Opn4KO melanocytes displayed a faster proliferation rate compared to Opn4WT melanocytes. Cell cycle population analysis demonstrated that OPN4KO melanocytes exhibited a faster cell cycle progression with reduced G0–G1, and highly increased S and slightly increased G2/M cell populations compared to the Opn4WT counterparts. Expression of specific cell cycle-related genes in Opn4KO melanocytes exhibited alterations that corroborate a faster cell cycle progression. We also found significant modification in gene and protein expression levels of important regulators of melanocyte physiology. PER1 protein level was higher while BMAL1 and REV-ERBα decreased in Opn4KO melanocytes compared to Opn4WT cells. Interestingly, the gene expression of microphthalmia-associated transcription factor (MITF) was upregulated in Opn4KO melanocytes, which is in line with a higher proliferative capability. Taken altogether, we demonstrated that OPN4 regulates cell proliferation, cell cycle, and affects the expression of several important factors of the melanocyte physiology; thus, arguing for a putative tumor suppression role in melanocytes.Skin melanocytes harbor a complex photosensitive system comprised of opsins, which were shown, in recent years, to display light- and thermo-independent functions. Based on this premise, we investigated whether melanopsin, OPN4, displays such a role in normal melanocytes. In this study, we found that murine  Melanocytes originate from neural crest cells during embryogenesis and are melanin-producing cells that inhabit the skin, but are also present in the inner ear, eyes, nervous system, and heart ,2,3,4. WThe microphthalmia-associated transcription factor (MITF) is a crucial player in melanocyte differentiation, development, survival, and proliferation as well as in the pigmentary responses. As a transcription factor, MITF regulates specific gene programs that lead to development and differentiation; thus, affecting a myriad of biological processes in melanocytes ,13. In aAn important feature of melanocytes is their sensitivity to UV and light stimulus responding with important physiological processes, mainly pigmentation. Most of the literature has focused on analyzing the endpoint of such response, i.e., pigmentation, proliferation, DNA damage, and others, while just a handful of studies have evaluated how melanocytes are actually able to sense light and UV radiation photons. Within this line of thought, opsins—light sensing molecules—known to be expressed in the eye, where they participate in visual and non-visual processes ,20,21,22Opn3 knockdown resulted in melanocyte apoptosis [Opn4WT) and non-functional (Opn4KO) OPN4 protein by evaluating cellular metabolism, proliferation, and cell cycle regulation.In recent years, the paradigm of opsins being light sensors was challenged by studies in murine melanocytes demonstrating that melanopsin can also detect thermal energy . In addipoptosis . OPN5 hapoptosis . In thisOpn4KO Melan-a melanocytes were generated using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique. Cells underwent phenotypic characterization and Sanger sequencing revealed a disruption of one Opn4 allele that rendered these cells OPN4 impaired, as previously described in detail [Opn4WT and Opn4KO cells were subject to Per1: Luc gene transfection as described previously [n detail . Opn4WT eviously  and were3 , 20 mM HEPES , 10% fetal bovine serum , 1% antibiotic/antimycotic solution , at pH 7.2. Cells were kept at 37 °C with 5% CO2 Geneticin  was used to guarantee selection only during maintenance. In experiments, cells were kept in the same medium with reduced (2%) FBS, and when kept in the absence of CO2, HEPES was increased to 50 mM. In maintenance and experimental conditions, phorbol 12- myristate 13-acetate  and all-trans retinal (100 nM) were added . Cell manipulation during the experiments was carried in the dark under red dim light .Cells were cultured in RPMI 1640 medium without phenol red , supplemented with 25 mM NaHCO4 Opn4WT or Opn4KO melanocytes were seeded per well in 96-well plates in 100 µL of experimental medium and kept for 24 h at 37 °C with 5% CO2. On the following day, 50 µL of XTT and electron coupling reagent (ECR) solution (7:1) were immediately added. Cells were kept at 37 °C with 5% CO2 for another 24 h, and then the absorbance was read in a spectrophotometer . Specific absorbance was calculated as (A450 sample - A450 blank) - A660 according to the manufacturer’s instructions .For XTT experiments, 2 × 10Opn4WT or Opn4KO melanocytes were seeded per well in 12-well plates in 2 mL of experimental medium and kept for 24 h at 37 °C with 5% CO2; 24, 48, 72, and 96 h later, the cells were harvested with Tyrode/EDTA solution and counted in a hemocytometer.One thousand https://www.doubling-time.com/compute.php (accessed on 22 June 2020). For gene expression, melanin quantification, and flow cytometry experiments, the same experimental setup was made and the cells were harvested with Tyrode/EDTA solution 4 days after seeding and processed as described below. Doubling time was calculated using the online tool available at g for 15 min, and the supernatant was collected. For intracellular melanin, cells were harvested using Tyrode/EDTA solution and centrifuged at 100× g for 5 min. NaOH was added to the cell pellet, heated at 80 °C for 60 min, then centrifuged at 1050× g for 15 min, and the supernatant was collected. Each sample was added in duplicate to wells of a 96-well flat-bottom plate, and total absorbance was measured at 475 nm. The values were interpolated in a standard curve of synthetic melanin , ranging from 1.5625 μg/mL to 40 μg/mL. Melanin content was expressed as the sum of intra- and extracellular melanin divided by cell number.At the end of the experiment, a 500 µL aliquot of the medium was collected in a tube containing 500 µL of 1M NaOH (in 10% DMSO), and heated at 80 °C for 60 min. Then, tubes were centrifuged at 1050× 2. Cells were harvested using Tyrode/EDTA solution, fixed using Cytoperm Cytofix solution  on ice for 30 min, and washed with Perm/Wash buffer. Then, approximately 105–5 × 105 cells were placed into each well of a 96-well round bottom plate, fixed again with Cytoperm Permeabilization Buffer Plus on ice for 10 min, and washed with Perm/Wash buffer. Cytofix/Cytoperm Buffer was again applied to the cells on ice for 5 min and cells were washed. Then, DNase (300 µg/mL) was added, cells were placed at 37 °C for 1 h, washed, resuspended in anti-BrdU antibody in Perm/Wash buffer , and kept at room temperature for 20 min. Cells were then washed, resuspended in 7-AAD solution (for DNA staining), and kept in staining buffer until the acquisition in Canto Flow Cytometry apparatus .For cell cycle evaluation, cell staining was based on the manufacturer’s instructions . In brief, cells were loaded with BrdU (an S phase marker) solution (10 mM) for 2 h and kept at 37 °C with 5% CO5–5 × 105 cells were added per well in 96-well round bottom plates and blocked with PBS containing 1% of bovine serum albumin (BSA) at room temperature for 30 min. Cells were washed and incubated overnight in PER1 , BMAL1 , or REV-ERBα  antibodies in Perm/Wash buffer. On the next day, cells were washed, and a secondary anti-rabbit antibody  was added at room temperature for 60 min. Cells were washed and resuspended in staining buffer, kept at 4 °C, and then read in a Canto Flow Cytometry . For BMAL1 and REV-ERBα staining, 0.5% Triton X-100 was added to allow nuclear permeabilization, which was not required for PER1 staining. At least 104 events were captured, cell doublets were excluded by analyzing FSC-H versus FSC-A. Non-stained controls were used to exclude cellular autofluorescence. Data was analyzed in FlowJO software . Percentage of positive cells and median intensity fluorescence (MIF) were exported and analyzed with PRISMA 7.0 .For protein evaluation, cells were harvested with Tyrode/EDTA solution and fixed with Cytoperm Cytofix solution  on ice for 30 min. Cells were washed with Perm/Wash buffer , approximately 10TURBO DNase . RNA concentration and quality (OD260/OD280) were assessed in a spectrophotometer . One μg of total RNA was subject to reverse transcriptase reaction using random primers and Superscript III, in addition to the reagents recommended by the enzyme manufacturer .The medium was removed and TRIzol  was added onto the cells, collected, and stored at −80 °C until processing. RNA was extracted using 1-bromo-3-chloropropane , precipitated with isopropanol , and washed with 75% molecular grade ethanol . RNA pellets were resuspended in DEPC water and genomic contamination was prevented using http://www.ncbi.nlm.nih.gov/genbank (accessed on 23 May 2020)), designed by Primer Blast (http://www.ncbi.nlm.nih.gov/genbank (accessed on 23 May 2020)) or Primer Quest , and synthesized by Integrated DNA Technologies . Rpl37a was used to normalize the expression values of the genes of interest.Twenty-five ng of cDNA was subject to quantitative PCR using species-specific primers  spanningPer1 and Bmal1 or Opn2 and Clock, multiplex reactions containing cDNA, primers, fluorescent probes, and Kapa Probe Fast Mix  were used and run in triplicates for each experimental cDNA sample. Reactions were carried out in an iQ5 thermocycler  in the following conditions: 3 min at 95 °C followed by 45 cycles of 15 s at 95 °C and 60 s at 60 °C. For the other genes, independent solutions were prepared with cDNA (25 ng), specific primers, and Kapa Sybr Fast Mix, and run in duplicates in an iQ5 thermocycler. Reactions were subject to the following conditions: 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, 60 s at 60 °C, and 80 cycles of 10 s at 55 °C with a gradual increase of 0.5 °C.For simultaneous analysis of Per1: Luc Opn4WT or Opn4KO melanocytes were seeded in 35 mm dishes in the experimental medium and kept at 37 °C with 5% CO2 for 24 h. On the next day, cells were treated with 200 nM dexamethasone or 10 µM FSK, 100 µM luciferin  was added, dishes were sealed with 35 mm round coverslips and parafilm , and placed in the Lumicycle equipment  in an incubator without CO2 positive pressure at 37 °C. Drugs remained in the medium until the end of the experiment. Bioluminescence was recorded and the temperature of the incubator was monitored  every 10 min.One hundred thousand t-test. Cellular growth was analyzed by two-way ANOVA followed by Bonferroni post-test. For bioluminescence analysis, the amplitude was calculated by subtracting the lowest values from the highest values within the first 24 h and analyzed by unpaired Student’s t-test. Data obtained from at least two independent experiments (N) were used for all statistical analyses. The number of samples (n) is shown in each figure legend. p values < 0.05 were used to reject the null hypothesis and were calculated in GraphPad Prism 7.0.XTT data, gene and protein expression, as well as cell cycle results, were analyzed by unpaired Student’s Opn4 deletion in normal melanocytes, which were gene-edited by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique and further validated by Sanger sequencing, as previously reported [We initially evaluated the impact of reported . Among tOpn4WT and Opn4KO melanocytes. We found that Opn4KO melanocytes displayed a higher cell number compared to Opn4WT cells after 3 days in culture, a difference maintained in the following day (Opn4KO melanocytes showed a faster doubling time (~34 h) compared to WTOpn4 normal melanocytes (~47 h). On the other