Extraction of high quality and high yield RNA from frozen EDTA blood.

Peripheral blood RNA profiling, which can reveal systemic changes in gene expression and immune responses to disease onset and progression, is a powerful tool for diagnosis and biomarker discovery. This technique usually requires high quality RNA, which is only obtainable from fresh blood, or frozen blood that has been collected in special RNA-stabilisation systems. The current study aimed to develop a novel protocol to extract high quality RNA from frozen blood that had been collected in the conventional EDTA tubes. We determined that thawing EDTA blood in the presence of cell lysis/RNA stabilisation buffers (Paxgene or Nucleospin) significantly improved RNA quality (RIN) from below 5 to above 7, which to date has not been shown possible. The EDTA-Nucleospin protocol resulted in 5 times higher yield than the EDTA-Paxgene-PreAnalytix method. The average RIN and mRNA expression levels of five different genes including 18 s, ACTB, MCP1, TNFa and TXNIP using this protocol were also indifferent to those from Paxgene blood, suggesting similar RNA quality and blood transcriptome. Moreover, the protocol allows DNA to be extracted simultaneously. In conclusion, we have developed a practical and efficient protocol to extract high quality, high yield RNA from frozen EDTA blood.

Alkylated EDTA potentiates antibacterial photodynamic activity of protoporphyrin.

Antibiotic resistance has garnered significant attention due to the scarcity of new antibiotics in development. Protoporphyrin IX (PpIX)-mediated photodynamic therapy shows promise as a novel antibacterial strategy, serving as an alternative to antibiotics. However, the poor solubility of PpIX and its tendency to aggregate greatly hinder its photodynamic efficacy. In this study, we demonstrate that alkylated EDTA derivatives (aEDTA), particularly C14-EDTA, can enhance the solubility of PpIX by facilitating its dispersion in aqueous solutions. The combination of C14-EDTA and PpIX exhibits potent antibacterial activity against Staphylococcus aureus (S. aureus) when exposed to LED light irradiation. Furthermore, this combination effectively eradicates S. aureus biofilms, which are known to be strongly resistant to antibiotics, and demonstrates high therapeutic efficacy in an animal model of infected ulcers. Mechanistic studies reveal that C14-EDTA can disrupt PpIX crystallization, increase bacterial membrane permeability and sequester divalent cations, thereby improving the accumulation of PpIX in bacteria. This, in turn, enhances reactive oxygen species (ROS) production and the antibacterial photodynamic activity. Overall, this effective strategy holds great promise in combating antibiotic-resistant strains.

Effect of photodynamic therapy with 5-aminolevulinic acid and EDTA-2Na against mixed infection of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

BACKGROUND: The increasing abundance of drug-resistant bacteria is a global threat. Photodynamic therapy is an entirely new, non-invasive method for treating infections caused by antibiotic-resistant strains. We previously described the bactericidal effect of photodynamic therapy on infections caused by a single type of bacterium. We showed that gram-positive and gram-negative bacteria could be killed with 5-aminolevulic acid and 410 nm light, respectively. However, clinically, mixed infections are common and difficult to treat. OBJECTIVE: We investigated the bactericidal effects of photodynamic therapy on mixed infections of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. METHODS: We compared bacterial growth with and without photodynamic therapy in vitro. Then, in vivo, we studied mixed infections in a mouse skin ulcer model. We evaluated the rates of ulcer area reduction and transitions to healing in treated and untreated mice. In addition, a comparison was made between PDT and existing topical drugs. RESULTS: We found that photodynamic therapy markedly reduced the growth of both methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, in culture, and it reduced the skin ulcer areas in mice. PDT was also more effective than existing topical medicines. CONCLUSION: This study showed that photodynamic therapy had antibacterial effects against a mixed infection of gram-positive and gram-negative bacteria, and it promoted skin ulcer healing. These results suggested that photodynamic therapy could be effective in both single- and mixed-bacterial infections.

Effect of sodium hypochlorite, ethylenediaminetetraacetic acid, and dual-rinse irrigation on dentin adhesion using an etch-and-rinse or self-etch approach.

