Amperometric determination of As(III) and Cd(II) using a platinum electrode modified with acetylcholinesterase, ruthenium(II)-tris(bipyridine) and graphene oxide.

The authors describe an amperometric biosensor for the determination As(III) and Cd(II) based on the inhibition of the enzyme acetylcholineesterase (AChE). A platinum electrode was modified with ruthenium(II)-tris(bipyridyl), graphene oxide and AChE and then showed redox peaks at 0.06 and 0.2 V vs Ag/AgCl in the presence of acetylthiocholine chloride (ATChCl). Amperometry unveiled a steady-state turnover rate with the release of thiocholine. In the presence of arsenic(III) and cadmium(II), AChE showed an inhibitive response at 0.214 and 0.233 V vs Ag/AgCl, respectively. The electrode exhibits a detection limit and linear range of 0.03 µM and 0.05-0.8 µM for As(III) and 0.07 µM and 0.02-0.7 µM for Cd(II), respectively. Type of inhibition and inhibition constants induced by As(III) and Cd(II) on the catalytic sites of AChE were determined from Dixon and Lineweaver-Burk plots. The modified electrode was applied to the determination of As3+ and Cd2+ in river, tap and waste water, and the results proved that the method is sensitive and can be an alternative to chromatographic and spectroscopic techniques. Graphical abstract Schematic presentation of Pt/Ru(II)-tris(bipy)-GO/AChE electrode in absence and presence of metal ions (As3+/Cd2+).

Discovery and Correction of Spurious Low Platelet Counts due to EDTA-Dependent Pseudothrombocytopenia.

BACKGROUND: Ethylene diamine tetraacetic acid dependent pseudothrombocytopenia (EDTA-PTCP) is a laboratory artifact that may lead to unnecessary evaluation and treatment of patients. The purpose of this article is to discuss how to identify EDTA-PTCP and correct spurious low platelet counts in clinical laboratories. METHODS: We use two criteria to screen for platelet aggregation: (1) an abnormal platelet count in EDTA-treated blood from a patient lacking clinical signs of a platelet disorder, and (2) an instrument flag for platelet clumps. EDTA-PTCP was confirmed by microscopic examination for platelet agglutination and by platelet counts that corrected with citrate sample. In addition, the time course of EDTA-PTCP was investigated in samples from 26 patients anticoagulated with EDTA-K2 and sodium citrate. Amikacin (5 mg/ml) was added to tubes with EDTA-K2 or sodium citrate from seven additional cases in order to confirm its dissociative effect on platelet aggregation. RESULTS: In our laboratory, the overall incidence of EDTA-PTCP was approximately 0.09%; and the duration was between 2 weeks and 6 months. EDTA-PTCP was time-dependent and occurred as early as 10 min after sample collection. Weaker agglutination could also occur in most corresponding citrate-treated samples. The dissociative effect of amikacin on platelet agglutination was case-specific and not concentration-dependent. CONCLUSIONS: The method of screening for platelet clumping with the help of XE5000 images is convenient. The decline in the platelet count is related to the length of time and the intensity of chelation. Amikacin supplement is not always effective for correcting platelet counts in vitro.

Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo'' to inhibition by the carbon monoxide-releasing molecule, CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration.

BACKGROUND: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO - a critical gasotransmitter - in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. METHODS: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli - cytochrome bd-I, cytochrome bd-II and cytochrome bo', to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24µM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. RESULTS: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo'. Cytochromes bo' and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. CONCLUSIONS: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. GENERAL SIGNIFICANCE: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Synergistic effects of EDTA and lysozyme on the properties of hydroxypropyl starch nano antibacterial films.