hand, mitochondrial metabolism of XTT, commonly used as a cellular proxy of proliferation and metabolism, did not show any difference between Opn4WT and Opn4KO melanocytes  cell population in the G0-G1 phase, but massively increased cell number in the S phase (300%), and a slightly increased (20%) in the G2/M phase compared to Opn4WT melanocytes  and ataxia telangiectasia and Rad3-related (Atr), which encode proteins that act on DNA damage response and are responsible for maintaining genome integrity [Atm expression was upregulated, while Atr was not affected in Opn4KO melanocytes compared to Opn4WT cells  is a member of the cyclins, which are known to be important regulators of the cell cycle due to their ability to bind and activate cyclin-dependent kinases (CDKs). It has been reported that Cyclin 1 expression increases during cell cycle progression reaching its highest levels in S and G2/M phases [Ccna1 expression in Opn4KO melanocytes compared to Opn4WT counterparts  gene encodes a serine/threonine-specific protein kinase that participates in cell cycle events in response to DNA damage, mainly leading to cell cycle arrest, DNA repair, and cellular death [2/M progression, and is activated by cyclin B. CHEK1 is known to regulate replication checkpoint in the G2/M phase and is required for S phase progression and cell survival [Chek1 expression was found in Opn4KO melanocytes in comparison to Opn4WT cells  in the absence of functional OPN4. We did not detect any difference in Opn2 mRNA expression between Opn4WT and Opn4KO cells  gene and protein levels, which results in retinoblastoma protein (pRb) hypophosphorylation. The consequence of such events is cell cycle arrest [As MITF is an important cell cycle regulator ,13, incre arrest ,71. BRAFe arrest . In murianocytes . Anotheranocytes . In a phe arrest .Opn4 seems to interact with Mitf, as removal of Opn4 resulted in increased Mitf expression, with important cell cycle alterations, which ultimately led to a faster proliferation. Such modifications may place Opn4 as a putative tumor suppressor gene in melanoma. Within this line of thought, we previously reported that Opn4KO melanocytes were more resistant to UVA-induced effects as daily low doses of UVA radiation reduced cellular growth only in Opn4WT melanocytes [anocytes . Collectanocytes  and fastOpn4 in murine melanocytes resulted in increased cellular growth and a faster cell cycle progression, which is associated with altered expression of gene/protein molecular clock components and increased Mitf expression, a master regulatory agent of melanocyte biology. A limitation of our study is a mechanistic view of how the lack of OPN4 affects Mitf expression and its downstream targets; however, in face of the phenotype and molecular clock gene alterations displayed by Opn4KO melanocytes, we suggest a putative role of Opn4 as a tumor suppressor gene in melanoma.Taken altogether, we have provided evidence that the lack of


Candida auris is a globally emerging multidrug-resistant fungal pathogen. Its pathogenicity-related signaling networks are largely unknown. Here, we characterized the pathobiological functions of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway in C. auris. We focused on adenylyl cyclase (CYR1), the PKA regulatory subunit (BCY1), and the PKA catalytic subunits (TPK1 and TPK2). We concluded that PKA acts both dependently and independently of Cyr1 in C. auris. Tpk1 and Tpk2 have major and minor roles, respectively, in PKA activity and functions. Both Cyr1 and PKA promote growth, thermotolerance, filamentous growth, and resistance to stress and antifungal drugs by regulating expression of multiple effector genes. In addition, Cyr1 and PKA subunits were involved in disinfectant resistance of C. auris. However, deletion of both TPK1 and TPK2 generally resulted in more severe defects than CYR1 deletion, indicating that Cyr1 and PKA play redundant and distinct roles. Notably, Tpk1 and Tpk2 have redundant but Cyr1-independent roles in haploid-to-diploid cell transition, which increases virulence of C. auris. However, Tpk1 and Tpk2 often play opposing roles in formation of biofilms and the cell wall components chitin and chitosan. Surprisingly, deletion of CYR1 or TPK1/TPK2, which resulted in severe in vitro growth defects at 37°C, did not attenuate virulence, and BCY1 deletion reduced virulence of C. auris in a systemic murine infection model. In conclusion, this study provides comprehensive insights into the role of the cAMP/PKA pathway in drug resistance and pathogenicity of C. auris and suggests a potential therapeutic option for treatment of C. auris-mediated candidemia. Candida auris is considered an emerging human pathogen of global concern. It was first isolated in Japan in 2009  pathway is generally required for the pathogenicity of human and plant fungal pathogens , the regulatory subunit of PKA (BCY1), and the catalytic subunits of PKA (TPK1 and TPK2) in C. auris and functionally analyzed them through reverse genetics and phenotypic analyses. Here, we found that PKA plays Cyr1-dependent and -independent roles in regulating temperature-dependent growth, the formation of pseudohyphae, stress responses, ploidy switching, and resistance to antifungal drugs and disinfectants in C. auris. Most importantly, we found that hyperactivation, but not inhibition, of the cAMP/PKA pathway significantly reduced the virulence of C. auris.In this study, we identified the genes encoding adenylyl cyclase . The four proteins in C. auris that had greatest identity to the corresponding protein in the C. albicans cAMP/PKA pathway were Cyr1 (B9J08_004540), Bcy1 (B9J08_002818), Tpk1 (B9J08_004030), and Tpk2 (B9J08_002788)  and a single regulatory subunit (Bcy1), as does the PKA complex from C. albicans.The goal of this study was to functionally characterize the cAMP/PKA pathway in 788) see . We conc10.1128/mBio.02729-21.1FIG S1http://www.candidagenome.org/). Download FIG S1, PDF file, 0.05 MB.Phylogenetic analyses of fungal adenylyl cyclases and PKA subunits. Phylogenetic trees of fungal orthologs for adenylyl cyclase (A), the PKA regulatory subunit (B), and PKA catalytic subunits (C and D) were retrieved from Candida Genome Database  or the hygromycin B resistance marker (CaHYG). These were introduced into the wild-type (WT) strain by electroporation (see cyr1Δ+CYR1) or reintegrated into its native locus   see .10.1128/mBio.02729-21.2FIG S2Candida auris. Target genes were replaced with gene deletion cassette containing the nourseothricin resistance gene (NATr) or the hygromycin resistance gene (HYGr) delivered by electroporation in C. auris . Homologous recombination strategies between the WT gene and the deletion cassette are illustrated graphically (left panels). Transformants were confirmed by diagnostic PCR (center panels) and Southern blot analysis (right panels). All primer sets were listed in CYR1 gene in WT was replaced with the NATr marker. The genomic DNA of WT and cyr1Δ mutants was digested by EcoRV for Southern blot analysis. (B) The BCY1 gene in WT was replaced with the NATr marker. The genomic DNA of WT and bcy1Δ mutants was digested by HincII for Southern blot analysis. (C) The TPK1 gene in WT was replaced with the NATr marker. The genomic DNA of WT and tpk1Δ mutants was digested by XhoI for Southern blot analysis. (D) The TPK2 gene in WT was replaced with the NATr marker. The genomic DNA of WT and tpk2Δ mutants was digested by HincII for Southern blot analysis. (E) The TPK1 gene in the tpk2Δ mutant was replaced with HYGr marker. The genomic DNA WT and tpk1Δ tpk2Δ mutants was digested by HincII for Southern blot analysis. Download FIG S2, PDF file, 0.2 MB.Construction and validation of gene deletion mutants in Copyright © 2021 Kim et al.2021Kim et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02729-21.3FIG S3cyr1Δ, bcy1Δ, tpk1Δ, and tpk2Δ mutants. Ectopic integration of a WT gene was confirmed by PCR using primer pairs in exon regions, and targeted integration of a WT gene into its native locus was confirmed by PCR using primers in the exon regions paired with primers outside the homologous region. All primer sets are listed in CYR1 gene in the cyr1Δ mutant (cyr1Δ+CYR1) (A), targeted integration of the BCY1 gene in the bcy1Δ mutant (bcy1Δ::BCY1) (B), targeted integration of the TPK1 gene in the tpk1Δ mutant (tpk1Δ::TPK1) (C), and targeted integration of the TPK2 gene in the tpk2Δ mutant (tpk2Δ::TPK2) (D). Each expected size is indicated under the corresponding electrophoresis image. Download FIG S3, PDF file, 0.09 MB.Validation of complemented strains for Copyright © 2021 Kim et al.2021Kim et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. auris exhibits much greater thermotolerance (can grow well up to 42°C) than other Candida species  and their complemented strains, both qualitatively and quantitatively, at a range of temperatures from 30 to 42°C. It was previously noted  than at low temperatures (25 and 30°C). We found that deletion of CYR1 markedly reduced the growth of C. auris compared to the WT strain, and this was most evident at 30°C. The growth rate was restored to that of the WT by complementation with the WT CYR1 gene  . However, we found that TPK1 expression levels did not change by TPK2 deletion (see TPK2 expression levels did not change by TPK1 deletion (see We next addressed whether the growth defects of the Δ mutant  and B. Wto 42°C) , which m42°C see . In cont mutants . In contactivity . Since ition see . Likewistion see . All of 10.1128/mBio.02729-21.4FIG S4TPK1 and TPK2 in Candida auris. (A) Growth curves of WT (B8441) and each gene deletion mutant were generated at 39 and 42°C in YPD medium. The strains are listed as follows: cyr1Δ (YSBA21), bcy1Δ (YSBA4), tpk1Δ (YSBA13), tpk2Δ (YSBA17), and tpk1Δ tpk2Δ (YSBA24). Each curve represents data from two-independent experiments. The OD600 was measured with a multichannel bioreactor (Biosan Laboratories) for 30 to 115 h based on the growth rate. (B) Expression analysis of TPK1 or TPK2 in tpk2Δ (YSBA17) or tpk1Δ (YSBA13) mutants. C. auris WT was used as a control. Cells were cultured overnight at 30°C in liquid YPD medium, subcultured to OD600 0.8, and extracted for total RNA. The expression level of each gene was normalized to that of ACT1, and the fold change was calculated relative to the basal expression level of each gene in the WT. Three independent biological experiments with three technical replicates were performed. The data represent means ± SEM. The data were analyzed statistically by the Student’s t-test using Prism 8.0 . Download FIG S4, PDF file, 0.3 MB.Growth analysis of the cAMP/PKA pathway mutants and expression analysis of Copyright © 2021 Kim et al.2021Kim et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the HSF1 (B9J08_001120) and heat shock proteins  in the WT and mutant C. auris strains. In all strains, expression of HSP12, HSP70, and HSP78 was significantly induced, from 5- to 20-fold, upon a temperature shift from 30 to 42°C  and methyl methane-sulfonate (MMS), can induce pseudohyphal growth  liquid medium supplemented with 100 mM HU for 24 h. At this time, a large number of WT cells had formed pseudohyphae , FLO8 (B9J08_000401), WOR1 (B9J08_004027), and UME6 (B9J08_000592) in the WT and mutants in response to HU. Expression levels of EFG1 were unaffected by HU in all strains, except that it was slightly reduced by HU in the tpk1Δ tpk2Δ mutant   , a membrC. auris strains. In cells of all the strains, when treated with 1.5 M NaCl, expression