The aim was to investigate the influence of endodontic irrigation solutions and protocols on the micro-tensile bond strength (µTBS) to dentin using an etch-and-rinse (ER) or self-etch (SE) adhesive approach. Eighty extracted human molars were ground to dentin. After pretreating for 27 min (21 min-3 min-3 min) with five different endodontic irrigation protocols (Group 1: NaOCl-EDTA-NaOCl; Group 2: NaOCl-NaOCl-EDTA; Group 3: NaOCl-NaCl-NaOCl; Group 4: Dual Rinse-Dual Rinse-Dual Rinse; Group 5: NaCl-NaCl-NaCl), an ER (Optibond FL, Kerr) or a SE (Clearfil SE Bond, Kuraray) adhesive system was applied. After light-curing, composite build-ups were made and cut into dentin-composite sticks. µTBS and failure modes were analyzed. Nonparametric statistical analyses (α = 0.05) were performed for comparison of the five groups within each type of adhesive as well as between the two adhesive systems used. The use of an ER instead of a SE adhesive system resulted in significantly higher µTBS for all irrigation protocols except for group 1 (NaOCl-EDTA-NaOCl) and 2 (NaOCl-NaOCl-EDTA). A statistical difference between the five different endodontic irrigation protocols was only found within the SE adhesive group, where group 1 (NaOCl-EDTA-NaOCl) achieved highest values. The use of an ER adhesive system cancels out the effect of the endodontic irrigation solution. The highest µTBS was achieved when using a NaOCl-EDTA-NaOCl-irrigation protocol in combination with Clearfil SE Bond, which shows that the selection of the endodontic irrigation should match the corresponding SE adhesive system.

Multianticoagulant Pseudothrombocytopenia in a Patient with Primary Carcinoma of the Liver with Hypersplenism.

BACKGROUND: Pseudothrombocytopenia (PTCP) is a relatively rare phenomenon in vitro, the mechanism is not completely clear, and there is no unified solution for it. How to identify and solve PTCP accurately is a challenge for laboratory personnel. METHODS: According to the patient's clinical manifestations, thrombocytopenia caused by hypersplenism was excluded. PTCP was confirmed by platelet volume histograms, scattergrams and platelet clumps on the blood smears. Commonly used alternative anticoagulants such as sodium citrate or heparin were used for platelet counting. The corrective effect of the platelet count was not good, so non-anticoagulant blood was collected and tested immediately, and blood smears were used to count platelets manually. RESULTS: The PTCP of the patient could not be solved using sodium citrate and heparin anticoagulation. By collecting non-anticoagulant blood and testing immediately, the platelet count returned to normal (180 x 109/L), which is consistent with the results of manual counting on the patient's blood smears (175 x 109/L). CONCLUSIONS: When PTCP is confirmed, commonly used alternative anticoagulants can be used. If these do not work, non-anticoagulant blood can be collected and tested immediately, and blood smears can be used to count platelets manually.

Understanding boron toxicity in aquatic plants (Salvinia natans and Lemna minor) in the presence and absence of EDTA.

Even though boron is a widely used element in various industries and a contributor to water pollution worldwide, few studies have examined the toxicity of boron in aquatic plants. EDTA is used to maintain aquatic plants cultures, however it is possible to modify the toxicity of metals. The objective of this study is to assess the toxicity of boron in aquatic plants and explore the impact of EDTA presence on the resulting toxic responses. Floating watermoss Salvinia natans and duckweed Lemna minor were exposed to concentrations ranging from 5 to 100 mg/L for 7 days and 1 to 60 mg/L for 3 days, respectively. Growth and photosynthetic activity parameters were investigated in the presence and absence of EDTA. Growth inhibitions in both aquatic plants were observed in a concentration-dependent manner, irrespective of the presence or absence of EDTA. For instance, based on the specific growth rate (leaves coverage), EC10 values for S. natans were calculated as 12.7 (9.9-15.3) mg/L and 8.0 (5.8-10.3) mg/L with and without EDTA, respectively. In the case of L. minor, EC10 values were calculated as 1.3 (0.8-1.89) mg/L and 2.0 (0.4-4.3) mg/L with EDTA without EDTA, respectively. Significant effects were also observed on the photosynthetic capacity, however there was no change in the increase of boron concentration. Generally, negligible effects of EDTA to the toxicity of boron were observed in the present study. By comparing toxicity results based on the presence and absence of EDTA, which is an essential element in the test medium, the results of this study are expected to be utilized for the ecological risk assessment of boron in aquatic ecosystems.

