Effect of herbal irrigants on surface roughness of intraradicular dentin using quantitative method of 3D surface texture analysis.

Replacing the conventional endodontic irrigants with herbal agents could avoid complications associated with using sodium hypochlorite (NaOCl). Endodontic irrigants alter the surface roughness of the dentinal wall surface, which affects sealer mechanical retention. This study aimed to assess the effect of experimental herbal Moringa oleifera and orange peel extract irrigant on intraradicular dentin (IRD) surface roughness using quantitative 3D surface analysis by scanning electron microscopy (SEM) regarding the smear layer assessment. Sixty human root sections were divided into four groups (n = 15): NaOCl combined with 17% ethylenediaminetetraacetic acid (EDTA); negative control (saline); moringa extract (MO); and orange oil (OO). SEM images were assessed quantitatively for surface roughness (Ra) in the coronal, middle, and apical IRD. The data were analysed by Kruskal-Wallis, Friedman, and Dunn's tests. All groups showed statistically significant differences (P = 0.007). MO exhibited significantly greater Ra values at the coronal, middle, and apical root levels than OO (P = 0.007, 0.009, and 0.046, respectively). There was no significant change in Ra values at various root levels within each group at P = 0.091, 0.819, 0.819, and 0.549 for the EDTA, saline, MO, and OO groups. Considerable (IRD) surface roughness analysis makes Moringa extract a promising herbal endodontic irrigant alternative to the NaOCl plus EDTA regimen.

Comprehensive Investigation of [Fe(EDTA)]--Functionalized Derivatives and their Supramolecular Adducts with Human Serum Albumin.

In recent years, the coordination chemistry of high-spin Fe(III) complexes has increasingly attracted interest due to their potential as effective alternatives to Gd(III)-based MRI contrast agents. This paper discusses the results from our study on Fe(III) complexes with two EDTA derivatives, each modified with either one (EDTA-BOM) or two (EDTA-BOM2) benzyloxymethylene (BOM) groups on the acetic arm(s). These pendant hydrophobic groups enable the complexes to form noncovalent adducts with human serum albumin (HSA), leading to an observed increase in relaxivity due to the reduction in molecular tumbling. Our research involved detailed relaxometric measurements and analyses of both 1H and 17O NMR data at varying temperatures and magnetic field strengths, which is conducted with and without the presence of a protein. A significant finding of this study is the effect of electronic relaxation time on the effectiveness of [Fe(EDTA-BOM)(H2O)]- and [Fe(EDTA-BOM2)(H2O)]- as diagnostic MRI probes. By integrating these relaxometric results with comprehensive thermodynamic, kinetic, and electrochemical data, we have thoroughly characterized how structural modifications to the EDTA base ligand influence the properties of the complexes.

Efficacy of root-end filling techniques using premixed putty type bioceramic cements: an ex vivo study.

OBJECTIVES: Currently, premixed putty-type bioceramic cements (PPBCs) have become popular materials for root-end fillings. This study investigated three root-end filling techniques using PPBCs and calcium silicate-based sealers including EDTA pretreatment. MATERIALS AND METHODS: Ninety root segments were prepared and standardized with an artificial fin and lateral canal, and assigned to three groups (n = 30). Root-end fillings were placed using BC-RRM Putty alone (Group PA), injection of BC sealer followed by BC-RRM Putty (Lid Technique: Group LT) or BC-RRM Putty with BC sealer coating (Deep putty packing technique: Group DP). Half of each group was pretreated with 17% EDTA. The radiographic images of the specimens were assessed by five graders and push-out bond strength tests were conducted. The data were analyzed with a general linear model including two-way ANOVA and chi-square test at a significance level of 5%. RESULTS: DP approach demonstrated significantly higher bond strength than LT (P < 0.05). However, there was no statistically significant difference in bond strength between PA and either DP or LT. EDTA pretreatment had no significant effect on push-out bond strength. Radiographically, for the main canal, PA and DP scored significantly higher than LT. In the fin, PA scored significantly higher than others (P < 0.05). CONCLUSION: Our study highlights variations in root-end filling techniques. Injecting a bulk of bioceramic sealer before the placement of PPBCs may reduce bond strength and radiopacity. The application of PPBCs alone or in the deep putty technique demonstrates potential for favorable outcomes. EDTA pretreatment did not enhance bond-strength. CLINICAL RELEVANCE: Careful selection and application of bioceramic materials and techniques in root-end fillings may influence the outcome of endodontic root-end surgery. When PPBCs and calcium silicate-based sealers are used together for root-end fillings, sealer followed by deep putty application may offer improved bond strength and radiographic fill compared to the lid technique.