of ENA1 (B9J08_002173) was enhanced ∼25-fold compared to the untreated cells. In WT cells, GPP1 (B9J08_004378) was enhanced approximately 12-fold compared to the untreated WT cells, while its expression was significantly reduced in response to salt treatment in the cyr1Δ and tpk1Δ tpk2Δ mutants compared to the WT cells  and GPD2 (B9J08_003684) was unaffected by salt treatment in the WT cells, but expression of both were strongly enhanced in salt-stressed tpk1Δ tpk2Δ mutant cells compared to those of the unstressed mutant , Gpp1 (glycerol-3-phosphate phosphatase), and Gpd1/2 (glycerol-3-phosphate dehydrogenases). This led us to assess the expression of the genes encoding these proteins in the WT cells . We suggd mutant . The enhC. auris to oxidative stress. Similar to the mutants’ response to osmotic stress, all of the mutant strains were generally more susceptible to oxidative stress agents   . One excf the WT . This is2·–) to H2O2 and catalase, glutathione (GSH)-GSH peroxidase (Gpx), thioredoxin (Trx), peroxiredoxin (Prx), and sulfiredoxin systems for detoxifying inorganic or organic peroxides. We found all of the corresponding orthologs to these proteins in C. auris. Therefore, we next addressed which oxidative defense system is regulated by the cAMP/PKA pathway in C. auris. In response to menadione, expression of SOD1 (B9J08_001318) was enhanced approximately 2- to 3-fold in WT, cyr1Δ, and tpk2Δ strains  was weakly enhanced in the WT mutant, but both basal and induced levels of CAT1 were significantly higher in cyr1Δ and tpk1Δ tpk2Δ mutants compared to the WT (GPX1 (B9J08_003442) and PRX1 (B9J08_003437) was not significantly enhanced by H2O2 in the WT strain or in the cyr1Δ, bcy1Δ, tpk1Δ, and tpk2Δ mutants, but strongly, approximately 8- to 18-fold, in the tpk1Δ tpk2Δ mutant (TSA1 (B9J08_000645), TRR1 (B9J08_001090), and TRX1 (B9J08_000543) was either not enhanced or only weakly so by H2O2 in the WT and cyr1Δ mutant strains. Nonetheless, the enhancement of expression of these genes in the tpk1Δ tpk2Δ mutant was significantly less than in the WT (SRX1 (B9J08_000169) was significantly enhanced (4-fold) by H2O2 in the WT strain but not significantly induced in cyr1Δ, bcy1Δ, tpk1Δ, and tpk1Δ tpk2Δ mutants  and calcofluor white (CFW). The tpk1Δ mutant was much more resistant to the dyes compared to the WT cells, while the cyr1Δ mutant was not. Deletion of TPK2 in the double mutant reduced the resistance of the tpk1Δ mutant to the WT level, or even exacerbated it , in the  mutants  and C. S mutants . The 3-m mutants , furthern the WT . Finally mutants  to E comCHS) and chitin deacetylase (CDA) genes is regulated by Bcy1 and Tpk1 (CHS and CDA are involved in chitin and chitosan biosynthesis (–C. auris: CHS1 (B9J08_003879), CHS2 (B9J08_005077), CHS3 (B9J08_004150), CHS4 (B9J08_004972), CHS5 (B9J08_000868), CHS6 (B9J08_004541), CHS7 (B9J08_001816), CHS8 (B9J08_002856), and CDA2 (B9J08_004841). Among these, we found that expression levels of CHS3, CHS4, CHS5, CHS7, and CHS8 were significantly less in the bcy1Δ mutant than in the WT and that expression of CHS3 and CDA2 was less in the tpk1Δ mutant than in the WT , and tpk1Δ (YSBA13) strains were cultured overnight at 30°C in YPD medium, subcultured to OD600 0.8, and extracted for total RNA. The expression level of each gene was normalized with that of ACT1, and the fold change was calculated relative to the basal expression level of each gene in WT. Three independent biological experiments with three technical replicates were performed. The data represent means ± SEM. The statistical significance of the difference was determined by one-way ANOVA with Bonferroni’s multiple-comparison test . Download FIG S6, PDF file, 0.03 MB.Expression analysis of Copyright © 2021 Kim et al.2021Kim et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Candida auris is a panresistant fungal species that does not respond to multiple classes of antifungal drugs , which is a target of FCZ. The expression level of ERG11 was significantly decreased in all deletion mutants compared to the WT , CDR2 (B9J08_002451), MDR1 (B9J08_003981), MDR2 (B9J08_000368), and TAC1 (B9J08_004819). We measured the expression level of CDR1, MDR1, and MDR2 in all strains, and observed that, generally, expression was greater in all deletion mutants than in the WT strain   . Collect10.1128/mBio.02729-21.7FIG S7C. auris. Each strain was adhered to a polystyrene plate for 90 min in RPMI medium at 37°C, nonadhered cells were removed, and the remaining cells were grown for 24 h at 37°C in RPMI medium. Biofilm formation was quantified by measuring the OD595 of cells that adhered to the bottom of the well after 24 h. Three independent biological experiments with three technical replicates were performed. Error bars indicate the SEM. The statistical significance of difference was determined by one-way ANOVA with Bonferroni’s multiple-comparison test . Download FIG S7, PDF file, 0.02 MB.Roles of the cAMP/PKA pathway in biofilm formation of Copyright © 2021 Kim et al.2021Kim et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Candida auris can survive very long time in health care environments and can be transmitted readily between patients  or iodine-based disinfectant (10% povidone-iodine), approximately 5 to 10% of C. albicans WT strain survived  showed significantly increased (∼2-fold) susceptibility to the disinfectants compared to WT  analysis confirmed that the white and pink colonies of the tpk1Δ tpk2Δ mutant represent haploid and diploid cells, respectively  were infected intravenously with the indicated inocula of C. auris WT cells (B8441). The cells were cultured overnight in YPD medium at 30°C, washed three times with distilled water, and resuspended in PBS for intravenous infection. Download FIG S8, PDF file, 0.02 MB.Survival rate of A/J mice infected with various inocula of Copyright © 2021 Kim et al.2021Kim et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. auris has only recently emerged as a fungal pathogen of global importance, and consequently, its pathogenicity-related signaling pathways are still largely unknown. We focused on the cAMP/PKA pathway as one of the most well-known signaling pathways in fungal pathogenicity and identified its major signaling components and their pathobiological functions in C. auris. These components include adenylyl cyclase, Cyr1, and the subunits of PKA—the catalytic subunits Tpk1 and Tpk2 and the regulatory subunit Bcy1.The pan-resistant C. auris, including growth, morphological transitions, response and adaptation to stress, control of its cell wall integrity, changes in ploidy from haploid to diploid, resistance to antifungal drugs and disinfectants, biofilm formation, and pathogenicity. We highlight two unique findings in this study. First, PKA has pathobiological functions that are both dependent and independent of Cyr1. Second, hyperactivation of the cAMP/PKA pathway attenuates the virulence of C. auris, but inhibition of the pathway does not  were not catastrophic, but markedly hampered normal growth (–PKA1 and PKA2) in the basidiomycetous fungal pathogen C. neoformans does not affect cell viability and growth rate at all . Ploidy variation is a common characteristic of fungal pathogens, providing genetic diversity and serving as a driving force for evolution  to antifungal drugs has led to its global importance. In particular, C. auris is markedly resistant to the azole family  characterization of potential Cyr1 upstream regulators, such as a G-protein-coupled receptor or heterotrimeric or small GTP-binding proteins; (ii) identification of the Cyr1-independent PKA upstream regulators; and (iii) elucidation of Cyr1 and PKA-dependent downstream signaling networks. Answering these research question may provide comprehensive understanding of the role of the cAMP/PKA pathway in pan-antifungal drug resistance and pathogenicity of C. auris.In conclusion, we have demonstrated here that the cAMP/PKA pathway is involved in diverse pathobiological functions, including growth, stress resistance, pseudohypha formation, drug and disinfectant resistance, cell wall integrity, biofilm formation, ploidy switching, and virulence of Care of animals and all experiments were conducted in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee of Yonsei University, and this committee approved all of the vertebrate studies.Candida auris strains used in this study are listed in d-glucose (YPD broth) or on plates containing 2% agar in YPD broth (YPD plates).10.1128/mBio.02729-21.9TABLE S1C. auris strains used in this study. Download Table S1, DOCX file, 0.02 MB.Copyright © 2021 Kim et al.2021Kim et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the CaNAT) or hygromycin B resistance marker (CaHYG) (CYR1 (B9J08_004540), BCY1 (B9J08_002818), TPK1 (B9J08_004030), and TPK2 (B9J08_002788). The gene deletion mutants bcy1Δ and tpk1Δ were generated with Cas9-ribonuleotideprotein-mediated transformation. Each gene disruption cassette containing either CaNAT or CaHYG selection marker was constructed by double-joint PCR , and incubated at 25°C with shaking for 1 h. Next, electrocompetent cells were centrifuged, washed once each with dH2O and 1 M sorbitol, and finally resuspended in 10 ml of 1 M sorbitol. Approximately 1 μg of disruption cassette dissolved in 5 μl dH2O was mixed with 40 μl of electrocompetent cells. Electroporation was performed using the C. albicans protocol on a GenePulsar Xcell (Bio-Rad). Cells were then allowed to recover in YPD broth at 30°C in a shaking incubator for 2 h, plated onto selective YPD agar medium containing 400 μg/ml nourseothricin or 1.8 mg/ml hygromycin B, and further incubated at 30°C for 2 to 3 days. The desired genotype of each positive nourseothricin or hygromycin B-resistant transformant was confirmed by diagnostic PCR and Southern blotting  or reintegrated into its native locus , were constructed as follows. First, each full-length gene fragment was amplified by Phusion-PCR by using genomic DNA of the WT B8441 strain as a template and each primer pair listed in CYR1, BCY1, TPK1, and TPK2 inserts into the plasmid pYM70 to produce the plasmids pHYG-CYR1, pHYG-BCY1, pHYG-TPK1, and pHYG-TPK2. For the targeted reintegration into its native locus, pHYG-CYR1, pHYG-BCY1, pHYG-TPK1, and pHYG-TPK2 were linearized by HpaI, AvrII, BglII, and AfeI, respectively, and introduced into each mutant by electroporation. The correct genotype of the complemented strain was confirmed by diagnostic PCR (see All gene deletion mutants were constructed by using the nourseothricin resistance marker ( (CaHYG)  flanked oint PCR . To amplvely see . For traB9J08_0028. The geications . Brieflyting see . To conf PCR see .10.1128/mBio.02729-21.10TABLE S2Table S2, DOCX file, 0.02 MB.Primers used in this study. Download Copyright © 2021 Kim et al.2021Kim et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. auris were inoculated into 50 ml of YPD broth and cultured overnight at 30°C in a shaking incubator. Cells were subcultured in 50 ml of fresh YPD broth until the optical density at 600 nm (OD600) was 0.6 to 0.8. At this point, the cells were collected by centrifugation, frozen in liquid nitrogen, and lyophilized. For stress response experiments, 10 ml was sampled for the zero time-point  and the remaining 30 ml was further incubated with indicated stress agents or conditions. Total RNA was isolated by the TRIzol extraction method using Easy-Blue (Intron). Complementary DNA (cDNA) was synthesized by reverse transcriptase (Thermo