathogenicity with respect to its human host . The preGenerally, the clade GR—1534 (50.4%), and G—895 (29.4%) characterized by the spike D614G, nucleocapsid R203K, and G204R variants were the most prevalent in our study. Clades G and GR from previous reports , 16 havehttps://epicov.org/epi3). Due to its high binding affinity (Kd 21.83 nM) to RdRp, Atazanavir has been identified as a potential COVID-19 therapeutic candidate [The leader protein, ORF1ab, cleaved into nonstructural proteins nsp1-nsp16) is essential for genome replication. RdRp is responsible for viral RNA replication, thus, due to this important role, it is expected that RdRp is well conserved. Interestingly, the present study corroborates earlier findings , 14 withandidate . Interesandidate , 16. is essenpro enzyme plays a vital role in the SARS-CoV-2 life cycle, replication, and processing of the carboxyl-terminus of nsp4 through nsp16 [pro was a candidate antiviral drug target during the outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV. Molecular docking application has identified aliskiren, dipyridamole, mopidamol, and rosuvastatin as potential antiviral candidates due to their relatively high binding energy to the ORF1ab 3CLpro domain [pro make it a suitable antiviral target as reported in previous studies [The 3CLgh nsp16 . The 3CLo domain . The sig studies , 20.The S glycoprotein mediates host cell-surface receptor binding via its S1 domain and induces host-membrane fusion through the S2 domain. This suggests its important role in viral-host tropism, transmission, and invasion . SeveralThe N phosphoprotein composed of the carboxyl- and N-terminal domain forms the ribonucleoprotein complex with the viral RNA; which enhances viral genome transcription, facilitates helical nucleocapsid formation, and membrane protein interaction during virion assemblage , 22. GenThe M protein which consists of three transmembrane domains determines the shape of the viral envelope, while the E protein facilitates viral assemblage and budding . The inthttp://www.chinadaily.com.cn/). Hence, the quest for a clinically approved and efficient therapeutic agent is still on and our study has been able to suggest potential targets for drugs or vaccine development.There is no substantial report to attribute the involvement of ORF10 in SARS-CoV-2 transmission and pathogenesis. The viral ORF3 and ORF10 proteins can synergistically attack heme on the host’s hemoglobin 1-β chain, thereby disintegrating iron to form porphyrin. This will result in reduced levels of hemoglobin carrying oxygen and carbon dioxide, interfering with the heme pathway, extreme poisoning, and inflammation of the hepatocytes . StudiesCurrently, the Pfizer-BioNTech, Moderna, and AstraZeneca’s COVID-19 vaccines have been authorized and recommended for use, while the Janssen and Novavax, among many other COVID-19 vaccines Phase 3 clinical trials are being planned or currently in progress . HoweverSARS-CoV-2 genomic instability shows that antiviral drugs targeting the viral proteins might not be as potent as drugs targeting the host’s cellular proteins and/or receptors. Hence, developing antiviral interventions with respect to the viral conserved domains and host proteins involved in viral invasion and replication might be a promising strategy. Africa is blessed with traditional plants that have been used over the years in managing and treating a wide spectrum of diseases; hence, the combination of traditional African medicine and other candidate antiviral drugs might have better therapeutic prospects. No doubt, the traditional plants and candidate medicines will require extensive clinical trials to ascertain the safety concerns, mechanism of action, adverse effects, and efficacy.Additional file 1. Supplementary file S1.Additional file 2. Supplementary file S2.


Diffuse gliomas are among the most common adult central nervous system tumors with an annual incidence of more than 16,000 cases in the United States. Until very recently, the diagnosis of these tumors was based solely on morphologic features, however, with the publication of the WHO Classification of Tumours of the Central Nervous System, revised 4th edition in 2016, certain molecular features are now included in the official diagnostic and grading system. One of the most significant of these changes has been the division of adult astrocytomas into IDH-wildtype and IDH-mutant categories in addition to histologic grade as part of the main-line diagnosis, although a great deal of heterogeneity in the clinical outcome still remains to be explained within these categories. Since then, numerous groups have been working to identify additional biomarkers and prognostic factors in diffuse gliomas to help further stratify these tumors in hopes of producing a more complete grading system, as well as understanding the underlying biology that results in differing outcomes. The field of neuro-oncology is currently in the midst of a “molecular revolution” in which increasing emphasis is being placed on genetic and epigenetic features driving current diagnostic, prognostic, and predictive considerations. In this review, we focus on recent advances in adult diffuse glioma biomarkers and prognostic factors and summarize the state of the field. Diffuse infiltrating gliomas are the second most common primary intracranial neoplasm, second only to meningioma. These tumors comprise approximately 22% of all central nervous system (CNS) neoplasms with more than 16,000 cases reported each year in the United States and a combined annual incidence of 5.13/100,000 individuals. In addition, glioblastoma (GBM) is the most common malignant CNS tumor, comprising 14.6% of all CNS tumors and 48.3% of all malignant CNS tumors with an average of 11,833 cases reported each year [IDH1 or IDH2 as the primary prognostic factor and molecular diagnostic criterion for adult astrocytomas [The 2016 WHO classification marked a significant departure from this previous morphology-alone classification, with the inclusion of chromosome 1p and 19q co-deletion as the “molecular signature” of oligodendroglioma and mutations in either ocytomas ,5. The aocytomas ,6. IDH1 R132 residue [Previous studies have shown in a grade-for-grade comparison within the astrocytoma group, IDH-mutant tumors have significantly longer progression-free survival (PFS) and overall survival (OS) compared to their IDH-wildtype counterparts ,8. As th residue ,10, immu residue ,12, with residue . IDH1/2 mutation, 2-hydroxyglutarate (2-HG), can be detected by magnetic resonance spectroscopy [ATRX and TP53 mutation to create the alternative lengthening of telomere (ALT) phenotype, as well as potentially promoting glial cell infiltration through HIF-1α and upregulating VEGF [Subsequent work also revealed that the oncometabolite produced as a result of the troscopy ,16,17, fing VEGF ,19,20,21ing VEGF ,23, althing VEGF . Additioing VEGF ,26.While IDH mutations have been established as one of the most important diagnostic and prognostic factors for adult diffuse gliomas, there remains significant intra-group heterogeneity in biologic behavior and clinical outcome in adult gliomas with significant survival outliers in all subtypes. As a result, a great deal of research effort has been spent searching for additional genetic and epigenetic prognostic factors to aid in further subdividing these tumors into more prognostically complete categories, as well as identifying new molecular targets for future therapies and understanding the underlying biology of these tumors. In hopes of addressing and better classifying these heterogenous tumors, research groups across the globe have reported a series of new molecular alterations and multivariate models. Additionally, numerous tumor entities have recently been discovered and defined by newly established recurrent molecular alterations . Recent th edition, including those currently being codified into clinical recommendations under the new Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) system [In this review, we discuss recent advances made in understanding molecular biomarkers and prognostic factors identified since the publication of the 2016 WHO Classification of Tumours of the Central Nervous System, Revised 4) system ,31. We dIDH1 or IDH2 confer a significant survival benefit in histologically lower-grade astrocytomas compared to their IDH-wildtype counterparts, and as such, is the most important prognostic factor in this group [TP53 and ATRX, respectively. With only rare exceptions noted, these mutations are thought to be mutually exclusive with the 1p/19q co-deletion [Lower-grade gliomas described here  include is group . IDH-mutdeletion ,36,37, adeletion ,39. The deletion , althougdeletion ,40,41,42CDK4 on chromosome 12q14 and CDKN2A on chromosome 9p21. CDK4 amplification and homozygous CDKN2A deletion have both been correlated with significantly shorter PFS and OS in multiple studies, and this effect was enhanced by the loss of 14q [RB1 gene, requiring either amplification of CDK4 (a cyclin-dependent kinase that inhibits RB1 activity) or homozygous deletion of CDKN2A  [CDK4 and CDKN2A alterations in all except one case. IDH-mutant astrocytomas with either CDK4 amplifications or CDKN2A deletions had significantly shorter survival  compared to IDH-mutant astrocytomas without these alterations  [RB1 with shorter survival is not as well defined. Alterations in the PI3K-AKT pathway, including PIK3R1 mutation, PIK3CA mutation, and PDGFRA amplification, have also been correlated with poor PFS and OS in cohorts of IDH-mutant astrocytomas [CDKN2A deletions as a factor predicting poor clinical outcome in this group but have suggested that there is not enough evidence to definitively include other alterations, including RB1 deletion or mutation, CDK4 and PDGFRA amplification, or PIK3R1 and PIK3CA mutations as independent prognostic factors at this time [Recent multi-dimensional genomic analyses ,43,44 has of 14q ,48,49,50hibitor) . Our ana = 0.02) . The assocytomas ,50. New his time .NRF2) and DJ1 are nuclear transcription factors that regulate intracellular antioxidants and phase II detoxification enzymes, protecting cells from oxidative damage. Increased nuclear Nrf2 and DJ1, as well as increased cytoplasmic levels of DJ1, are associated with better prognosis. However, increased cytoplasmic levels of Nrf2 have been shown to correlate with poor prognosis by an incompletely understood mechanism in IDH-mutant lower-grade astrocytoma cohorts [IGFBP2) and X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) [MYCN has also been correlated with shorter survival in some studies [EMP3, GSX2, EMILIN3, PPIC, and CHI3L1 [In other studies, isolated partial or complete loss of chromosome 19q with intact 1p has been shown to be associated with a better clinical outcome in IDH-mutant lower-grade astrocytomas but not in their GBM counterparts . Nuclear cohorts . IDH-mut1 (XAF1) ,57,58,59 studies , althoug studies . Others  studies  and wors studies . Multipld CHI3L1 ,63,64, ad CHI3L1 ,67,68,69IDH1 R132H, in that it is a continuous variable instead of binary, thus a threshold of 15% (approximately 470 megabases) for whole-genome CNV was established for clinical prognostic purposes [Multiple studies have also indicated that IDH-mutant lower-grade astrocytomas have significantly lower overall copy number variation , expressed as the sum of all gains and losses spread in a seemingly random pattern without regions of definitive “molecular signature” across the entire genome, compared to their IDH-mutant GBM counterparts ,67 or IDpurposes . CDK4 amplification or CDKN2A deletion and poor outcomes tend to have elevated CNV [CDK4 amplification or CDKN2A deletion [Higher overall CNV correlated with worse prognosis within the IDH-mutant astrocytomas, irrespective of other histologic or genetic alterations. Cases