Hydroxypropyl starch (HPS) nano antibacterial films incorporating Ethylene Diamine Tetraacetic Acid (EDTA) and lysozyme (LY) were fabricated via solvent casting method. The synergistic effects of EDTA and LY on the microstructure, component interactions, color, optical, mechanical, barrier and antibacterial properties of HPS nano antibacterial films were evaluated. The results indicated that EDTA and LY were well dispersed in the matrix of the HPS nano antibacterial films, the film-forming substrates have good compatibility, resulting in a dense multi-layer structure of the HPS nano antibacterial films. The addition of EDTA and LY increased the color parameters (L*, a*, b* and △E*) of the HPS nano antibacterial films. The synergistic effects of EDTA and LY significantly decreased the light transmission of the HPS nano antibacterial films. The presence of EDTA and LY increased the tensile strength (TS) and the elongation at break (EAB) of the HPS nano antibacterial films. The TS and EAB of E2.5L1 reached the highest values of 6.329 MPa and 50.24 %, respectively. The incorporation of EDTA and LY had positive effects on the improvement of water vapor permeability (WVP) and oxygen permeability (OP). The WVP and OP of E2.5L1 reached the highest values of 0.9350 × 10-12 g cm/cm2•s•Pa and 0.297 × 10 -2 g m/m2 •d, respectively. In addition, EDTA and LY had significant synergistic effects on the antibacterial activity against S. aureus (Gram-positive bacteria) and E. coli (Gram-negative bacteria). E2.5L1 exhibited the highest antibacterial activity and the inhibition zone diameters of S. aureus and E. coli were 3.69 mm and 4.28 mm, respectively. The HPS nano antibacterial films incorporating EDTA and LY are potential functional packaging materials.

Reversal of heavy arterial calcification in a rat model of chronic kidney disease using targeted ethylene diamine tetraacetic acid-loaded albumin nanoparticles.

Background: Elastin degradation and severe calcification in the medial layer of the vessel wall, known as medial arterial calcification (MAC), is typical in the aging population and patients with metabolic disorders, such as diabetes and chronic kidney disease (CKD). We have previously reported that ethylene diamine tetraacetic acid (EDTA) delivery to the site of calcification can be achieved by tagging nanoparticles with an elastin antibody that recognizes explicitly damaged elastin, and such systemic therapy can remove focal calcium deposits from the calcified arteries in CKD rodent model. The current study aims to test whether heavy calcification seen throughout arterial tree and kidneys in CKD can be reversed with nanoparticle therapy. Methods: Thirty healthy male Sprague-Dawley rats weighing approximately 300 g, were placed on an adenine diet for 21 non-consecutive days to induce kidney failure, followed by daily vitamin D3 (VitD3) injections for 4 sequential days to cause severe calcification throughout the cardiovascular system and kidneys. DiR-dye loaded and elastin antibody conjugated albumin nanoparticles were used to confirm the targeting of nanoparticles to the calcification area. The rats were divided into two groups for targeted removal of calcification starting at day 7 of the last doses of VitD3. The experimental group received biweekly IV injections of anti-elastin antibody conjugated EDTA loaded human serum albumin nanoparticles (EDTA-HSA-El-Ab NPs), while the sham controls received blank nanoparticles (Blank-HSA-El-Ab NPs) (5 injections in total). Micro-computed tomography (microCT) was used to analyze the extent of calcification. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry studies were performed for osteogenic markers, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), and tissue non-specific alkaline phosphatase (TNAP). For comparison, aortic ring organ cultures from healthy rats were treated with high phosphate to induce calcification in vitro, and then they were treated with EDTA. Human calcified femoral arteries were also treated ex vivo with EDTA-HSA-EL-Ab NPs to test if nanoparticles remove heavy calcification. Results: EDTA-loaded nanoparticles that specifically target degraded elastin reversed existing heavy mineral deposits in arteries, as per elemental calcium analysis (124.161±34.410 µg Ca per mg of the dry aorta in Blank-HSA-El-Ab NPs vs. 100.520±19.131 µg in EDTA-HSA-El-Ab NPs group, P=0.04) and microCT (object volume, 129.001±37.785 vs. 29.815±24.169 mm3, P=0.0005). The reversal of aortic calcification was accompanied by a significant reduction of bone-associated mRNA expression of BMP2 and RUNX2 (P=0.01). Immunohistochemistry studies corroborated RT-PCR results, showing a reduction of BMP2 and RUNX2 stains in the vessel wall. The rat aortic ring culture study also showed similar results, where osteogenic genes (BMP2, RUNX2) and proteins (BMP2, RUNX2, TNAP) were suppressed upon reversal of calcification with EDTA (P=0.001). We also show ex vivo reversal of human femoral artery calcification by microCT (calcium intensity: untreated, 57.721±28.551 vs. day 6 of treatment, 5.441±3.615, P=0.01) by EDTA nanoparticle therapy. Conclusions: This is the first study showing the removal of calcium from heavily calcified arteries by using intravenous targeted EDTA therapy. Such therapy also reversed vascular smooth muscle cell osteoblastic transition and apoptosis in the arterial tissue, thereby potentially creating an environment for suitable tissue repair.