COMPARING THE EFFECTS OF DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID AND LITHIUM HEPARIN ON HEMATOLOGIC VALUES IN BLANDING'S TURTLES (EMYDOIDEA BLANDINGII), PAINTED TURTLES (CHRYSEMYS PICTA), AND COMMON SNAPPING TURTLES (CHELYDRA SERPENTINA).

Hematology is a routine component of clinical management in veterinary patients. Anticoagulant choice can profoundly influence morphologic assessment of erythrocytes, leukocytes, thrombocytes, and their subsequent quantification. Previous chelonian studies suggest that lithium heparin (LH) is a superior anticoagulant due to hemolysis resulting from dipotassium ethylenediaminetetraacetic acid (dEDTA) in some species. The aim of this study was to compare the effects of dEDTA and LH on hematologic values in Blanding's turtles (Emydoidea blandingii, n = 35), painted turtles (Chrysemys picta, n = 34), and common snapping turtles (Chelydra serpentina, n = 36). We collected samples from free-ranging turtles and immediately divided whole blood into LH and dEDTA tubes. Packed cell volume, total solids, erythrocyte sedimentation rate, white blood cell counts, and differential leukocyte counts were determined. Hemolysis was observed macro- and microscopically in dEDTA samples from painted turtles and common snapping turtles. Packed cell volume and heterophil:lymphocyte was lower and erythrocyte sedimentation rate was higher in LH samples from painted turtles (p, 0.05). In snapping turtles, the PCV, number of monocytes, and number of eosinophils was lower in LH samples (p, 0.05). In Blanding's turtles, the number of eosinophils and basophils was higher in LH samples, while heterophil counts were lower (p, 0.05). Anticoagulant choice created constant and proportional bias for multiple analytes in a species-dependent fashion. LH is the recommended anticoagulant for hematology in painted turtles and common snapping turtles. Either LH or dEDTA may be used in Blanding's turtles, though anticoagulant-specific reference intervals may be necessary.

The efficacy of 2780 nm Er,Cr;YSGG and 940 nm Diode Laser in root canal disinfection: A randomized clinical trial.

OBJECTIVES: Effective disinfection of the root canals is the cornerstone of successful endodontic treatment. Diminishing the microbial load within the root canal system is crucial for healing in endodontically treated teeth. The aim of this study was to evaluate the effect of 2780 nm Er,Cr:YSGG and 940 nm diode lasers on the eradication of microorganisms from single-rooted teeth with asymptomatic apical periodontitis. MATERIALS AND METHODS: Thirty participants conforming to the inclusion criteria were randomly divided into 3 groups according to the disinfection protocol used; Conventional group: 2.5% Sodium Hypochlorite (NaOCl) and 17% EDTA solution NaOCl/EDTA, Dual laser group: 2780 nm Erbium, chromium: yttrium scandium-gallium-garnet (Er,Cr:YSGG) laser and 940 nm diode laser Er,CrYSGG/Diode, and Combined group: 17% EDTA and 940 nm diode laser EDTA/Diode. Bacterial samples were collected before and after intervention. The collected data were statistically analyzed using Friedman's test and Kruskal-Wallis test (P ≤ 0.05). RESULTS: The results of the study showed that both dual laser Er,CrYSGG/Diode and combined laser EDTA/Diode groups showed significantly less mean Log10 CFU/ml of aerobic and anaerobic bacterial counts than the conventional NaOCl/EDTA group. CONCLUSIONS: In this study we evaluated in vivo the bactericidal efficacy of three disinfection protocols for endodontic treatment of single-rooted teeth with apical periodontitis. The results indicated that both dual laser Er,CrYSGG/Diode and combined laser EDTA/Diode groups provide superior bactericidal effect compared to the conventional NaOCl/EDTA group. CLINICAL RELEVANCE: The integration of lasers into root canal disinfection protocols has demonstrated significant bacterial reduction which might promote healing and long-term success.

Effect of final irrigation protocols with chitosan nanoparticle and genipin on dentine against collagenase degradation: An ex-vivo study.