Scientific) with purified total RNA and subsequently used for quantitative PCR using specific primer pairs for each gene with the CFX96 real-time system (Bio-Rad). Expression of ACT1 served as the control for normalization. Statistical differences between samples were analyzed by one-way analysis of variance (ANOVA) using Bonferroni’s multiple-comparison test. All experiments were performed in triplicate from three biological replicates.WT and cAMP/PKA mutant strains of C. auris. To do this, C. auris cells grown overnight at 30°C were serially diluted 10-fold four times  and then spotted onto YPD plates. For growth measurements, plates were incubated at 30, 37, 39, and 42°C. Growth was assessed qualitatively by photographing the plates 1 to 2 days later.To analyze the growth and sensitivity to various stresses of WT and cAMP/PKA mutant strains of tert-butyl hydroperoxide (an organic peroxide), menadione (a superoxide anion generator), or diamide (a thiol-specific oxidant). Membrane-destabilizing stress was imposed using SDS, and cell wall-destabilizing stress was imposed using CFW and CR, fludioxonil, fluconazole, amphotericin B, or flucytosine for antifungal drug susceptibility. Cells were incubated at 30°C and photographed 1 to 6 days after treatment.Various stresses were imposed by adding to the media chemical agents that would impose stress to the cells. Osmotic stress was supplied as sorbitol. Cation and salt stress were imposed with NaCl or KCl; these were supplied in either glucose-rich YPD or in glucose-deficient media (YPD without dextrose). Oxidative stress was supplied with hydrogen peroxide, C. auris were grown overnight to their stationary phase at 30°C in 50 ml of YPD broth. Cells were pelleted, resuspended in 1 ml of 10 mM sodium phosphate buffer , and lysed by disruption with glass beads at 4°C, and the cell debris was pelleted by centrifugation for 20 min. The supernatant was used for the PKA activity assay. The PKA assay was performed either in the presence or absence of 10 μM cAMP using a PKA kinase activity kit (EZNO).Cells of WT and cAMP/PKA mutant strains of C. auris, cells were grown for 24 h in YPD broth containing 100 mM HU . Next, cells were stained with 100 μg/ml fluorescein isothiocyanate (FITC)-conjugated WGA, 25 μg/ml CFW, or 30 μg/ml EY for 30 min in the dark. Stained cells were observed and photographed by fluorescence microscopy (Olympus BX51). The fluorescence of at least 50 individual cells was measured with ImageJ/Fiji software.To measure chitin and chitosan content of cells of WT and cAMP/PKA mutant strains of 2 was added to each. The samples were incubated at room temperature for 15 min; 200 μl of 12.5% ammonium sulfamate was added to each sample, which were then incubated for another 5 min at room temperature. Next, 200 μl of 0.25% MBTH was added to each sample, followed by a 30-min incubation at 37°C, the addition of 200 μl of 0.5% FeCl3, and a further 5 min of incubation at 37°C. Finally, the optical density of each sample was measured at 595 nm.The chitosan content of the WT and mutant cells was measured quantitatively with a 3-methyl-2-benzothiazolinone hydrazone (MBTH)-based chemical assay , 81. CelC. auris strain cells were cultured overnight at 30°C in YPD medium, washed twice with H2O, and then resuspended in H2O. For the EUCAST  MIC test, we used a cell concentration corresponding to an OD600 of 1.0. We added 100 μl of the cell suspension to 10 ml of morpholinepropanesulfonic acid (MOPS)-buffered RPMI 1640 media (pH 7.4 with 0.165 M MOPS and 2% glucose for EUCAST) and loaded into 96-well plates containing 2-fold serially diluted drugs. The 96-well plates were incubated at 35°C for 2 days, and the cell density in each well was measured at OD595  and iodine-based disinfectant (10% povidone iodine) against C. auris WT and cAMP/PKA mutants, we followed the protocol of the European Standard EN 13624:2013 with modifications. Briefly, 106 cells in 100 μl of 0.3% bovine albumin solution was added in 900 μl of each disinfectant. After the 30 s of contact time, 100 μl of the resulting suspension was transferred to an e-tube containing 900 μl of appropriate neutralizing solution . After 5 min of neutralization, 3 μl of cell suspension was spread on the YPD medium to count the CFU of each strain. As a control group, cells treated only with 0.3% bovine albumin solution without disinfectant treatment were spread on the YPD medium to count the CFU of each strain. The percent survival rate of each strain was calculated by comparing the colony pop-up ratio between the control group and the disinfectant-treated group.To quantitatively assess fungicidal activity of chlorine-based disinfectants  for 1 h at 37°C, and then washed twice with PBS. The cells were stained in 500 μl of 100-μg/ml propidium iodide for 30 min at room temperature, washed with PBS, and resuspended in PBS. These cells were used for DNA content analysis. In each experiment, at least 30,000 cells were used for the flow cytometry analysis.Cells from WT and cAMP/PKA mutant strains of in vivo virulence of WT and cAMP/PKA mutant strains of C. auris were examined in a murine model. The fungal cells were incubated overnight at 30°C in YPD broth and washed three times with PBS. Cell concentrations were measured and adjusted to 108 cells/ml in PBS. To confirm the CFU and the viability of the inoculum, the diluted cells were plated onto YPD agar plate and incubated at 37°C for 24 h. Six-week-old female mice of the A/J strain were acquired for this study . After arrival, the mice were habituated for 1 week. The fungal cells were injected intravenously as follows: mice were restrained, and their tails were placed in warm (40°C) water to expand the lateral veins. The cell suspension (100 μl) was injected. Survival was monitored daily. A 10% decrease of a mouse’s body weight or abnormal behavior (tilting or whining) was deemed a humane endpoint to the experiment, and the mouse was sacrificed. Statistical analysis was performed with log-rank (Mantel-Cox) test for the murine survival curve.The We will provide any strain and materials used in this study upon request.


The β-carbolines in our diet, mainly including harman and norharman, are a group of biologically active, naturally occurring plant-derived alkaloids. Fragrant sesame seed oil is one of the most popular flavor edible oils in China. Considering that sesame seeds are roasted at 200–240 °C during the processing of flavor sesame seed oils, it is meaningful to investigate the levels of β-carboline compounds in various sesame seed oils. In this work, the levels of β-carbolines (harman and norharman) in different types of sesame seed oils in China  have been determined systematically. The results showed that the levels of total β-carbolines in pressed fragrant sesame oils (700.5~2423.2 μg/kg) were higher than that in ground fragrant sesame oils (660.4~1171.7 μg/kg). Roasting sesame seeds at high temperatures (200–240 °C) led to higher levels of β-carbolines (660~2400 μg/kg) in fragrant sesame seed oils. In addition, the loss of tryptophan might be attributed to the formation of β-carbolines in sesame seeds during the roasting process. In general, fragrant sesame seed oils  contain higher levels of β-carbolines due to the formation of harman and norharman during the roasting sesame seed process. HarIn the past decades, harman and norharman have been detected in many processed and stored foods, including cookies, maize, soy, coffee and even fermented alcoholic beverages ,7,8,9. AEdible oils, especially vegetable oils, are one of the most important food ingredients in daily life. The average consumption of edible oil is around 28 kg per year in China. However, less attention has been paid to the levels of harman and norharman in edible vegetable oils. Indeed, edible vegetable oils, including soybean oil, palm oil, canola oil, sunflower oil, rapeseed oil and peanut oil, are produced through chemical refining  or pressing  . Thus, aSesame seed oil  is one of the most popular flavor oils in China; about 400,000 tons of flavor sesame oil are consumed in China every year. Considering the processing of flavor sesame oil (roasting the sesame seed at 200–240 °C) and bioactivity of β-carbolines (harman and norharman), it is meaningful to investigate the level of β-carboline compounds in sesame oils, including pressed fragrant sesame oil, ground fragrant sesame oil and cold-pressed sesame oil. In this work, the level of β-carbolines (harman and norharman) in different sesame seed oils in China has been investigated, and the effect of different processing methods on the levels of harman and norharman in sesame seed oils has been discussed. The effects of roasting temperature and time on the levels of β-carboline compounds (harman and norharman) in sesame seed oils have been investigated as well.Based on sesame seed oil processing methods, sesame seed oils in China can be divided into four types, including: (1) pressed fragrant sesame seed oil, (2) ground fragrant sesame seed oil, (3) cold-pressed sesame seed oil and (4) refined sesame seed oil ,16. ThesThe contents of β-carbolines (harman and norharman) in a batch of different pressed fragrant sesame oils (seven samples) were determined . The resThe contents of β-carbolines (harman and norharman) in a batch of different ground fragrant sesame seed oils (seven samples) were determined . The resCold-pressed sesame seed oils are produced without sesame seed roasting pretreatment (at higher temperature) before the pressing process. Limited commercial resources could be found in China due to its non-flavor. In fact, cold-pressed sesame seed oils can be used as a good cooking oil for its non-flavor.The contents of harman and norharman in two cold-pressed sesame seed oil samples were also determined . The resBased on the above results, it can be concluded that the contents of norharman (6.2 μg/kg–1230.7 μg/kg) were higher than the contents of harman (1.8 μg/kg–1197.7 μg/kg) in all tested sesame seed oil samples see . The levIndeed, roasting of sesame seeds at 200–240 °C for 30–60 min is required at the first stage of pressed fragrant sesame seed oil and ground fragrant sesame seed oil processing  20,21],,21], whiTo investigate the effect of sesame seed roasting temperature on the formation of β-carboline compounds (harman and norharman) in sesame seed oils, a model roasting process was conducted at different roasting temperatures , and the results were summarized in In most cases, the levels of norharman in the total β-carbolines was higher than the levels of harman at different roasting temperatures (200–240 °C) . It was Moreover, it was observed that the sequence of the oil color of different sesame seed oils after roasting at different temperatures followed the same order: 240 °C > 220 °C > 200 °C, and prolonging of roasting time was attributed to the color-deepening of sesame seed oils . The aboIn addition, the fatty acid compositions of sesame seed oil at different roasting temperatures (200–240 °C) were determined . The sesThe formation mechanism of β-carboline compounds (harman and norharman) in sesame seed oil was also discussed. In fact, harman and norharman, as non-polar heterocyclic aromatic amines, are usually assigned as pyrolysis products of amino acids at higher temperatures (>150 °C) , while tCommercial sesame seed oils, including pressed fragrant sesame seed oils (n = 7) and ground fragrant sesame seed oils (n = 7), were purchased from a local supermarket in China. Sesame seeds were