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Ethylenediaminetetraacetic acid (EDTA) is a divalent cation chelator and chemical preservative that has been shown to be the active ingredient of the popular DNA preservative DESS. EDTA may act to reduce DNA degradation during tissue storage by sequestering divalent cations that are required by nucleases naturally occurring in animal tissues. Although EDTA is typically used between pH 7.5 and 8 in preservative preparations, the capacity of EDTA to chelate divalent cations is known to increase with increasing pH. Therefore, increasing the pH of EDTA-containing preservative solutions may improve their effectiveness as DNA preservatives. To test this hypothesis, we stored tissues from five aquatic species in 0.25 M EDTA adjusted to pH 8, 9, and 10 for 12 months at room temperature before DNA isolation. For comparison, tissues from the same specimens were also stored in 95% ethanol. DNA extractions performed on tissues preserved in EDTA pH 9 or 10 resulted in as great or greater percent recovery of high molecular weight DNA than did extractions from tissues stored at pH 8. In all cases examined, percent recovery of high molecular weight DNA from tissues preserved in EDTA pH 10 was significantly better than that observed from tissues preserved in 95% ethanol. Our results support the conclusion that EDTA contributes to DNA preservation in tissues by chelating divalent cations and suggest that preservative performance can be improved by increasing the pH of EDTA-containing DNA preservative solutions.

Abstract Background Blood-based analysis of circulating tumor DNA (ctDNA) is a promising tool for cancer screening, monitoring relapse/recurrence and evaluating response to treatment. Although plasma is widely used to obtain ctDNA, biorepositories worldwide possess a huge number of serum samples and comparative studies on the use of serum vs plasma as ctDNA sources are essential. Methods We analyzed cell-free DNA (cfDNA) from matched EDTA-plasma and serum samples from healthy donors and patients with colorectal or lung cancer, and used targeted next-generation sequencing to evaluate mutation detection efficiency and reproducibility. Matched samples from healthy individuals were spiked with reference oligonucleotides and sequenced using the Ion-S5 Oncomine-Pan-Cancer panel. Detection efficiency in matched samples from patients with cancer was evaluated using 2 distinct gene panels and compared to mutations found in tissue-biopsy samples at diagnosis. Results Mean total cfDNA was 55% higher in serum samples and the presence of longer DNA fragments was significantly increased in serum compared with plasma samples (P = 0.0001 to 0.015). Spiked mutated nucleotides were detected in both samples, but allele frequencies (AF) were approximately half in serum compared with plasma, suggesting ctDNA from serum was more diluted by DNA of noncancerous origins. Matched samples from patients with cancer revealed that up to 44.8% of mutations with low AF were missed in serum samples and concordance rates with somatic mutations found in tissue biopsy at diagnosis was better in plasma samples. Conclusion The use of serum in retrospective studies should consider the limitations for detecting low AF mutations. Plasma is clearly preferable for prospective clinical applications of liquid biopsy.

Cryopreservation is the gold standard for preserving high molecular weight (HMW) DNA (>10 kb) in tissue samples. However, frozen tissues are typically thawed either before or during DNA extraction, which can lead to substantial DNA degradation. In this study, we thawed the previously frozen tissues of 10 marine species (five fishes and five invertebrates) in the preservatives EDTA (250 mM, pH 10) or ethanol (EtOH, 95%) and maintained them in their respective preservatives overnight at 4°C before DNA extraction. We then compared the recovery of HMW DNA in these extracts to extracts prepared directly from frozen tissues. To evaluate the effect of these treatments on HMW DNA recovery, we determined the percentage of high molecular weight DNA (%HMW) and yield of HMW DNA normalized by tissue weight (nY) in each DNA extract. The average %HMW values for eight of the 10 species and the average nY values for five of the 10 species were significantly higher in extracts from EDTA-treated tissues compared to extracts from untreated frozen tissues. For all 10 species, we observed no significant decreases in average %HMW or nY values in extracts of EDTA-thawed tissues compared to those extracted directly from frozen tissues. In contrast, EtOH treatment did not significantly improve the average %HMW or nY values in extracts from tissues of nine of the 10 species when compared to extracts prepared directly from frozen tissues. Therefore, investigators may consider EDTA treatment as a simple method for improving HMW DNA recovery from frozen tissues.