AIM: Endodontic irrigants may affect the mechanical and chemical properties of dentine. This study evaluated the effects of various final irrigation protocols including the use of chitosan nanoparticle (CSnp) and cross-linking with genipin on the (1) mechanical and (2) chemical properties of dentine against enzymatic degradation. METHODOLOGY: CSnp was synthesized and characterized considering physiochemical parameters and stability. The root canals of 90 single-rooted teeth were prepared and irrigated with NaOCl. Dentine discs were obtained and divided into groups according to the following irrigation protocols: Group NaOCl+EDTA, Group NaOCl+CSnp, Group NaOCl+EDTA+CSnp, Group NaOCl+CSnp+Genipin, Group NaOCl+EDTA+CSnp+Genipin and Group distilled water. (1) Mechanical changes were determined by microhardness analysis using Vickers-tester. (2) Chemical changes were determined by evaluating molecular and elemental compositions of dentine using Fourier transform infrared spectroscopy (FTIR) analysis and scanning electron microscope (SEM)/energy dispersive X-ray spectroscopy (EDS) analysis, respectively. All analyses were repeated after the discs were kept in collagenase for 24 h. Data were analysed with repeated measures analysis of variance and Bonferroni correction for microhardness analysis, and Kruskal-Wallis and Wilcoxon tests for FTIR and SEM/EDS analyses (p = .05). RESULTS: (1) Collagenase application did not have a negative effect on microhardness only in Group NaOCl+EDTA+CSnp+Genipin when compared with the post-irrigation values (p > .05). Post-collagenase microhardness of Group NaOCl+EDTA+CSnp and Group NaOCl+CSnp+Genipin was similar to the initial microhardness (p > .05). (2) After collagenase, Amide III/ PO 4 3 - ratio presented no change in Group NaOCl+EDTA+CSnp, Group NaOCl+CSnp+Genipin and Group NaOCl+EDTA+CSnp+Genipin (p > .05), while decreased in other groups (p < .05). Collagenase did not affect CO 3 2 - / PO 4 3 - ratio in the groups (p > .05). There were no changes in the groups in terms of elemental level before and after collagenase application (p > .05). CONCLUSIONS: CSnp and genipin positively affected the microhardness and molecular composition of dentine. This effect was more pronounced when CSnp was used after EDTA.

COMPARING THE EFFECTS OF LITHIUM HEPARIN AND DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID ON HEMATOLOGIC VALUES IN PRAIRIE RATTLESNAKES (CROTALUS VIRIDIS) AND LAKE ERIE WATER SNAKES (NERODIA SIPEDON INSULARUM).

Anticoagulants prevent clotting of blood samples and preserve cellular morphology for hematologic evaluations, but studies comparing anticoagulants are limited in snakes. The objective of this study was to evaluate the effects of lithium heparin (LH) and dipotassium ethylenediaminetetraacetic acid (EDTA) on hematologic values in prairie rattlesnakes (PR; Crotalus viridis, n = 16) and Lake Erie water snakes (LEWS; Nerodia sipedon insularum, n = 21). Venipuncture was performed and blood samples were immediately aliquoted into LH and EDTA microtainers. Packed cell volume (PCV), total solids (TS), 100-cell differential counts, and Avian Leukopet white blood cell counts (WBC) were determined for each anticoagulant. Passing-Bablok regression and Bland-Altman plots revealed that anticoagulant choice did not constantly or proportionally bias the values of any WBC parameter. Mixed models demonstrated that blood anticoagulated with EDTA had higher PCV in PR (P = 0.04) and TS in both species (P < 0.05). However, the magnitude of the differences attributable to anticoagulant choice was relatively small and likely not clinically important. Hemolysis was not appreciated in any samples. Our findings demonstrate that LH and EDTA are equally appropriate for use in PR and LEWS, but may require separate reference values.

Dislodgement resistance and structural changes of tricalcium silicate-based cements after exposure to different chelating agents.

