Energy Transfer Between i-Motif DNA Encapsulated Silver Nanoclusters and Fluorescein Amidite Efficiently Visualizes the Redox State of Live Cells.

The redox regulation, maintaining a balance between oxidation and reduction in living cells, is vital for cellular homeostasis, intricate signaling networks, and appropriate responses to physiological and environmental cues. Here, a novel redox sensor, based on DNA-encapsulated silver nanoclusters (DNA/AgNCs) and well-defined chemical fluorophores, effectively illustrating cellular redox states in live cells is introduced. Among various i-motif DNAs, the photophysical property of poly-cytosines (C20)-encapsulated AgNCs that sense reactive oxygen species (ROS) is adopted. However, the sensitivity of C20/AgNCs is insufficient for evaluating ROS levels in live cells. To overcome this drawback, the ROS sensing mechanism of C20/AgNCs through gel electrophoresis, mass spectrometry, and small-angle X-ray scattering is primarily defined. Then, by tethering fluorescein amidite (FAM) and Cyanine 5 (Cy5) dyes to each end of the C20/AgNCs sensor, an Energy Transfer (ET) between AgNCs and FAM is achieved, resulting in intensified green fluorescence upon ROS detection. Taken together, the FAM-C20/AgNCs-Cy5 redox sensor enables dynamic visualization of intracellular redox states, yielding insights into oxidative stress-related processes in live cells.

Tailed-Hoogsteen Triplex DNA Silver Nanoclusters Emit Red Fluorescence upon Target miRNA Sensing.

MicroRNAs (miRNAs) are small RNA molecules, typically 21‒22 nucleotides in size, which play a crucial role in regulating gene expression in most eukaryotes. Their significance in various biological processes and disease pathogenesis has led to considerable interest in their potential as biomarkers for diagnosis and therapeutic applications. In this study, a novel method for sensing target miRNAs using Tailed-Hoogsteen triplex DNA-encapsulated Silver Nanoclusters (DNA/AgNCs) is introduced. Upon hybridization of a miRNA with the tail, the Tailed-Hoogsteen triplex DNA/AgNCs exhibit a pronounced red fluorescence, effectively turning on the signal. It is successfully demonstrated that this miRNA sensor not only recognized target miRNAs in total RNA extracted from cells but also visualized target miRNAs when introduced into live cells, highlighting the advantages of the turn-on mechanism. Furthermore, through gel-fluorescence assays and small-angle X-ray scattering (SAXS) analysis, the turn-on mechanism is elucidated, revealing that the Tailed-Hoogsteen triplex DNA/AgNCs undergo a structural transition from a monomer to a dimer upon sensing the target miRNA. Overall, the findings suggest that Tailed-Hoogsteen triplex DNA/AgNCs hold great promise as practical sensors for small RNAs in both in vitro and cell imaging applications.

Silver Nanoclusters Serve as Fluorescent Rivets Linking Hoogsteen Triplex DNA and Hairpin-Loop DNA Structures.

Greater understanding of the mutual influence between DNA and the associated nanomaterial on the properties of each other can provide alternative strategies for designing and developing DNA nanomachines. DNA secondary structures are essential for encapsulating highly emissive silver nanoclusters (DNA/AgNCs). Likewise, AgNCs stabilize secondary DNA structures, such as hairpin DNA, duplex DNA, and parallel-motif DNA triplex. In this study, we found that the fluorescence of AgNCs encapsulated within a Hoogsteen triplex DNA structure can be turned on and off in response to pH changes. We also show that AgNCs can act as nanoscale rivets, linking two functionally distinctive DNA nanostructures. For instance, we found that a Hoogsteen triplex DNA structure with a seven-cytosine loop encapsulates red fluorescent AgNCs. The red fluorescence faded under alkaline conditions, whereas the fluorescence was restored in a near-neutral environment. Hairpin DNA and random DNA structures did not exhibit this pH-dependent AgNCs fluorescence. A fluorescence lifetime measurement and a small-angle X-ray scattering analysis showed that the triplex DNA-encapsulated AgNCs were photophysically convertible between bright and dark states. An in-gel electrophoresis analysis indicated that bright and dark convertibility depended on the AgNCs-riveted dimerization of the triplex DNAs. Moreover, we found that AgNCs rivet the triplex DNA and hairpin DNA to form a heterodimer, emitting orange fluorescence. Our findings suggest that AgNCs between two cytosine-rich loops can be used as nanorivets in designing noncanonical DNA origami beyond Watson-Crick base pairing.