with ated CNV , althougdeletion ,42, suggdeletion  and mutadeletion ,52, suggdeletion . In addideletion ,72. cIMPdeletion .In short, numerous targeted and large-scale studies have now demonstrated that molecular features are superior to histologic criteria in terms of diagnosis and prognosis of lower-grade IDH-mutant astrocytomas ,66,73,74IDH1 or IDH2 and co-deletion of chromosomes 1p and 19q, regardless of other histologic features [ATRX or TP53 mutation [CIC and FUBP1  and the promoter region of TERT (TERTp) [TERTp (most frequently C228T or C250T) do not have a significant prognostic utility, due to the ubiquity of these mutations in adult oligodendrogliomas [CIC and FUBP1 mutations within the oligodendroglioma category is currently unclear.Oligodendrogliomas  are now features ,76. Thesmutation ,37,39, amutation ; althougmutation . Whole 1mutation ,80 and imutation . These t (TERTp) ,84,85,86ogliomas ,88, althogliomas ,90. The NOTCH1, PIK3CA, PIK3R1, and ARID1A [CDKN2A has also been associated with poor outcomes in patients with anaplastic oligodendroglioma [Oligodendrogliomas have a minimum whole-genome CNV level of approximately 5.1%, which is accounted for by the losses of 1p and 19q, as well as frequent loss of chromosomes 4, 14, 15, and 18 . In our d ARID1A ,45,93. Lroglioma . A numberoglioma ,96,97,98Although some studies have recently called the role of histologic features into question, in IDH-mutant 1p/19q co-deleted oligodendrogliomas histologic features remain an important prognostic factor with increased mitotic figures, microvascular proliferation (MVP), and necrosis dividing grade II from grade III ,100,101.IDH1 or IDH2 carries the most significant prognostic value [th decade compared to 7th decade, respectively) [TP53 and ATRX mutation as well as lower rates of TERTp mutation, PTEN mutation, and EGFR amplification [IDH mutations are found in a minority of all glioblastoma specimens, although this molecular signature is far more frequent in what was called “secondary glioblastoma” in previous grading schemes ,106,107.ic value ,7  ,105,106,CDK4 and CDKN2A/B alterations than their IDH-mutant lower-grade glioma counterparts, which corresponds to their worse clinical outcomes in terms of PFS and OS, but unlike within the lower-grade cohorts, we found no statistically significant prognostic effect of harboring these alterations in GBM [MET amplification, PDGFRA amplification, and CDKN2A deletion are associated with poor prognosis within some IDH-mutant GBM cohorts [SNRX1, NRF2, and DJ2, which may have prognostic implications in IDH-mutant GBM cohorts in addition to IDH-mutant lower-grade astrocytomas [Multiple studies have demonstrated that the histologic features separating IDH-mutant glioblastoma from IDH-mutant lower-grade astrocytoma (microvascular proliferation and/or necrosis) remain important prognostic factors, as IDH-mutant glioblastoma has significantly shorter median recurrence-free and overall survival intervals than IDH-mutant grade II or III astrocytomas , and IDHs in GBM . Other g cohorts ,72,75,94ocytomas .CCND2 gene, which may be a predictor of favorable prognosis [CCND2 gene amplification has been associated with gemistocytic histology and, in some cases, slower cell proliferation [CCND2 amplification is not always correlated to increased transcripts or protein expression [CCND2 amplification may be mediated through pep5, a Cyclin D2-derived peptide, which has been shown in some cancers to inhibit cell growth or cause cell death [CDK4 and CDK6. CDK4 and MDM2 (possibly MDM4 as well) are frequently co-amplified due to location at the breakpoint-enriched region of 12q14-15 [In previous work, we have shown high-level amplification of the 12p13 locus containing the rognosis . CCND2 gferation . CCND2 apression , and thell death ,119. The12q14-15 . This co12q14-15 ,121. Like IDH-mutant lower-grade gliomas, more complex multi-gene and multi-CNV prognostic schemes have also been devised for the IDH-mutant GBM category ,68,69, aEGFR amplification, combined gain of chromosome 7 with loss of chromosome 10 (7+/10−), TERTp mutation, and tumors with these three factors have similar outcomes to IDH-wildtype glioblastoma, while lower-grade tumors without these characteristic alterations have significantly better clinical outcomes [diffuse astrocytic glioma, IDH-wildtype with molecular features of glioblastoma“, for IDH wildtype astrocytomas lacking the histologic features of glioblastoma but having one or more of these “molecular grade IV” features [Like their IDH-mutant counterparts, IDH-wildtype lower-grade astrocytomas  include outcomes ,128,129.features , and thifeatures .MYB and BRAF V600E mutation, which have both been shown to correlate with longer OS in select grade II IDH-wildtype gliomas [There are, however, heterogenous subsets within this IDH-wildtype astrocytoma group, which show longer median survival, with a handful of proposed genetic alterations responsible for the better prognosis, including amplification of  gliomas . In addi gliomas .NF1, BRAF, and FGFR1 mutations, as well as frequent ATRX alterations and CDKN2A/B homozygous deletions [An additional subset of cases that may be included here is anaplastic astrocytoma with piloid features (AAPF) or high-grade astrocytoma with piloid features. These IDH-wildtype tumors are frequently found in the cerebellum with pilocytic astrocytoma-like features with increased peripheral invasion, increased mitotic figures, microvascular proliferation, and necrosis, and have a somewhat better prognosis than IDH-wildtype glioblastoma. These cases have a distinct methylation profile, and characteristically have mutations in the MAPK pathway, including eletions ,134,135.EGFR amplification, PTEN mutation, and TERTp mutation with significantly less frequent ATRX and TP53 mutations [TERTp mutation occurs with similar frequency in IDH-wildtype glioblastoma and lower-grade astrocytoma, EGFR amplification and 7+/10− appear to occur more frequently in IDH-wildtype GBM [Comprising the majority of “primary” or “de novo” glioblastoma (those occurring without a documented precursor lesion), IDH-wildtype GBMs  characteutations ,90,107. type GBM ,138.EGFR alterations and lower frequency of CDK4 amplification or homozygous CDKN2A/B deletions. Other studies have shown that co-amplification of CDK4 and MDM2, with or without chromosome 1, gain results in worse overall survival while isolated chromosome 1 or 19 gain without CDK4/MDM2 co-amplification results in somewhat better survival in IDH-wildtype glioblastomas [PIK3CA mutations [EGFR, PTEN, and CDKN2A alterations [PTEN protein loss in IDH-wildtype LGGs, within the IDH-wildtype GBMs, loss of this protein conferred a significantly better prognosis in one study [While carrying an overall dismal prognosis, subsets of tumors with longer overall survival have been shown, with less frequent lastomas . PIK3CA utations  and comberations  conferrene study . The simne study .EGFR, 7+/10−, and TERTp mutations may have similar prognostic significance in IDH-wildtype GBM as in IDH-wildtype lower-grade tumors in both univariate and multivariate analysis [Recently, we have shown that alterations in analysis  . There analysis ,140, ouranalysis  . O6-Methylguanine-DNA methyltransferase (MGMT) in glioblastoma cases. MGMT promoter methylation status is an important predictive marker in terms of response to alkylating chemotherapeutic agents, specifically in IDH-wildtype GBM. The value of this in IDH-mutant GBM is less certain, although many studies demonstrate that MGMT promoter methylation is equally important in these tumors [MGMT promoter methylation may in part depend on the presence of TERTp mutation [MGMT promoter methylation has also been shown to correlate with methylation of the XAF1 tumor suppressor gene promoter, leading to decreased XAF1 expression. XAF1 expression correlated inversely with patient survival (similar to its prognostic role in IDH-mutant LGG); thus, XAF1 may have a unique, yet undescribed biologic function in astrocytomas [MGMT promoter methylation analysis is important in the vast majority of these cases, it may be of limited value in cases without at least one of the cIMPACT-NOW update 3 factors [MSH6 mutations occurring during TMZ therapy [Current guidelines recommend reflex testing for methylation of the promoter region of e tumors ,148,149,mutation ,148,149.ocytomas . Our own factors . Other s therapy .An additional area of investigation in GBM is immunotherapy, especially in light of the finding that the majority of current standardized GBM therapies (including radiotherapy and TMZ) tend to have an immunosuppressive effect. Similar to other systemic malignancies, programmed cell death-1 (PD-1) blockers and other immune checkpoint inhibitors have been utilized as monotherapy and in conjunction with other standard therapies to reverse local immunosuppression with mixed results. While most studies did not identify a benefit of anti-PD-1 therapy in randomized cohorts of GBM patients, others showed survival benefits in select groups, including some subsets of patients with hypermutated GBMs receiving neoadjuvant anti-PD-1 therapy ,154,155.BRAF V600E mutations, suggesting a possible connection to pleomorphic xanthoastrocytoma [BRAF V600E mutation  is also found more frequently in other glioma subtypes, including pediatric diffuse gliomas and non-infiltrating tumors such as pilocytic astrocytoma and ganglioglioma. Recent studies have shown a response to BRAF inhibitors in some of these cases [Of note, there are several histologically-defined variants of IDH-wildtype glioblastoma with characteristic genomic alterations and variation in outcome. Epithelioid glioblastoma is an IDH-wildtype variant with a relatively poor prognosis that harbors frequent rocytoma ,159,160,rocytoma . The BRAse cases ,163,164.EGFR amplification and TERT promoter mutation, but more frequent TP53 and ATRX mutation [EGFR amplification [Giant cell GBM is another IDH-wildtype histologic variant with slightly better outcomes than other variants . Cohortsmutation . Gliosarmutation . These tfication .TP53 and ATRX mutation and characteristic lack of Olig2 expression. However, caution should be exercised in assigning grade IV based solely on the presence of an H3 K27M mutation since studies have shown its presence in lower grade tumors such as pilocytic astrocytoma and glioneuronal tumors or tumors in non-midline locations that do not necessarily have poor clinical outcomes [IDH-wildtype tumors that show loss of ATRX immunohistochemical staining, frequently show mutations in histone H3 . These moutcomes ,180,181.This review highlights the speed of discovery of a vast variety of molecular alterations, as well as a better understanding of previously known genetic aberrations in diffuse gliomas. With wider adoption of high-throughput genetic sequencing, discoveries are being made at a faster pace than ever before, and a majority of the mutations we discuss here have been described in only the last 5–10 years. There are clearly survival outliers in all glioma classifications, some of which can be explained based on molecular factors reviewed here, while others certainly remain to be discovered. Codification of molecular markers and further stratification based on genetic and epigenetic features within these five main adult diffuse glioma groups discussed here and in other CNS tumors remains. While histologic examination is essential for initial tumor classification, expanded molecular testing will no doubt become the de facto standard of care.EGFR, members of the RB and PI3K-AKT pathways, and IDH1/2 in the form of inhibitors, antibodies, vaccines, RNA-based therapies, and immunotherapy [For over a decade, efforts have been made to create therapies aimed at many of the molecular targets discussed in this review, including otherapy ,183,184.otherapy ,185,186.