Hydrothermally Stimulated Molecular Interfaces for Augmented Electron Delocalization in Wet-Chemical Phosphorus Recovery from Incineration Ash of Sewage Sludge.

Wet-chemically recovering phosphorus (P) from sewage sludge incineration ash (SSIA) has already become a global initiative to address P deficit, but effectively isolating P from these accompanying metals (AMs) through adsorption in a SSIA-derived extract remains elusive. Here, we devised a hydrothermal stimulus-motivated thermodynamic and kinetic enhancement to gain anionic ethylenediaminetetraacetic acid (EDTA) molecular interfaces for AM enclosure to resolve this conundrum. A new dosage rule based on the EDTA coordination ratio with AMs was established for the first time. Upon hydrothermal extraction at 140 °C for 1 h, the P extraction efficiency reached 96.7% or higher for these obtained SSIA samples, and then exceptional P sequestration from these EDTA-chelated AMs was realized by the peculiar lanthanum (La)-based nanoadsorbent (having 188.86 mg P/g adsorbent at pH ∼ 3.0). Relevant theoretical calculations unraveled that these delocalized electrons of tetravalent EDTA molecules boosted the enclosure of liberated AMs, thereby entailing a substantially increased negative adsorption energy (-408.7 kcal/mol) of P in the form of H2PO4- through intruding lattice-edged carbonates to coordinate La with monodentate mononuclear over LaCO5(1 0 1). This work highlights the prospect of molecular adaptation of these common extractants in wet-chemical P recovery from various P-included wastes, further sustaining global P circularity.

Dual- or single rinse? The tubular sealer penetration of endodontic chelating agents.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Novel [68Ga/177Lu]Ga/Lu-AZ-093 as PSMA-Targeting Agent for Diagnosis and Radiotherapy.

Prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer cells can serve as a target for imaging and radioligand therapy (RLT). Previously, [68Ga]Ga-P16-093, containing a Ga(III) chelator, N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), displayed excellent PSMA-targeting properties and showed a high tumor uptake and retention useful for diagnosis in prostate cancer patients. Recently, [177Lu]Lu-PSMA-617 has been approved by the U.S. food and drug administration (FDA) for the treatment of prostate cancer patients. Derivatives of PSMA-093 using AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), as the chelator, were designed as alternative agents forming complexes with both diagnostic and therapeutic radiometals, such as gallium-68 (log K = 22.18) or lutetium-177 (log K = 21.85). The aim of this study is to evaluate AAZTA-Gly-O-(methylcarboxy)-Tyr-Phe-Lys-NH-CO-NH-Glu (designated as AZ-093, 1) leading to a gallium-68/lutetium-177 theranostic pair as potential PSMA targeting agents. Synthesis of the desired precursor, AZ-093, 1, was effectively accomplished. Labeling with either [68Ga]GaCl3 or [177Lu]LuCl3 in a sodium acetate buffer solution (pH 4-5) at 50 °C in 5 to 15 min produced either [68Ga]Ga-1 or [177Lu]Lu-1 with high yields and excellent radiochemical purities. Results of in vitro binding studies, cell uptake, and retention (using PSMA-positive prostate carcinoma cells line, 22Rv1-FOLH1-oe) were comparable to that of [68Ga]Ga-P16-093 and [177Lu]Lu-PSMA-617, respectively. Specific cellular uptake was determined with or without the competitive blocking agent (2 µM of "cold" PSMA-11). Cellular binding and internalization showed a time-dependent increase over 2 h at 37 °C in the PSMA-positive cells. The cell uptakes were completely blocked by the "cold" PSMA-11 suggesting that they are competing for the same PSMA binding sites. In the mouse model with implanted PSMA-positive tumor cells, both [68Ga]Ga-1 and [177Lu]Lu-1 displayed excellent uptake and retention in the tumor. Results indicate that [68Ga]Ga/[177Lu]Lu-1 (68Ga]Ga/[177Lu]Lu-AZ-093) is potentially useful as PSMA-targeting agent for both diagnosis and radiotherapy of prostate cancer.