Impaired release of corticosterone from adrenals contributes to impairment of circadian rhythms of activity in hyperammonemic rats.

Patients with liver cirrhosis may present impaired sleep-wake and circadian rhythms, relative adrenal insufficiency and altered hypothalamus-pituitary-adrenal gland (HPA) axis. The underlying mechanisms remain unclear. Circadian rhythms are modulated by corticosteroids which secretion is regulated by HPA axis. Hyperammonemia alters circadian rhythms of activity and corticosterone in rats. The aims were: (1) assessing whether corticosterone alterations are responsible for altered circadian rhythm in hyperammonemia: (2) to shed light on the mechanism by which corticosterone circadian rhythm is altered in hyperammonemia. The effects of daily corticosterone injection at ZT10 on circadian rhythms of activity, plasma corticosterone, adreno-corticotropic hormone (ACTH) and hypothalamic corticotropic releasing hormone (CRH) were assessed in control and hyperammonemic rats. ACTH-induced corticosterone release was analyzed in cultured adrenal cells. Corticosterone injection restores the corticosterone peak in hyperammonemic rats and their activity and circadian rhythm. Plasma ACTH and CRH in hypothalamus are increased in hyperammonemic rats. Corticosterone injection normalizes ACTH. Chronic hyperammonemia impairs adrenal function, reduces corticosterone content and ACTH-induced corticosterone release in adrenals, leading to reduced feedback modulation of HPA axis by corticosterone which contributes to impair circadian rhythms of activity. Impaired circadian rhythms and motor activity may be corrected in hyperammonemia and hepatic encephalopathy by corticosterone treatment.

Efficacy of Different Root Canal Irrigating Solutions in Removing Smear Layer: A Scanning Electron Microscopic Study.

Background The aim of the present investigation was to evaluate and compare the efficacy of frequently used chemical agents in terms of their capacity to eliminate the smear layer after instrumentation, as observed via scanning electron microscopy (SEM). Materials and methods Sixty extracted single-rooted mandibular premolar teeth, each with roots 15 mm in length, were used in this study. The teeth were divided into one control group and four study groups, each containing 12 teeth. In Control Group 0, teeth were irrigated with 3 ml of saline only. In Group 1, teeth were irrigated initially with 3% sodium hypochlorite (NaOCl) and then given a final rinse with 3 ml of 17% ethylenediaminetetraacetic acid (EDTA) for one minute. In Group 2, teeth were irrigated with 3% NaOCl and given a final rinse with 3 ml of a mixture of tetracycline, acid, and detergent (MTAD, BIOPURE) for one minute. In Group 3, teeth were irrigated with saline and given a final rinse with 3 ml of 17% EDTA for one minute. In Group 4, teeth were irrigated with saline and given a final rinse with 3 ml of MTAD for one minute. One-half of each tooth was chosen and prepared for scanning electron microscopic (SEM) examination at the cervical, middle, and apical thirds. These were observed at magnifications of up to 1,000 times to check for the presence or absence of a smear layer. The data were analyzed using the Kruskal-Wallis test and post-hoc Dunn's test. Results All of the root canal irrigation protocols exhibited superior efficacy compared to the control group in the elimination of the smear layer. Group 2 (3% NaOCl with MTAD) showed the lowest mean scores, compared to all the groups, followed by Group 1 (3% NaOCl with 17% EDTA). MTAD was more effective than EDTA. The smear layer was effectively removed from the apical third, followed by the middle and coronal thirds of the root. Conclusion Initial irrigation with 3% NaOCl and one-minute final irrigation with 3 ml MTAD was the most effective root irrigant, and particularly indicated in teeth with infection of the apical third.

The effect of EDTA solution on corneal endothelial cells in rabbits.