purchased from a local supermarket in China. Acetonitrile (HPLC grade) was purchased from Thermo Fisher Scientific . Ammonium hydroxide and hydrochloric acid (HPLC grade) were obtained from Kemiou Chemical Reagent Co. Ltd. . Methyl alcohol, acetic acid and n-hexane were of HPLC grade, and other chemicals were of analytical reagent grade. Oasis MCX solid-phase extraction cartridge  was purchased from Waters . The water used was Wahaha purified water purchased from a local supermarket. The standards AαC, MeAαC, DMIP, Trp-P-1, Trp-P-2, Glu-P-2, MeIQ, MeIQx, IQ, PhIP, 4,8-DiMeIQx and 7,8-DiMeIQx were purchased from Toronto Research Chemicals . Harman , norharman  and 4,7,8-TriMeIQx were purchased from Alta scientific .n-hexane three times. The obtained mixture (oil–hexane) was then rotationally vaporized and blown with nitrogen to remove the remaining n-hexane. The resulting sesame seed oil was centrifuged to remove impurity particles. All samples were prepared as triplicates.Sesame seeds (about 1000 g) were roasted using an automatic electric heater  at different temperatures  for the duration of 5 min, 10 min, 20 min and 30 min at each temperature with constant stirring. The roasted sesame seeds (100 g) were crushed with a mortar and then extracted with The fatty acid composition was determined by conversion of oil to fatty acid methyl esters, prepared following the standard IUPAC method 2.301. Fatty acid compositions of sesame seed oils were analyzed by an Agilent Technologies 6890 N gas chromatograph (GC) equipped with a 30.0 m × 250 µm × 0.25 µm BPX-70 capillary column and detected using a flame ionization detector (FID). Samples (1 µL) were injected under the following conditions: the nitrogen flow rate was 1.0 mL/min, the oven was programmed from the set temperature of 170 to 210 °C at 2 °C/min, the split ratio was 50:1, the GC injection temperature was 250 °C, and the detector temperature was 300 °C.Free amino acids were analyzed by a Biochrom 30 amino acid analyzer . The extraction and analysis method was according to Song with modification [Heterocyclic aromatic amines (HAAs) were determined by LC-MS based on the literature . Oil samv/v). All extracts were transferred to the MCX column for enrichment and purification. Then it was washed with 10 mL water, 10 mL methanol and 10 mL methanol/ammonia/water  mixed solution. Finally, 10 mL methanol/ammonia  solution was used for elution. The eluent was collected and evaporated to dryness under nitrogen. The residue was dissolved in 10 mL 5% formic acid/acetonitrile  mixture and filtered through a 0.45 μm microporous filter for LC/MS analysis.A solid phase extraction column cartridge Oasis MCX (150 mg/6 mL) was flushed in advance with 10 mL of methanol and 10 mL 0.1 mol/L HCl–methanol mixed solution  was used to analyze the HAAs of the sample extract. Chromatographic separation was performed on an Agilent ZORBAX Eclipse XDB-C18 column  maintained at 35 °C. The gradient elution was achieved with a binary mobile phase of 5% formic acid/5 mM ammonium formate aqueous solution (A) and 5% formic acid/5mM ammonium formate methanol solution (B) at a flow rate of 0.4 mL/min. The gradient elution program was as follows: 0–0.01 min, 5%B; 0.01–1.00 min, 5%B; 1.00–1.10 min, 5–60%B; 1.10–5.00 min, 60–80%B; 5.00–6.00 min, 80–95%B; 6.00–8.00 min, 95%B; 8.00–8.10 min, 95–5%B; 8.10–8.20 min, 5%; 8.20–10.00 min adjust mobile phase balance to initial state. The single injection volume was set at 5 μL.MS analysis was carried out with positive electrospray ionization (ESI+). Multiple reaction monitoring (MRM) conditions were automatically optimized. The capillary voltage was 5.5 kV, and the ion source temperature was 550 °C. The MRM parameters for 14 HAAs and internal standard are summarized in p < 0.05.All experiments were carried out in triplicate, and the mean and standard deviation (SD) for each of the determinations were calculated and reported. Figure preparation was performed using Origin Pro software . The differences between groups were tested by ANOVA and Duncan’s multiple range tests. Means were compared, and they were considered significant when In this work, the levels of β-carbolines (harman and norharman) in different types of sesame seed oils in China, including pressed fragrant sesame seed oil, ground fragrant sesame seed oil, refined sesame seed oil and cold-pressed sesame seed oil, were investigated systematically. The results showed that the levels of the total β-carbolines (harman and norharman) in pressed fragrant sesame seed oils were higher than those in ground fragrant sesame seed oils, and they were much higher than those in cold-pressed or refined sesame seed oils. Model roasting sesame seeds at higher temperatures (200–240 °C) for 5–30 min led to higher levels of β-carbolines harman and norharman (660~2400 μg/kg) in fragrant sesame seed oils. The loss of tryptophan might be attributed to the formation of β-carbolines (harman and norharman) in sesame seeds during the roasting process at higher temperature. This study will be meaningful to prove a correlation between the contents of β-carbolines in fragrant sesame seed oils and the roasting processes of sesame seeds, and whether fragrant sesame seed oils will be a dietary supplement of β-carbolines (harman and norharman).


In addition, XYW treatment dramatically reversed the reduced activity of superoxide dismutase and the decreased level of glutathione, while also lowering levels of malondialdehyde, an inflammatory mediator (nitric oxide), and pro-inflammatory cytokines (interleukin-6 and 1β) in the serum and cortex of OB rats. Mechanistically, XYW induced marked upregulation of mRNA and protein expression levels of NFE2L2, KEAP1, GPX3, HMOX1, SOD1, NQO1, OGG1, PIK3CA, p-AKT1/AKT1, NTRK2, and BDNF, and downregulation of ROS in the cortex and hippocampus via the activation of the NFE2L2/KEAP1, PIK3CA/AKT1, and NTRK2/BDNF pathways. These findings suggest that XYW exert antidepressant-like effects in OB rats with depression-like symptoms, and these effects are mediated by the alleviation of oxidative stress and the enhancement of neuroprotective effects through the activation of the PIK3CA-AKT1-NFE2L2/BDNF signaling pathways.Numerous studies have revealed that oxidative stress is closely associated with the occurrence and development of depression. Xiaoyao Pills (XYW) are included in the Chinese Pharmacopoeia and are frequently used for treating anxiety and depression by smoothing the liver, strengthening the spleen, and nourishing the blood. However, the antidepressant effects of XYW have not yet been thoroughly investigated. The objective of our study was to investigate the antidepressant-like effects of XYW and the underlying molecular mechanism in the olfactory bulbectomized (OB) rat model of depression using the open field test (OFT), sucrose preference test (SPT), splash test (ST), and novelty suppressed feeding test (NSFT). Results showed that XYW (0.93 and 1.86 g·kg Depression is one of the most common mental illnesses and is characterized by low mood, sleep disturbances, loss of energy, change in appetite, constipation, decreased concentration, and risk of suicide . It has Bupleurum chinense DC., Angelica sinensis Diels., Paeonia lactiflora Pall., Atractylodes macrocephala Koidz., Mentha haplocalyx Briq., Poria cocos Wolf., Glycyrrhiza uralensis Fisch., Zingiber officinale Rosc. in ratios of 9:9:9:9:1.5:9:4.5:9, respectively). Because it is included in the qi stagnation, blood stasis, and a deficiency of the spleen-qi  are commonly used as complementary and alternative therapies for the treatment of depression. Recently, numerous studies have demonstrated that TCMs are effective in relieving symptoms of depression in both patients and animal models via various complex mechanisms . Xiaoyaopleen-qi . Xiaoyaopleen-qi , improvipleen-qi , promotipleen-qi , reversipleen-qi , and redpleen-qi , 2015. AThe NFE2L2/Kelch-like ECH associated protein-1 (KEAP1) pathway is a major regulator of redox homeostasis . NFE2L2 23H28O11). According to previous literature (23H28O11), liquiritin (C21H22O9), saikosaponin B2 (C42H68O13) and atractylenolide Ⅱ (C15H20O2).Xiaoyao Pills is composed of eight Chinese herbal medicines with the characteristics of complex composition. However, the Chinese Pharmacopoeia only provides content determination for paeoniflorin  was used and the chromatographic separation conditions were as follows: mobile phase: 0.05% (V/V) phosphoric acid (A) + acetonitrile (B) ; detection wavelength: 230 nm , 210 nm , 210 nm , 230 nm ; column temperature: 30°C; flow rate: 1.0 ml·min−1; injection volume: 10 μL. Stock solutions of XYW was prepared by dissolving 1.0 g of analyte in 100 ml dilute methanol. The content of paeoniflorin (C23H28O11), liquiritin (C21H22O9), saikosaponin B2 (C42H68O13) and atractylenolide II (C15H20O2) in XYW was determined.The analysis was performed by high-performance liquid chromatography (HPLC) . Hypersil GOLDThe XYW  and fluoxetine hydrochloride (FLX)  were dissolved in pure water solution preparation , malondialdehyde  , and GSH/glutathione disulfide  levels were assessed using commercially available kits . Nitric oxide (NO) assay kit . Rat interleukin 6 (IL6) ELISA Kit , rat interleukin 1β (IL1B) ELISA Kit , rabbit anti-NTRK2 antibody , rabbit anti-AKT1 antibody , rabbit anti-p-AKT1 antibody , rabbit anti-HMOX1 antibody , rabbit anti-PIK3CA antibody , rabbit anti-GAPDH antibody , rabbit anti-SOD1 antibody , rabbit anti-NFE2L2 antibody , rabbit anti-brain-derived neurotrophic factor (BDNF) antibody  and anti-rabbit IgG HRP-linked antibody .Male Sprague-Dawley rats weighing 180–200 g were purchased from Chengdu Dashuo Biotechnology Company . Before commencing the experiment, the animals were randomly housed in cages at room temperature (25 ± 2°C) with a 12-h light/dark cycle. They had full access to food and water and were acclimatized to the environment for 3 days. The experiments were conducted in accordance with the guidelines of the Committee for Animal Care and Use of Laboratory Animals, College of Pharmacy, Chengdu University of Traditional Chinese Medicine .Olfactory bulbectomy was performed according to previously used methods  but with minor modifications. Briefly, rats were anesthetized, and the skull covering the olfactory bulbs was exposed using a midline incision. Then, 2 mm burr holes were drilled 8 mm anterior to the bregma and 2 mm lateral to the midline. The top of the rats’ heads was shaved and fixed to a brain stereotaxic instrument. The incisor bar was set at 4.5 mm below the interaural line. Both olfactory bulbs were obliterated and aspirated, the holes were filled with glass-ionomer cement, and the scalp was subsequently sutured. Sham-operated rats underwent the same procedure but without the bulb obliteration/aspiration. The animals received daily intramuscular injections of penicillin  for 3 days post-surgery to prevent infection and were subsequently housed individually in polypropylene cages. The experiments were continued after 15 days of recovery, after which the OB rats were selected using the open field test. The experimental procedure is illustrated in −1) were intragastrically administered XYW daily for 30 days after the 15-days recovery from surgery. Animals in the FLX group (0.01 g·kg−1) were intragastrically administered FLX daily for 30 days after the 15-days recovery from surgery. Animals in the sham and OB groups received equivalent volumes of water daily to ensure isocaloric intake.Animals in