The nuclear factor erythroid 2-related factor 2 (NRF2)–Kelch-like ECH-associated protein 1 (KEAP1) regulatory pathway plays an essential role in protecting cells and tissues from oxidative, electrophilic, and xenobiotic stress. By controlling the transactivation of over 500 cytoprotective genes, the NRF2 transcription factor has been implicated in the physiopathology of several human diseases, including cancer. In this respect, accumulating evidence indicates that NRF2 can act as a double-edged sword, being able to mediate tumor suppressive or pro-oncogenic functions, depending on the specific biological context of its activation. Thus, a better understanding of the mechanisms that control NRF2 functions and the most appropriate context of its activation is a prerequisite for the development of effective therapeutic strategies based on NRF2 modulation. In line of principle, the controlled activation of NRF2 might reduce the risk of cancer initiation and development in normal cells by scavenging reactive-oxygen species (ROS) and by preventing genomic instability through decreased DNA damage. In contrast however, already transformed cells with constitutive or prolonged activation of NRF2 signaling might represent a major clinical hurdle and exhibit an aggressive phenotype characterized by therapy resistance and unfavorable prognosis, requiring the use of NRF2 inhibitors. In this review, we will focus on the dual roles of the NRF2-KEAP1 pathway in cancer promotion and inhibition, describing the mechanisms of its activation and potential therapeutic strategies based on the use of context-specific modulation of NRF2.  Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor and a key modulator of cellular antioxidant responses that regulates the expression of genes encoding antioxidant enzymes with a protective role against different types of oxidative changes. NRF2 in combination with its own negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), has become the center of a debate regarding whether NRF2 suppresses the tumor promotion or, conversely, exerts pro-oncogenic functions. Based on this, the present review will describe the role of NRF2 in cancer prevention and promotion, discussing potential advantages and disadvantages derived from its therapeutic modulation in cancer prevention and treatment. As it is known, under non-stressed conditions, NRF2 is constitutively poly-ubiquitinated by the CUL3-KEAP1 E3 ubiquitin ligase complex and subjected to degradation through the proteasome pathway ,5. The iNFE2L2 gene that belongs to “Cap’N’Collar” type of basic region leucine zipper factor family (CNC-bZIP) [Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcriptional factor encoded by the NC-bZIP) . Human NNC-bZIP) ,28. Neh2NC-bZIP) ,30,31,32NC-bZIP) ,33. The NC-bZIP) . The NehNC-bZIP) . The NehNC-bZIP) ,37. In aNC-bZIP) . A schemKEAP1 is a 69-kDa protein, which belongs to the BTB-Kelch family of proteins . All theHistorically, the NRF2-KEAP1 pathway has been the focus of extensive research aimed at assessing its potential role in human chronic diseases characterized by alterations of the redox homeostasis such as diabetes, cardiovascular diseases, neurodegenerative diseases and cancer. Among them, effort have been made to explore the chemopreventive properties of naturally occurring as well as synthetic compounds functioning as NRF2 activators or KEAP1 inhibitors in vitro and in vivo. The identification of the molecular mechanisms underlying NRF2 modulation has driven a renovated interest in the field of basic and clinical cancer research, fostering a growing number of studies. However, it is now increasingly recognized that NRF2 can exert oncogenic as well as oncosuppressive functions, so that the development of effective therapeutic approaches based on NRF2 modulation requires a careful evaluation of the specific context of its activation including not only the histotype, stage, and genetic background of a specific tumor but also the therapeutic scheme of administration and the target population that might benefit from treatment. In the following section, we will describe some of the most relevant NRF2 activators and their use in cancer treatment. Activation of the NRF2 system is complex and can follow canonical and non-canonical pathways. A difficulty in identifying activators and inhibitors of NRF2 or KEAP1 as modulators of inflammation and potential protectors against oxidative stress and carcinogenesis, is the dual nature of the NRF2-KEAP1 protein-protein interaction ,60,61,62Collectively, NRF2-inducers fall into two classes—electrophilic and non-electrophilic —with theThe largest class of chemicals so far identified with the ability to activate NRF2 through KEAP1 inhibition is represented by triterpenoids . These mH-1,2-dithiole-3-thione) has been shown to increase the nuclear accumulation of NRF2, an effect mediated in part by the activation of the ERK 1/2 pathway [The compound D3T . Several investigators focused on the molecular targets of curcumin, including NRF2, on curcumin action, and its uses as a neuroprotective agent against toxicants inducing oxidative stress but also as an antitumor agent [A 2016 review systematically examined the activation of NRF2 by dietary factors, but also extended these findings to discuss how diet changes may restrict the nutritional utility of some activators . A naturor agent ,92,93. Tor agent . Other cor agent ,96. Silior agent , and to or agent . From prFoods containing compounds with positive effects on human health are sometimes referred to as “nutraceuticals.” One chemical class found in cruciferous vegetables is represented by the isothiocyanates, that include compounds such as sulforaphane, isolated from broccoli, cauliflower, cabbage, and Brussel sprouts. The role of sulforaphane in ameliorating various health-related disorders has received a lot of attention, and some of its biological effects were described in 1992 . A recenCurcuma longa (turmeric), was shown to induce the expression of antioxidant enzymes such as glutathione S-transferase, aldose reductase, and HO-1 through NRF2-KEAP1 signaling [Whereas new findings indicate that NRF2 plays a dual role in cancer  genes expression and reduces migration and invasion capacity of NSCLC cells [NOTCH1 that in turn induces the expression of genes promoting EMT [RhoA gene, which is a critical factor for growth and metastasis, while NRF2 down regulation inhibits proliferation of BC cells [Bcl-xL and MMP-9 genes expression [Epithelial to mesenchymal transition (EMT) is a biological process that contributes to cancer metastasis and tissue invasion ,208. DurLC cells . Overexpting EMT . NRF2 alBC cells . Furtherpression . In contpression . Additiopression . Moreovepression ,219. TakMYC or KRAS): the first involves direct transactivation of key metabolic enzymes [G6PD (Glucose-6-Phosphate Dehydrogenase) and PGD (Phospho Gluconate Dehydrogenase), or enzymes that regenerate glycolytic intermediates that can be diverted into the oxidative PPP branch, such as TKT (Transketolase) and TALDO1  [G6PD, PGD and TKT expression, through yet unknown epigenetic mechanisms involving the histone deacetylase HDAC4 [G6PD in HCC cells, an event that positively correlated with grading, metastasis number and poor prognosis in HCC patients [G6PD, PGD, TKT, TALDO1 and other NADPH-producing enzymes was reported in A549 NSCLC cells with sustained PI3K/AKT pathway activation [TKT gene, inducing its expression in HCC cells (SMMC and MHCC97/L) [TKT promoter was also reported in MEFs and A549 NSCLC cells, suggesting a direct transactivation mechanism [NRF2 overexpression or KEAP1 KD can upregulate the mRNA and protein levels of G6PD and TKT, enhancing tumor proliferation, migration and invasion of MCF7 and MDA-MB-231 BC cells by promoting EMT through G6PD/HIF-1α activation of Notch1 signaling, while NRF2 silencing or KEAP1 overexpression reverted these changes [MTHFD2 (Methylenetetrahydrofolate Dehydrogenase 2) or in the TCA cycle, such as IDH1 (Isocitrate Dehydrogenase 1) and ME1  [MTHFDL1 (Methylenetetrahydrofolate Dehydrogenase 1-like) can increase the NADPH levels in HCC cells, supporting proliferation and redox homeostasis [It is well known that cancer cells reprogram their central metabolism to meet the energetic needs imposed by their uncontrolled growth. Initial studies mainly focused on the Warburg effect, while subsequent work also investigated changes in the one-carbon and fatty acids metabolism, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA) and glutamine catabolism ,220,221. enzymes , while t enzymes , Notch [ enzymes ,224, AHR enzymes ,226 and  enzymes . It is k enzymes ,229,230. enzymes ,234,235. enzymes . Secondl enzymes ,238. Impolase-1) ,215,239.se HDAC4 . Also mipatients . Insteadtivation , in agretivation ,182,242.echanism . Also, onzyme 1) ,239,243,eostasis . PHGDH (Phosphoglycerate Dehydrogenase), PSAT1 (Phosphoserine Aminotransferase 1), PSPH (Phosphoserine Phosphatase) and SHMT1-2 (Serine Hydroxymethyltransferase 1-2) in a panel of NSCLC cells [SLC6A9, SLC36A4, SLC38A1, and SLC38A3) codying for AATs (aminoacid transporters) involved in the uptake of glycine and glutamine. Notably, AATs inhibition sensitized autophagy-deficient CRC cells but not wild-type cells to apoptosis induced by glutamine withdrawal [SLC7A11 (solute carrier family 7 member 11) gene upregulation, leading to an increase in the xCT mRNA and protein levels and marked glutamate release. Of note, these changes were phenocopied by NRF2 overexpression or KEAP1 KD and reverted by its overexpression [NRF2 and SLC7A11 expression was found to be positively correlated across 947 cancer cell lines from the CCLE dataset [NRF2 KD markedly decreased SLC7A11 expression and glutamate extrusion in Hs578T and MDA-MB-231 BC