This study aimed to evaluate the dislodgement resistance and structural changes of different mineral trioxide aggregate cements (MTA) like Pro-Root MTA, Ortho MTA, and Retro MTA after exposure to sodium hypochlorite (NaOCl), NaOCl-Ethylenediaminetetraacetic acid (EDTA), 1-hydroxyethylidene-1, 1-bisphosphonate (Dual Rinse HEDP), and NaOCl-Maleic acid (MA). The root canal spaces of 150 dentine slices were obturated using tricalcium silicate cements and divided into 3 groups (n = 50): Group1: ProRoot MTA, Group2: Retro MTA, and Group3: Ortho MTA. The samples in each group were further subdivided into four experimental (n = 10) and one control groups (n = 10): 2.5% NaOCl-17% EDTA, Dual Rinse HEDP, 2.5% NaOCl-7% Maleic acid, 2.5% NaOCl, distilled water (control). The dislodgement resistance and structural changes of cements were measured. Use of DR HEDP resulted in higher dislodgement resistance compared to17% EDTA and 7% MA in the samples obturated with Ortho MTA and Pro-Root MTA (p<0.001). In Retro MTA group, samples treated with DR HEDP and 17% EDTA had higher dislodgment resistance compared to 7% MA (p<0.001). On microstructural and elemental analysis of all the three MTA cements, samples treated with 17% EDTA and 7% MA were more amorphous and granular when compared to DR HEDP, which was pettle shaped. Calcium level was decreased more in samples treated with 17% EDTA and 7% MA when compared to DR HEDP.

Extrusion bond strength and disinfection of E. faecalis from canal dentin using synchronized microbubble photodynamic activation and photon-induced photoacoustic streaming.

OBJECTIVE: This study aimed to assess the antimicrobial effectiveness of a new disinfection regimen Curcumin photosensitizer (CP), Synchronized Microbubble Photodynamic Activation (SYMPA), Photon-induced photoacoustic streaming (PIPS), and its impact on the extrusion bond strength (EBS) of root filling material to canal dentin. MATERIALS AND METHODS: Root canal treatments of sixty mandibular incisors were performed, and an overnight culture of E. faecalis was cultivated in the BHI medium. A volume of 1 mL was introduced into twenty root canals. All the samples were randomly allocated into 4 groups based on the irrigation used: -Group 1: 2.5% NaOCl+17% EDTA (Control), Group 2: CP+17% EDTA, Group 3: SYMPA+17% EDTA, and Group 4: PIPS+17% EDTA. The viable bacterial count was estimated, and 15 specimens from each group were obturated, followed by sectioning in 1-mm thick slices. The EBS was measured using a universal testing machine, and failure modes were analyzed using a stereomicroscope at 40x magnification. Means and standard deviations (SD) of the survival rate of E. faecalis and EBS of root filling to the dentin were analyzed using ANOVA Tukey multiple comparison t-tests (p=0.05). RESULTS: Group 1 (2.5% NaOCl+17% EDTA) demonstrated the highest survival rate of E. faecalis. Group 3, in which SYMPA+17% EDTA was used to sterilize the canal, unveiled the lowest survival rate (1.55±0.11 CFU/mL). Similarly, the coronal section of Group 3 specimens (8.67±0.43MPa) demonstrated the highest EBS. However, an apical section of Group 1 samples (2.81±0.11MPa) displayed the lowest outcome of bond integrity. Intergroup comparison analysis revealed that Group 4 (PIPS+17% EDTA) and Group 2 (CP+17% EDTA) samples demonstrated comparable values of bond integrity and bacterial survival. CONCLUSIONS: Canal disinfection using the synchronized microbubble photodynamic activation (SYMPA) technique proved to be a promising alternative technique in decreasing the count of E. faecalis and improving extrusion bond strength of gutta percha to canal dentin.

Novel multiplexed alkali enzyme lysis coupled with EDTA pretreatment for RNA virus extraction from wastewater sludge: Optimization, recovery, and detection.

RNA viruses are readily enriched in wastewater sludge owing to adsorption by extracellular polymeric substances (EPS) during wastewater treatment, causing pathogenicity. However, conventional wastewater extraction methods often fail to fully extract these viruses from sludge. In this study, three methods: enzymatic (ENP), alkaline (ALP), and ethylenediaminetetraacetic acid (EDTA) pretreatments were applied to sludges and promote the RNA virus extraction from sludge. Our results show that the total recovery rate of RNA viruses increased by 87.73% after ENP pretreatment, whereas ALP pretreatment inhibited virus extraction. The highest recovery rate of viruses from sludge, reaching 296.80%, was achieved with EDTA pretreatment (EDP) coupled with ENP. Notably, the most significant increase was observed in the abundance of Astroviruses, which increased from 7.60 × 107 to 7.86 × 108 copies/g TSS after EDP + ENP treatment. Our investigations revealed that virus extraction was affected by a class of short-wavelength protein substances, as opposed to tryptophan or tyrosine, which were eluted by proteins with beef paste buffer by substitution after EDP + ENP treatment. The results of this study provide essential insights for sludge-based epidemiology with the required sensitivity for managing the extraction of RNA epidemic viruses to control viral transmission.

