Role In Oxidative Stress Response Research Articles

Methionine Restriction Reduces Lung Cancer Progression and Increases Chemotherapy Response.

Targeting tumor metabolism through dietary interventions is an area of growing interest, and may help to improve the significant mortality of aggressive cancers, including non-small cell lung cancer (NSCLC). Here we show that the restriction of methionine in the aggressive KRAS/Lkb1-mutant NSCLC autochthonous mouse model drives decreased tumor progression and increased carboplatin treatment efficacy. Importantly, methionine restriction during early stages of tumorigenesis prevents the lineage switching known to occur in the model, and alters the tumor immune microenvironment (TIME) to have fewer tumor-infiltrating neutrophils. Mechanistically, mutations in LKB1 are linked to anti-oxidant production through changes to cystathionine-β-synthase (CBS) expression. Human cell lines with rescued LKB1 show increased CBS levels and resistance to carboplatin, which can be partially rescued by methionine restriction. Furthermore, LKB1 rescued cells, but not mutant cells, show less G2-M arrest and apoptosis in high methionine conditions. Knock-down of CBS sensitized both LKB1 mutant and non-mutated lines to carboplatin, again rescuing the carboplatin resistance of the LKB1 rescued lines. Given that immunotherapy is commonly combined with chemotherapy for NSCLC, we next wanted to understand if T cells are impaired by MR. Therefore, we examined the ability of T cells from MR and control tumor bearing mice to proliferate in culture and found that T cells from MR treated mice had no defects in proliferation, even though we continued the MR conditions ex vivo. We also identified that CBS is most highly correlated with smoking, adenocarcinomas with alveolar and bronchiolar features, and adenosquamous cell carcinomas, implicating its roles in oxidative stress response and lineage fate in human tumors. Taken together, we have shown the importance of MR as a dietary intervention to slow tumor growth and improve treatment outcomes for NSCLC.

Novel Roles of the Greatwall Kinase Rim15 in Yeast Oxidative Stress Tolerance through Mediating Antioxidant Systems and Transcriptional Regulation.

The Greatwall-family protein kinase Rim15 is associated with the nutrient starvation response, whereas its role in oxidative stress responses remains unclear. Here, acetic acid and peroxide were used as two oxidative stress elicitors. The antioxidant indicator assay under acetic acid stress revealed the impaired growth in rim15Δ related to the regulation of antioxidant systems. Comparative transcriptome analysis revealed that differentially expressed genes (DEGs) are predicted to be mostly regulated by oxidative stress-responsive transcriptional factor Yap1. Among the DEGs, acetic acid stress-induced genes were found, and YAP1 disruption also inhibited their induction. The deletion of Rim15 or the Rim15 kinase domain in yap1Δ did not further decrease the gene expression, suggesting that Rim15 functions together with Yap1 in regulating acetic acid stress-induced genes, which requires Rim15 kinase activity. Additionally, Rim15 regulated H2O2 stress tolerance through partially similar but special mechanisms in that Rim15 kinase activity impacted acetic acid and H2O2 stress tolerance in different degrees, indicating the different mechanisms underlying Rim15-mediated redox regulation against different stressors. These results benefit the better understanding of stress signaling pathways related to Rim15. Given that Rim15 and some of its target genes are conserved across eukaryotes, these results also provide a basis for studies of oxidative stress-related processes in other organisms.

Molecular mechanism of tolerance evolution in Candida tropicalis to inhibitory compounds derived from corn stover hydrolysis