the XYW group  = sucrose intake/(sucrose intake + water intake) × 100%The splash test was performed in a standard rat cage with no bedding. The rats were sprayed with a 2 ml 10% sucrose solution on the dorsal while in their home cage to induce grooming behavior. We recorded the total grooming time during a 5-min period .The rats were deprived of food for 48 h before testing. For the test, the rats were placed in an open field (60 × 60 × 20 cm) that had a small amount of food placed at the center. The time taken for the rat to bite the food was recorded as the feeding latency where rats were given up to 5 min .After the behavioral tests were completed, abdominal aorta bleeds were performed, and plasma was collected by centrifugation at 3,000 rpm for 15 min. Both the hippocampus and cortex tissues were immediately dissected on an ice-plate and frozen with liquid nitrogen. All tissue samples were then stored at −80°C.The levels of SOD, MDA, GSH, and GSH/GSSG in the serum and cortex were assessed using commercially available kits according to manufacturer instructions. Briefly, the cortex tissue was homogenized with 0.9% saline in an ice-bath and centrifuged . The supernatant and serum were applied to estimate the antioxidant content.The levels of NO in the serum were determined using a commercially available NO assay kit according to manufacturer instructions. The optical density was measured at 540 nm using a micro-plate reader. The levels of IL6 and IL1B in the serum and cortex were determined using commercially available ELISA kits according to manufacturer instructions. Briefly, the cortex tissue was homogenized with 0.9% saline in an ice-bath and centrifuged . The supernatant and serum were applied to estimate the IL6 and IL1B content. Optical density was measured at 450 nm using a micro-plate reader.−ΔΔCt method.Total RNA was used to synthesize complementary DNA using a FastQuant RT kit . The amplification reactions were performed in 96-well reaction plates with a 20 μL reaction volume . The gene primer sequences for Actb, Nfe2l2, Keap1, Gpx3, Hmox1, Nqo1, and Ogg1 used in this study are listed in Ak; Cell Signaling Technology; Cat. No. #4060S), rabbit anti-HMOX1 , rabbit anti- PIK3CA , rabbit anti-GAPDH , rabbit anti-SOD1 , rabbit anti-NFE2L2 , and rabbit anti-BDNF  antibodies overnight at 4°C and then incubated with anti-rabbit IgG HRP-linked antibody  at 37°C for 1.5 h. Detection was performed using a ChemiDoc XRS+  image analysis system.RIPA lysate buffer containing 1 mM phenylmethanesulfonyl fluoride and 1 mM phosphatase inhibitor cocktail was added to each sample to collect the total protein. The total protein concentration of each sample was determined using the bicinchoninic acid assay method, and all samples were adjusted to have the same concentration. The prtein samples were mixed with a 5× loading buffer and denatured at 95°C. The proteins were separated using sodium dodecylsulfonate-polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene fluoride membranes. The membranes were probed with rabbit anti-NTRK2 , rabbit anti-AKT1 , rabbit anti-p-AKT1 , and the tissue sections were subsequently rewarmed. The tissue slides were incubated for 30 min at 37°C in the dark with DHE  stain, which was diluted with phosphate-buffered saline. The slides were placed on a shaker and washed three times for 5 min each time. After drying the slide slightly, the DAPI dye solution was added dropwise, and the core was stained for 10 min at room temperature. The sections were observed under a fluorescence microscope , and the images were captured. The nucleus was stained blue, and the level of ROS in the cells was judged based on the intensity of red fluorescence. Image acquisition was performed at ×200 magnification. The relative expression of ROS was calculated according to the relative cumulative optical density (IOD), which was calculated by the formula: IOD = ROS cumulative optical density/nuclear stain cumulative optical density.t-test. Differences were considered statistically significant at p < 0.05.All statistical analyses were performed using SPSS 21 . Data are presented as means ± standard deviations. All analyses were performed using a one-way analysis of variance (SNK was used for pairwise between group comparisons) or According to National Pharmacopoeia Committee (2020 edition), the content of paeoniflorin should not be less than 4.0 mg in 1.0 g of condensed pills. The characteristic map (chromatogram) of Xiaoyao Pills is shown in p < 0.05) and a significant decrease in grooming time in the ST (p < 0.001) compared with the sham group, whereas the FLX and XYW (0.93 and 1.86 g·kg−1) treatment groups showed a dramatic reversal of depression-like behaviors .On day 15, OB rats showed a significant increase in horizontal movements in the OFT (p < 0.001) and feeding latency in the NSFT (p < 0.05), whereas grooming time in the ST (p < 0.001) and sucrose preference in the SPT (p < 0.05) decreased, which suggested that olfactory bulbectomy induced continuous depression-like behaviors. However, FLX treatment significantly alleviated the depression-like behavior of OB rats in the OFT only (p < 0.05), whereas XYW (0.93 and 1.86 g·kg−1) treatment significantly reversed the depression-like behaviors in the OFT, ST, NSFT, and SPT . Moreover, the trend of weight gain in OB rats was less than that in the sham rats, whereas the drugs restored that (−1) can significantly improve the depression-like behaviors of rat caused by olfactory bulbectomy, suggesting that it has anti-depression-like effects.In contrast with the sham group, on day 30, OB rats showed a significant increase in both horizontal movements in the OFT (red that . The behp < 0.05), whereas the ratio of GSSG/GSH was significantly higher (p < 0.05); these were significantly alleviated by treatment with FLX and XYW (0.93 and 1.86 g·kg−1). Similarly, the OB rats exhibited lower GSH activity (p < 0.05) and higher MDA (p < 0.05) levels in the cortex (p < 0.05) and XYW  effectively reduced MDA levels without reversing the levels of GSH. Our examination of the production of cytokines to detect whether further inflammation occurred due to oxidative stress revealed significant elevation in the levels of NO, IL6, and IL1B in both the serum and cortex of OB rats. Moreover, treated rats also showed alleviation and enhanced expression of the inflammatory mediator, NO, and proinflammatory cytokines, IL6 and IL1B  administration can restore the damage caused by central and peripheral oxidative stress and minimize further inflammation, which demonstrates the neuroprotective effect of XYW.Taken together, these results confirm that XYW (0.93 and 1.86 g·kgp < 0.01). Similarly, XYW (1.86 g·kg−1) and FLX treated rats showed distinctly less red fluorescence in the cortex and hippocampal CA3 and DG regions (p < 0.01). Our results further indicate that high doses of XYW eliminate ROS to reduce the oxidative stress level in the cortex and hippocampus of OB rats.An increased ROS level is an important indicator of oxidative stress. To visually observe oxidative stress levels, we conducted immunofluorescence detection of ROS in the cortex and CA3 and DG regions of the hippocampus. A DHE dye solution was used to tag superoxide anion  were significantly downregulated (p < 0.05) in the cortex of OB rats. Consistent with previous findings, treatment with FLX and XYW (0.93 and 1.86·g kg−1) showed marked antioxidant effects. FLX treatment significantly restored transcription of Nfe2l2, Hmox1, Nqo1 and Gpx3 mRNA (p < 0.05), and XYW (0.93 and 1.86 g·kg−1) treatment restored transcription of Nfe2l2, Keap1, Hmox-1, Nqo1 and CPX3 mRNA (p < 0.05). In the hippocampus of OB rats (p < 0.05), whereas the downregulated Hmox1 and Nfe2l2 mRNA were restored by XYW (0.93 and 1.86 g·kg−1) treatment (p < 0.05). Furthermore, Ogg1 (−1) treatment (p < 0.05).As shown in  OB rats , there wre, Ogg1 , a specip < 0.05), whereas FLX and XYW (0.93 and 1.86 g·kg−1) treatments accelerated the production of HMOX1 and SOD1  treatment; however, this was not statistically significant. These findings suggest that during states of excessive oxidative stress, the NFE2L2/KEAP1 pathway is suppressed, so that downstream antioxidants, such as HMOX1, NQO1, GPX3 and SOD1 are reduced, which heightens the risk for DNA damage. Moreover, XYW treatments reverse the downregulated NFE2L2/KEAP1 pathway to produce central antioxidant effects.In terms of protein level, HMOX1 and SOD1 were significantly reduced in the cortex and hippocampus of OB rats (p < 0.05), whereas the expression of AKT1 showed a decreasing trend. Similarly, PIK3CA in the hippocampus of OB rats showed a decreasing trend and AKT1 was significantly lower (p < 0.05) compared with the sham rats (−1) improved the protein levels of PIK3CA and AKT1 to some extent . FLX and XYW (0.93 and 1.86 g·kg−1) treatment dramatically restored the reduction in protein expression of NTRK2 and BDNF (p < 0.05), which demonstrated the neuroprotective effects of the treatments. Our results suggest that oxidative stress damage caused by olfactory bulbectomy is accompanied by the suppression of the NTRK2/BDNF pathway, and XYW is able to reverse this effect to promote the expression of BDNF and provide neuroprotection.BDNF has been well established as a specific indicator of depression-related diseases. Furthermore, the production of BDNF is closely related to the activation of the PIK3CA/AKT1 and CREB1/BDNF pathways . The actDepression is a common chronic disease, which seriously affects the physical health and quality of life of patients. XYW, a clinically prescribed medication, has been shown to induce specific antidepressant effects; however, the underlying mechanism remains unclear. Olfactory bulbectomy is a recognized animal model of depression that induces changes in physiological functions and behaviors in the animal that resemble symptoms commonly observed in patients with depression . Our finPrevious reports have confirmed that neuroinflammation is involved in the pathogenesis of depression and may be a potential therapeutic target for depression . In our in vivo and vitro  continuing to be the most widely used class of antidepressants . However pathway . These t pathway . These bBoth the cortex and hippocampus are two important brain regions that are associated with the pathogenesis of depression. The cortex is a significant nerve center that regulates thinking and behavior, whereas the hippocampus is responsible for memory formation, storage, and emotional adjustment . Recent Overall, we demonstrated that by removing the olfactory bulb, it is possible to induce continuous oxidative stress in the cortex and hippocampus, which results in increased levels of ROS and MDA, reduced activity of SOD, HMOX1, NQO1, GSH, and GPX3, and increased lipid peroxidation. Moreover, we find that the PIK3CA/AKT1, NFE2L2/KEAP1, and NTRK2/BDNF pathways are suppressed, whereas XYW induces the clearance of excessive ROS and enhances the activation of the PIK3CA-AKT1/NFE2L2/BDNF pathway, which results in antidepressant and neuroprotective effects. Although determining the underlying mechanisms requires further investigation, our research highlights that NFE2L2 antioxidant signaling is important in depression and raises the possibility that the Chinese herbal compound, XYW, has promise as an effective treatment for depression.Since there may be a risk of non-specific effects at this dose level, it will be important to further study this extract at lower dose levels.