Porcine corneas were decellularized for future use in corneal regeneration by using various washing steps with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) detergent, Benzonase, and Ethylenediaminetetraacetic acid (EDTA). The quality of the decellularized corneas was assessed by quantitative and qualitative measurement of DNA content, glycosaminoglycans (GAGs), immunofluorescent staining for Collagen (Col) I, V, Keratocan, Fibronectin, Laminin, Lumican and Decorin, and Transmission Electron Microscopy (TEM) to observe the structure of the collagen fibrils was used. The decellularization process showed 99.5% DNA content reduction in the corneas and a similar pattern was observed in the preservation of GAGs. Hematoxylin & Eosin (H&E) and immunofluorescent staining showed no presence of cell nuclei, while Alcian blue staining confirmed the presence of GAGs. Col I, V, Keratocan, Fibronectin, Laminin, Lumican and Decorin were still present in the decellularized corneas and TEM microscopy further confirmed the similar patterns of the collagen fibrils in the decellularized, compared to the native corneas. This pilot study showed our method is effective in decellularizing porcine corneas, with a very high amount of DNA being removed, while the GAGs being preserved to an acceptable extent, and the structure and pattern of the collagen fibrils maintained.

This study aimed to compare the effectiveness of initial irrigation with sodium hypochlorite (NaOCl) and final irrigation with QMix, 40% citric acid, and 17% ethylenediaminetetraacetic acid (EDTA) on smear layer removal and dentin erosion. Forty extracted human mandibular premolar teeth were randomly divided into four groups (n = 10) according to the type of final irrigants used: 17% EDTA, QMix, citric acid, and control (normal saline). Canals were mechanically prepared using ProTaper Next instruments to an apical size of X3. Subsequently, the roots were sectioned in a buccolingual direction. Scanning electron microscopy (SEM) was used to assess the presence of the smear layer and the amount of dentin erosion in the coronal, middle, and apical thirds of the root canals. In regards to smear layer removal, there was a significant difference between the control group and the other tested groups. Moreover, it was significantly higher in the coronal and middle thirds than in the apical third. However, there were no significant differences between the groups of EDTA, QMix, and citric acid. Concerning dentin erosion, citric acid produced significantly more dentin erosion than the other tested groups. Final irrigation with solutions had a higher ability to remove the smear layer in the coronal and middle thirds compared to the apical third. Of all the solutions tested, 40% citric acid had the most pronounced impact on dentin erosion, followed by 17% EDTA and QMix.

Pollenkitt, a sticky substance produced by the tapetal cells of pollen grains, plays a crucial role in pollen functionality, yet it remains an understudied component in plant biology. In this study, we investigated the role of pollenkitt in onion ( Allium cepa ) pollen, focusing on its effects on pollen germination, DNase activity, fatty acid composition, and ultrastructure. Our findings reveal that pollenkitt is essential for successful onion pollen germination and tube growth on the stigma, as its removal significantly inhibited these processes. Additionally, we demonstrated that onion pollenkitt exhibits DNase activity, degrading plasmid DNA in a concentration-dependent manner, with EDTA effectively inhibiting the degradation. Gas chromatography identified 20 fatty acids in pollenkitt, with a predominance of unsaturated fatty acids. Using scanning electron microscopy (SEM), we showed structural differences between pollen grains with and without pollenkitt, and we observed the process of pollenkitt removal from the surface of pollen grains by water washing. These results offer valuable insights for future research aimed at optimizing pollen-mediated gene-editing systems and highlight the importance of considering pollenkitt in such applications.

Amblyomma sculptum is a species of tick in the family Ixodidae, with equids and capybaras among its preferred hosts. In this study, the acaricidal activity of the essential oil (EO) from Piper aduncum and its main component, Dillapiole, were evaluated against larvae of A. sculptum to establish lethal concentration values and assess the effects of these compounds on tick enzymes. Dillapiole exhibited slightly greater activity (LC[sub.50] = 3.38 mg/mL; 95% CI = 3.24 to 3.54) than P. aduncum EO (LC[sub.50] = 3.49 mg/mL; 95% CI = 3.36 to 3.62) against ticks. The activities of α-esterase (α-EST), β-esterase (β-EST), and glutathione-S-transferase (GST) enzymes in A. sculptum larvae treated with Dillapiole showed a significant increase compared to the control at all concentrations (LC[sub.5], LC[sub.25], LC[sub.50] and LC[sub.75]), similar results were obtained with P. aduncum EO, except for α-EST, which did not differ from the control at the highest concentration (LC[sub.75]). The results of the acetylcholinesterase (AChE) activity show an increase in enzyme activity at the two lower concentrations (LC[sub.5] and LC[sub.25]) and a reduction in activity at the two higher, lethal concentrations (LC[sub.50] and LC[sub.75]) compared to the control. These results suggest potential mechanisms of action for these natural acaricides and can provide guidance for the future development of potential plant-derived formulations.