cells, improving cell viability upon glucose depletion while in the same conditions NRF2 activation by DMF impaired cell viability. Thus, despite the enhanced activation of the NRF2-xCT axis might efficiently preserve the redox homeostasis of BC cells under OS, it might also decrease their metabolic flexibility, unveiling a specific vulnerability that can be therapeutically exploited [BRAF activation (BRAFV600E), Khamari et al., explored potential metabolic changes promoting resistance (A375RIV1 cells) or sensitivity (A375-v cells) to the BRAF inhibitor Vemurafenib, by using an in vivo long-term treated xenograft mouse model. Here, A375RIV1 cells exhibited a strong activation of the NRF2 signaling, followed by an increased expression of genes involved in ROS scavenging , GSH synthesis  and NADPH-generation , compared to the A375-v counterpart. Of note, NRF2 KD by siRNA decreased the protein content of its target genes in A375RIV1 cells, partly reversing their resistance to Vemurafenib [KEAP1 gene can mediate glutamine addiction in both mouse (KPK) and human KRAS-driven LUAD cell lines. Here, NRF2 increased activation led to enhanced xCT/SLC7A11 expression, causing an imbalance in the TCA cycle and sensitization of KPK cells to pharmacologic or metabolic glutamine depletion. Importantly, the glutaminase inhibitor CB-839, impaired cell growth in a panel of tumor cells including melanoma, colon, renal, bone, squamous and urinary-tract cancers with KEAP1 LOF mutations while the use of KI696, a small-molecule activator of NRF2, conversely sensitized KEAP1 WT cancer cells previously refractory to CB-839 [SLC1A5 in HeLa cells through the ATF4 transcription factor [KEAP1-/- mice and human ESCC cells, revealed that NRF2 causes metabolic reprogramming by enhancing the expression of the SLC1A4 glutamine transporter and other metabolic enzymes [KEAP1 KD by siRNA [In line of principle NRF2 can also regulate the redox balance of cancer cells via transcriptional induction of metabolic enzymes or membrane channels that control the availability of cysteine, glutamate and glycine, essential precursors in the GSH synthesis. In this regard, NRF2 was shown to enhance the expression of key enzymes for serine/glycine biosynthesis such as LC cells . Also, athdrawal . Other wthdrawal ,248,249.pression . In a la dataset , especiaxploited . In a sturafenib . Thus, turafenib . In thiso CB-839 . Thus, oo CB-839 . Intrigun factor  while a  enzymes . Lastly,by siRNA . TherefoCPT1 and CPT2, two isoforms of the enzyme carnitine palmitoyltransferase (CPT) that catalyzes the rate-limiting step of FAO [NRF2 expression, Wu et al., showed that NRF2-null mice exhibited increased hepatic mRNA levels of the enzymes fatty acid synthase (FASN), fatty acid desaturase , stearoyl-CoA desaturase (SCD1), fatty acid elongases , acetyl-CoA carboxylase-1 (ACC1) and ATP-citrate lyase (ACLY), while the opposite was seen in KEAP1-KO mice suggesting that NRF2 restrains lipogenesis and desaturation to prevent NADPH depletion [Interestingly, NRF2 has been found to exert opposite changes in the metabolism of fatty acids, since a repression of FAS (fatty acid synthesis), but a stimulation of FAO (fatty acid oxidation) has been reported in isolated mitochondria, MEFs (mouse embryonic fibroblasts) and tissues of transgenic mice . Despitep of FAO . Anotherepletion . On the epletion . In concTumors are complex entities composed by heterogeneous cell populations that dynamically adapt to their microenvironment driven by genetic/epigenetic alterations and metabolic rewiring ,266,267.Brucea javanica [GCLC, GCLM, HMOX-1, and NQO1, promoting ROS accumulation and apoptosis [HO-1 and NQO-1. Notably, Brusatol enhanced the antitumor activity of Gemcitabine in vivo by reducing the growth rate of PANC-1 xenografts implanted in BALB/c nude mice [Dichroa febrifuga, which is rapidly emerging as one of the most promising NRF2 inhibitors. Indeed Halofuginone has been shown to impair proliferation, migration and invasion of HepG2 HCC cells [Despite the increasing demand for negative modulators of NRF2, selective inhibitors are neither currently available nor under clinical trial evaluation. Yet, alternative strategies have been explored based on the pharmacologic manipulation of various NRF2 modulators through natural and synthetic compounds ,303. Hisjavanica ,305,306,javanica ,308. Thejavanica ,310,311.javanica . Also, Bjavanica . Also, Kpoptosis . Finallyude mice . SimilarCC cells  and MCF7CC cells ,318, to CC cells , or to iCC cells . In a reCC cells . It shouHMOX-1, AKR1B10 and MRP5, was significantly reduced and the chemoresistance attenuated [Other works have explored the potential use of Chrysin, a natural flavone found in many plant extracts such as honey, propolis, and blue passion flowers, endowed with anticancer effects against different tumors ,323. In tenuated . In anottenuated . Rabdosia rubescens, represents another promising compound whose potent antitumor effects have been described in leukemia [HMOX1, NQO1 genes and their encoded antioxidant proteins, leading to ROS-dependent apoptosis, in vitro and in vivo [Plumbago zeylanica L., has received substantial attention as a potent inducer of apoptosis in pancreatic, lung, breast and prostate cancer cells caused by ROS overproduction [HMOX1 in H838 and H460 NSCLC cells with LOF mutations of the KEAP1 gene, suggesting that AEM1 might preferentially target tumor cells with constitutive NRF2 activation [KEAP1 mutations, enhancing the cytotoxic effects of Doxorubicin, Carboplatin and Paclitaxel without affecting the non-tumorigenic BEAS2B cells. Importantly, the therapeutic efficacy of ML385 as a single agent and in combination with carboplatin was also confirmed in orthotopic lung cancer xenografts subcutaneously implanted in nude mice [Streptomyces sp. after HTS screening of almost 10000 culture broth samples. Of note, K-563 abrogated the expression of NRF2 target genes such as GCLC, GCLM, AKR1C1 (reductase family 1 member C1), ME1, NQO1 and TXNRD1 in A549 NSCLC cells and in the human gallbladder cancer (GBC) cell line TGBC24TKB, without altering the NRF2 protein levels, its ARE-binding ability, or its nuclear localization. As a result, K-563 promoted ROS accumulation and synergized with either Cisplatin or Etoposide in A549 NSCLC cells, suppressing also cell proliferation in GBC cells (TGBC24TKB) without affecting normal human lung cells (BEAS-2B) [HMOX1 and GCLC genes, promoting increased caspase-3 and PARP cleavage. Besides, 4f suppressed the growth of THP-1 xenografts seeded onto the CAMs of chicken eggs and also impaired blood vessels formation in vivo in a gelatin sponge assay, suggesting that 4f might be a promising treatment in advanced AML [Oridonin, a diterpenoid isolated from the herb leukemia , BC [327leukemia . Very re in vivo . Also Ploduction ,330. Witoduction . In anotoduction . Extensioduction . In a laoduction . Importaoduction . Interesoduction . Lately,oduction . Thus, Ttivation . Similarude mice . MoreoveBEAS-2B) . In the nced AML . In summHMOX1 and NQO1 and increased ROS production [Scutellaria baicalensis Georgi, has emerged as a promising anticancer agent, due to its chemosensitizing ability in Doxocycline-resistant MCF7 BC cells [Trans Retinoic Acid (ATRA) can induce RARα expression, which in turn forms a protein complex with NRF2 and antagonizes its transactivation [α, since ATRA was ineffective when parental NB4 cells were pre-treated with the RARα antagonist Ro-41-5253 or when applied to mutant NB4-R2 cells lacking RARα expression [An alternative strategy to hamper the pro-tumoral effects of NRF2 is through the modulation of functional regulators that ultimately converge on this signaling pathway. This approach offers at least two significant advantages. First, it relies on already established drugs with proven anticancer activity and, more importantly, it does not require selective NRF2 inhibitors. Of note, recent work supports the applicability of this strategy. For example, LGK-974, a specific inhibitor of the O-acyltransferase porcupine (PORCN) used to disrupt the WNT signaling, was recently found to prevent NRF2 nuclear translocation and its expression in HepG2 cells, presumably by impairing the WNT3A-dependent formation of the GSK-3/β-TrCP protein complex. As a result, the HepG2 cells were sensitized to otherwise non-toxic radiation doses, due to downregulation of NRF2 target genes such as oduction . Also WoBC cells . FurtherBC cells . Other wBC cells . In a laBC cells . Hence, BC cells . AdditioBC cells . Also, RBC cells . Initialtivation ,348, sentivation . Other epression .Since its discovery in 1994  NRF2 has