Assessing metal extraction from metalliferous waste: A study using deep eutectic solvents and chelating agents vs. ethylenediaminetetraacetic acid.

Conventional methods of metal recovery involving solvents have raised environmental concerns. To address these concerns and promote sustainable resource recovery, we explored the use of deep eutectic solvents (DES) and chelating agents (CA) as more environmentally friendly alternatives. Goethite and blast oxide slag dust (BOS-D) from heap piles at their respective sites and characterised via ICP-MS. The greatest extraction of critical metals was from goethite, removing 38% of all metals compared to 21% from the blast oxide slag. Among the tested CA, nitrilotriacetic acid (NTA) was the most effective, while for DES, choline chloride ethylene glycol (ChCl-EG) demonstrated superior performance in extracting metals from both blast oxide slag dust and goethite. The study further highlighted the selectivity for transition metals and metalloids was influenced by the carboxyl groups of DES. Alkaline metals and rare earth lanthanides extractions were favoured with DES due to improved mass transfer and increased denticity, respectively. In comparison to ethylenediaminetetraacetic acid (EDTA), typically used for metal extraction, CA and DES showed comparable extraction efficiency for Fe, Cu, Pb, Li, Al, Mn, and Ni. Using these greener chelators and solvents for metal extraction show significant promise in enhancing the sustainability of solvometallurgy. Additional conditions e.g., temperature and agitation combined with a cascading leaching process could further enhance metal extraction potential.

Dystrophic calcinosis: structural and morphological composition, and evaluation of ethylenediaminetetraacetic acid ('EDTA') for potential local treatment.

BACKGROUND: To perform a detailed morphological analysis of the inorganic portion of two different clinical presentations of calcium-based deposits retrieved from subjects with SSc and identify a chemical dissolution of these deposits suitable for clinical use. METHODS: Chemical analysis using Fourier Transform IR spectroscopy ('FTIR'), Raman microscopy, Powder X-Ray Diffraction ('PXRD'), and Transmission Electron Microscopy ('TEM') was undertaken of two distinct types of calcinosis deposits: paste and stone. Calcinosis sample titration with ethylenediaminetetraacetic acid ('EDTA') assessed the concentration at which the EDTA dissolved the calcinosis deposits in vitro. RESULTS: FTIR spectra of the samples displayed peaks characteristic of hydroxyapatite, where signals attributable to the phosphate and carbonate ions were all identified. Polymorph characterization using Raman spectra were identical to a hydroxyapatite reference while the PXRD and electron diffraction patterns conclusively identified the mineral present as hydroxyapatite. TEM analysis showed differences of morphology between the samples. Rounded particles from stone samples were up to a few micron in size, while needle-like crystals from paste samples reached up to 0.5 µm in length. Calcium phosphate deposits were effectively dissolved with 3% aqueous solutions of EDTA, in vitro. Complete dissolution of both types of deposit was achieved in approximately 30 min using a molar ratio of EDTA/HAp of ≈ 300. CONCLUSIONS: Stone and paste calcium-based deposits both comprise hydroxyapatite, but the constituent crystals vary in size and morphology. Hydroxyapatite is the only crystalline polymorph present in the SSc-related calcinosis deposits. Hydroxyapatite can be dissolved in vitro using a dosage of EDTA considered safe for clinical application. Further research is required to establish the optimal medium to develop the medical product, determine the protocol for clinical application, and to assess the effectiveness of EDTA for local treatment of dystrophic calcinosis.