Corneal disease threatens vision globally. Among corneal diseases, calcific band keratopathy has severe effects on vision owing to its unique location. Currently, ethylene diamine tetraacetic acid (EDTA) chelation remains the most important treatment. However, only the safety of low-dose topical EDTA eye drops is well established in humans. Therefore, the purpose of this study was to determine the safe dose range of EDTA for calcific band keratopathy surgery and its toxic effects on rabbit eyes. Rabbits were administered different doses of EDTA solutions (0.50, 0.20, 0.10, 0.05, and 0.01 M) for twenty minutes. In day seven, the rabbits were euthanized and pathological examination was performed for cornea. We found severe corneal edema in 0.50 M group, while milder edema in lower-concentration treated groups. Followed by corneal thickness measurement, the measured values increase to the peak in post-operative three day (0.20 M group) or one day (lower-concentration groups), then decreased. Groups comparison shown significant difference between BSS control group and higher concentration groups (0.20 M and 0.10 M) (P < 0.001) in observation period, but no significance was observed between low concentration and control group in the day seven after surgery (P > 0.05). Confocal microscopy examination suggested, the number of corneal endothelial cells significantly decreased from 3428.6 ± 180.3 cells/mm2 to 2808 ± 80.6 cells/mm2 in the 0.50 M group, while the lower-concentration groups showed lesser toxic effects on corneal endothelial cells. Finally, our histological examination demonstrated inflammation in each experimental group and dose-dependent, compared with control group. Our study found 0.05 M and 0.01 M EDTA solutions had no obvious toxic effect on the corneal endothelium compared with higher concentration. However, further study of EDTA side effect by clinical trials, and therapeutic effect observation with different concentration are necessary.

Comparative evaluation of canal cleanliness at apical third using Self-Adjusting File and Wave One File with different irrigants: an in vitro scanning electron microscopic study.

Aim: To evaluate the cleaning ability of two single-file rotary systems- Self-Adjusting File (SAF) and Wave one (WO) systems in removing the smear layer using sodium hypochlorite (NaOCl) + ethylene diamine tetraacetic acid (EDTA) and NaOCl + Qmix as irrigants at apical one-third of the root canal. Methods: Forty extracted human mandibular premolars were selected and working length was determined. The canal was manually instrumented up to a number 25 size K-file. The roots were divided into the following groups with 10 samples each - Group 1 using SAF: Group 1a- 3% NaOCl + 17% EDTA, Group 1b- 3% NaOCl + Qmix. Group2 using WO: Group 2a- 3% NaOCl + 17% EDTA, Group 2b- 3% NaOCl + Qmix. In the SAF group, the irrigation was performed continuously using the special irrigation apparatus. In the WO group, syringe irrigation was done followed by final irrigant activation using passive ultrasonic irrigation (PUI). The roots were sectioned longitudinally and subjected to scanning electron microscopic (SEM) examination. The amount of smear layer was evaluated using a five score index at the apical third level. Statistical analysis was performed using the Chi-square test. Results: Group 1 (SAF) showed better canal cleanliness at apical third compared to Group 2 (WO) with both irrigant combinations and the results were statistically significant (p<0.05). 3% NaOCl + Qmix was equally as effective as 3% NaOCl + 17% EDTA in removing the smear layer with no significant difference between them. Conclusion: Within the limitation of this study, SAF in combination with 3% NaOCl + Qmix or 3% NaOCl + 17% EDTA should be used for removing smear layer in critical areas of the root canal.

Endometrial receptivity defects MUC-1 and COX-2 polymorphisms in endometriosis.

The endometrium produces MUCIN-1 (MUC-1) and cyclooxygenase-2 (COX-2), which are essential for implantation. MUC-1 is required for adhesion, while COX-2 is necessary for decidualization. Variations or polymorphisms in MUC-1 and COX-2 can lead to changes in endometrial receptivity. This study investigated the relationship between MUC-1 and COX-2 polymorphisms and endometrial receptivity in endometriosis patients. Blood DNA samples were collected from 35 patients with endometriosis and 32 healthy patients between days 19 to 24 of their menstrual cycle (secretory phase). MUC-1 polymorphism was determined using the Amplification Refractory Mutation System (ARMS), and COX-2 gene polymorphism was assessed using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The frequency distribution of gene polymorphisms between the two groups was compared using bivariate analysis. There were seven genotypic combinations of MUC-1 and COX-2: AAGC; AAGG; GACC; GAGC; GAGG; GGGC; GGGG. The AAGC genotype combination test was significant, with an OR=6.43 (95% CI:1.09-7.62) and p=0.01. In conclusion, combining MUC-1 and COX-2 (AAGC) genotypes results in endometrial receptivity defects in endometriosis.