Background: Acute kidney injury (AKI) constitutes a multi-factorially caused condition, which significantly affects kidney function and can lead to elevated risk of morbidity and mortality. Given the rising scientific evidence regarding vitamin D's (VitD's) multisystemic role, the connection between AKI and VitD is currently being studied, and the complex relation between them has started to be unraveled.Methods: A systematic review had been conducted to identify the pathogenetic relation of VitD and AKI and the potential role of VitD as a biomarker and therapeutic–renoprotective factor.Results: From 792 articles, 74 articles were identified that fulfilled the inclusion criteria. Based on these articles, it has been found that not only can VitD disorders (VitD deficiency or toxicity) cause AKI but, also, AKI can lead to great disruption in the metabolism of VitD. Moreover, it has been found that VitD serves as a novel biomarker for prediction of the risk of developing AKI and for the prognosis of AKI's severity. Finally, animal models showed that VitD can both ameliorate AKI and prevent its onset, suggesting its renoprotective effect.Conclusion: There is a complex two-way pathogenetic relation between VitD disorders and AKI, while, concomitantly, VitD serves as a potential novel predictive–prognostic biomarker and a treatment agent in AKI therapy. Acute kidney injury results in further systemic disorders and is related to elevated morbidity and mortality . There aThe pathophysiology of AKI can be classified into: (1) prerenal azotemia , (2) intrinsic or intrarenal AKI (direct damage to kidney's structure) due to acute tubular necrosis (ATN), vascular damage, and glomerular damage, 3) interstitial AKI , or 4) postrenal AKI (obstruction of urinary tract) .via UV radiation–induced transformation of 7-dehydroxycholesterol (7DHC) to cholecalciferol, which then undergoes consecutive hydroxylations in the liver and kidneys, accordingly, which leads to the synthesis of 1,25(OH)2D , enhances innate immunity and changes the cytokine and chemokine profile of acquired immunity from proinflammatory to anti-inflammatory, and protects against autoimmune response and cancer 2D . However25(OH)2D , 9. Bried cancer .The aim of the present systematic review is to unravel the relationship between AKI and VitD in both patients and experimental models. We assumed that this is a two-way relationship, insinuating that on the one hand, VitD levels may play a role in AKI, and inversely, AKI induces changes in VitD levels. In this perspective, we examined whether hypo- or hypervitaminosis causes AKI and, inversely, whether AKI causes hypo- or hypervitaminosis. It was actually pointless to search for active comparators. Moreover, we examined possible implications of VitD as a predictive and prognostic marker for AKI, as well as its role in AKI treatment.In May 2020, a bibliographic search was conducted in PubMed, Scopus, and Embase regarding the relation between VitD and AKI. The terms employed were “vitamin D AND AKI,” “vitamin D AND ARF,” and “vitamin D AND acute kidney injury.” Additionally, a manual search of the reference lists of other studies had been conducted, to detect subsequent material that could be included in this systemic review.Then, the articles were screened, based on:Removing duplicatesRelativity to the subjectDate of publicationValidity of methods and conclusionsArticles that did not meet the above criteria were excluded.There were 756 articles identified in the three databases. Moreover, 36 articles have been included from screening the reference lists of the articles. After the exclusion of 336 duplicates and 340 irrelevant studies, the remaining 117 articles were analyzed based on the eligibility for full access and the inclusion criteria. Finally, 74 articles matched our search criteria .via various mechanisms, lead to either D hypo- or hypervitaminosis. Moreover, both VitD deficiency and VitD toxicity can lead to the development of AKI.These articles reveal a two-way relation between VitD and AKI. Acute kidney injury can, Given the fact that our knowledge concerning the relationship between VitD and AKI comes from small series, cases, and experimental models, extrapolation of our results to the general population may be risky and biased. Moreover, introducing VitD in the treatment of AKI is also questionable on a clinical basis since there are no prospective randomized trials available and our knowledge is a result of animal experiments. Finally, more clinical data are available on the use of VitD levels as biomarkers concerning both the prediction and the prognosis of AKI. However, despite the fact that there are nine clinical studies on this matter, those studies are observational (either prospective or retrospective), and no data coming from randomized controlled trials are available.Acute kidney injury is the significant depletion of renal function, affecting the normal renal enzymatic activity, and consequently disrupts VitD. Usually, the decreased kidney function causes VitD deficiency, but in some cases, VitD toxicity is also observed .2D synthesis, and therefore negatively contributes to VitD metabolism tabolism , 12. Mortabolism  and to dtabolism , 11.via acting on FGF-receptor (FGFR)/Klotho coreceptor complex, by (1) downregulating the luminal sodium/phosphate cotransporter in the proximal convoluted tubules, (2) suppressing kidneys' 1-hydroxylase, and (3) inducing 24-hydroxylase, which catalyzes the first step of VitD catabolism, causing hypophosphatemia and significantly lower levels of active VitD  channel and the transient receptor potential vanilloid-5 (TRPV5). Moreover, sKl has a renoprotective effect via (1) the transforming growth factor-β1 (TGF-β1)–dependent anti-fibrotic mechanism and (2) the tumor necrosis factor (TNF)/nuclear factor-κB (NFκB)–dependent anti-inflammatory mechanism (Fibroblast growth factor-23 (FGF-23), a phosphaturic factor, induces phosphaturia ive VitD , 14. The2D D and 1,25(OH)2D . Additio2D . Klotho'2D .2D was observed, which was greater in patients who developed hypercalcemia D and 1,25(OH)calcemia . These r−/− mice, suggesting that angiotensin II is responsible for the increased renal damage. Contrarily, when VDR agonists were administrated, proteinuria, podocytes' damage, mesangial dilation, macrophage infiltration, oxidative stress damage, proinflammatory and profibrogenic factors, and extracellular matrix protein and neutral lipid accumulation were reduced, proposing that VDR depletion worsens renal injury  induced AKI] by deteriorating the renal vascular condition and it can accelerate the AKI-to-CKD progression, d Klotho –21.Buttar et al. presented a case of an 86-year-old woman with mild chronic disease and decreased levels of 25(OH)D, who developed rhabdomyolysis and secondary AKI after sitagliptin administration. The patient was on chronic atorvastatin therapy, and a possible interaction between these two drugs is proposed .In current clinical practice, most assays estimate total 25(OH)D, which cannot distinguish the three different forms of 25(OH)D . This approach has many limitations since there are many variables that are influenced by physiologic and pathophysiologic conditions. The VDBP's affinity is affected by both hyperlipemic conditions and the three common variants of VDBP's gene, GC1F (group-specific component-1f), GC1S, and GC2 . Many ef2D can act as novel biomarkers of AKI D but, also, 1,25(OH)s of AKI .Braun et al. found that 25(OH)D could serve as an independent predictor of AKI since serum 25(OH)D deficiency (<15 ng/ml) and insufficiency (15–30 ng/ml) are associated with greater risk of AKI. Moreover, serum 25(OH)D can also act as independent prognostic biomarker of 30-day mortality since its deficiency and insufficiency are linked to elevated risk of 30-day death D can act as a predictor of AKI, given that patients with serum 25(OH)D <10.9 ng/ml had greater risk of AKI than VitD-sufficient patients. Moreover, lower levels of serum 25(OH)D were more common in non-survivor patients, and serum 25(OH)D acted as a prognostic biomarker of mortality  .r = −0.45) and mortality, while 25(OH)D can only correlate with sepsis severity (r = −0.42). Also, VDBP, 1,25(OH)2D, and FGF-23 are predictive biomarkers of AKI, while FGF-23 served as biomarker of sepsis severity too (r = 0.35) D could serve as an independent prognostic biomarker of sepsis severity ( = 0.35) .Sahin et al. found that VitD acts as an independent predictive biomarker of contrast-induced nephropathy (CIN)–AKI, even in multivariable models .Chaykovska et al. found that neither urinary VDBP (uVDBP) nor uVDBP/sCr could predict AKI development, but they served as biomarkers of need-of-dialysis, mortality, major adverse renal events (MAREs) (only uVDBP), and non-elective hospitalization. After adjustments, the predictive value of uVDBP was confirmed and was independent of well-known CIN risk factors, such as anemia, already-existing kidney injury, heart failure, and diabetes .Vicente-Vicente et al. found that uVDBP can, potentially, predict the risk of gentamicin-induced AKI, in order to prevent its manifestation, given that increased uVDBP is associated with chronic proclivity to gentamicin nephrotoxicity .2D (it is the only dependent biomarker) act as independent biomarkers of development of the ESRD stage, even after various multivariable models of adjustments D, bioavailable 25(OH)D, total 25(OH)D, and 1,25(OH)ustments .2D, 25(OH)D, and  × 1,000 ratio. As for the prediction of AKI, only 1,25(OH)2D and  × 1,000 ratio were significant, while as for the prognosis, these two biomarkers had been negatively correlated with AKI stage stratification . None of the markers could predict mortality, while 25(OH)D showed neither predictive nor prognostic effect c effect .via univariate and multivariate models of adjustments. However, hCAP-18 showed no significant predictive effect regarding AKI. As for the prognostic value of free and bioavailable 25(OH)D, only the former acted as a biomarker of 90-day mortality, while the latter showed no significant prognostic value , free 25(OH)D and bioavailable 25(OH)D, and the predictive value of hCAP-18. As for the prognosis, hCAP-18 measured on ICU day 1 is an independent biomarker of sepsis and 90-day mortality, as had been found ic value .Many studies have been performed mainly with rats to define the potential use of VitD as treatment for AKI .via an MMP-dependent inflammatory mechanism . Ersan et al. studied the effect of paricalcitol on MMPs expression and, subsequently, on IRI progress. Pre-treatment with paricalcitol resulted in amelioration of IRI-AKI echanism .via inhibition of Toll-like receptor-4 (TLR-4) and interferon-γ (IFN-γ), (2) a reduction of Na+/H+ exchanger-1 (NHE-1 exchanger), (3) a pro-autophagic action via elevating Beclin-1 expression and LC3II/LC3I ratio, (4) an anti-apoptotic action via elevating Bax/Bcl-2, cytochrome c and caspase-3 expression, and (5) an inhibitory action on G1 cell cycle arrest via reducing insulin-like growth factor-binding protein-7 (IGFBP-7) and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) expression  on IRI-AKI and found that it can ameliorate AKI through: (1) an anti-inflammatory mechanism pression .In another study, Kapil et al. also investigated the protective role of VitD pre-treatment in IRI-AKI and showed a renoprotective effect in IRI against oxidation and lipid peroxidation mediated by peroxisome proliferator–activated receptor-γ (PPAR-γ)- .via reducing the expression TLR-4 and MCP-1  inhibition on IRI-AKI and found that this inhibition attenuates AKI pression . This efpression . Additiopression .via an anti-inflammatory mechanism mediated by the inhibition of TLR-4 expression and the suppression of NFκB signaling, by increasing IκB in a TNF-α-dependent way , superoxide dismutase (SOD)-1, and SOD-2 and via decreasing nitric oxide synthase (iNOS), p47phox, and gp91phox  and (2) an anti-apoptotic mechanism -induced AKI and found that it can attenuate AKI through: (1) an anti-oxidative mechanism echanism .via reduction of TLR-4 and (2) an anti-apoptotic mechanism via elevation of Bcl-2 (it is anti-apoptotic) and via decrease of caspase-3, PUMA (it is a pro-apoptotic member of the Bcl-2 family), and miR-155 (it targets Bcl-2 and blocks its expression)  an anti-inflammatory mechanism ression) . Regardiression) .via increasing Bcl-2 and via decreasing P-p53 and p21, p-Bad, Bax, and caspase-3 expression, and (3) increasing the cell proliferation via elevating the expression of p27kip1 and decreasing that of cyclin-dependent kinase-2 (CDK2) and Cyclin E. The underlying mechanism of these actions includes the inhibition of TGF-β1 and p53 signaling pathways and the elevation of the p27kip1 signaling pathway  inhibiting EMT, (2) reducing apoptosis  pathway .Another study on the effect of paricalcitol on cisplatin-induced AKI demonstrated that the ameliorated AKI was correlated with reduced lipid peroxidation and ferroptotic cell death due to the direct binding of VDR to the glutathione peroxidase 4 (GPX4) promoter, which induces the expression of GPX4, affecting the induction of ferroptosis .AR blocker), on cisplatin-induced AKI. Their administration led to attenuation of AKI via a VDR-, ET-1-, and ETAR-dependent mechanism, which includes the signaling cascade of Pnf-κBp65, TNF-α, and TGF-β1. The combined administration of both drugs led to an enhanced therapeutic effect. These results propose a merger of VitD and endothelin-1 signaling pathways, which is promising as a therapeutic option for cisplatin-induced AKI ting AKI . Howeverting AKI . So, Vitvia reducing caspase-3 and (2) a decrease of Cyp24α1 and VDBP expression and an increase of Cyp27b1, Cyp2R1, and VDR expression, (3) an anti-oxidative and anti-inflammatory mechanism via decreasing IL1-β, IL1R1 (IL-1 receptor 1), IL-6, IL6R (IL-6 receptor), IFN-γ, IFNGR1 (IFN-γ receptor 1), IL17A, and IL17RA (IL-17 receptor A) and via elevating GSH, chloramphenicol acetyltransferase (CAT), Gpx, IL10 (and its gene), IL22, and IL22RA (IL-22 receptor A)  an anti-apoptotic mechanism eptor A) . The effeptor A) .via the reduction of the expression of heme oxygenase 1 (HO-1) and its regulators, NrF2 and BACH1, and (2) an anti-inflammatory action via the reduction of NFκB, TNF-α, and IL-10 , MCP-1, and IL-1β, (2) an anti-oxidative mechanism via elevating SOD and via decreasing 8-epi-PGF2α and nitrotyrosine, (3) reducing vimentin, proliferating cell nuclear antigen (PCNA), and caspase-3, and (4) elevating CYP24  an anti-inflammatory mechanism ng CYP24 .Although VitD seems to ameliorate AKI, its administration should be performed with extreme caution. Hypervitaminosis D can significantly afflict kidney function by inducing hypercalcemia and hyperphosphatemia. Specifically, hypercalcemia leads to the development of nephrogenic diabetes insipidus, which affects water homeostasis by causing polyuria and diuresis. Therefore, there is evident loss of water, leading to hypovolemia, which causes AKI and concomitantly deteriorates the hypercalcemia. So, there is a continuous circle of hypercalcemia causing hypovolemia-induced AKI and vice versa –51. More2 or D3 ingestion/supplementation, (2) extravagant calcitriol ingestion or pharmaceutical administration, (3) elevated ectopic production of calcitriol , (4) milk-alkali syndrome (MAS), and (5) depleted catabolism of calcitriol due to mutations of CYP24A1 genes  excessive VitDA1 genes .VitD toxicity due to immoderate administration of VitD supplements or overfortified milk is a global phenomenon , potentiAlso, VitD toxicity–induced AKI has been observed due to anabolic steroid and VitD supplement abuse , dispensAdditionally, in another study, 33 patients with osteoporosis treated with 0.75 μg/day eldecalcitol developed hypercalcemia-induced AKI since the discontinuation of eldecalcitol ameliorated the situation . FurtherMoreover, VitD toxicity–induced AKI is related with granulomatous diseases , MAS, subclinical hyperparathyroidism, and immune reconstitution syndrome.2D by macrophages within the sarcoid granulomas, the process of which is without systemic regulation  infection, who was in highly active antiretroviral therapy (HAART) because of plasma viral load increase. This 48-year-old male patient, after the initiation of HAART, developed various hypercalcemia symptoms, accompanied by hypervitaminosis D , which progressed to coma and AKI. IRIS can be due to many opportunistic infections, but the most common infection is M. tuberculosis, which is incriminated as the cause of VitD-induced hypercalcemia, which leads to AKI  due to s to AKI . All casIt can be concluded that there is a two-way relation between AKI and VitD. Specifically, disruptions of VitD—both hypovitaminosis and hypervitaminosis—can lead to the development of AKI, while also, AKI can contribute to dysregulation of VitD's homeostasis and function.On this ground, due to this two-way causality relation, VitD is examined on whether its forms can act as a novel biomarker of AKI. Many studies have confirmed VitD's significant role as a predictive (it can help to determine the risk of developing AKI) and prognostic  biomarker.via genomic and non-genomic actions.Finally, VitD is found to be a potentially important therapeutic factor for AKI due to its multisystemic functions, which include regulation of many enzymic mechanisms However, many prospective studies and trials need to be conducted, in order to: (1) fully determine the complex relation between VitD and AKI, (2) find the best cutoff points with the most significant statistic importance, and (3) determine the therapeutic protocols of VitD as treatment of AKI and its potential adverse effects.Despite the necessity of future studies, VitD is a very promising biomarker and a potential treatment for AKI, which is one the most prominent health problems nowadays.The original contributions generated in the study are included in the article/MP and TP: conceptualization and supervision. SG and MP: data curation and investigation. SG: formal analysis and writing—original draft. All authors methodology and writing—review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Resveratrol exhibited the strongest lipid-reducing activity, which was accompanied by the improvement of lifespan, oxidative stress, and aging, without affecting worm development. Whole-genome expression microarrays demonstrated that resveratrol affected fat mobilization, fatty acid metabolism, and unfolded protein response of the endoplasmic reticulum (UPRER), mimicking the response to calorie restriction. Apigenin induced the oxidative stress response and lipid mobilization, while vanillic acid affected the unfolded-protein response in ER. In summary, our data demonstrates that phenolic compounds exert a lipid-reducing activity in C. elegans through different biological processes and signaling pathways, including those related with lipid mobilization and fatty acid metabolism, oxidative stress, aging, and UPR-ER response. These findings open the door to the possibility of combining them in order to achieve complementary activity against obesity-related disorders.Supplementation with bioactive compounds capable of regulating energy homeostasis is a promising strategy to manage obesity. Here, we have screened the ability of different phenolic compounds  and phenolic acids ( Obesity is characterized by a misbalance between food intake and energy consumption, which leads to an excess of fat accumulation and an increased body weight . The incDifferent strategies have been suggested to reduce the excessive accumulation of fat characteristic of obesity. In this sense, the identification and characterization of bioactive compounds (BACs) capable of regulating energy homeostasis represent a potential tool for the management and prevention of obesity-related diseases ,5,6. AmoCaenorhabditis elegans (C. elegans) has become a popular model for studying the mechanisms of different diseases and physiological processes, including obesity, aging, development, locomotive activity, and neurodegenerative disorders [C. elegans has been demonstrated to constitute a powerful model for exploring the genetic basis of fatty acid synthesis and the regulation of fat storage [C. elegans, this nematode constitutes a powerful in vivo model for the screening of food ingredients and the identification of bioactive molecules with healthy properties, including the ability of modulating fat storage, together with the characterization of the biological mechanism involved in this activity [isorders ,12,13. I storage . Due to activity ,15,16.p-coumaric, ellagic, ferulic, gallic, and vanillic acids) on C. elegans fat accumulation and healthspan. Whole-genome transcriptome analyses