Transcriptome analysis revealed that jasmonic acid biosynthesis/signaling is involved in plant response to Strontium stress.

Strontium (Sr) has become an increasing global threat for both environment and human health due to its radioactive isotope, Sr-90 which can be found in the nuclear-contaminated soils and water. Although excessive Sr has been known to be toxic to plant growth and development, the molecular mechanisms underlying plant response to Sr stress, especially on the transcription level, remains largely unknown. To date, there is no published genome-wide transcriptome data available for the plant responses to Sr toxicity. Therefore, we aimed to gain insight on the molecular events occurring in plants in Sr toxicity condition by comparing the genome-wide gene expression profiles between control and Sr-treated plants using RNA-seq analysis. A total of 842 differentially expressed genes (DEGs) were identified in response to Sr stress compared to the control. Based on the analysis of DEGs using Gene Ontology (GO), DEGs were significantly enriched in the GO terms of response to salicylic acid (SA), response to jasmonic acid (JA), and defense response to bacterium. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that DEGs were mainly involved in metabolic processes including phenylpropanoid biosynthesis and alpha-linolenic acid metabolism, which is known as a precursor of JA biosynthesis. Furthermore, MapMan analysis revealed that a number of genes related to the biotic stress such as pathogenesis-related protein (PR) genes were highly up-regulated under Sr stress. Taken together, this study revealed that JA biosynthesis and/or signaling might be associated with plant response to Sr stress, and play important roles to maintain proper growth and development under Sr stress.

Strontium stress disrupts miRNA biogenesis by reducing HYL1 protein levels in Arabidopsis.

Strontium (Sr) is an emerging environmental pollutant that has become a major global concern after the nuclear accident at the Fukushima Daiichi Nuclear Power Plant in 2011. Although many studies have demonstrated the harmful effects of Sr on plant growth and development at the physiological level, knowledge regarding how plants sense and respond to Sr stress at the molecular level is limited. Recent studies have suggested that microRNAs (miRNAs) function as key regulators of plant growth and development as well as in the responses of plants to environmental stresses, including salinity, drought, cold, nutrient starvation, and heavy metals. In this study, we examined the global expression profile of miRNAs under Sr stress using small RNA sequencing analysis in Arabidopsis to better understand the molecular basis of plant responses to Sr stress. To identify specific Sr-responsive miRNAs, we performed comparative miRNA expression profiling analysis using control, CaCl2-, and SrCl2-treated seedlings. Compared to the control treatment, the expressions of most miRNAs were considerably decreased in the Sr-treated seedlings. However, under Sr stress, the expressions of primary miRNAs (pri-miRNAs) and their target genes were significantly increased; the protein levels of HYPONASTIC LEAVES 1 (HYL1), one of the core components of the microprocessor complex, were strongly reduced despite the increased HYL1 mRNA expression. In addition, hyl1-2 mutant plants were shown to be more sensitive to Sr stress than wild-type plants. Collectively, our results strongly suggested that Sr stress may be associated with the disruption of miRNA biogenesis by reducing the protein level of HYL1, which is required to maintain proper growth and development for plants. Our findings further indicated that some miRNAs may play important roles in plant responses to Sr stress.

Noncanonical Head-to-Head Hairpin DNA Dimerization Is Essential for the Synthesis of Orange Emissive Silver Nanoclusters.

