Ethyl Methanesulfonate Research Articles

Transcriptional Regulation and Gene Mapping of Internode Elongation and Late Budding in the Chinese Cabbage Mutant lcc.

Two important traits of Chinese cabbage, internode length and budding time, destroy the maintenance of rosette leaves in the vegetative growth stage and affect flowering in the reproductive growth stage. Internodes have received much attention and research in rice due to their effect on lodging resistance, but they are rarely studied in Chinese cabbage. In Chinese cabbage, internode elongation affects not only the maintenance of rosette leaves but also bolting and yield. Budding is also an important characteristic of Chinese cabbage entering reproductive growth. Although many studies have reported on flowering and bolting, studies on bud emergence and the timing of budding are scarce. In this study, the mutant lcc induced by EMS (Ethyl Methane Sulfonate) was used to study internode elongation in the seedling stage and late budding in the budding stage. By comparing the gene expression patterns of mutant lcc and wild-type A03, 2280 differentially expressed genes were identified in the seedling stage, 714 differentially expressed genes were identified in the early budding stage, and 1052 differentially expressed genes were identified in the budding stage. Here, the transcript expression patterns of genes in the plant hormone signaling and clock rhythm pathways were investigated in relation to the regulation of internode elongation and budding in Chinese cabbage. In addition, an F2 population was constructed with the mutants lcc and R500. A high-density genetic map with 1602 marker loci was created, and QTLs for internode length and budding time were identified. Specifically, five QTLs for internode length and five QTLs for budding time were obtained. According to transcriptome data analysis, the internode length candidate gene BraA02g005840.3C (PIN8) and budding time candidate genes BraA02g003870.3C (HY5-1) and BraA02g005190.3C (CHS-1) were identified. These findings provide insight into the regulation of internode length and budding time in Chinese cabbage.

Liver-on-chip model and application in predictive genotoxicity and mutagenicity of drugs

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.

Characterization and Mapping of a Rolling Leaf Mutant Allele rlT73 on Chromosome 1BL of Wheat.

Leaf rolling is regarded as an important morphological trait in wheat breeding. Moderate leaf rolling is helpful to keep leaves upright and improve the photosynthesis of plants, leading to increased yield. However, studies on the identification of genomic regions/genes associated with rolling leaf have been reported less frequently in wheat. In this study, a rolling leaf mutant, T73, which has paired spikelets, dwarfism, and delayed heading traits, was obtained from a common wheat landrace through ethyl methanesulfonate mutagenesis. The rlT73 mutation caused an increase in the number of epidermal cells on the abaxial side and the shrinkage of bulliform cells on the adaxial side, leading to an adaxially rolling leaf phenotype. Genetic analysis showed that the rolling leaf phenotype was controlled by a single recessive gene. Further Wheat55K single nucleotide polymorphism array-based bulked segregant analysis and molecular marker mapping delimited rlT73 to a physical interval of 300.29-318.33 Mb on the chromosome arm 1BL in the Chinese Spring genome. We show that a point mutation at the miRNA165/166 binding site of the HD zipper class III transcription factor on 1BL altered its transcriptional level, which may be responsible for the rolling leaf phenotype. Our results suggest the important role of rlT73 in regulating wheat leaf development and the potential of miRNA-based gene regulation for crop trait improvement.

Frequency and spectrum of viable mutations in mungbean (Vigna radiata (L.) Wilczek)

The mutagenic effect of gamma rays (200Gy, 300Gy, 400Gy, 500Gy, and 600 Gy), ethyl methane sulphonate (0.2%, 0.3%, 0.4%, 0.5%, and 0.6%) and sodium azide (1 mM, 2 mM and 3 mM) on frequency and spectrum of viable mutations in two mungbean varieties viz., WGG-42 and LGG-460 were investigated. A wide spectrum of viable mutations for stature, leaf, flowering/maturity duration, cotyledonary leaf, pod, seed size, seed color, and colour stem were identified in the M2 generation. The spectrum of viable mutants includeda total of 94 mutants in gamma gamma-irradiated population, 132 mutants in EMS and 45 mutants in SA treatments in WGG-42. In LGG-460, a total of 91 mutants in gamma gamma-irradiated population, 118 mutants in EMS treated population, and 45 mutants in SA treatments were observed. Viable mutation frequency increased with an increase in dose/concentration of gamma rays, EMS, and SA treatments in both genotypes. In both the genotypes, EMS showed the highest frequency of viable mutations than gamma rays and SA treatments. These viable mutants could be exploited as a variety or donor for bringing desirable traits into the otherwise well-adapted cultivars.

Mutagenesis-Derived Resistance to Black Point in Wheat.

Black point, a severe global wheat disease, necessitates deploying resistant cultivars for effective control. However, susceptibility remains prevalent among most wheat cultivars. Identifying new sources of resistance and understanding their mechanisms are crucial for breeding resistant cultivars. This study pinpointed black point resistance in an ethyl methane sulfonate (EMS)-mutagenized wheat population of Wanyuanbai 1 (WYB) and analyzed resistant mutants using RNA-Seq. The findings revealed the following: (i) wyb-18, among 10,008 EMS-mutagenized lines, exhibited robust resistance with significantly lower black point incidence under artificial Bipolaris sorokiniana inoculation in 2020 and 2021 (average incidence of 5.2% over 2 years), markedly reduced compared with WYB (50.9%). (ii) wyb-18 kernels displayed black point symptoms at 12 days after inoculation (dai), 3 days later than WYB. At 15 dai, wyb-18 kernels had isolated black spots, unlike WYB kernels, where the entire embryo turned black. (iii) wyb-18 showed heightened antioxidant enzyme activity, including peroxidase, catalase, and superoxide dismutase. (iv) Analysis of 543 differentially expressed genes between wyb-18 and WYB at 9 dai identified enrichment in the MAPK signaling pathway through KEGG analysis. Ten genes in this pathway exhibited upregulated expression, while one was downregulated in wyb-18. Among these genes, PR1, WRKY11, SAPK5, and TraesCS1A02G326800 (chitin recognition protein) consistently showed upregulation in wyb-18, making them potential candidates for black point resistance. These results offer valuable germplasm resources for breeding and novel insights into the mechanisms of black point resistance.

Investigation of the Genotoxic Effect of Fluoxetine Hydrochloride in Drosophila melanogaster

This study aimed to determine the potential genotoxic effect of fluoxetine hydrochloride (FLX-HCl), an antidepressant commonly used for treating depression, using Somatic Mutation and Recombination Test (SMART). Third-¬instar Drosophila melanogaster larvae transheterozygous for the mutations multiple wing hair (mwh) and flare (flr3) were chronically fed in a medium containing different concentrations of FLX-HCl (0.1, 0.5, 1, and 2 mg/mL) in the experimental group. Distilled water, 0.1 mM ethyl methane sulfonate (EMS), and 2% dimethyl sulfoxide (DMSO) were used in negative, positive, and solvent control groups, respectively. The survival percentages were calculated by determining the number of individuals surviving when the larvae completed their development in the experimental and control groups. In all application groups, the wings of 40 individuals with both normal and serrate wing phenotypes were examined under a microscope, and genetic changes were evaluated by counting the mutant clones in the wings. The data obtained show that 1 and 2 mg/mL concentrations of FLX-HCl caused toxic effects in D. melanogaster individuals. Additionally, FLX-HCl showed a negative genotoxic effect at 0.1 mg/mL concentration, insignificant at 0.5 mg/mL concentration, and positive at 1 and 2 mg/mL concentrations in terms of total mutation evaluation and clone induction frequency in D. melanogaster individuals.

Effect of chemical mutagen (Ethyl methane sulfonate) on Money plant (Epipremnum aureum)

Epipremnum aureum, commonly known as pothos, is a hardy indoor foliage ornamental crop. Mutation breeding in Golden pothos is needed to meet the demand for more valuable and highly sought-after cultivars. The present study aimed to exploit variability on various parameters viz. new leaf emergence, survival percentage, plant height, length and width of leaves. The experiment was set up using a Complete Randomized Design (CRD) with 8 treatments and 3 replications. Rooted cuttings of Golden pothos were treated with varying concentrations of Ethyl Methane Sulfonate (EMS) for 4 hours, and a control treatment was included. The results showed that all the treated cuttings with EMS delays the new leaf emergence period compared to the control. Compared to the control, the minimum number of new leaf emergences was found in EMS-treated plants. The maximum survival percentage (100%) was recorded in 0.1%, 0.5% and control treatment. The lowest plant height (15.3 cm ) was observed in treatment T4 (1.5 % EMS) treated plants. However, maximum plant height was observed in the control treatment at 150 days of interval. In comparison to the control, EMS decreased the length and width of leaves at higher dosages. The minimum leaf length and width, i.e. 2.40 cm and 1.20 cm, were recorded in plants treated with EMS at 2%, while maximum length and width were observed in control and low EMS-treated plants. The study showed that ethyl methane sulfonate affected plant development characteristics and showed some dwarfness of money plants. This appears to be the first report of EMS treatment on pothos.

Genetic study for seed germination and shattering in Euphorbia lagascae in response to different seed treatments

Euphorbia lagascae Spreng is a promising emerging oilseed crop, with its seed oil accounting for approximately 50% of the seed weight. Euphorbia oil contains a significant amount of vernolic acid, comprising two-thirds of its composition, which boasts various industrial applications, including acting as a stabilizer-plasticizer and natural dye. However, this species was known to have a high degree of seed-shattering and a low germination rate, which act as two important barriers to large-scale production and exploitation. Therefore, the objective of this study is to determine the genetic control of seed germination and seed-shattering traits in order to develop a reliable pipeline that would be applicable for industries and breeders to select superior E. lagascae lines and design a robust breeding scheme in a short time at reduced labor costs. For this objective, five different wild-type genotypes of E. lagascae that demonstrated high germination potential were crossed with an ethyl methanesulfonate (EMS) mutant genotype that produces non-shattering capsules. The F2 populations from two successful crosses (A and B) were separated into three different treated groups for seed germination evaluation and to study the segregation of 200 individuals per F2 population. The three treatments were: light, gibberellic acid (GA3), and control treatment. Consequently, plants treated with approximately 250 μmol/m2/s of light showed significant improvement in germination up to 75% in cross A and 82.4 % in cross B compared with the control plants and the group treated with 0.05% GA3. According to the chi-square test results, the inheritance pattern of seed germination in response to light treatment follows a 3:1 segregation ratio between germinated and non-germinated seeds, indicating a dominant gene action in the F2 generation. The same conclusion was followed for the shattering trait in the group treated with light, which was also simply inherited as a 3:1 ratio for shattering vs. non-shattering capsules. Our results emphasize the importance and significance of light treatment in producing uniform populations through acceptable germination and shattering resistance of the mutant genotypes of E. lagascae. This is the first report of light treatment that significantly improved seed germination of E. lagascae, which may enhance efforts in the development of this new industrial crop as a feedstock for vernolic acid production.

Construction of ethyl methane sulfonate mutant library in G. arboreum and rapid identification of mutant genes via repeated re-sequencing

Cotton (Gossypium) is the leading economic crop and provides most of renewable spinning fibers for the worldwide textile industry. The diploid Asiatic cotton (G. arboreum), with spinnable fiber and relatively simple genome, is an ideal model for cotton functional genomics and fiber biology. In this study, we constructed an ethyl methane sulfonate (EMS) mutant library of Asiatic cotton. Stable mutants with various phenotypic variations in leaf, flower, fiber and plant architecture were obtained in M1 generation. Genome-wide mutation analysis revealed high-density mutations distributed evenly on whole genome. Using repeated re-sequencing of M1 mutant plants, the candidate genes of 10 out of 15 mutants were identified. The functions of GaYUC4 and GaPDX1, candidate genes for bar blade and yellow leaf vein, respectively, were further confirmed using virus induced gene silencing in Asiatic and/or upland cottons. Our work paved way for expansion of Asiatic cotton mutant library and subsequent applications in cotton functional genomics and cotton breeding.

Ascertaining Lethal Dose 50 (LD50) and Simultaneous Effect of Ethyl Methane Sulphonate (EMS) and Sodium Azide (SA) On Seedling Characters in Mungbean Genotypes ‘Pusa 1031’ and ‘Pusa 1431’

Background: Finding effective dose is the most crucial step before commencing any mutagen treatment. The present study determined the lethal median dose in two mungbean genotypes, ‘Pusa 1031’ and ‘Pusa 1431’, using two different chemical mutagens namely Ethyl Methane Sulphonate and Sodium Azide. Methods: A pair of mungbean genotypes “Pusa 1031” and “Pusa 1431” were subjected to varying concentrations of two chemical mutagens i.e., Ethyl Methane Sulphonate (EMS) (10-100 mm with a ten mm different) and Sodium Azide (SA) (0.01 mm - 0.05 mm) followed by germinating the seeds in trays containing soil rite. Result: EMS treatment in two genotypes displayed LD50 values as 58.81 mM and 45.04 mM for the genotypes ‘Pusa 1031' and ‘Pusa 1431’, respectively. Lethal dose 50 was determined as 0.047 mM for both genotypes when treated with sodium azide. Seedling characters exhibited a linear response with dose augmentation for both the chemical mutagens, despite displaying similar LD50 values both genotypes exhibited remarkable differences in seedling parameters when treated with sodium azide.

Determination of the Effects of Different EMS Doses Applied to Seeds of Chickpea and Lentil Varieties on Some Seedling Characteristics

In this study, it was aimed to determine the effects of 11 different doses of Ethyl Methane Sulphonate (EMS) applied to the seed to create variation in the M1 generation of chickpea (Gökçe) and lentil (Şakar) genotypes during germination and seedling development periods and to determine the lethal dose that caused a 50% reduction in plant emergence rate. The research was conducted under the greenhouse and laboratory conditions of the Faculty of Agriculture of Dicle University in 2019/2020. The study was conducted according to a randomized block design with three replicates. For the M1 generation, seeds of each genotype in the elite stage were treated with EMS solution at 0 (control), 10, 20, 30, 40, 40, 50, 60, 60, 70, 70, 80, 90, and 100 mM (1000 seeds for each dose) and then sown in the greenhouse. A total of 132 tubes were sown with 30 seeds for each dose, and the effective EMS dose was determined for each genotype based on the traits examined in the developing seedlings. In the study, it was concluded that 11 different EMS doses applied to the seeds of chickpea and lentil varieties had negative effects on seedling development in the M1 generation, and increasing EMS doses from the control caused a decrease in all traits examined. With increasing EMS doses, plant emergence was observed in both chickpea and lentil up to 60 mM dose, while no germination was observed at 60 mM dose, and the dose rate varied according to species and varieties. The dose that caused a 50% decrease in the plant emergence rate in Gökçe chickpea and Şakar lentil varieties was determined as the LD50 dose. Accordingly, it was determined that the LD50 dose was 30 mM for Gökçe chickpea variety and 60 mM for Şakar lentil variety.

Mutagenic Effect of Ethyl Methane Sulfonate on Some Fiber Species

Jute, kenaf and mesta are prominent fibrous plants with significant industrial and nutritional value for optimization of their cultivation process can have substantial agricultural and economic implications. The study was conducted on exploring the impact of ethyl methane sulfonate (EMS) presoaking treatments on the germination and the sensitivity of fiber crops to mutagens. This research aimed to evaluate the germination potential of jute, kenaf and mesta variety using various concentrations of EMS presoaking treatments, and analyze the sensitivity and induced variability in the M1 generation. Seeds were presoaked in four different concentrations of EMS. The subsequent parameters, including germination, root length, and shoot length, were measured at three crucial growth stages: 12, 24, and 36 days after sowing (DAS). A probit analysis was conducted to determine the LD50 value, which signifies the concentration at which 50% of the population exhibits an adverse effect. Moreover, significant variances were detected in germination rates, root elongation, and shoot growth across the different presoaking treatments. The findings emphasize the sensitivity of these fibrous plants to EMS concentrations, with increasing concentrations having a detrimental effect on growth parameters. This information can be instrumental for breeders and agriculturists looking to optimize germination in these crops. Bangladesh J. Nuclear Agric, 37(2): 19-27, 2023

Ethyl Methanesulfonate Mutant Library Construction in Tartary Buckwheat with Agronomic Trait and Flavonoid Screening for Germplasm Innovation

As a nutrient-rich multigrain crop, buckwheat is a typical “medicinal food homology” crop that is rich in flavonoids, including rutin and various vitamins. It has positive anti-oxidant and anti-tumour properties and lowers blood pressure. However, due to strict self-crossing characteristics, slow progress has been made in Tartary buckwheat (TB) cross-breeding, resulting in the slow breeding of new varieties of new TB varieties, which has limited the improvement of yield and quality. Therefore, mutant breeding is a rapid and effective technique for broadening and innovating TB breeding. In recent years, improving qualities related to yield, lodging resistance, and stability have become key points in TB breeding. Based on the above findings, excellent, potentially valuable TB lines with rich phenotypes were obtained for the TB mutation library via ethyl methanesulfonate (EMS), laying a foundation for creating new TB germplasms. In this study, we systematically investigated more than 10 agronomic traits of JQ2 and JQ4 mutants, including plant type, leaf colour, grain type, grain colour, grain number per plant, grain length, grain width, grain weight per plant, and 1000-grain weight. The results show that the maximum number of grains per plant was 1956, the weight was 32.84 g, and the 1000-grain weight was 30.89 g. The maximum number of grains per JQ4 plant was 2308, and the weight was 44.82 g. The maximum 1000-grain weight was 24.7 g. Among the 295 JQ2 mutants and 153 JQ4 mutants, 10 flavonoids (orientin, morin, quercetin, kaempferol, luteolin, naringin, hesperetin, myricetin, hesperidin, and rutin) were detected with near infrared spectroscopy (NIR). The mutants were divided into five groups according to the flavonoid content of the JQ2 mutants, of which the first group included 31 individual lines. and the second to fifth groups included 70, 69, 72, and 53 lines, respectively. The JQ4 mutants were divided into four classes, of which 41, 50, 32, and 30 were individual lines, respectively, with the highest rutin content being 82.06 mg/g. In summary, through systematic analysis and screening of the agronomic traits and flavonoid contents of JQ2 and JQ4 mutant seeds, we obtained three lines with a high 1000-grain weight, including two JQ2 mutant lines (30.89 g) and one JQ4 mutant line, which reached 24.70 g and ten lines with high grain weight per plant. This included 8 JQ2 mutants and 2 JQ4 mutants, as well as 72 high-rutin mutants (including 31 lines from JQ2 and 41 lines from JQ4 mutants). These elite lines provide the material basis for creating TB germplasms with excellent qualities and cultivation characteristics.

Dauer juvenile recovery transcriptome of two contrasting EMS mutants of the entomopathogenic nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode Heterorhabditis bacteriophora, symbiotically associated with enterobacteria of the genus Photorhabdus, is a biological control agent against many insect pests. Dauer Juveniles (DJ) of this nematode are produced in industrial-scale bioreactors up to 100 m3 in liquid culture processes lasting approximately 11days. A high DJ yield (> 200,000 DJ·mL-1) determines the success of the process. To start the mass production, a DJ inoculum proceeding from a previous monoxenic culture is added to pre-cultured (24h) Photorhabdus bacteria. Within minutes after contact with the bacteria, DJ are expected to perceive signals that trigger their further development (DJ recovery) to reproductive hermaphrodites. A rapid, synchronized, and high DJ recovery is a key factor for an efficient culture process. In case of low percentage of DJ recovery, the final DJ yield is drastically reduced, and the amount of non-desired stages (males and non-fertilized females) hinders the DJ harvest. In a preliminary work, a huge DJ recovery phenotypic variability in H. bacteriophora ethyl methanesulphonate (EMS) mutants was determined. In the present study, two EMS-mutant lines (M31 and M88) with high and low recovery phenotypes were analyzed concerning their differences in gene expression during the first hours of contact with Photorhabdus supernatant containing food signals triggering recovery. A snapshot (RNA-seq analysis) of their transcriptome was captured at 0.5, 1, 3 and 6h after exposure. Transcripts (3060) with significant regulation changes were identified in the two lines. To analyze the RNA-seq data over time, we (1) divided the expression profiles into clusters of similar regulation, (2) identified over and under-represented gene ontology categories for each cluster, (3) identified Caenorhabditis elegans homologous genes with recovery-related function, and (4) combined the information with available single nucleotide polymorphism (SNP) data. We observed that the expression dynamics of the contrasting mutants (M31 and M88) differ the most within the first 3h after Photorhabdus supernatant exposure, and during this time, genes related to changes in the DJ cuticle and molting are more active in the high-recovery line (M31). Comparing the gene expression of DJ exposed to the insect food signal in the haemolymph, genes related to host immunosuppressive factors were not found in DJ upon bacterial supernatant exposure. No link between the position of SNPs associated with high recovery and changes in gene expression was determined for genes with high differential expression. Concerning specific transcripts, nine H. bacteriophora gene models with differential expression are provided as candidate genes for further studies.

Abstract A052: ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell

Abstract Objective: Ovulation is a major risk factor of epithelial ovarian cancer (EOC), presumably acts from the cancer initiation stage. High-grade serous carcinoma (HGSC) is the most prevalent and fatal EOC, mainly arising from fallopian tube fimbrial epithelium (FTE). TP53 missense mutations are considered the earliest event of carcinogenesis since they form “p53 signatures” in FTE and were found in virtually all HGSC. Previously, we discovered that a subset of ovulatory follicular fluids (FF) harbored high levels of ROS, which were both mutagenic and oncogenic, causing DNA double-strand breaks (DSB) and tumorigenesis. However, DSB is unlikely the cause of TP53 mutations which are mostly CG: TA base substitutions involving deamination. We hypothesized that during ovulation FF exposes the fallopian tube fimbria and causes TP53 mutations by activating the activation-induced cytidine deaminase (AID). Materials and Methods: By conducting an HPRT gene mutation assay, we determined the mutagenic activity of FF on FTE cells with or without AID-knockdown. AID gene activation and protein nuclear translocation were analyzed in human FTE tissues and cells with different degrees of transformation, as well as in the oviducts of mice under controlled ovulation. The binding of AID to the TP53 gene and the subsequent deamination were determined by ChIP-PCR and uracil-qPCR, respectively. Results: After FF exposure, the mutation frequency of HPRT in FTE cells increased by 4.1 folds, at levels 40% higher than the ethyl methane sulfonate control and could be diminished by antioxidants or knockdown of the AID gene. AID activation, including mRNA and protein inductions and nuclear translocation, was evident in FTE cells and tissue after FF exposure and in mouse oviducts after ovulation. Two waves of AID activations were noted, peaking at 45 minutes (early activity) and 8.5 hours (late activity) after ovulation or FF exposure. TNF-α and ROS were found to be responsible for the activities in FF. Mechanistically, the early activity worked through the MAPK, while the late activity worked through the NF-κB pathway. The induced AID in both activities physically binds to and biochemically deaminates the TP53 gene irrelative to the known mutational hotspots of HGSC. However, it did not act on PTEN, the other prevalent tumor suppressor gene in HGSC. Meanwhile, ovulatory FF also activates the putative tumor-initiating and stemness gene LGR6 in FTE at locations where AID was also activated. Conclusion: The serial studies characterized the mutagenic activities of ovulatory FF, which acts on the LGR6-expressing cells in the fallopian tube fimbria and targets the TP53 gene. The study also identified ROS as the mutagen and pointed to strategies of ovulation inhibition and antioxidation for the prevention of EOC. Citation Format: Tang-Yuan Chu, Sanchana Subramani, Hsuan-Shun Huang. ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A052.

Assessment of the genotoxicity of tert-butyl alcohol in an in vivo thyroid comet assay.

Chronic exposure to high (20,000 ppm) concentrations of tert-butyl alcohol (TBA) in drinking water, equivalent to ~2100 mg/kg bodyweight per day, is associated with slight increases in the incidence of thyroid follicular cell adenomas and carcinomas in mice, with no other indications of carcinogenicity. In a recent toxicological review of TBA, the U.S. EPA determined that the genotoxic potential of TBA was inconclusive, largely based on non-standard studies such as in vitro comet assays. As such, the potential role of genotoxicity in the mode of action of thyroid tumors and therefore human relevance was considered uncertain. To address the potential role of genotoxicity in TBA-associated thyroid tumor formation, CD-1 mice were exposed up to a maximum tolerated dose of 1500 mg/kg-day via oral gavage for two consecutive days and DNA damage was assessed with the comet assay in the thyroid. Blood TBA levels were analyzed by headspace GC-MS to confirm systemic tissue exposure. At study termination, no significant increases (DNA breakage) or decreases (DNA crosslinks) in %DNA tail were observed in TBA exposed mice. In contrast, oral gavage of the positive control ethyl methanesulfonate significantly increased %DNA tail in the thyroid. These findings are consistent with most genotoxicity studies on TBA and provide mechanistic support for non-linear, threshold toxicity criteria for TBA. While the mode of action for the thyroid tumors remains unclear, linear low dose extrapolation methods for TBA appear more a matter of policy than science.

A prototrophic suppressor of a Vibrio fischeri D-glutamate auxotroph reveals a member of the periplasmic broad-spectrum racemase family (BsrF).

D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in Vibrio fischeri, we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.

A novel transcription factor FnMYB4 regulates pigments metabolism of yellow leaf mutants in Fragaria nilgerrensis

The strawberry species Fragaria nilgerrensis Schlechtendal ex J. Gay, renowned for its distinctive white, fragrant peach-like fruits and strong disease resistance, is an exceptional research material. In a previous study, an ethyl methane sulfonate (EMS) mutant library was established for this species, resulting in various yellow leaf mutants. Leaf yellowing materials are not only the ideal materials for basic studies on photosynthesis mechanism, chloroplast development, and molecular regulation of various pigments, but also have important utilization value in ornamental plants breeding. The present study focused on four distinct yellow leaf mutants: mottled yellow leaf (MO), yellow green leaf (YG), light green leaf (LG), and buddha light leaf (BU). The results revealed that the flavonoid content and carotenoid-to-chlorophyll ratio exhibited a significant increase among these mutants, while experiencing a significant decrease in chlorophyll and carotenoid contents compared to the wild type (WT). To clarify the regulatory mechanisms and network relationships underlying these mutants, the RNA-seq and weighted gene co-expression network (WGCNA) analyses were employed. The results showed flavonoid metabolism pathway was enriched both in MO and YG mutants, while the chlorophyll biosynthesis pathway and carotenoid degradation pathway were only enriched in MO and YG mutants, respectively. Subsequently, key structural genes and transcription factors were identified on metabolic pathways of three pigments through correlation analyses and quantitative experiments. Furthermore, a R2R3-MYB transcription factor, FnMYB4, was confirmed to be positively correlated with flavonoid synthesis through transient overexpression, virus-induced gene silencing (VIGS), and RNA interference (RNAi), accompanying by reoccurrence and attenuation of mutant phenotype. Finally, dual-luciferase (LUC) and yeast one-hybrid assays confirmed the binding of FnMYB4 to the FnFLS and FnF3H promoters, indicating that FnMYB4 positively regulates flavonoid synthesis. In addition, correlation analyses suggested that FnMYB4 also might be involved in chlorophyll and carotenoid metabolisms. These findings demonstrated the pivotal regulatory role of FnMYB4 in strawberry leaf coloration.