Highly sensitive and accurate measurement of underivatized phosphoenolpyruvate in plasma and serum via EDTA-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate quantification of PEP in plasma or serum poses a significant challenge owing to its strong polarity and metal affinity. In this study, a sensitive method for the direct determination of PEP in plasma and serum based on ethylenediaminetetraacetic acid (EDTA)-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed. Superior chromatographic retention and peak shapes were achieved using a zwitterionic stationary-phase HILIC column with a metal-inert inner surface. Efficient dechelation of PEP-metal complexes in serum/plasma samples was achieved through the introduction of EDTA, resulting in a significant enhancement of the PEP signal. A PEP isotopically labelled standard was employed as a surrogate analyte for the determination of endogenous PEP, and validation assessments proved the sensitivity, selectivity, and reproducibility of this method. The method was applied to the comparative quantification of PEP in plasma and serum samples from mice and rats, as well as in HepG2 cells, HEK293T cells, and erythrocytes; the results confirmed its applicability in PEP-related biomedical research. The developed method can quantify PEP in diverse biological matrices, providing a feasible opportunity to investigate the role of PEP in relevant biomedical research.

Photodynamic Inactivation in agriculture: combating fungal phytopathogens resistant to conventional treatment.

Botrytis cinerea is a severe threat in agriculture, as it can infect over 200 different crop species with gray mold affecting food yields and quality. The conventional treatment using fungicides lead to emerging resistance over the past decades. Here, we introduce Photodynamic Inactivation (PDI) as a strategy to combat B. cinerea infections, independent of fungicide resistance. PDI uses photoactive compounds, which upon illumination create reactive oxygen species toxic for killing target organisms. This study focuses on different formulations of sodium-magnesium-chlorophyllin (Chl, food additive E140) as photoactive compound in combination with EDTA disodium salt dihydrate (Na2EDTA) as cell-wall permeabilizer and a surfactant. In an in vitro experiment, three different photosensitizers (PS) with varying Chl and Na2EDTA concentrations were tested against five B. cinerea strains with different resistance mechanisms. We showed that all B. cinerea mycelial spheres of all tested strains were eradicated with concentrations as low as 224 µM Chl and 3.076 mM Na2EDTA (LED illumination with main wavelength of 395 nm, radiant exposure 106 J cm-2). To further test PDI as a Botrytis treatment strategy in agriculture a greenhouse trial was performed on B. cinerea infected bell pepper plants (Capsicum annum L). Two different rates (560 or 1120 g Ha-1) of PS formulation (0.204 M Chl and 1.279 M Na2EDTA) and a combination of PS formulation with 0.05% of the surfactant BRIJ L4 (560 g Ha-1) were applied weekly for 4 weeks by spray application. Foliar lesions, percentage of leaves affected, percentage of leaf area diseased and AUDPC were significantly reduced, while percentage of marketable plants were increased by all treatments compared to a water treated control, however, did not statistically differ from each other. No phytotoxicity was observed in any treatment. These results add to the proposition of employing PDI with the naturally sourced PS Chl in agricultural settings aimed at controlling B. cinerea disease. This approach seems to be effective regardless of the evolving resistance mechanisms observed in response to conventional antifungal treatments.

Hypertonic saline- and detergent-accelerated EDTA-based decalcification better preserves mRNA of bones.

Ethylenediaminetetraacetic acid (EDTA), a classically used chelating agent of decalcification, maintains good morphological details, but its slow decalcification limits its wider applications. Many procedures have been reported to accelerate EDTA-based decalcification, involving temperature, concentration, sonication, agitation, vacuum, microwave, or combination. However, these procedures, concentrating on purely tissue-outside physical factors to increase the chemical diffusion, do not enable EDTA to exert its full capacity due to tissue intrinsic chemical resistances around the diffusion passage. The resistances, such as tissue inner lipids and electric charges, impede the penetration of EDTA. We hypothesized that delipidation and shielding electric charges would accelerate EDTA-based penetration and the subsequent decalcification. The hypothesis was verified by the observation of speedy penetration of EDTA with additives of detergents and hypertonic saline, testing on tissue-mimicking gels of collagen and adult mouse bones. Using a 26% EDTA mixture with the additives at 45°C, a conventional 7-day decalcification of adult mouse ankle joints could be completed within 24 h while the tissue morphological structure, antigenicity, enzymes, and DNA were well preserved, and mRNA better retained compared to using 15% EDTA at room temperature. The addition of hypertonic saline and detergents to EDTA decalcification is a simple, rapid, and inexpensive method that doesn't disrupt the current histological workflow. This method is equally or even more effective than the currently most used decalcification methods in preserving the morphological details of tissues. It can be highly beneficial for the related community.