DNA secondary structures, such as dimers and hairpins, are important for the synthesis of DNA template-embedded silver nanoclusters (DNA/AgNCs). However, the arrangement of AgNCs within a given DNA template and how the AgNC influences the secondary structure of the DNA template are still unclear. Here, we introduce a noncanonical head-to-head hairpin DNA nanostructure that is driven by orange-emissive AgNCs. Through detailed in-gel analysis, sugar backbone switching, inductively coupled plasma mass spectrometry, small-angle X-ray scattering, and small angle neutron scattering, we show that the orange-emissive AgNCs mediate cytosine-Ag-cytosine bridging between two six-cytosine loop (6C-loop) hairpin DNA templates. Unlike green, red, or far-red emissive AgNCs, which are embedded inside a hairpin and duplex DNA template, the orange-emissive AgNCs are localized on the interface between the two 6C-loop hairpin DNA templates, thereby linking them. Moreover, we found that deoxyribose in the backbone of the 6C-loop at the third and fourth cytosines is crucial for the formation of the orange-emissive AgNCs and the head-to-head hairpin DNA structure. Taken together, we suggest that the specific wavelength of AgNCs fluorescence is determined by the mutual interaction between the secondary or tertiary structures of DNA- and AgNC-mediated intermolecular DNA cross-linking.

The structural shift of a DNA template between a hairpin and a dimer tunes the emission color of DNA-templated AgNCs.

The scaffolding DNA sequence and the size of silver nanoclusters (AgNCs), confined in a DNA template are the key parameters in determining the fluorescent properties of DNA-stabilized silver nanoclusters (DNA/AgNCs). In addition, we suggest here that the structural shift of a DNA hairpin-dimer is as important as the DNA sequence in determining the emission wavelength of DNA/AgNCs. Furthermore, we show that the structural shift post AgNC formation can be triggered by incubation time and pre-AgNC formation under salt conditions. As an important factor in predicting the emission properties of DNA/AgNCs, the modulation of DNA secondary structures with either sequence changes or ionic conditions can be applied for the dual-color detection system of a target molecule. Particularly, the dual-color detection method may increase the reliability of DNA/AgNC sensors for miRNAs.

A potential oral anticancer drug candidate, Moringa oleifera leaf extract, induces the apoptosis of human hepatocellular carcinoma cells.

It has previously been reported that cold water-extracts of Moringa oleifera leaf have anticancer activity against various human cancer cell lines, including non-small cell lung cancer. In the present study, the anticancer activity of M. oleifera leaf extracts was investigated in human hepatocellular carcinoma HepG2 cells. By the analysis of apoptotic signals, including the induction of caspase or poly(ADP-ribose) polymerase cleavage, and the Annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, it was demonstrated that M. oleifera leaf extracts induce the apoptosis of HepG2 cells. In the hollow fiber assay, oral administration of the leaf extracts significantly reduced (44-52%) the proliferation of the HepG2 cells and A549 non-small cell lung cancer cells. These results support the potential of soluble extracts of M. oleifera leaf as orally administered therapeutics for the treatment of human liver and lung cancers.

Cesium Toxicity Alters MicroRNA Processing and AGO1 Expressions in Arabidopsis thaliana.

MicroRNAs (miRNAs) are short RNA fragments that play important roles in controlled gene silencing, thus regulating many biological processes in plants. Recent studies have indicated that plants modulate miRNAs to sustain their survival in response to a variety of environmental stimuli, such as biotic stresses, cold, drought, nutritional starvation, and toxic heavy metals. Cesium and radio-cesium contaminations have arisen as serious problems that both impede plant growth and enter the food chain through contaminated plants. Many studies have been performed to define plant responses against cesium intoxication. However, the complete profile of miRNAs in plants during cesium intoxication has not been established. Here we show the differential expression of the miRNAs that are mostly down-regulated during cesium intoxication. Furthermore, we found that cesium toxicity disrupts both the processing of pri-miRNAs and AGONOUTE 1 (AGO1)-mediated gene silencing. AGO 1 seems to be especially destabilized by cesium toxicity, possibly through a proteolytic regulatory pathway. Our study presents a comprehensive profile of cesium-responsive miRNAs, which is distinct from that of potassium, and suggests two possible mechanisms underlying the cesium toxicity on miRNA metabolism.

Soluble extract from Moringa oleifera leaves with a new anticancer activity.