Candida tropicalis is distinguished from Saccharomyces cerevisiae by its great xylose utilization ability, maximizing its capability to ferment both hexoses and xylose to produce high-value products, and its ability to overcome the problem of end-product inhibition derived from lignocellulosic hydrolysis. However, little or no information about the molecular evolution mechanism of C. tropicalis to inhibitory compounds (acetic acid, phenol, and furfural) derived from lignocellulosic hydrolysis is known. In this study, the adaptation of a C. tropicalis strain SHC-03 to corn stover hydrolysate after mutagenesis through stepwise evolution resulted in multi-generation evolved strains after 60 rounds of evolution using the C. tropicalis strain SHC-03 as the parental strain. The evolved strain, generation B-60, (GB-60) showed the highest tolerance (15.81 g/L) and resistance as well as the lowest subcellular damage to inhibitory compounds derived from corn stover hydrolysis. Comparative transcriptome and genomic analyses between the evolved strain GB-60 and the control strain SHC-03 revealed enhanced functional and mutated genes including CTRG_03726, CTRG_01923, CTRG_04945, CTRG_05115, CTRG_03102, CTRG_05127, CTRG_00952 and CTRG_03034 involved in increased cell wall biosynthesis and remodeling, pentose phosphate pathway, metabolic and carbon pathways, detoxification of inhibitory compounds, and the multidrug/multixenobiotic resistance (MDR/MXR) transporters. These genes also played active roles in oxidative stress response, mitochondrial stress response, ER stress response, vacuole stress response and autophagy, DNA repair, and chromatin protection resulting in less ROS accumulation, as well as lower damage to some subcellular organelles in the evolved strain. The information from this study will provide guidelines for the development of engineering strains with high tolerance towards inhibitors derived from corn stover hydrolysis.

A combined transcriptomic and physiological approach to understanding the adaptive mechanisms to cope with oxidative stress in Fusarium graminearum.

In plant-pathogen interactions, oxidative bursts are crucial for plants to defend themselves against pathogen infections. Rapid production and accumulation of reactive oxygen species kill pathogens directly and cause local cell death, preventing pathogens from spreading to adjacent cells. Meanwhile, the pathogens have developed several mechanisms to tolerate oxidative stress and successfully colonize plant tissues. In this study, we investigated the mechanisms responsible for resistance to oxidative stress by analyzing the transcriptomes of six oxidative stress-sensitive strains of the plant pathogenic fungus Fusarium graminearum. Weighted gene co-expression network analysis identified several pathways related to oxidative stress responses, including the DNA repair system, autophagy, and ubiquitin-mediated proteolysis. We also identified hub genes with high intramodular connectivity in key modules and generated deletion or conditional suppression mutants. Phenotypic characterization of those mutants showed that the deletion of FgHGG4, FgHGG10, and FgHGG13 caused sensitivity to oxidative stress, and further investigation on those genes revealed that transcriptional elongation and DNA damage responses play roles in oxidative stress response and pathogenicity. The suppression of FgHGL7 also led to hypersensitivity to oxidative stress, and we demonstrated that FgHGL7 plays a crucial role in heme biosynthesis and is essential for peroxidase activity. This study increases the understanding of the adaptive mechanisms to cope with oxidative stress in plant pathogenic fungi. IMPORTANCE Fungal pathogens have evolved various mechanisms to overcome host-derived stresses for successful infection. Oxidative stress is a representative defense system induced by the host plant, and fungi have complex response systems to cope with it. Fusarium graminearum is one of the devastating plant pathogenic fungi, and understanding its pathosystem is crucial for disease control. In this study, we investigated adaptive mechanisms for coping with oxidative stress at the transcriptome level using oxidative stress-sensitive strains. In addition, by introducing genetic modification technique such as CRISPR-Cas9 and the conditional gene expression system, we identified pathways/genes required for resistance to oxidative stress and also for virulence. Overall, this study advances the understanding of the oxidative stress response and related mechanisms in plant pathogenic fungi.

Signaling pathways of oxidative stress response: the potential therapeutic targets in gastric cancer.