Eco-friendly approach to decrease the harmful effects of untreated wastewater on growth, yield, biochemical constituents, and heavy metal contents of carrot (Daucus carota L.).

Here, the impact of irrigation using untreated wastewater (WW) on carrots (Daucus carota L.) was examined. We hypothesized that the addition of ethylenediaminetetraacetic acid (EDTA), dry algal powder (Spirulina platensis or Chlorella vulgaris), and Salix alba leaves powder would function as chelators for harmful contaminants in wastewater. The findings showed that irrigation of carrot plants with the sampled untreated wastewater led to significant decreases in the shoot lengths, fresh, dry weights of shoots and roots at stage I, the diameter of roots, pigment content, carotenoids, total soluble carbohydrate content, and soluble protein content. Furthermore, a significantly increased level of proline, total phenols, and the activities of polyphenol oxidase (PPO), peroxidase (POX), superoxide dismutase (SOD), and catalase (CAT) was identified in stage I samples. In contrast to the stage I, the length of the roots, the number of leaves on each plant, wet and dry weights of the stage II roots were all greatly enhanced. In spite of the increased yield due to the wastewater irrigation, carrot roots irrigated with wastewater had significantly more cadmium (Cd), nickel (Ni), cobalt (Co), and lead (Pb) than is considered safe. Our data clearly show that the application of Spirulina platensis, Chlorella vulgaris, EDTA, and leaves powder of salix was able to alleviate the toxicity of wastewater on carrot plants. For example, we recorded a significant decrease in the accumulation of carrot's Cd, Ni, Co, and Pb contents. We conclude that the treatments with Spirulina platensis and Chlorella vulgaris can be utilized as eco-friendly tools to lessen the damaging effects of wastewater irrigation on carrot plants.

Effect of EDTA Activation on Blood Clot Structure in Regenerative Endodontics: A Scanning Electron Microscopy Study.

INTRODUCTION: EDTA plays a crucial role in regenerative endodontic therapy (RET) because of its significant biological effects. However, EDTA is also recognized as the preferred anticoagulant for hematologic tests. Thus, this study aimed to assess the influence of different EDTA activation techniques on the morphology of blood clots after conditioning the root canal dentin. METHODS: Forty extracted human teeth were prepared to simulate immature teeth and divided into the following 5 groups: (1) saline solution (negative control), (2) EDTA 17% + saline solution (CNI), (3) CNI + ultrasonic activation, (4) CNI + Easy clean activation, and (5) CNI + XP-endo Finisher activation. After irrigation, the roots were cleaved, and the root canals were filled with human blood to clot formation. The morphology and density of erythrocytes, platelets, and the fibrin network were observed using a scanning electron microscope. The fibrin network density was classified using a 4-point scale. Data were analyzed using the Friedman test and the Kruskal-Wallis test with Bonferroni adjustment (α = 5%). RESULTS: All groups exhibited consistent blood clot morphology characterized by a high density of erythrocytes, platelets, and white blood cells throughout the entire length of the root canal. The negative control group showed statistically significant high scores of fibrin density compared with the CNI group in all root thirds (P < .05). However, there was no statistical difference in the scores for the fibrin network density between the groups irrigated with EDTA with and without activation (P > .05). CONCLUSIONS: EDTA may impair the fibrin network formation compared with the saline group. However, EDTA activation did not significantly change the effects on the blood clot in contact with the conditioned intraradicular dentin.

In Vitro Evaluation of Smear Layer and Debris Removal and Antimicrobial Activity of Different Irrigating Solutions.