Moringa oleifera has been regarded as a food substance since ancient times and has also been used as a treatment for many diseases. Recently, various therapeutic effects of M. oleifera such as antimicrobial, anticancer, anti-inflammatory, antidiabetic, and antioxidant effects have been investigated; however, most of these studies described only simple biological phenomena and their chemical compositions. Due to the increasing attention on natural products, such as those from plants, and the advantages of oral administration of anticancer drugs, soluble extracts from M. oleifera leaves (MOL) have been prepared and their potential as new anticancer drug candidates has been assessed in this study. Here, the soluble cold Distilled Water extract (4°C; concentration, 300 µg/mL) from MOL greatly induced apoptosis, inhibited tumor cell growth, and lowered the level of internal reactive oxygen species (ROS) in human lung cancer cells as well as other several types of cancer cells, suggesting that the treatment of cancer cells with MOL significantly reduced cancer cell proliferation and invasion. Moreover, over 90% of the genes tested were unexpectedly downregulated more than 2-fold, while just below 1% of the genes were upregulated more than 2-fold in MOL extract-treated cells, when compared with nontreated cells. Since severe dose-dependent rRNA degradation was observed, the abnormal downregulation of numerous genes was considered to be attributable to abnormal RNA formation caused by treatment with MOL extracts. Additionally, the MOL extract showed greater cytotoxicity for tumor cells than for normal cells, strongly suggesting that it could potentially be an ideal anticancer therapeutic candidate specific to cancer cells. These results suggest the potential therapeutic implications of the soluble extract from MOL in the treatment of various types of cancers.

Metabolic roles of carotenoid produced by non-photosynthetic bacterium Gordonia alkanivorans SKF120101.

Carotenoids produced by non-photosynthetic bacteria protect organisms against lethal photodynamic reactions and scavenge oxygenic radicals. However, the carotenoid produced by Gordonia alkanivorans SKF120101 is coupled to reducing power generation. SKF120101 selectively produces carotenoid under light conditions. The growth yield of SKF120101 cultivated under light conditions was higher than that under dark condition. In the cyclic voltammetry, both upper and lower voltammograms for neutral red (NR) immobilized in intact cells of SKF120101 were not shifted in the condition without external redox sources but were commonly shifted downward by glucose addition and light. Electric current generation in a biofuel cell system (BFCS) catalyzed by harvested cells of SKF120101 was higher under light than dark condition. The ratio of electricity generation to glucose consumption by SKF120101 cultivated in BFCS was higher under light than dark condition. The carotenoid produced by SKF120101 catalyzes production of reducing power from light energy, first evaluated by the electrochemical technique used in this research.

Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK via PKCα-mediated MKP-1 induction.

AIMS: Hyperplasia of vascular smooth muscle cells (VSMCs) after blood vessel injury is one of the major pathophysiological mechanisms associated with neointima. Tauroursodeoxycholate (TUDCA) is a cytoprotective agent in a variety of cells including hepatocytes as well as an inducer of apoptosis in cancer cells. In this study, we investigated whether TUDCA could prevent neointimal hyperplasia by suppressing the growth and migration of VSMCs. METHODS AND RESULTS: Transporters of TUDCA uptake in human VSMCs (hVSMCs) were analysed by RT-PCR and western blot. A knock-down experiment using specific si-RNA revealed that TUDCA was incorporated into hVSMCs via organic anion transporter 2 (OATP2). TUDCA reduced the viability of hVSMCs, which were mediated by inhibition of extracellular signal-regulated kinase (ERK) by induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) via protein kinase Cα (PKCα). The anti-proliferative effect of TUDCA was reversed by treatment with 7-hydroxystaurosporine, an inhibitor of PKC, and by the knock-down of MKP-1. In addition, TUDCA suppressed hVSMC migration, which was mediated by reduced matrix metalloproteinase-9 (MMP-9) expression by ERK inhibition, as well as reduced viability of hVSMCs. Rats with carotid artery balloon injury received oral administration of TUDCA; this reduced the increase in ERK and MMP-9 caused by balloon injury. TUDCA significantly decreased the ratio of intima to media by reducing proliferation and inducing apoptosis of the VSMCs. CONCLUSION: TUDCA inhibits neointimal hyperplasia by reducing proliferation and inducing apoptosis of smooth muscle cells by suppression of ERK via PKCα-mediated MKP-1 induction.