Gastric cancer is one of the top causes of cancer-related death globally. Although novel treatment strategies have been developed, attempts to eradicate gastric cancer have been proven insufficient. Oxidative stress is continually produced and continually present in the human body. Increasing evidences show that oxidative stress contributes significantly to the development of gastric cancer, either through initiation, promotion, and progression of cancer cells or causing cell death. As a result, the purpose of this article is to review the role of oxidative stress response and the subsequent signaling pathways as well as potential oxidative stress-related therapeutic targets in gastric cancer. Understanding the pathophysiology of gastric cancer and developing new therapies for gastric cancer depends on more researches focusing on the potential contributors to oxidative stress and gastric carcinogenesis.

Identification and characterization of a novel SoxB2 gene in Litopenaeus vannamei and its potential roles in response to ammonia-N stress and post-exposure recovery

Increased concentrations of ammonia-N can cause seriously physiological harm to crustaceans, especially farmed shrimp (Litopenaeus vannamei). Ammonia-N is generally removed by water change, and will gradually accumulate for unconsumed feed. However, the underlying adaptation mechanism of shrimp under such environmental conditions is still not fully understood. A novel Sox gene expressed differentially in previous comparative transcriptome studies was characterized in this study, designated as LvSoxB2–1. The LvSoxB2–1 encodes a protein of 420 amino acids, sharing the highest identity with the Chinese mitten crab (Eriocheir sinensis) Sox14b. The expression level of LvSoxB2–1 was higher in fertilized egg and cleavage stage of early embryonic development and declined in the blastula stage, which showed no significant difference compared with the subsequent stage. During larval development, LvSoxB2–1 increased significantly in nauplius stage III and zoea stage I and then restored to the initial level. LvSoxB2–1 was highly expressed in the gills, followed by eyestalks and brain ganglion. Results of subcellular localization have shown that LvSoxB2–1 is a nuclear-localized protein. In the ammonia-N stress and post-exposure recovery experiments, LvSoxB2–1 mRNA significantly increased in the gills, hepatopancreas, and hemocytes. The total hemocyte counts (THC) of shrimp was decreased through LvSoxB2–1 RNA interference (RNAi). Moreover, the inhibition of LvSoxB2–1 significantly exacerbated the hepatopancreas pathology and reduced the survival rates of shrimp exposed to ammonia-N. Dual-luciferase reporter assay confirmed the activation of the Wnt/β-catenin pathway by LvSoxB2–1. Under ammonia-N stress and recovery after exposure, LvSoxB2–1 silencing could decrease the expression of Wnt/β-catenin signaling molecules (β-catenin and Pangolin) and Rh protein in L. vannamei, with the higher malonyldialdehyde (MDA) and protein carbonylation (PCO) levels and hemolymph ammonia-N and urea contents. These results suggested that LvSoxB2–1 played positive roles in oxidative stress responses and ammonia-N excretion through the Wnt/β-catenin pathway.

Transcriptome responses of lactic acid bacteria isolated from kimchi under hydrogen peroxide exposure

In this study, five species of lactic acid bacteria (LAB) isolated from kimchi were analyzed in terms of their potential antioxidant activity. Latilactobacillus curvatus WiKim38, Companilactobacillus allii WiKim39, and Lactococcus lactis WiKim0124 exhibited higher radical scavenging activity, reducing power, and lipid peroxidation inhibition than the reference strain and tolerated hydrogen peroxide (H2O2) exposure up to a concentration of 2.5 mM. To investigate the antioxidant mechanism of LAB strains, transcriptomic and proteomic signatures were compared between the H2O2-exposed and untreated group using RNA sequencing and two-dimensional protein gel electrophoresis. Across all LAB strains, cell membrane responses and metabolic processes were the most prominent in the main categories of gene ontology classification, indicating that cellular components and interactions play an important role in oxidative stress responses. Thus, LAB strains isolated from kimchi could be considered for potential use in functional food production and in antioxidant starter cultures.

Biological Functions of Selenoprotein Glutathione Peroxidases (GPXs) and their Expression in Osteoarthritis.

In order to further study the biological functions of glutathione peroxidases (GPXs) and their expression level in patients with osteoarthritis (OA), we fully explored the potential relationship between GPXs and OA. This will provide new ideas for basic biological studies and therapeutic strategies for OA patients. In this study, bioinformatics techniques were used to explore the biological functions of five GPXs. The core genes related to the biological functions of GPXs were identified by constructing a protein-protein interaction network (PPI). In addition, we utilized microarray data in public databases to analyze the expression levels of GPXs in OA patients and healthy controls. Finally, we used quantitative real-time polymerase chain reaction (qRT-PCR) to detect the expression of GPXs in OA patients and controls to validate our bioinformatic analysis results. Enrichment analysis showed GPXs were mainly enriched in the glutathione metabolic pathway and participate in the biological process of oxidative stress response, and further play an antioxidant role. The PPI network indicated that superoxide dismutase 1 (SOD1), superoxide dismutase 2(SOD2) and catalase (CAT) were the core proteins of this network. GPX1 was regulated by the greatest number of miRNAs. Experiments showed that the expression of GPX1 was elevated in OA patients compared with controls. GPXs play an important antioxidant role in oxidative stress response. The expression of GPX1 was elevated in peripheral blood mononuclear cells (PBMCs) of OA patients. The changes of GPXs in OA patients may regulate the level of oxidative stress, which may influence synovial lesions and chondrocyte apoptosis.

B” and C subunits of PP2A regulate the levels of reactive oxygen species and superoxide dismutase activities in Arabidopsis

The serine-threonine protein phosphatases PP2A regulate many cellular processes, however their role in oxidative stress responses and defence is less known. We show the involvement of its C (catalytic) and B” (a regulatory) subunits. The c3c4 (C subunit) and fass (B”) subunit mutants and Col wt of Arabidopsis were used. Controls and treatments with the PP2A inhibitor microcystin-LR (MCY-LR) and reactive oxygen species (ROS) inducer diquat (DQ) were employed. ROS levels of primary roots were largely genotype dependent and both C and B” subunit mutants had increased sensitivity to MCY-LR and DQ indicating the involvement of these subunits in oxidative stress induction. Superoxide dismutases (SOD), mainly the Cu/Zn-SOD isoform, as key enzymes involved in ROS scavenging are also showing altered (mostly increased) activities in both c3c4 and fass mutants and have opposite relations to ROS induction. This indicates that the two types of subunits involved have partially different regulatory roles. In relation to this, control and MCY-LR/DQ treated B” subunit mutants were proven to have altered levels of phosphorylation of histone H2AX. γH2AX, the phosphorylated form indicates double stranded DNA damage during oxidative stress. Overall we point out the probable pivotal role of several PP2A subunits in the regulation of oxidative stress responses in plants and pave the way for future research to reveal the signaling pathways involved.

A thioredoxin peroxidase protects Pieris rapae from oxidative stress induced by chlorantraniliprole exposure.

Chlorantraniliprole (CAP) is an insecticide widely used to control the small white butterfly (SWB), Pieris rapae. Exposure to CAP can cause oxidative injury in SWB; however, it is unclear if antioxidant enzymes are involved in the defense process. In this study, a thioredoxin peroxidase (PrTPX1) gene was identified from SWB by using a homology search method. The gene encoded a 195 amino-acid PrTPX1 protein. Sequence characteristics and phylogenetic analysis indicated that PrTPX1 was a typical "2-Cys" TPX, and the PrTPX1 gene consisted of four exons and three introns. Reverse transcription-quantitative polymerase chain reaction analysis indicated that the messenger RNA levels of PrTPX1 were highest in third-, fourth- and fifth-instar larval stages and in the larval midgut. Treatment with sublethal doses (LD20 and LD50 ) of CAP for 6, 12, 18, and 24 h resulted in increased H2 O2 concentration in SWB larvae, indicating insecticide-induced oxidative stress. The transcriptional levels of PrTPX1 were significantly enhanced in larvae exposed to CAP. Recombinant PrTPX1 protein was expressed in Escherichia coli. Enzymatic assay revealed that the protein displayed antioxidant activity and was able to protect against oxidative challenge. These results indicated that PrTPX1 plays an important role in oxidative stress responses and may contribute to the CAP tolerance in SWB.

Trehalose biosynthetic gene otsB of Corynebacterium glutamicum is regulated by whcE in response to oxidative stress.

The gene whcE of Corynebacterium glutamicum plays a positive role in oxidative stress responses and the WhcE protein interacts with SpiE. By utilizing 2D-PAGE analysis, we identified the otsB gene to be under the control of whcE. The transcription of otsB, encoding trehalose 6-phosphatase, was stimulated by oxidative stress, and whcE and spiE were involved in diamide-mediated transcriptional stimulation. The ΔotsB strain was created and found to be sensitive to the thiol-specific oxidant diamide, suggesting a role of the gene in stress responses. Genes located upstream of otsB, such as NCgl2534 and otsA, formed an operon and purified WhcE was able to bind to the promoter region of the operon (PNCgl2534), but the binding was only possible in the presence of the oxidant diamide. In addition, the transcriptional activation of PNCgl2534 by WhcE was demonstrated in in vivo assays and the transcription was stimulated in cells exposed to the oxidant diamide. These findings indicate that WhcE is a transcriptional activator, and otsB, which is involved in trehalose biosynthesis, has a role in oxidative stress responses in C. glutamicum.

Melatonin confers enhanced polyamine metabolism and cell tolerance in Vitis vinifera against oxidative damage: Quantitative proteomic evidence

Melatonin (MLT) exhibited pleiotropic effects in multiple plant physiological processes; however, its role in the polyamine metabolism and quality performance of postharvest Vitis vinifera berry remain unknown. In the present study, MLT was applied to postharvest Vitis vinifera cv. Kyoho berry, which delayed berry abscission, berry rotten, and promoted cell tolerance to oxidative stress. Notably, the malondialdehyde content of berries was reduced to 40.8 % on the application of 200 μmol L−1 MLT. Furthermore, tandem mass tags (TMT)-based quantitative proteomic analysis identified 5,156 proteins from berry skin, among which 158 proteins showed quantitatively differences in response to MLT application. These differentially expressed proteins were mainly upregulated in the arginine and proline metabolism pathways, lysine degradation, and ascorbate and aldarate metabolism pathways. The abundance of proteins involved in polyamine metabolisms, including N-carbamoylputrescine amidase, spermidine synthase, and aldehyde dehydrogenases were also upregulated. Moreover, MLT application enhanced contents of arginine, proline, 4-aminobutanoic acid, and polyamines, which played an essential role in oxidative stress response. Further, the transcript level of key enzymes involved in polyamine metabolism, including VvADC, VvODC, VvNAC, VvSPDS and VvCuAO were up-regulated on MLT application. Overall, the role of MLT in regulating the polyamine metabolism and subsequently in cell tolerance against oxidative damage was scientifically established for the first time for Vitis vinifera. The empirical findings of this study may propose new insights on MLT application in postharvest berry physiology and provide theoretical regulation for the preservation of berries, perhaps other fruits.

Small RNAs as a New Platform for Tuning the Biosynthesis of Silver Nanoparticles for Enhanced Material and Functional Properties.