Impact of anti-coagulant choice on blood elongational behavior.

Blood is a highly complex fluid with rheological properties that have a significant impact on various flow phenomena. In particular, it exhibits a non-Newtonian elongational viscosity that is comparable to polymer solutions. In this study, we investigate the effect of three different anticoagulants, namely EDTA (ethylene diamine tetraacetic acid), heparin, and citrate, on the elongational properties of both human and swine blood. We observe a unique two stage thinning process and a strong dependency of the characteristic relaxation time on the chosen anticoagulant, with the longest relaxation time and thus the highest elongational viscosity being found for the case of citrate. Our findings for the latter are consistent with the physiological values obtained from a dripping droplet of human blood without any anticoagulant. Furthermore, our study resolves the discrepancy found in the literature regarding the reported range of characteristic relaxation times, confirming that the elongational viscosity must be taken into account for a full rheological characterization of blood. These results have important implications for understanding blood flow in various physiological, pathological and technological conditions.

EDTA enables to alleviate impacts of metal ions on conjugative transfer of antibiotic resistance genes.

Various heavy metals are reported to be able to accelerate horizontal transfer of antibiotic resistance genes (ARGs). In real water environmental settings, ubiquitous complexing agents would affect the environmental behaviors of heavy metal ions due to the formation of metal-organic complexes. However, little is known whether the presence of complexing agents would change horizontal gene transfer due to heavy metal exposure. This study aimed to fill this gap by investigating the impacts of a typical complexing agent ethylenediaminetetraacetic acid (EDTA) on the conjugative transfer of plasmid-mediated ARGs induced by a range of heavy metal ions. At the environmentally relevant concentration (0.64 mg L-1) of metal ions, all the tested metal ions (Mg2+, Ca2+, Co2+, Pb2+, Ni2+, Cu2+, and Fe3+) promoted conjugative transfer of ARGs, while an inhibitory effect was observed at a relatively higher concentration (3.20 mg L-1). In contrast, EDTA (0.64 mg L-1) alleviated the effects of metal ions on ARGs conjugation transfer, evidenced by 11 %-66 % reduction in the conjugate transfer frequency. Molecular docking and dynamics simulations disclosed that this is attributed to the stronger binding of metal ions with the lipids in cell membranes. Under metal-EDTA exposure, gene expressions related to oxidative stress response, cell membrane permeability, intercellular contact, energy driving force, mobilization, and channels of plasmid transfer were suppressed compared with the metal ions exposure. This study offers insights into the alleviation mechanisms of complexing agents on ARGs transfer induced by free metal ions.

Iron nanoparticles in combination with other conventional Fe sources remediate mercury toxicity-affected plants and soils by nutrient accumulation in bamboo species.