Enrichment of CO2-fixing bacteria in cylinder-type electrochemical bioreactor with built-in anode compartment.

Bacterial assimilation of CO2 into stable biomolecules using electrochemical reducing power may be an effective method to reduce atmospheric CO2 without fossil fuel combustion. For the enrichment of the CO2-fixing bacteria using electrochemical reducing power as an energy source, a cylinder-type electrochemical bioreactor with a built-in anode compartment was developed. A graphite felt cathode modified with neutral red (NR-graphite cathode) was used as a solid electron mediator to induce bacterial cells to fix CO2 using electrochemical reducing power. Bacterial CO2 consumption was calculated based on the variation in the ratio of CO2 to N2 in the gas reservoir. CO2 consumed by the bacteria grown in the electrochemical bioreactor (2,000 ml) reached a maximum of approximately 1,500 ml per week. Time-coursed variations in the bacterial community grown with the electrochemical reducing power and CO2 in the mineral-based medium were analyzed via temperature gradient gel electrophoresis (TGGE) of the 16S rDNA variable region. Some of the bacterial community constituents noted at the initial time disappeared completely, but some of them observed as DNA signs at the initial time were clearly enriched in the electrochemical bioreactor during 24 weeks of incubation. Finally, Alcaligenes sp. and Achromobacter sp., which are capable of autotrophically fixing CO2, were enriched to major constituents of the bacterial community in the electrochemical bioreactor.

Knockdown of the Dickkopf 3 gene induces apoptosis in a lung adenocarcinoma.

The expression of the Wnt-antagonist Dickkopf gene (DKK) is downregulated in several types of tumors as a consequence of epigenetic DNA modification; four DKK members, DKK1, DKK2, DKK3, and DKK4, have been identified. In this study, we investigated another function of DKK3 in non-small cell lung cancer H460 cells, in which DKK3 was hypermethylated (44%) but still expressed, by interfering with DKK3 expression using DKK3-silencing RNA (SiRNA). We found that knockdown of DKK3 expression by DKK3 SiRNA transfection led to the detachment of H460 cells from the bottom of the culture plate and caused apoptosis. The expression of cyclin-dependent kinases D1 and E were increased by DKK3 knockdown, indicating that cells with blocked DKK3 expression entered the apoptotic pathway. We also found that the intracellular level of reactive oxygen species was higher in cells with blocked DKK3 expression than in normal H460 cells, and levels of p53, p21, and Bax were also increased by the gene knockdown. These results indicate that DKK3 acts as an antiapoptotic molecule by decreasing the intracellular level of reactive oxygen species.

PTEN/pAkt/p53 signaling pathway correlates with the radioresponse of non-small cell lung cancer.

The sensitivity or resistance of cancer cells and normal tissues to ionizing radiation plays an important role in the clinical setting of lung cancer treatment. However, to date the exact molecular mechanisms of intrinsic radiosensitivity have not been well explained. In this study, we compared the radiosensitivity or radioresistance in two non-small cell lung cancers (NSCLCs), H460 and A549, and investigated the signaling pathways that confer radioresistance. H460 cells showed a significant G(2)/M arrest after 12 h of irradiation (5 Gy), reaching 60% of G(2)/M phase arrest. A549 cells also showed a significant G(2)/M arrest after 12 h of exposure; however, this arrest completely disappeared after 24 h of exposure. A549 has higher methylated CpG sites in PTEN, which is correlated with tumor radioresistance in some cancer cells, than H460 cells, and the average of the extent of the methylation was approximately 4.3 times higher in A549 cells than in H460 cells. As a result, PTEN expression was lower in A549 than in H460. Conducting Western blot analysis, we found that PTEN acted as a negative regulator for pAkt, and the pAkt acted as a negative regulator for p53 expression. According to the above results, we concluded that the radiosensitivity shown in H460 cells may be due to the higher expression of PTEN through p53 signaling pathway.