Mixed food waste valorization using a thermostable glucoamylase enzyme produced by a newly isolated filamentous fungus: A sustainable biorefinery approach

Food waste is a lucrative source of complex nutrients, which can be transformed into a multitude of bioproducts by the aid of microbial cell factories. The current study emphasizes isolating Glucoamylase enzyme (GA) producing strains that can effectively break down mixed food waste (MW), which serves as a substrate for biomanufacturing. The screening procedure relied heavily on the growth of isolated fungi on starch agar media, to specifically identify the microbes with the highest starch hydrolysis potential. A strain displayed the highest GA activity of 2.9 ± 0.14 U/ml which was selected and identified as Aspergillus fumigatus via molecular methods of identification. Exposure of the A. fumigatus with 200 mM Ethyl methanesulphonate (EMS) led to a 23.79% increase compared to the wild-type GA. The growth conditions like cultivation temperature or the number of spores in the inoculum were investigated. Further, maximum GA activity was exhibited at pH 5, 55 °C, and at 5 mM Ca2+ concentration. The GA showed thermostability, retaining activity even after long periods of exposure to temperatures as high as 95 °C. The improvement of hydrolysis of MW was achieved by Taguchi design where a maximum yield of 0.57 g g−1 glucose was obtained in the hydrolysate. This study puts forth the possibility that mixed food waste, despite containing spices and other microbial growth-inhibitory substances, can be efficiently hydrolyzed to release glucose units, by robust fungal cell factories. The glucose released can then be utilized as a carbon source for the production of value-added products.

In silico Identification of Alternatively Spliced Variants from Transcriptome Data of Rice Lines Exhibiting Complete Panicle Exsertion

Alternative splicing (AS) is a molecular mechanism governing gene expression, particularly in plants, wherein a single gene can yield multiple mRNA transcripts, thereby diversifying the resulting protein isoforms. Panicle exsertion (PE) is an important trait associated with grain yield in rice. We deployed the mRNA sequencing (transcriptome) data from incompletely exserted panicle genotype, BPT-5204 and its ethyl methanesulphonate (EMS) mutant lines viz., CPE-109 and CPE-110 exhibiting completely exserted panicle for identification of Alternative Splicing (AS) events. Our systematic analysis using rMATS package revealed 414 and 368 genes alternatively spliced upon the comparison of CPE-109 with BPT-5204 and CPE-110 with BPT-5204 respectively. We identified alternative 3′ splice site (A3SS) as the predominant AS type upon comparison of CPE-109 with BPT-5204 (51.20% A3SS) and CPE-110 with BPT-5204 (48.91% A3SS) at panicle initiation stage. In total 23 and 19 genes emanated multiple transcripts via., AS. Remarkably, upon comparison of splicing data of CPE-109 with BPT-5204 and CPE-110 with BPT-5204, we found three common genes namely, Os07g0406600 encoding DDHD domain containing protein, Os10g0442100 encoding tRNA methyltransferase and Os04g0675800 encoding H0103C06.10 protein, that generated more than two transcripts via., AS. These genes can be further validated for determining its role in panicle exsertion through gene expression studies, overexpression and functional characterization.