In this study, we aimed to compare the effectiveness of various chelating agents, ethilenediaminetetraacetic acid (EDTA), citric acid (CA), and etidronic acid (HEDP) mixed in two different forms, in removing the smear layer and promoting the penetration of an endodontic sealer into the dentinal tubules of extracted single-rooted teeth. The study used 75 teeth divided into five groups: 17% EDTA, 10% CA, 9% HEDP + NaOCl, 9% HEDP + distilled water (DW), and a control (DW) group. Scanning electron microscopy was used to assess smear layer removal and confocal laser microscopy was used to evaluate tubular sealer penetration at different depths from the apical tip. Sealer penetration was highest with 17% EDTA and 10% CA as compared with the other agents (p<0.001). At the cervical third, the sealer penetration for EDTA, HEDP + NaOCl, and HEDP + DW groups were significantly different than those in DW (p = 0.020). For the middle third, EDTA, CA, and HEDP + NaOCl groups were significantly higher than those of the DW group (p<0.001). Cervical-level values were significantly higher than apical-level values for HEDP + NaOCl, HEDP + DW, and DW (p<0.001). Smear layer removal was lower with 9% HEDP + DW than with 17% EDTA and 10% CA at all depths (p<0.001). A significancy in smear layer removal was observed between 10% CA and control (p = 0.015) in middle depth. Within the limitations of this study, highest values were seen in EDTA and CA in terms of sealer penetration and smear layer removal. In the light of these findings, the use of strong chelating agents highlights better clinical efficiency than dual-rinse or single HEDP irrigation.

Leontopodium alpinum is a source of raw material for food additives and skin health. The purpose of this study was to investigate the application of Leontopodium alpinum callus culture extract (LACCE) to prevent blue light damage to the skin. We screened and identified the blue light-damage-protecting activities and mechanisms of ten components of LACCE, including chlorogenic acid (A), isoquercitrin (B), isochlorogenic acid A (C), cynaroside (D), syringin (E), isochlorogenic acid (F), cynarin (G), rutin (H), leontopodic acid A (I), and leontopodic acid B (J), using a novel blue light-induced human foreskin fibroblast (HFF-1) cell injury model. The study examined the cytotoxicity of ten ingredients using the cell counting kit-8 (CCK-8) assay, and selecting concentrations of 5, 10, and 20 μM for experiments with a cell viability above 65%. We explored the effects and mechanisms of action of these LACCE components in response to blue light injury using Western blotting and an enzyme-linked immunosorbent assay. We also measured ROS secretion and Ca[sup.2+] influx. Our study revealed that leontopodic acid A effectively boosted COI-1 expression, hindered MMP-1 expression, curbed ROS and Ca[sup.2+] endocytosis, and reduced OPN3 expression. These results provide theoretical support for the development of new raw materials for the pharmaceutical and skincare industries.

In this study, we developed gelatin-based films for active packaging with the ability to inhibit E . coli . We created these novel biodegradable gelatin-based films with a nisin-EDTA mix. FT-IR, TGA, and SEM analysis showed that nisin interacted with the gelatin by modifying its thermal stability and morphology. The use of nisin (2,500 IU/mL) with concentrations of Na-EDTA (1.052 M stock solution) distributed in the polymer matrix generated a significant decrease in the growth of E . coli when compared to the control. In freshly made films (t 0 ), the growth of E . coli ATCC 25922 was reduced by approximately 3 logarithmic cycles. Two weeks after the films were made, a reduction in antimicrobial activity was observed in approximately 1, 1 and 3 logarithmic cycles of the films with 5%, 10% and 20% of the compound (nisin/Na-EDTA) distributed in the polymer matrix, respectively. This evidences an antimicrobial effect over time. Also, biodegradation tests showed that the films were completely degraded after 10 days. With all these results, an active and biodegradable packaging was successfully obtained to be potentially applied in perishable foods. These biodegradable, gelatin-based films are a versatile active packaging option. Further research on the barrier properties of these films is needed.