Polyamine as a signaling molecule for controlling an adaptive mutation.

In the absence of exogenous polyamines, the polyamine-deficient Escherichia coli mutant shows not only a characteristic dual-phase growth with abnormal growth, growth arrest, and normal growth after mutation, but also a higher expression of the SOS genes than the polyamine-proficient wild type. The interval of the growth arrest is inversely regulated in a polyamine concentration-dependent manner. These results indicate that the polyamines can act as a signal not only for provoking an adaptive mutation, but also for hastening generation of an adaptive mutation.

Spontaneous liberation of intracellular polyhydroxybutyrate granules in Escherichia coli.

Despite receiving much attention as a biodegradable substitute for conventional non-biodegradable plastics, the commercial use of polyhydroxybutyrate (PHB) remains limited because of its high production cost. In order to reduce the recovery/purification cost, which forms over half of the total production cost, we have developed a new cultivation method which enables spontaneous liberation of PHB by modulation of the initial inoculum size and the medium composition in recombinant Escherichia coli harboring Alcaligenes eutrophus phbCAB genes. In flask cultivation using a low cell inoculum and 2x LB medium containing 21% glucose, autolysis of 80.2% as well as yields of 85.2 g/l of PHB and a PHB content of 99.0% (w/w) were obtained. The glucose conversion rate was 0.43. The strategies developed in this study can minimize complex and unfavorable efforts required for efficient recovery/purification processes, thereby enabling biodegradable plastic to be produced by the recombinant E. coli so as to compete with conventional non-biodegradable plastic.

RpoS-mediated growth-dependent expression of the Escherichia coli tkt genes encoding transketolases isoenzymes.

Escherichia coli tktA and tktB genes encode two transketolase isoenzymes involved in the pentose-phosphate pathway, In this study, two reporter lacZ fusions, tktA- and tktB-lacZ, were constructed to examine their transcriptional regulation on the E. coli chromosome. The tktA gene was induced in the exponential growth phase and suppressed in the stationary growth phase. However, the genetic elimination of the rpoS, whose product is an alternative sigma factor (RpoS), derepressed the tktA gene expression in the stationary growth phase, indicating that the RpoS sigma factor negatively regulates the tktA gene expression in the stationary growth phase. On the contrary, the tktB gene expression showed the highest value in the stationary growth phase and the RpoS positively regulated the tktB gene expression in the stationary growth phase. We also verified the role of the RpoS affecting the regulation of the tktA and tktB gene expression by the reverse transcription (RT)-PCR experiments. These results suggest that the differential growth-dependent expressions of the tktA and tktB genes are caused by the RpoS action.

GAD 67KD antisense in colon cancer cells inhibits cell growth and sensitizes to butyrate and pH reduction and H2O2 and gamma-radiation.

In eukaryotes, glutamate decarboxylase (GAD) expression was found in brain, kidney, and several kinds of tumor tissues. But its function has been emphasized only as a neurotransmitter-synthesizer, the role in controlling intracellular physiology is poorly understood. According to our studies, when GAD 67KD expression in colon cancer HT-29 cell was repressed by antisense DNA, the cell proliferation was significantly inhibited. GAD 67KD antisensed cells exhibited the low glutathione and high reactive oxygen species level. More importantly, these cells were extremely sensitive to butyrate or pH reduction, both of which naturally cause metabolic stress in the colon lumen, as well as H2O2 and ionizing radiation. These data indicate that GAD 67KD regulates the intracellular redox potential and is important for resistance to acidic or oxidative stress. So, based on these results, we suggest that inhibition of GAD 67KD expression has potentially important implications for overcoming the drug resistance of cancer cells.

Abnormal growth of polyamine-deficient Escherichia coli mutant is partially caused by oxidative stress-induced damage.

Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli. In this study, we constructed a new polyamine-deficient E. coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions. We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth. Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including z.rad;O(2)(-), and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner. As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type. Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant. Finally, we confirmed that chloromethyl-2('),7(')-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells. These results showed that abnormal growth of the polyamine-deficient E. coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth.