Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains.

Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid-based reporter assays in budding yeast using a RNR3 promoter-driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid-based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid-based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. KEY POINTS: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast.

Enhancing bauxite residue properties for plant growth: Gypsum and organic amendment effects on chemical properties of soil and leachate.

Here, we assess the effects of gypsum and local organic waste as amendments to non-weathered, filter-pressed bauxite residue (BR) to improve its properties and support plant growth. In addition, we monitored the leachate quality of the amended BR under progressive leaching that simulated precipitation conditions in Northern Brazil. Free-draining column tests consisting of BR amended with gypsum and organic waste, at 5% and 10% w/w, respectively, were leached for 8 weeks to assess the effects on the chemical composition of BR and the leachates. Adding gypsum to BR reduced the exchangeable sodium (Na) percentage (ESP) from approximately 79%-48%, whereas adding only organic waste had smaller effects on ESP (from ∼79% to âˆ¼ 70%). The mean leachate pH ranged from 8.7 to 9.4 for the gypsum, and organic waste amended BR, while this was 10.3 in the leachate of the unamended BR. The treatments had similar trends of electrical conductivity throughout the experiments and were below 2 dS/cm after 8 weeks, when ∼1.700 mm simulated precipitation had leached. Aluminium (Al), Arsenic (As), and Vanadium (V) concentrations in leachates of BR with gypsum, either alone or in combination with organic waste, were significantly lowered than in leachate of non-amended BR. By contrast, metal concentrations increased if organic waste was added to BR. We conclude that amending BR with gypsum, in combination with organic waste, significantly improves the chemical properties of the solid phase and achieved rehabilitation goals for SAR and EC of the leachates after 8 weeks of leaching. However, despite high leaching rates, rehabilitation goals for pH and ESP were not achieved with gypsum either alone or combined with organic waste.

Butyrylcholinesterase level as an independent prognostic factor for overall survival in patients on maintenance hemodialysis: a single-center retrospective study.

BACKGROUND: We investigated whether butyrylcholinesterase (BChE) was independently related to the overall survival (OS) of patients on maintenance hemodialysis (MHD). METHODS: Baseline information, serum BChE level, and other laboratory data were collected from 295 patients on MHD in a single HD hospital in 2018. We retrospectively investigated the mortality of these patients after 38 months. We assessed the prognostic markers such as the Geriatric Nutritional Risk Index (GNRI), Erythropoiesis Resistance Index (ERI), and Simplified Creatinine Index (SCI) of each patient. The primary objective was to examine the impact of BChE on OS. The secondary objective included the designation of a risk score in predicting the OS. RESULTS: We evaluated 284 patients. The median value of the serum BChE level was 206 IU/L. Of 284 patients evaluated, eighty-six patients died; all had a higher ERI and a lower serum BChE level, SCI, and GNRI than the surviving patients. The optimal cutoff values of the BChE level, GNRI, ERI, and SCI for OS were 166 IU/L, 90.0, 8.00, and 20.6, respectively. The multivariate Cox regression analysis showed that the age, HD vintage, dialysis dose, GNRI of < 90.0, and serum BChE level of < 166 IU/L (hazard ratio, 2.03; P = 0.003) were the independent prognostic factors. We designed a risk score consisting of the GNRI and serum BChE level. The predictive value of our risk score was superior to that of GNRI alone. CONCLUSION: The serum BChE level could be an independent prognostic factor for patients on MHD.

Association between female urinary urgency and aponeurotic ptosis: Results from a community-based cross-sectional study in Japan.