OBJECTIVE: The aim of this in vitro study was to compare the smear layer and debris removal and antimicrobial activity of two dual-action irrigating solutions for continuous chelation (Triton; Brasseler, Savannah, USA and Dual Rinse HEDP; Medcem GmbH, Weinfelden, Switzerland) with a dual step irrigation protocol with sodium hypochlorite (NaOCl) followed by ethylenediaminetetraacetic acid (EDTA). METHODS: Thirty single-rooted single-canal teeth were divided into three groups (n=10) and irrigated with Triton, Dual Rinse HEDP mixed with 6% NaOCl and 6% NaOCl/17% EDTA. The teeth were observed under a scanning electron microscope (SEM) to assess the canal wall cleanliness. In addition, 80 dentine discs were contaminated with Candida albicans and 80 discs with Enterococcus faecalis and irrigated with Triton, Dual Rinse HEDP mixed with 6% NaOCl and 6% NaOCl/17% EDTA or not treated (n=20). Fifteen discs were used to evaluate colony-forming units, while 5 discs were analysed by SEM. Data were analysed using the Shapiro- Wilk, Kruskal-Wallis and One-Way ANOVA tests. RESULTS: Triton was statistically more effective than Dual Rinse HEDP and NaOCl/EDTA in removing debris (p<0.05), except with NaOCl/EDTA in the coronal third. Triton was more effective than Dual Rinse HEDP in removing the smear layer from the apical and middle thirds (p<0.05). All the irrigation protocols significantly re- duced the number of E. faecalis. The Triton group showed the lowest number of remaining C. albicans (p<0.05). CONCLUSION: Triton was the most effective irrigation solution in removing debris and as effective as NaOCl/ EDTA in removing the smear layer. Triton showed the highest efficacy against C. albicans. New irrigating solutions that provide continuous chelation may provide an alternative to current irrigation protocols.

Potential low-impact immunogen for the production of anti-bothropic serum: Bothrops alternatus venom treated with Na2EDTA.

This study proposes an alternative method using Na2EDTA to neutralize B. alternatus venom and using it as an immunogen from the start of inoculation to minimize side effects and enhance antivenom production. To achieve this, 1.8 mg/mL of B. alternatus venom (B.aV) was treated with Na2EDTA, and any extra chelate was eliminated by filtering the resulting solution through a Sephadex G-25 column. Two groups of BALB/c mice were immunized subcutaneously on days 1, 15 and 30 with B.aV/Na2EDTA (45, 90, 135 µg/mouse) or B.aV (15, 30, 45 µg/mouse), respectively. Both formulations were emulsified with Freund's adjuvant (complete first and incomplete-booster). Blood samples were collected from each mouse on days 14, 29, 41, and 50 post-first immunization, and serum was separated for antibody detection. Animals were then sacrificed and lungs removed for histological analysis (hematoxylin-eosin). Immunoblotting analysis revealed that the sera from mice inoculated with B.aV/Na2EDTA (anti-B.aV/Na2EDTA) recognized the major venom proteins (20-66 kDa) similarly to the sera from mice inoculated with B.aV (anti-B.aV). The enzyme-linked immunosorbent assay results indicated that the anti-B.aV/Na2EDTA had a higher titer (5.76 × 104) than those the anti-B.aV (1.92 × 104). Additionally, sera from animals immunized with B.aV/Na2EDTA significantly neutralized proteolytic, indirect hemolytic and coagulant activity (p < 0.05). Finally, histological examination of the lungs of mice inoculated with B.aV/Na2EDTA showed normal appearance, while animals inoculated with B.aV showed interstitial lung injury (p < 0.05). In conclusion, the B.aV/Na2EDTA formulation, free of excess Na2EDTA, proved to be a promising candidate as an immunogen for antivenom production.

Citric Acid Conditioning as an Alternative to EDTA for Growth Factors Release and Stem Cell Response in Regenerative Endodontics: A Systematic Review of In Vitro Studies.

INTRODUCTION: Citric acid (CA) conditioning may be a promising alternative to ethylenediaminetetraacetic acid (EDTA) in regenerative endodontic procedures, as reported to improve growth factors' release from dentin. This review systematically investigated the effect of CA conditioning on the growth factors release from dentin and cell behavior compared to EDTA conditioning. METHODS: Searches were conducted (PubMed/MEDLINE, Scopus, Web of Science, Embase, SciELO, Cochrane Library, and grey literature) until May-2023. Only in vitro studies that evaluated the effects of CA on growth factors' release from dentin and cell behavior outcomes compared to EDTA were included. The studies were critically appraised using a modified Joanna Briggs Institute's checklist. Meta-analysis was unfeasible. RESULTS: Out of the 335 articles screened, nine were included. Among these, three studies used dentin discs/roots from permanent human teeth; the rest combined them with stem cells. 10% CA for 5 or 10 minute was the most used protocol. Meanwhile, EDTA concentrations ranged from 10% to 17%. In eight studies examining the release of growth factors, five reported a significant release of transforming growth factor-ß after dentin conditioning with 10% CA compared to 17% EDTA. Regarding cell behavior (6 studies), three studies assessed cell viability. The findings revealed that 10% CA conditioning showed cell viability similar to those of 17% EDTA. Additionally, in two out of three studies, it was observed that 10% CA conditioning did not affect cell morphology. The studies had a low risk of bias. CONCLUSIONS: The use of 10% CA to condition dentin for 5-10 minutes resulted in a notable transforming growth factor -ß1 release, but its cell responses were similar to those of EDTA.