Genetic engineering of nanoparticle biosynthesis in bacteria could help facilitate the production of nanoparticles with enhanced or desired properties. However, this process remains limited due to the lack of mechanistic knowledge regarding specific enzymes and other key biological factors. Herein, we report on the ability of small noncoding RNAs (sRNAs) to affect silver nanoparticle (AgNP) biosynthesis using the supernatant from the bacterium Deinococcus radiodurans. Deletion strains of 12 sRNAs potentially involved in the oxidative stress response were constructed, and the supernatants from these strains were screened for their effect on AgNP biosynthesis. We identified several sRNA deletions that drastically decreased AgNP yield compared to the wild-type (WT) strain, suggesting the importance of these sRNAs in AgNP biosynthesis. Furthermore, AgNPs biosynthesized using the supernatants from three of these sRNA deletion strains demonstrated significantly enhanced antimicrobial and catalytic activities against environmentally relevant dyes and bacteria relative to AgNPs biosynthesized using the WT strain. Characterization of these AgNPs using electron microscopy (EM), energy-dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) revealed that the deletion of these small RNAs led to changes within the supernatant composition that altered AgNP properties such as the surface chemistry, surface potential, and overall composition. Taken together, our results demonstrate that modulating specific sRNA levels can affect the composition of supernatants used to biosynthesize AgNPs, resulting in AgNPs with unique material properties and improved functionality; as such, we introduce sRNAs as a new platform for genetically engineering the biosynthesis of metal nanoparticles using bacteria. Many of the sRNAs examined in this work have potential regulatory roles in oxidative stress responses; further studies into their targets could help provide insight into the specific molecular mechanisms underlying bacterial biosynthesis and metal reduction, enabling the production of nanoparticles with enhanced properties.

The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure.

The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.

Expression and distribution of glutathione transferases in protoscoleces of Echinococcus granulosus sensu lato

Glutathione transferases (GSTs) belong to a diverse superfamily of multifunctional proteins involved in metabolic detoxification. In helminth parasite, GSTs are particularly relevant since they are also involved in host immunomodulation. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least three phylogenetically distant cytosolic GSTs: EgGST1 and EgGST2 previously grouped within Mu and Sigma classes, respectively; and EgGST3 related to both Omega and Sigma classes. To better characterize E. granulosus s.l. GSTs, herein their expression and distribution were assessed in the pre-adult protoscolex (PSC) parasite stage. Potential transcriptional regulatory mechanisms of the corresponding EgGST genes were also explored. Firstly, the transcription of the three EgGSTs was significantly induced during the early stages of the murine model of infection, suggesting a potential role during parasite establishment. EgGST1 was detected in the parenchyma of PSCs and its expression increased after H2O2 exposure, supporting its role in detoxification. EgGST2 was mainly detected on the PSCs tegument, strategically localized for potential immunoregulation functions due to its Sigma-class characteristics. In addition, its expression increased after anthelmintic treatment, suggesting a role in chemotherapy resistance. Finally, the Omega-related EgGST3 was localized throughout the entire PSC body, including suckers and tegument, and since its expression also increased after H2O2 treatment, a potential role in oxidative stress response could also be ascribed. On the other hand, known cis-acting regulatory motifs were detected in EgGST genes, suggesting similar transcription processes to other eukaryotes. The results herein reported provide additional data regarding the roles of EgGSTs in E. granulosus s.l. biology, contributing to a better understanding of its host-parasite interaction.

Tracing the Evolution of Plant Glyoxalase III Enzymes for Structural and Functional Divergence

Glyoxalase pathway is the primary route for metabolism of methylglyoxal (MG), a toxic ubiquitous metabolite that affects redox homeostasis. It neutralizes MG using Glyoxalase I and Glyoxalase II (GLYI and GLYII) enzymes in the presence of reduced glutathione. In addition, there also exists a shorter route for the MG detoxification in the form of Glyoxalase III (GLYIII) enzymes, which can convert MG into D-lactate in a single-step without involving glutathione. GLYIII proteins in different systems demonstrate diverse functional capacities and play a vital role in oxidative stress response. To gain insight into their evolutionary patterns, here we studied the evolution of GLYIII enzymes across prokaryotes and eukaryotes, with special emphasis on plants. GLYIII proteins are characterized by the presence of DJ-1_PfpI domains thereby, belonging to the DJ-1_PfpI protein superfamily. Our analysis delineated evolution of double DJ-1_PfpI domains in plant GLYIII. Based on sequence and structural characteristics, plant GLYIII enzymes could be categorized into three different clusters, which followed different evolutionary trajectories. Importantly, GLYIII proteins from monocots and dicots group separately in each cluster and the each of the two domains of these proteins also cluster differentially. Overall, our findings suggested that GLYIII proteins have undergone significant evolutionary changes in plants, which is likely to confer diversity and flexibility in their functions.