The issue of mercury (Hg) toxicity has recently been identified as a significant environmental concern, with the potential to impede plant growth in forested and agricultural areas. Conversely, recent reports have indicated that Fe, may play a role in alleviating HM toxicity in plants. Therefore, this study's objective is to examine the potential of iron nanoparticles (Fe NPs) and various sources of Fe, particularly iron sulfate (Fe SO4 or Fe S) and iron-ethylene diamine tetra acetic acid (Fe - EDTA or Fe C), either individually or in combination, to mitigate the toxic effects of Hg on Pleioblastus pygmaeus. Involved mechanisms in the reduction of Hg toxicity in one-year bamboo species by Fe NPs, and by various Fe sources were introduced by a controlled greenhouse experiment. While 80 mg/L Hg significantly reduced plant growth and biomass (shoot dry weight (36%), root dry weight (31%), and shoot length (31%) and plant tolerance (34%) in comparison with control treatments, 60 mg/L Fe NPs and conventional sources of Fe increased proline accumulation (32%), antioxidant metabolism (21%), polyamines (114%), photosynthetic pigments (59%), as well as root dry weight (25%), and shoot dry weight (22%), and shoot length (22%). Fe NPs, Fe S, and Fe C in plant systems substantially enhanced tolerance to Hg toxicity (23%). This improvement was attributed to increased leaf-relative water content (39%), enhanced nutrient availability (50%), improved antioxidant capacity (34%), and reduced Hg translocation (6%) and accumulation (31%) in plant organs. Applying Fe NPs alone or in conjunction with a mixture of Fe C and Fe S can most efficiently improve bamboo plants' tolerance to Hg toxicity. The highest efficiency in increasing biochemical and physiological indexes under Hg, was related to the treatments of Fe NPs as well as Fe NPs + FeS + FeC. Thus, Fe NPs and other Fe sources might be effective options to remove toxicity from plants and soil. The future perspective may help establish mechanisms to regulate environmental toxicity and human health progressions.

Cryo-storage of porcine hides at the industrial scale for tissue engineering and regenerative medicine application.

BACKGROUND: The industrial scale cryo-storage of raw tissue materials requires a robust, low-cost and easy-to-operate method that can facilitate the down-stream process. OBJECTIVE: The study was aimed to develop the multifunctional protective solutions (MPS) for transportation at ambient conditions and also subsequent cryo-storage below -20 degree C of raw porcine hides for tissue engineering and regenerative medicine. MATERIALS AND METHODS: Protective solutions with antimicrobial activity and proteinase-inhibiting activity were developed and tested for its efficacy in preserving the extracellular matrix of porcine dermis from microbial spoilage, proteolytic degradation, freeze damage and excessive dehydration during shipping and cryo-storage. The MPSs contained phosphate-buffered saline with ethylene diamine tetra acetic acid (EDTA) added as chelator and proteinase inhibitor, as well as glycerol or maltodextrin (M180) as cryoprotectants. RESULTS: MPSs prepared with EDTA and glycerol or M180 had significant antimicrobial activity and proteinase-inhibiting activity during the period of shipping and handling. Glycerol and M180 prevented eutectic salt precipitation and excessive freeze dehydration upon cryo-storage of porcine hides. Without glycerol or M180, hides could be freeze-dehydrated to the low hydration at ~0.4 g/g dw, and formed irreversible plications after freezing. A critical hydration (0.8~0.9 g/g dw) was observed for the extracellular matrix of porcine dermis, and dehydration to a lower level could impose enormous stress and potential damage. The soaking of porcine hides in MPSs decreased water content as glycerol and M180 entered into dermis. Upon equilibration, the glycerol content in the tissue was about 94% of the incubating glycerol solution, but the M180 content in the tissue was only about 50% of the incubating M180 solution, indicating that M180 did not get into the entire aqueous domain within dermis. MPSs reduced ice formation and increased the unfrozen water content of porcine raw hides upon cryo-storage. CONCLUSION: MPSs prepared with EDTA and glycerol or M180 have antimicrobial activity and proteinase-inhibiting activity, which can be used for transportation and cryo-storage of raw hides at the industrial scale. Glycerol at 7.5% w/v and M180 at 20% w/v were sufficient to prevent freeze damage and excessive freeze dehydration. Doi.org/10.54680/fr24310110312.

Novel EDTA mediated ethanol protein precipitation method and the application for polysorbate quantification in high protein concentration biopharmaceuticals.

Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10-250 mg/mL).

Characterization of unique EDTA-insensitive methylthioalkylmalate synthase from Eutrema japonicum and its potential application in synthetic biology.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum, commonly referred to as Japanese wasabi. Eutremajaponicum MAMs (EjMAMs) were expressed in an Escherichiacoli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l-methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform.

Effective oxidation decomplexation of Cu-EDTA and Cu2+ electrodeposition from PCB manufacturing wastewater by persulfate-based electrochemical oxidation: Performance and mechanisms.