of the most effective compounds have revealed different biological mechanisms underlying the fat-reducing activity of these molecules.Here, we have investigated the effect of different flavonols (myricetin and kaempferol), flavanones (naringin and hesperidin), flavones (apigenin and luteolin), epicatechin, and other non-flavonoid phenolic compounds (curcumin and resveratrol), as well as phenolic acids  and total lipid content quantified by TLC  [In pathways , constitpathways ,17. Althal cells . These dal cells . Due to al cells ,14,19. Tal cells ,21,22 an elegans ,24,25, a elegans ,24,25. BC. elegans lifespan, [C. elegans is less studied. Besides, differences in the protocols and doses used sometimes make difficult the comparison among studies. The present study provides a screening of the potential lipid-reducing effect of different phenolic compounds in C. elegans during larvae development, and clarifies the molecular mechanisms involved for the three with the strongest activity.In particular, several studies have reported the activity of some flavonoids and phenolic acids that, either supplemented as extracts or as single compounds, prolong ifespan, ,31,32,33ifespan, ,27,29. HC. elegans lipid homeostasis, by determining their effect on lipid accumulation. The mean value, calculated as the fluorescence mean value per pixel, together with the integrated density and the volume of the worms were examined. DMSO-treated worms and Orlistat-treated worms were used as controls [C. elegans lipid content at least at one of the doses tested, with the exception of epicatechin, which promoted an increase in the fat proportion of the worms at the three doses tested. In most cases, this reduction in lipid content was accompanied by a significant decrease in worm volume, as a consequence of the lower fat accumulation in the intestine.We initially screened the potential role of different phenolic compounds modulating controls . All pheC. elegans lipid content, we calculated the Cohen’s d effect size average for each treatment, considering all experiments and doses (10–100–500 µM) evaluated  , we performed the Cohen’s –100 μM) B. In thiC. elegans during its development from L1 larvae to adulthood, and this effect was induced in a dose-dependent manner. The results observed with Nile Red were confirmed by Oil Red O , which is often used as a marker of health or rate of aging [C. elegans fluorescence by microscopy in intact animals revealed that treatment with resveratrol from L1 to L4 caused a significant reduction of the lipofuscin pigment in comparison with the DMSO control worms (p = 0.0019)  E. The ef authors ,51,52,53Finally, we have also demonstrated that the beneficial effects of resveratrol treatment were independent of effects on the worm development. We observed that worms treated with resveratrol until day 1 of adulthood (day 3 of treatment) showed a reduction in total size, compared with DMSO control nematodes F. HoweveC. elegans fat content with no effects on the worm development, and that this activity is accompanied by a reduction in the cellular aging and the production of ROS, leading to an increased lifespan of the nematode.In conclusion, these results show that treatment with resveratrol during L1 to adulthood reduces the The life-expanding activity of resveratrol has been previously attributed to its ability to activate the sirtuin sir-2.1, a member of the Sir-2 family of NAD+-dependent protein deacetylases that regulate nematode aging ,58,59. HC. elegans to this molecule. Gene-level differential expression analysis, performed by Affymetrix Transcriptome Analysis, revealed a total of 31 genes upregulated and 156 genes downregulated  biological processes and KEGG signaling pathways . These rF49E12.10 and F49E12.9 or drd-1 (dietary restriction down regulated), ortholog of human FAXDC2 (fatty acid hydroxylase domain containing 2). This protein, highly expressed in liver and adipose tissue, has oxidoreductase activity and is involved in fatty acid and sterol biosynthetic processes. Moreover, resveratrol downregulates some important genes involved in lipid biosynthesis and fatty acid elongation, such as fat-7, an ortholog of human Stearoyl-CoA desaturase (SCD). This gene codes for an, Δ9 desaturase, a key enzyme in the de novo lipogenic pathway, responsible of the formation of monounsaturated fatty acids from saturated fatty acids by catalyzing the insertion of a double bond to the ninth carbon of saturated C16 and C18 substrates, a needed reaction for the synthesis of triacylglycerol (TAG) and membrane phospholipids and sphingolipids [Fat-7 and fat-6 are lipid biosynthesis genes required for lipid droplet expansion, and their expression promotes increased fat stores [C. elegans to trans-trimethoxy resveratrol (TMR) [acdh-1 and acdh-2, orthologs of human ACADSB (acyl-CoA dehydrogenase short/branched chain), that encode proteins with medium chain acyl-CoA dehydrogenase activity and oxidoreductase activity, or cpt-4, a carnitine palmitoyl transferase ortholog of human CPT1A, CPT1B, and CPT1C genes. Remarkably, fat-7, acdh-1, acdh-2, and cpt-4 have been previously described as fasting response genes [The genes with the strongest downregulation were golipids . Fat-7 at stores , so downol (TMR) , supportse genes ,65, as pC. elegans [Y51H4A.5, F09C8.1 (orthologs of human PLB1), and F25E2.3 (ortholog of human ACOT8). PLB1 is a membrane-associated phospholipase that catalyzes the hydrolysis of membrane glycerophospholipids [F56A4.3 and gst-14, orthologs of different human glutathione S-transferases, and the glutathione peroxidase 5 (gpx-5), which would demonstrate the activity of this polyphenol reducing the oxidative stress status. All these changes in gene expression were confirmed by qPCR  genes, involved in the non-canonical unfolded protein response (UPR) [ER) GO processes (ER-related genes (abu/pqn) genes, but also the heat-shock protein encoding genes hps-16 and hsp-70 (F44E5.5), orthologs of human HSPB2/HSPB7/CRYAA and HSPA14/HSPA4/HSPH1, respectively.Viswanathan and colleagues have reported that resveratrol induces an overexpression of se (UPR) ,69. In orocesses . Thus, wed genes , includiaarg-4, ortholog of the human GANAB, that is a key glycoprotein quality control protein in endoplasmic reticulum, where it removes glucose residues from immature glycoproteins, and whose expression has been associated with cellular remodeling during weight regain [Endoplasmic reticulum HSP levels increase in response to enhanced protein damage, as a consequence of exposure to environmental stress conditions, such as presence of oxidants. Besides, these proteins play an important role in aging and longevity ,71. Finat regain .ER) [ER-related genes can be explained by the reduced fat synthesis and decreased oxidative stress induced by resveratrol compared with untreated control worms.The ER is the major subcellular compartment for protein and lipid biosynthesis. Changes in fatty acid desaturation or sterol levels, together with aberrant phospholipid (PL) composition can severely disrupt homeostasis of the endoplasmic reticulum (ER), activating the unfolding-protein response (UPRER) ,74. ThusC. elegans exposed to resveratrol is dominated by genes associated with a reduced fat synthesis and an elevated lipid oxidation, which explains the reduced fat content of the treated worms, mimicking the calorie restriction response. As a consequence, endoplasmic reticulum-UPR and glutathione metabolism stress responses are downregulated by this polyphenol, which might be responsible for the antioxidant, antiaging, and the life-expanding effect observed in resveratrol-treated nematodes.In conclusion, the transcriptional response of C. elegans [A recent study has reported the fat-reducing activity of six dietary flavonoids, including apigenin, luteolin, kaempferol, and myricetin in  elegans . In that elegans . In our p value < 0.05; −1.5 ≥ logFC ≥ 1.5; As it can be observed in lipl-5 , LIPF , and LIPM (lipase family member M), that encodes for a lipase that catabolizes TAG to free fatty acids and glycerol in the response to nutritional and immune stresses [lipl-5 expression is induced by starvation, activating lipid catabolism [lipl-5 gene product participates in lipid homeostasis in both well-fed and starved conditions, with mitochondrial lipids being particularly affected by lipl-5 mutation [Among them, apigenin induced a strong upregulation of lipl-5 , ortholostresses ,76,77. Atabolism . Macedo ncx-8, ortholog of the human SLC8B1 (solute carrier family 8 member B1), which is involved in calcium:sodium antiporter activity and lipid storage [pgp-9, ortholog of human ABCB1 (ATP binding cassette subfamily B member 1). Changes in lipl-5 and ncx-8 genes were confirmed by qPCR , or different genes of the cytochrome P450 family. Thus, apigenin downregulated cyp-35C1, cyp-37b1, cyp-14A2, and cyp-33C8, and induced the expression of cyp-13A6. The cytochrome (CYP) P450 enzymes (P450s) are phase I detoxification enzymes that catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds [cyp-35A1, cyp-35A2, cyp-35A3, cyp-35A4, and cyp-35A5 (gpx-3 (C11E4), ortholog of the human glutathione peroxidase 3 (GPX3) gene, that encodes for a phase II antioxidant enzyme crucial to maintain redox homeostasis [cyp-13A and gpx-3 genes, together with the downregulation of cyp-33C8, were confirmed by qPCR  in comparison with DMSO control worms A.gpx3, ortholog of the human glutathione peroxidase 3 gene (GPX3)  , that encyp-37b1, ortholog of human cytochrome P450 family 4 members CYP4A11, CYP4B1, and CYP4F3, which encodes an oxidoreductase expressed in the worm intestinal cells. This gene has been found to be modulated in response to food deprivation, and its activity has been related to endogenous fatty acid metabolism and fat mobilization