AIMS: To investigate the relationship between urinary urgency (UU) and aponeurotic ptosis (AP) in a health promotion project. METHODS: This cross-sectional study analyzed 658 women in Aomori, Japan. The presence of UU was evaluated using the Overactive Bladder Symptom Score. The distance from the light reflex on the cornea to the upper eyelid (margin reflex distance-1 [MRD-1]) was measured. AP was defined as MRD-1 of less than 2.0 mm. The daily salt intake amount was estimated using the dietary questionnaire. Daily salt intake was defined as excessive if it was 10 g/day or higher. Independent factors of UU and AP were analyzed via multivariable logistic regression model. RESULTS: The number of women with UU and AP was 193 and 65, respectively. Similar background differences were observed in age, cardiovascular disease history, renal function, hypertension, diabetes mellitus, dyslipidemia, and daily salt intake between participants with UU and those with AP. Participants with UU had a higher prevalence of AP (19% vs. 6.2%) than those without. Moreover, more than 50% of the women with AP experienced UU. Multivariable logistic analysis on UU showed that age (≥65 years), diabetes mellitus, daily salt intake (≥10 g/day), and AP (odds ratio, 2.07, p = .014) were independent factors. Multivariable analysis on AP revealed that age (≥65 years), hypertension, daily salt intake (≥10 g/day), and UU were independent factors. CONCLUSIONS: AP was an independent indicator of UU, in addition to excessive daily salt intake. Women with AP may tend to intake excessive salt and experience UU.

Quantitative monitoring of circulating tumor DNA in patients with advanced pancreatic cancer undergoing chemotherapy.

According to cancer genome sequences, more than 90% of cases of pancreatic ductal adenocarcinoma (PDAC) harbor active KRAS mutations. Digital PCR (dPCR) enables accurate detection and quantification of rare mutations. We assessed the dynamics of circulating tumor DNA (ct-DNA) in patients with advanced PDAC undergoing chemotherapy using dPCR. KRAS G12/13 mutation was assayed by dPCR in 47 paired tissue- and ct-DNA samples. The 21 patients were subjected to quantitative ct-DNA monitoring at 4 to 8-week intervals during chemotherapy. KRAS mutation was detected in 45 of those 47 patients using tissue DNA. In the KRAS mutation-negative cases, next-generation sequencing revealed KRAS Q61K and NRAS Q61R mutations. KRAS mutation was detected in 23/45 cases using ct-DNA (liver or lung metastasis, 18/19; mutation allele frequency [MAF], 0.1%-31.7%; peritoneal metastasis, 3/9 [0.1%], locally advanced, 2/17 [0.1%-0.2%]). In the ct-DNA monitoring, the MAF value changed in concordance with the disease state. In the 6 locally advanced cases, KRAS mutation appeared concurrently with liver metastasis. Among the 6 cases with liver metastasis, KRAS mutation disappeared during the duration of stable disease or a partial response, and reappeared at the time of progressive disease. The median progression-free survival was longer in cases in which KRAS mutation disappeared after an initial course of chemotherapy than in those in which it was continuously detected (248.5 vs 50 days, P < .001). Therefore, ct-DNA monitoring enables continuous assessment of disease state and could have prognostic utility during chemotherapy.

Photoinduced electron transfer detection method for identifying UGT1A1*28 microsatellites.

During development of a novel detection method for the UDP-glucuronosyl transferase 1A1 (UGT1A1)*28, the fluorescence intensity of a dye conjugated to cytosine (C) at the end of a DNA strand decreased upon hybridization with guanine (G). This phenomenon is referred to as photoinduced electron transfer (PeT). Using this phenomenon, we devised a method for the naked-eye detection of UGT1A1*28 (thymine-adenine (TA)-repeat polymorphism). Fluorescently labeled single-stranded DNA (ssDNA) oligonucleotides (probes) were designed and hybridized with complementary strand DNAs (target DNAs). Base pair formation at the blunt end between fluorescently labeled C (probe side) and G (target side), induced dramatic fluorescence quenching. Additionally, when the labeled-CG pair formed near the TA-repeat sequence, different TA-repeat numbers were discriminated. However, obtaining enough target DNA for this probe by typical polymerase chain reaction (PCR) was difficult. To enable the practical use of the probe, producing sufficient target DNA remains problematic.