Clade III cytokinin response factors share common roles in response to oxidative stress responses linked to cytokinin synthesis.

Cytokinin response factors (CRFs) are transcription factors that are involved in cytokinin (CK) response, as well as being linked to abiotic stress tolerance. In particular, oxidative stress responses are activated by Clade III CRF members, such as AtCRF6. Here we explored the relationships between Clade III CRFs and oxidative stress. Transcriptomic responses to oxidative stress were determined in two Clade III transcription factors, Arabidopsis AtCRF5 and tomato SlCRF5. AtCRF5 was required for regulated expression of >240 genes that are involved in oxidative stress response. Similarly, SlCRF5 was involved in the regulated expression of nearly 420 oxidative stress response genes. Similarities in gene regulation by these Clade III members in response to oxidative stress were observed between Arabidopsis and tomato, as indicated by Gene Ontology term enrichment. CK levels were also changed in response to oxidative stress in both species. These changes were regulated by Clade III CRFs. Taken together, these findings suggest that Clade III CRFs play a role in oxidative stress response as well as having roles in CK signaling.

Screening and functional identification of antioxidant microRNA-size sRNAs from Spirulina platensis using high-throughput sequencing.

MiRNA-size small RNAs, abbreviated as sRNAs, are increasingly being discovered as research progresses and omics technologies development in prokaryotes. However, there is a paucity of data concerning whether or not sRNAs exist in cyanobacteria and regulate the resistance to oxidative stress. In this investigation, small RNA libraries were constructed from the control, 50-nM and 100-nM H2O2 treatments of Spirulina platensis. By high-throughput sequencing, 23 candidate sRNAs showed significantly differential expression under oxidative stress, among which eight sRNAs were identified with the similar expression patterns as the sequencing results by real-time qPCR. By nucleic acid hybridisation, the corresponding expression changes also demonstrated that sequencing results of sRNAs were feasible and credible. By bioinformatics prediction and structure identification, 43 target genes were predicted for 8 sRNAs in plant miRNA database, among which 29 were annotated into the genome and related metabolic pathways of S. platensis. By COG functional classification and KEGG pathway analysis, 31 target genes were predicted to be directly or indirectly involved in the defence mechanism of H2O2 stress. Thirteen target genes displayed reversely changing patterns compared with those of their sRNAs under H2O2 treatment. These findings provide compelling evidence that these sRNAs in S. platensis play a crucial role in oxidative stress responses, and thus provide a theoretical reference for improving the stress-triggering physiological regulation.

Heme oxygenase 1 plays a crucial role in swamp eel response to oxidative stress induced by cadmium exposure or Aeromonas hydrophila infection.

Oxidative stress contributes a lot to initiation and progression of pathological conditions. Heme oxygenase 1 (HO1), a cytoprotective enzyme, is usually upregulated to alleviate oxidative stress in vivo. The function of teleost HO1 in the response to oxidative stress induced by heavy metal exposure and in pathogenic bacterial infection remains uncertain. In the present study, both complementary DNA and genomic sequence of a HO1-like gene cloned from the liver of swamp eel (Monopterus albus) are reported. Sequence analysis showed that the putative amino acid sequence contained a conserved heme oxygenase signature and displayed higher similarity to HO1 genes of other teleosts. Expression profile of swamp eel HO1 was investigated in healthy tissues and in tissues following stimulation with pathogenic bacteria (Aeromonas hydrophila) or cadmium chloride (CdCl2) exposure. Results demonstrated that HO1 messenger RNA (mRNA) was highly expressed in the liver and relatively less in other tissues. Bacterial infection with A. hydrophila significantly changed HO1 mRNA expression in the liver, spleen, and kidney, and the mRNA expression of HO1 and Nrf2 in the liver was elevated after the fish were exposed to CdCl2. Subsequently, the swamp eel HO1 was subcloned into a pET28a expression vector and transformed into Escherichia coli BL21 (DE3). Recombinant HO1 (rHO1) was successfully induced by 0.1mmol/l IPTG and purified by Ni-NTA His Bind Resin purification system. To determine whether the rHO1 could confer stress tolerance in vitro, the viability of control and HO1-expressing E. coli under CdCl2 stress was compared by spot assay. The rHO1 protein significantly increased survival rates of the bacterial hosts. To evaluate whether intraperitoneal injection with rHO1 protected the liver of swamp eel against A. hydrophila-induced oxidative stress, mRNA expression of HO1, Nrf2, hepcidin, and IL-1β as well as the oxidative stress-related parameters (ROS and total antioxidant capacity (T-AOC)) in the liver were examined. The results showed that exogenous rHO1 could significantly upgrade the mRNA expression of HO1 and hepcidin, coupled with increased ROS and T-AOC levels. However, Nrf2 and IL-1β expression levels were significantly downregulated and upregulated, respectively. These results suggested that HO1 should not only play a protective role in oxidative stress response and its adverse effects deserved further investigation.

Abstract B05: Tumor evolution and oxidative stress in recurrent human papillomavirus-mediated oropharyngeal squamous cell carcinoma

Abstract Background: Human papillomavirus (HPV)-mediated head and neck squamous cell carcinomas (HPVmHNSCC) are more responsive to platinum-based concurrent chemoradiation treatment than non-HPVmHNSCC. Previously, our group demonstrated that recurrent HPVmHNSCC are genomically more characteristic of HPV-negative than non-recurrent HPVmHNSCC. Moreover, we observed a propensity for NFE2L2 hotspot mutations among HPVmHNSCC that recurred. Since NFE2L2 is a transcription factor regulating oxidative stress, we hypothesized that genes involved with the management of oxidative stress would be upregulated in recurrent HPVmHNSCC. Methods: We performed whole-exome sequencing (WES) and RNA-Seq on matched primary and metachronous HPVmHNSCC from the Universities of Pittsburgh (UPitt) and Washington (UW). Somatic nucleotide (SNVs) and copy number variants (CNVs) were quantified. Clonal evolution analyses were performed. Differential expression and gene set enrichment (GSEA) were quantified. Results: RNA-Seq was available for 10 tumor pairs and WES for 7/10. Consistent with prior WES data, KMT2D was the most frequently mutated gene. We found two unique NFE2L2 hotspot mutations in one paired set. The primary tumor had an NFE2L2H150Y mutation while the recurrent tumor had an NFE2L2E79K mutation. Metachronous tumors possessed significantly more deleterious somatic alterations in genes involved with regulation of oxidative stress and NFE2L2 networks compared to the primary tumors. Clonality analyses revealed a propensity for subclonal mutations in oxidative-stress related genes. RNA-Seq analysis revealed differential expression of genes involved in mitochondrial oxidative stress (MAP3K9) and oxidoreductase activity (CYP24A1, HEPHL1). Moreover, 6/8 enriched gene sets among the metachronous recurrent tumors by GSEA were involved with oxidative stress response, mitochondrial genes, and electron transport. Conclusions: Here, using WES and RNA-Seq of pairs of primary and metachronous HPVmHNSCC we identified increased expression of and somatic alterations in oxidative stress regulatory pathways in recurrent HPVmHNSCC that appear to occur later in tumor evolution. One possible explanation for this is that treatment selects for tumor clones that can withstand the oxidative environment induced by chemoradiation. Ultimately, understanding the role of oxidative stress response under therapy may be critical to mitigating recurrence and treating recurrences with a favorable genomic profile. Citation Format: Daniel Faden, Richard A. Harbison, Qing Zhang, Jeffrey Delrow, Umamaheswar Duvvuri. Tumor evolution and oxidative stress in recurrent human papillomavirus-mediated oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B05.