in C. elegans [gpx-3 and cyp-37B1 genes by vanillic acid were confirmed by qPCR , confirming the in vivo antioxidant capacity of this compound in C. elegans.Moreover, vanillic acid induced the downregulation of some cytochrome P450-related genes, such as  elegans . The exp by qPCR B and miggpdh-1 (C. elegans hypodermis and intestine. Gpdh-1 corresponds to the ortholog of the human Glycerol-3-Phosphate dehydrogenase (GPD1) gene and GPD1L (glycerol-3-phosphate dehydrogenase 1 like). GPD1 is member of the NAD-dependent glycerol-3-phosphate dehydrogenase family which catalyzes the reversible redox reaction of dihydroxyacetone phosphate (DHAP) and NADH to glycerol-3-phosphate (G3P) and NAD+ [C.elegans, deletion of gpdh-1 results in decreased stress resistance, so its upregulation by vanillic acid might be related with the antioxidant capacity of this phenolic compound, which might also underly the fat-reducing activity observed by Nile Red and Oil Red O staining methods.On the other hand, vanillic acid significantly upregulated gpdh-1 B, which and NAD+ . Thus, tand NAD+  and fattand NAD+ . Moreoveand NAD+ . In C.elER) was observed after vanillic acid treatment, as it can be observed by the significant upregulation of the genes encoding for the heat-shock proteins hsp-16.2, hsp-70 (or F44E5.5) and hsp-16.48, orthologs of human HSPB1, HSPA8, and HSPB2, respectively (hsp-70 (F44E5.5) by vanillic acid was confirmed by qPCR  w by qPCR B. The UPtabolism . In C. embalance . Thus, tC. elegans gene expression, mainly affecting to genes involved in the antioxidant and stress resistance response, which might underlie the fat-reducing activity observed by this phenolic compound. Besides, the changes in the lipid content after treatment induced the activation of the UPRER, highlighting the importance of this organelle in the regulation of lipid synthesis and metabolism. Although further research in needed to investigate the effect of vanillic acid on mammalian metabolism, our data suggest that this compound, together with the phenolic compounds resveratrol and apigenin, could be useful in the prevention of the excessive fat accumulation characteristic of obesity. Besides, the different mechanisms involved in the fat-reducing activity of each phenolic compound  powder ; p-Coumaric acid ≥98.0% ; Ellagic acid powder from tree bark ≥95% ; trans-Ferulic acid >99%, ; Gallic acid 97.5-102.5% (titration), ; Vanillic acid purum ≥97.0% . All reagents were dissolved in dimethyl sulfoxide (DMSO).Chemicals were obtained from SIGMA Aldrich : Hesperidin analytical standard, ≥97.0% ; Naringin ≥95% ; Apigenin ≥97% (TLC) from parsley powder ; Luteolin analytical standard, ≥97.0% ; Kaempferol ≥90% ; Myricetin analytical standard, ≥98% ; Resveratrol ≥99% ; Curcumin from C. elegans was cultured as previously described [Caenorhabditis Genetics Center  was grown at 20 °C on Nematode Growth Medium (NGM) with Escherichia coli OP50 as normal nematode diet.escribed ,94. N2 BE. coli OP50 was seeded and plates were incubated in darkness at room temperature until dry [All assays were performed in triplicate in 6-well cell culture plates with 4 mL of NGM per well. Orlistat supplemented plates  were used as fat reduction control ,34. Suppntil dry . For all experiments, age-synchronized L4 worms were obtained by standard hypochlorite treatment of gravid animals. The eggs were allowed to hatch overnight in M9 medium and about 750 L1 larvae per well were transferred onto plates and grown during 46 h until L4 stage, in which worms were collected and experiments were performed.Nile Red , a dye for neutral lipids, staining was performed as previously described ,25,96 wiORO staining was performed as described ,24. The ROS levels in vivo were measured using the fluorescent dye dihydroethidium , Sigma-Aldrich, St. Louis, MO, USA), as described previously ,97 with Synchronized 500 L1 larvae were transferred onto plates containing DMSO (control) or resveratrol (200 µM) and were grown until the L4 stage. Worms were collected, washed, and mounted onto 2% agarose pads with a 1% of sodium azide. Lipofuscin pigment was determined as marker of aging by autofluorescence .For all conditions tested, approximately 300 animals were fixed and stained. Fluorescent images of Nile Red stained worms were captured at 100× magnification on a Nikon SMZ18 research stereomicroscope equipped with an epi-fluorescence system and a DS-FI1C refrigerated color digital camera . Images were taken at the same conditions and integration time under a GFP filter . For the ORO analysis, images were also captured at 100× magnification with a Nikon SMZ18 research stereomicroscope equipped with a Nikon DS-Fi2 high-definition color camera. The dihydroethidium (DHE)-labeled ROS formation and the lipofuscin autofluorescence were detected by measuring the fluorescence intensity using a Nikon Eclipse 80i epi-fluorescent microscope, equipped with a TRITC filter  and the DAPI filter (with excitation at 340–380 nm and emission at 435–485 nm), respectively . In all cases, the image analysis of the Nile Red, ORO, DHE, and lipofuscin assays was performed using ImageJ software as previously described . The meaC. elegans lifespan analyses were performed in the same manner for all treatments at 20 °C. Synchronized L1 larvae were transferred to NGM plates containing DMSO (control group) or resveratrol 200 µM for 46 h, to allow C. elegans to develop to L4 stage. Four replicates were used per condition. At least 50–65 L4 larvae per replicate were transferred then onto new plates containing 40 µM of 5-fluoro-2-deoxyuridine , with no additional treatments. Surviving or dead animals were counted daily, until all nematodes died. Worms were scored as dead when they failed to respond to a gentle touch with a platinum wire.2) was calculated with the Nikon NIS-ELEMENTS Software .Egg lying was observed in young adult nematodes (day 3 of growth) grown on NGM agar plates supplemented or not with the polyphenol. The images were taken at 135× magnification using a Nikon SMZ18 stereomicroscope equipped with a Nikon DS-Fi1C high-definition color camera. Worm size (µMC. elegans fat content (quantified by Nile Red), the Cohen’s d effect size [p corresponds to the pooled standard deviation:In order to compare the effect of the different compounds and doses used in reducing the ect size ,99,100 wC. elegans N2 strain was extracted using Trizol® RNA isolation reagent  according to the manufacturer’s instructions. Concentration and purity of RNA were determined at 260/280 nm using a NanoDrop ND-1000 spectrophotometer . Then, 1000 ng of RNA was treated with DNAse  according to the manufacturer’s protocol. For the quantitative gene expression analyses, DNA-free RNA was reverse-transcribed into cDNA following the protocol previously described [Total RNA from escribed ,25.pmp3 and tba1 genes from Life Technologies (TaqMan Gene Expression Assays), which were used as housekeeping gene controls [−∆∆Ct method [Gene expression analyses were performed by quantitative real-time PCR (qPCR) using the TaqMan Universal PCR master mix and specific probes  from Appcontrols . Gene ext method .C. elegans was evaluated using the Experion™ Automated Electrophoresis System . RNAs were then hibridizated in Affymetrix GeneChip®C. elegans Gene 1.0 ST arrays using the Affymetrix technology . Background correction and normalization were performed by algorithm Robust Multiarray Analysis (RMA) and then evaluated using the Transcriptome Analysis Console (TAC) v3. (Affymetrix). Probesets were filtered by ANOVA p value (p < 0.05) and fold change (−2.0 ≤ FC ≥ 2.0). Then, the biological interpretation of the experiment was performed using the Exploratory Gene Association Networks (EGAN) program, from UCSF . Biological effect categories were selected by Gene Ontology Process and KEGG tools. Gene-gene functional interaction networks were performed with GeneMANIA [Microarray hybridization was performed by the Genomics Unit of the Center for Applied Medical Research . Three microarrays were used for each condition. Initially, the quality of total RNA from N2 eneMANIA .C. elegans body fat reduction (Nile Red and Oil Red O) between treatments and control condition (NGM group), together with oxidative stress (DHE) and lipofuscin determinations, were evaluated by a hierarchical ANOVA test, where replicates were nested in treatments, followed by multiple comparison  tests. For lifespan assays, log-rank (Mantel-Cox test) between resveratrol and control (NGM) treatments were performed. Real-time PCR data was analysed using Wilcoxon test comparing each treatment to its control. All tests were performed using StataSE v12 software .C. elegans during larvae development, with resveratrol, apigenin, and vanillic acid being the most effective ones. Whole-genome expression analyses demonstrate that this activity is mediated by changes in the expression of genes involved in lipid synthesis, oxidation, and mobilization, but also involves other crucial biological processes, such as the cellular response to oxidative stress and UPRER, evidencing the importance of these processes in metabolism. Although further research would be necessary using mammalian obesity models, our data supports the potential application of phenolic compounds, including phenolic acids, as bioactive compounds in the prevention of the excessive accumulation of fat characteristic of metabolic diseases. Furthermore, given the different mechanism of action, our results would open the possibility of new studies in which the synergy between these phenolic compounds could be verified, which may be relevant for the maintenance of the energy homeostasis in obesity-related diseases.In conclusion, our study describes the lipid-reducing activity of different phenolic compounds and phenolic acids in