Understanding the effects induced by carcinogens on primary colonic epithelial cells and how to counteract them might help to prevent colon cancer, which is one of the most frequent and aggressive cancers. In this study, we exposed primary human colonic epithelial cells (HCoEpC) to Benzo[a]pyrene (B[a]P) and found that it led to an increased production of pro-inflammatory cytokines and activated ERK1/2 and mTOR. These pathways are known to be involved in inflammatory bowel disease (IBD), which represents a colon cancer risk factor. Moreover, B[a]P reduced autophagy and mitophagy, processes whose dysregulation has been clearly demonstrated to predispose to cancer either by in vitro or in vivo studies. Interestingly, all the effects induced by B[a]P could be counteracted by 3,4-Dihydroxyphenylethanol , the most powerful anti-inflammatory and antioxidant compound contained in olive oil. This study sheds light on the mechanisms that could be involved in colon carcinogenesis induced by a chemical carcinogen and identifies a safe natural product that may help to prevent them. It has been now clearly ascertained that chronic inflammation represents a risk factor for the development of cancer, as about 20% of all human malignancies seem to be linked to chronic inflammatory conditions . RegardiThe exposure to environmental and food-borne mutagens plays an important role in colon cancer development . Benzoa. B[a]P, 2 incubator. In 2D experiments, HCoEpC were seeded at a density of 5 × 104 cells/well in 6-well plate and treated with 5 µM Benzo[a]pyrene (B[a]P) . Depending on the experiment:−Cells were grown for 96 h and 6 days, adding Benzo[a]pyrene every other day;−Pre-treatments with 1 µM Hydroxytyrosol (H)  were performed for 45 min before adding B[a]P; −HCoEpC were incubated with 1 µM UC2288 , a p21 inhibitor, for 6 days, refreshing medium supplemented with the inhibitor every other day;−In order to evaluate autophagy, cells treated or not (CT) with Benzo[a]pyrene for 6 days were incubated or not with 20 nM Bafilomycin A1  for the last 4 h.Human colonic epithelial cells (HCoEpC) were purchased from iXCells Biotechnologies. HCoEpC were cultured in Epithelial Cell Growth Medium  at 37 °C in a 5% CO3 cells/well in BIOFLOATTM 96-well plates provided by faCellitate. After seeding, plates were centrifuged at 500× g for 5 min and, subsequently, maintained at 37 °C in a 5% CO2 incubator to allow spheroid growth. Then, spheroids were tested under the same experimental conditions of 2D cultures.HCoEpC 3D cultures were established by seeding 3 × 10DMSO was added as vehicle in untreated control cells, in all the experiments.3 cells/well were seeded in 96-well plates and treated with 5, 10 and 20 µM B[a]P for 96 h and 6 days; in the presence or absence of 1 µM UC2288 for 6 days; 1 µM Hydroxytyrosol in the presence or absence of 5µM B[a]P. Cell viability was assayed using a cell proliferation kit, MTT, (Roche), following the manufacturer’s instructions. Absorbance was evaluated by Absorbance 96 reader . To assess cell viability, 1 × 10Supernatants from HCoEpC, untreated (CT) and treated with Benzo[a]pyrene and/or Hydroxytyrosol for 6 days, were collected and Interleukin-6 (IL-6), Interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), CXCL13, CCL2 and Cathepsin S were measured by Magnetic Luminex assay using a human premixed multi-analyte kit , following the manufacturer’s instructions. Monocytes isolated from human peripheral blood mononuclear cells (PBMCs) of healthy donors, as previously described , were cu6 macrophages were cultured in a 24-well plate with (25% vol) supernatants from HCoEpC treated with B[a]P or not (CT). Supernatants were concentrated by centrifuge using Microcon-10 kDa Centrifugal Filters (Millipore) according to the manufacturer’s instructions, subsequently resuspended in RPMI and then added to macrophages cultures. After 24 h, cells were washed and incubated for 30 min at 4 °C with CD86 antibody . Cells were analyzed with FACSCalibur, using CELLQuest software . Cells were gated according to their forward scatter and side scatter properties. At least 10 × 103 events were acquired for each sample.For immunofluorescence, 1 × 10TM 4–12% Bis-Tris Plus, Invitrogen, Waltham, MA, USA) and transferred to a nitrocellulose membrane (Amersham TM). Membranes were incubated for 30 min in a blocking solution  and then with primary antibodies for 1 h at room temperature or overnight at 4 °C. Membranes were washed three times in PBS Tween-20 0.1% and then incubated for 30 min with the proper secondary antibody conjugated to horseradish peroxidase. Then, membranes were washed three times in PBS Tween-20 0.1% and protein detection was performed through a chemiluminescence kit WesternBright ECL . Densitometric analysis was carried out using ImageJ software (https://imagej.nih.gov/ij/ (accessed on 13 January 2022)).Cells in 2D cultures were detached with Accutase  for 10 min at 37 °C in cell incubator, while spheroids in 3D cultures were harvested and then washed twice in PBS. Cell pellets were lysed in ice in RIPA buffer  containing protease and phosphatase inhibitors. A total of 10 µg of proteins for each sample was loaded on a precast polyacrylamide gel  1:500; rabbit polyclonal anti-SQSTM1/p62  1:4000; mouse monoclonal anti-phosphoERK1/2  1:500; rabbit polyclonal anti-ERK1 and anti-ERK2  1:500; rabbit polyclonal anti-p21  1:800; rabbit polyclonal anti-LC3 I/II  1:1000; rabbit monoclonal anti-phospho4EBP1  1:500; mouse monoclonal anti-4EBP1  1:500; mouse monoclonal anti-β Actina  1:10,000.Goat polyclonal anti-mouse 1:15,000  and anti-rabbit 1:15,000  were used as secondary antibodies.4 HCOEPC were seeded on cover glasses in 24-well plate and treated with Benzo[a]pyrene (5 µM) and/or Hydroxytyrosol (1 µM) for 6 days. Then, cells were washed in PBS and fixed in Paraformaldehyde 2%  for 30 min. After washing in PBS, cells were permeabilised with Triton-X 100 0.15% for 5 min, washed in PBS and then incubated with BSA 3%-Glycine 1% (in PBS) for additional 30 min. Subsequently, cells were incubated with rabbit polyclonal anti-SQSTM1/p62  primary antibody for 1 h at room temperature and then washed in PBS. Next, cells were incubated with Fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit  for 30 min and, following several washes in PBS, nuclei were stained with DAPI for 1 min. Subsequently, cover glasses were washed in PBS and mounted with PBS:Glycerol (1:1) on microscope slides. Finally, analysis was performed with Olympus BX53  fluorescence microscope at 20× magnification.To perform immunofluorescence, 2 × 10TM Green-FM  fluorescent dye (200 nM) to cell culture medium for the last 30 min at 37 °C. Then, cells were washed in PBS and cover glasses mounted with PBS:Glycerol (1:1) on microscope slides. Cells were analyzed with Apotome Axio Observer Z1 inverted microscope  equipped with an AxioCam MRM Rev.3 (Germany) at 40× magnification.HCoEpC were grown on cover glasses, treated with Benzo[a]pyrene (5 µM) and/or Hydroxytyrosol (1 µM) for 6 days, and mitochondrial labelling was performed by adding MitoTrackert-test. Differences were considered significant if p-value was < 0.05 (*) and <0.01 (**).Results are shown as the mean ± standard deviation (SD) of three independent experiments. Statistical analysis was performed by Student’s The toxicity of B[a]P towards primary human colonic epithelial cells (HCoEpC) was first investigated. Indeed, although previous papers showed that this compound is cytotoxic to several cancer cell lines, primary cells ,25 and iFor this purpose, we treated HCoEpC with different B[a]P concentrations (5–20 μM) for up to 6 days and evaluated cell viability by MTT assay. We observed a dose- and time-dependent reduction in cell survival compared to the untreated control A. Based Among the pro-inflammatory pathways activated in IBD and playing a key role in the control of cytokine release, there are ERK1/2 and PI3K/AKT/mTOR. Therefore, we evaluated the phosphorylation of ERK1/2 and 4EBP1 mTOR target, in HCoEpC exposed or not to 5 µM B[a]P for 96 h and 6 days. As shown in Hydroxytyrosol (H), also known as DPE, is a polyphenol contained in the olive oil that has been shown by ours  and otheNext, given the antioxidant and anti-inflammatory properties of Hydroxytyrosol, here we evaluated the possibility to counteract the effects induced by B[a]P on HCoEpC by using this compound. We found that Hydroxytyrosol was able to reduce both the constitutive cytokine and chemokine release and the one induced by B[a]P, with the exception of CCL2 C. MoreovThe reduction in bulk and selective autophagy, particularly mitophagy, has been demonstrated to facilitate cancer onset, mostly due to the accumulation of toxic materials and ROS . MoreoveThus, these data indicate that Hydroxytyrosol could also efficiently revert these pro-tumorigenic effects induced by B[a]P.Colon cancer represents a leading cause of death in industrialized countries, due to its high incidence and mortality , as in tA link between diet and colon cancer development could be also represented by inflammation ,42, a reAnother process whose dysregulation has been shown to play a role in promoting cancer onset , includiHere, we found that all of the above-described pro-carcinogenic effects were induced by B[a]P in primary human colonic epithelial cells. Indeed, B[a]P increased the production of pro-inflammatory cytokines, sustained the activation of pro-inflammatory pathways and impaired the autophagic and mitophagic flux. Of note, we revealed that the inflammatory cytokines were able to activate macrophages that, in turn, could amplify inflammation, resulting in a tumor-promoting microenvironment that could exacerbate the risk of cancer development ,49. AmonInterestingly, in this study we found that the pro-tumorigenic effects induced by B[a]P could be efficiently counteracted by pre-treatment with Hydroxytyrosol , suggesting that this natural compound, contained in olive oil, could play an important role in preventing tumorigenesis by chemical carcinogens such as B[a]P. Interestingly, as B[a]P and olive oil are contained in food and can both come into direct contact with colonic epithelial cells, it is important to evaluate their effects, either as single agents or in combination, on normal primary epithelial cells. DPE has been demonstrated by our laboratory to be toxic against colon cancer cells and safe for normal colonic epithelial cells ; therefoAlthough the ability of olive oil in preventing colon carcinogenesis mediated by B[a]P has been previously demonstrated in vivo ,54, the