Complex wastewater matrices such as printed circuit board (PCB) manufacturing wastewater present a major environmental concern. In this work, simultaneous decomplexation of metal complex Cu-EDTA and reduction/electrodeposition of Cu2+ was conducted in a persulfate-based electrochemical oxidation system. Oxidizing/reductive species were simultaneously produced in this system, which realized 99.8% of Cu-EDTA decomplexation, 94.5% of Cu2+ reduction/electrodeposition under the conditions of original solution pH = 3.2, electrode distance = 3 cm, [Na2S2O8]0 = 5 mM, current density = 12 mA/cm2, and reaction time = 180 min. The total treatment cost is as low as 0.80 USD/mol Cu-EDTA. Effective mineralization (74.1% total organic carbon removal) of the solution was obtained after 3 h of treatment. •OH and SO4•- drove the Cu-EDTA decomplexation, destroying the chelating sites and finally it was effectively mineralized to CO2, H2O and Cu2+. The mechanisms of copper electrodeposition on the stainless steel cathode and persulfate activation by the BDD anode were proposed based on the electrochemical measurements. The electrodes exhibited excellent reusability and low metal (total iron and Ni2+) leaching during 20 cycles of application. This study provide an effective and sustainable method for the application of the electro-persulfate process in treating complex wastewater matrices.

Improvement of performance for flexible film dosimeter by incorporating additive agents.

Objective.To evaluate the reduction in energy dependence and aging effect of the lithium salt of pentacosa-10,-12-diynoic acid (LiPCDA) films with additives including aluminum oxide (Al2O3), propyl gallate (PG), and disodium ethylenediaminetetracetate (EDTA).Approach. LiPCDA films exhibited energy dependence on kilovoltage (kV) and megavoltage (MV) photon energies and experienced deterioration over time. Evaluations were conducted with added Al2O3and antioxidants to mitigate these issues, and films were produced with and without Al2O3to assess energy dependence. The films were irradiated at doses of 0, 3, 6, and 12 cGy at photon energies of 75 kV, 105 kV, 6 MV, 10 MV, and 15 MV. For the energy range of 75 kV to 15 MV, the mean and standard deviation (std) were calculated and compared for the values normalized to the net optical density (netOD) at 6 MV, corresponding to identical dose levels. To evaluate the aging effect, PG and disodium EDTA were incorporated into the films: sample C with 1% PG, sample D with 2% PG, sample E with 0.62% disodium EDTA added to sample D, and sample F with 1.23% disodium EDTA added to sample D.Main results. Films containing Al2O3demonstrated a maximum 15.8% increase in mean normalized values and a 15.1% reduction in std, reflecting a greater netOD reduction at kV than MV energies, which indicates less energy dependence in these films. When the OD of sample 1-4 depending on the addition of PG and disodium EDTA, was observed for 20 weeks, the transmission mode decreased by 8.7%, 8.3%, 29.3%, and 27.3%, respectively, while the reflection mode was 5.4%, 3.0%, 37.0%, and 34.5%, respectively.Significance. Al2O3effectively reduced the voltage and MV energy dependence. PG was more effective than disodium EDTA in preventing the deterioration of film performance owing to the aging effect.

Comprehensive chromatographic assessment of forced degraded in vitro transcribed mRNA.

Heightened interest in messenger RNA (mRNA) therapeutics has accelerated the need for analytical methodologies that facilitate the production of supplies for clinical trials. Forced degradation studies are routinely conducted to provide an understanding of potential weak spots in the molecule that are exploited by stresses encountered during bulk purification, production, shipment, and storage. Consequently, temperature fluctuations and excursions are often experienced during these unit operations and may accelerate mRNA degradation. Here, we present a concise panel of chromatography-based stability-indicating assays for evaluating thermally stressed in vitro transcribed (IVT) mRNA as part of a forced degradation study. We found that addition of EDTA to the mRNAs prior to heat exposure reduced the extent of degradation, suggesting that transcripts may be fragmenting via a divalent metal-ion mediated pathway. Trace divalent metal contamination that can accelerate RNA instability is likely carried over from upstream steps. We demonstrate the application of these methods to evaluate the critical quality attributes (CQAs) of mRNAs as well as to detect intrinsic process- and product-related impurities.