Effect of photobiomodulation therapy on TGF-ß release from dentin, migration and viability of dental pulp stem cells in regenerative endodontics treatment: An ex vivo study.

BACKGROUND AND AIM: Regenerative endodontic procedures (REPs) are oriented by the principles of tissue engineering, incorporating dental pulp stem cells (DPSC), crucial growth factors like Transforming growth factor-ß (TGF-ß1), and scaffolds to facilitate the regeneration of dental pulp tissues. The present study aimed to investigate the effect of photobiomodulation (PBM) therapy, using an 808 nm diode laser on cellular modulation mechanisms in REPs. METHOD AND MATERIAL: A total of 108 human dentin discs obtained from intact single root teeth were randomly assigned into six groups (n = 8): 1. Positive control (EDTA), 2. PBM-1 (3 J/cm2), 3. PBM-2 (5 J/cm2), 4. EDTA+PBM-1, 5. EDTA+PBM-2, and 6. Negative control (NaOCl). Then, an extract solution was prepared from each disc and the concentration of released TGF-ß1 from the discs was measured using enzyme-linked immunosorbent assay (ELISA). Moreover, the extract solution was added to DPSC culture medium to evaluate cell viability and migration through MTT assay and scratch test, respectively. RESULT: The group exposed to PBM-1 showed the highest cell viability, while treatment with EDTA and EDTA+PBM-2 decreased cellular viability. Also, the PBM-treated groups showed significantly higher release of TGF-ß1 compared to the negative control. EDTA and EDTA+PBM-1 showed the highest release among all the groups. No significant difference was found between EDTA and EDTA+PBM-1, as well as between PBM-1 and PBM-2. Moreover, the PBM-1 group exhibited the highest migration after 24 h, which was significantly greater than other groups, except for the PBM-2 group. CONCLUSION: According to the obtained data, 808 nm mediated-PBM (3 J/cm2), both independently and in conjunction with EDTA, enhanced the release of TGF-ß1 from dentin and improved cell viability and migration of DPSCs. It seems that, PBM under the specific parameters employed in this study, could be an effective adjunctive therapy in REPs.

Antibacterial Synthetic Nanocelluloses Synergizing with a Metal-Chelating Agent.

Antibacterial materials composed of biodegradable and biocompatible constituents that are produced via eco-friendly synthetic strategies will become an attractive alternative to antibiotics to combat antibiotic-resistant bacteria. In this study, we demonstrated the antibacterial properties of nanosheet-shaped crystalline assemblies of enzymatically synthesized aminated cellulose oligomers (namely, surface-aminated synthetic nanocelluloses) and their synergy with a metal-chelating antibacterial agent, ethylenediaminetetraacetic acid (EDTA). Growth curves and colony counting assays revealed that the surface-aminated cellulose assemblies had an antibacterial effect against Gram-negative Escherichia coli (E. coli). The cationic assemblies appeared to destabilize the cell wall of E. coli through electrostatic interactions with anionic lipopolysaccharide (LPS) molecules on the outer membrane. The antibacterial properties were significantly enhanced by the concurrent use of EDTA, which potentially removed metal ions from LPS molecules, resulting in synergistic bactericidal effects. No antibacterial activity of the surface-aminated cellulose assemblies was observed against Gram-positive Staphylococcus aureus even in the presence of EDTA, further supporting the contribution of electrostatic interactions between the cationic assemblies and anionic LPS to the activity against Gram-negative bacteria. Analysis using quartz crystal microbalance with dissipation monitoring revealed the attractive interaction of the surface-aminated cellulose assembly with LPS Ra monolayers artificially produced on the device substrate.