An elevated atmospheric CO2 concentration, 1000 ppm CO2, significantly increased the accumulation of deoxynivalenol in infected plants. Furthermore, growth in cool growing conditions, 20 °C/18 °C, day and night, respectively, resulted in the highest amounts of pathogen biomass and toxin accumulation in both inoculated wheat and corn. Warm temperatures, 25 °C/23 °C, day and night, respectively, suppressed pathogen growth and toxin accumulation, with reductions as great as 99% in corn. In wheat, despite reduced pathogen biomass and toxin accumulation at warm temperatures, the fungal pathogen was more aggressive with greater disease damage and toxin production per unit biomass. Disease outcomes were also pathogen strain specific, with complex interactions between host, strain, and growth conditions. However, we found that atmospheric CO2 and temperature had essentially no significant interactions, except for greatly increased deoxynivalenol accumulation in corn at cool temperatures and elevated CO2. Plants were most susceptible to disease damage at warm and cold temperatures for wheat and corn, respectively. This work helps elucidate the complex interaction between the abiotic stresses and biotic susceptibility of wheat and corn to Fusarium graminearum infection to better understand the potential impact global climate change poses to future food security.This work details the impact of atmospheric CO Fusarium graminearum (F. graminearum) is a devastating mycotoxigenic fungal pathogen that can cause disease in cereal crops such as wheat and corn , reduced F. graminearum growth but caused strain specific increases in mycotoxin production [2,F. graminearum radial growth was inhibited on artificial media [2 on F. graminearum aggressiveness, disease severity, and mycotoxin accumulation was shown to be dependent on both the interacting F. graminearum strain and wheat host variety [2 because they have different photosynthetic systems. Warmer temperatures and elevated CO2 have a greater effect on C3 photosynthetic crops, such as wheat, than C4 photosynthetic crops, such as corn [2 due to photorespiration [2 within the bundle sheath at the primary site of C4 photosynthesis, effectively eliminating photorespiration regardless of atmospheric temperature and CO2 concentration [2, while corn does not. Thus, altering the pathogen host/growth medium.The effects of abiotic variable treatments on disease and mycotoxin accumulation are likely influenced by the medium/host. Changes in wheat nutritional content, due to growth at elevated [COoduction . At eleval media , but conal media . Additio variety ,19,20. F as corn . Unlike piration , C4 planntration ,24. ThusF. graminearum disease and mycotoxin contamination potential. To test this hypothesis, we compared disease development and DON contamination in a full-factorial experimental design using two F. graminearum strains  and 1000 ppm (elevated)), and two temperature treatments (20 °C/18 °C (cool) and 25 °C/23 °C (warm)) temperature conditions.Therefore, we hypothesized that it would be essential to include diverse interacting organisms and abiotic factors to fully understand the combined effects on the outcome of Triticum aestivum) cultivar developed in collaboration with the National Aeronautics and Space Administration (NASA) for growth in space, but has since also been established as a model wheat cultivar for FHB studies [Zea mays) variety originating from Canadian landrace which is adapted to cooler temperatures and has been used experimentally to evaluated Fusarium ear rot [F. graminearum infection in wheat and corn will help elucidate the potential impact global climate change poses to future food security.Furthermore, we independently conducted these comparisons in two different hosts (wheat and corn). To accommodate the size of the experiment and utilize the same temperature treatments for both crops, we used two model varieties. Apogee is a full-dwarf hard red spring wheat  × 2 (strain: 13MN1-6 and 12SD6-2) full-factorial analysis of variance (ANOVA), significant contributing factors to differences in the area under the disease progression curve (AUDPC) in wheat included strain, temperature, and the interaction between strain and CO2. Visual disease progression was greater for the NA2 (12SD6-2) strain than the NA1 (13MN1-6) strain . Additionally, elevated CO2 significantly increased disease progression for the NA2 strain, but not the NA1 strain (p = 0.04). Interestingly, quantitative polymerase chain reaction (PCR) estimates of relative F. graminearum DNA to wheat DNA (designated as Fg/Ta relative biomass or Fg biomass) did not correspond with visual disease. Only temperature was a significant contributing factor, and F. graminearum biomass was 2 to 3.5-fold higher at the cool temperature treatment (20 °C/18 °C) in comparison to the warm treatment (p < 0.0001) (2 did not affect F. graminearum biomass accumulation in wheat.According to the 2 (temperature: 20 °C/18 °C and 25 °C/23 °C) × 2 (CO 0.0001) b. Elevat2, and the interaction between strain and temperature (p < 0.0001). However, this strain-specific difference was temperature dependent and although on average the NA2 strain resulted in more DON contamination, the difference was only significantly different at the cool temperature treatment (p = 0.03). The warm temperature treatment resulted in significantly less DON contamination (p < 0.0001), and elevated CO2 caused significantly greater DON contamination in inoculated wheat . Consistent with DON contamination levels, the amount of DON produced per unit fungal biomass for the NA2 strain was 40% more than the NA1 strain (p = 0.0092). However, both strains had greater DON per unit biomass at the warm temperature treatment (p = 0.0002). There was no significant impact of elevated CO2 on DON production per unit biomass for either of the strains in infected wheat.Differences in wheat DON contamination levels were significantly affected by strain, temperature, COperature a. Overalp < 0.01). Furthermore, the NA1 strain caused 22% more disease compared to the NA2 strain (p < 0.01), which was the inverse of observations in wheat.Strain and temperature significantly contributed to visual disease symptoms in corn according to the full-factorial ANOVA a. Unlikep = 0.0001). F. graminearum biomass was also greater at elevated CO2 (p = 0.036), but the effect was dependent on the strain. While the biomass of the NA2 strain was unaffected by elevated CO2, the NA1 fungal biomass nearly doubled at elevated CO2 (p = 0.0005), regardless of temperature treatment. As with disease symptoms, the amount of F. graminearum biomass was 46% less in corn at the warm temperature treatment in comparison to the cool temperature, and the effect of temperature was significantly more severe for the NA1 strain (p = 0.0001).Overall, the relative NA1 biomass was also significantly greater than the NA2 biomass , at the cooler temperature treatment (p < 0.0001), and at elevated CO2 (p = 0.02). The highest amount of DON was in corn at the cool temperature treatment, particularly with the NA1 strain, which produced significantly more DON than the NA2 strain (p < 0.0001). The difference in DON contamination of corn between the temperature treatments was the most dramatic, with DON content being 99% and 94% less at the warmer treatment for the NA1 and NA2 strain, respectively. At elevated CO2, DON contamination was not significantly impacted at the warm temperature treatment but was approximately 1.4 and 23 times greater in cool temperature treated corn inoculated with the NA1 and NA2 strain, respectively (p = 0.03).Factors contributing to a significant difference in corn DON contamination included strain, temperature, CO and CO2 a. DON coF. graminearum biomass was significantly less in corn at the warm temperature treatment (p = 0.0019). However, neither F. graminearum strain nor CO2 concentration affected DON production per unit biomass in corn.Furthermore, DON production per unit 2 exacerbated Fusarium disease outcomes, are consistent with previous reports. Elevated CO2 was shown to increase disease susceptibility in wheat, resulting in increased disease damage and DON contamination in a host cultivar and pathogen strain dependent manner [2 can also significantly alter plant nutritional content, particularly in C3 photosynthetic crops [F. graminearum growth but increased DON per unit biomass, as previously observed in F. graminearum infected grain from wheat grown at elevated CO2 [2 , two CO2 concentrations (400 ppm and 1000 ppm), and two F. graminearum strains (13MN1-6 and 12SD6-2). The wheat cultivar Apogee, and the corn cultivar Gaspe Flint, were grown in Conviron PGR15 environmentally controlled growth chambers . Apogee and Gaspe Flint both grow well under similar control conditions (between 18–25 °C) allowing for simultaneous growth, and the short stature of the Gaspe Flint cultivar was ideal for the limited vertical space within the growth chamber. Eight wheat seeds, or four corn seeds, were sown in a 20 × 15 cm plastic pot, filled with approximately 4 L of SunGrow Horticulture potting mix . After one week the plants were culled to 5 plants per pot for wheat, and 2 plants per pot for corn. Growth chamber conditions were set to either ambient CO2  or elevated CO2 , with 50 ± 10% relative humidity and a 14 h photoperiod (550 μmol m−2 s−1 photosynthetic photon flux density). Chamber temperatures were set to either 20 °C/18 °C, day and night, or 25 °C/23 °C, day and night, respectively. Plants were watered daily, and pot positions were randomized weekly within the growth chamber. Additionally, a biweekly fertilization supplement, using soluble Peters 20-20-20  was applied until anthesis, or pollination, for wheat and corn, respectively.To evaluate the effects of elevated COF. graminearum isolates, 13MN1-6 and 12SD6-2, representing an NA1 and NA2 strain [5 mL−1 microconidia suspension was produced by the addition of 0.04% Tween 20 in sterile water , and subsequently used for inoculations.Two 2 strain , respect2 strain , but bot2 strain . Media p2 strain . In brien = 12) following previously reported methodology [Apogee was inoculated at flowering, anthesis, with 10 µL of the conidial suspension into single florets  injecting the inoculum into the side of the ear following previously reported methodology [Gaspe Flint corn was inoculated 5 d after manual pollination with 1 mL of the conidial suspension into each cob  in liquid media, in planta the mycotoxin deoxynivalenol (DON) was the overwhelmingly predominant form [Mycotoxins, were extracted from 1 g of ground infected plant tissues, derivatized, and analyzed via GC-MS, on an Agilent 7890 gas chromatograph  fitted with a HP-5MS column  and a 5977 mass detector following previously reported methodology . Though ant form . TherefoF. graminearum DNA to plant host DNA via a quantitative polymerase chain reaction (qPCR), following the previously reported protocols [F. graminearum DNA to host DNA was determined by dividing the geometric mean of initial DNA concentration (N0) from the Fusarium probes by the geometric mean of N0 from the host probes. The amount of DON relative to F. graminearum DNA was estimated by dividing the µg g−1 DON by the relative pathogen biomass, as quantified by qPCR.The relative amount of fungal biomass in the inoculated tissues was assessed using the ratio of rotocols ,45. Four2. Data from the wheat and corn hosts were analyzed separately. Details of factor combinations and significant interactions can be found within the figures.Results were evaluated by a 2 × 2 × 2 full factorial analysis of variance , to determine the significant differences in the disease and mycotoxin contamination of hosts due to the effects of temperature and elevated CO2 was determined to significantly increase the accumulation of the mycotoxin deoxynivalenol in infected plants. Furthermore, infected plants in cool growing conditions had the highest amounts of pathogen biomass and toxin accumulation in both wheat and corn. Warm temperatures suppressed pathogen growth and toxin accumulation, with reductions as great as 99% in corn. In wheat, despite reduced pathogen biomass and toxin accumulation at warm temperatures, F. graminearum was more aggressive with greater disease damage and toxin production per unit biomass. However, we found that atmospheric CO2 and temperature had essentially no significant interactions, except for greatly increased deoxynivalenol accumulation in corn at cool temperatures and elevated CO2. This work helps elucidate the complex interaction between abiotic stresses and biotic susceptibility to Fusarium graminearum infection in wheat and corn to better understand the potential impact global climate change poses to future food security.Elevated CO