Applying EDTA in Chelating Excess Metal Ions to Improve Downstream DNA Recovery from Mine Tailings for Long-Read Amplicon Sequencing of Acidophilic Fungi Communities.

The hostile environment of mine tailings contains unique microbial life capable of bioleaching. The metagenomic analysis of such an environment provides an in-depth understanding of the microbial life and its potential, especially in biomining operations. However, DNA recovery from samples collected in those environments is challenging due to the presence of metal ions that interfere with the DNA analysis. A varied concentration of EDTA (4-13 µg/µL) to chelate the metal ions of enriched tailing samples prior to DNA extraction was performed. The results show that 9 µg/µL of EDTA was effective in most samples. However, the increasing concentration of EDTA negatively affected the DNA recovery. The sequencing of the successfully extracted DNA revealed a diverse range of fungal genera, some of which have not been previously reported in tailing or bioleaching applications. The dominant genera include Fodinomyces, Penicillium, Recurvomuces, Trichoderma, and Xenoacremonium; their traits were determined using the FungalTraits database. This study demonstrates the need to include a preliminary metal-chelating step using EDTA before DNA extractions for samples collected from metal-rich environments. It further showed the need for optimization but provided a benchmark range, particularly for tailings. However, we caution that a further EDTA removal step from the extracted DNA should be included to avoid its interferences in downstream applications.

Antimicrobial Efficacy of Morinda citrifolia, Nisin, and 2% Chlorhexidine Against Enterococcus faecalis: An In-Vitro Study.

Aim The aim of this study was to evaluate and compare the antimicrobial activity of 2% chlorhexidine gluconate (2% CHX), Morinda citrifolia (M. citrifolia), and nisin (NI) all in gel forms against Enterococcus faecalis (E. faecalis)-infected root canals. Methodology Forty single-rooted mandibular premolars extracted for orthodontic reasons were decoronated and chemomechanical preparation of the root canal was performed. After sterilization, the samples were inoculated with E. faecalis for one week and grouped according to the medicament used namely, saline as the control group (Group-A), 2% CHX (Group-B), M. citrifolia (Group-C), and NI (Group-D). After 7days of incubation, in order to evaluate the effectiveness of the intracanal medicaments on the canal wall and its radicular dentin, the specimens dentin chips were retrieved and inoculated on brain heart infusion (BHI) blood agar plates from each tube and incubated at 37°C for 24 hours to obtain bacterial colony forming unit (CFU) count. The data was statistically analyzed using one-way ANOVA test and multiple comparisons among different groups were complemented by post hoc Tukey test. Results The CFU count indicating the number of viable bacterial colonies was found to be highest in Group-A (saline). Group-B (CHX 2%) showed the least CFUs followed by Group-D (NI) and Group-C (M. citrifolia). Conclusion In an attempt to overcome the disadvantages and toxic effects of a few commercially available intracanal medicaments and irrigants, the present study was aimed at using herbal extracts to evaluate and compare their antimicrobial efficacy with the commercially available medicaments against E. faecalis. Nisin was an effective antimicrobial agent and its action was found to be comparable with CHX.

Mechanistic insights into Ca-alginate gel-associated membrane fouling affected by ethylene diamine tetraacetic acid (EDTA).

While transparent exopolymer particles (TEP) is a major foulant, and ethylene diamine tetraacetic acid (EDTA) is a strong chelating agent frequently used for fouling mitigation in membrane-based water treatment processes, little has been known about TEP-associated membrane fouling affected by EDTA. This work was performed to investigate roles of EDTA addition in TEP (Ca-alginate gel was used as a TEP model) associated fouling. It was interestingly found that, TEP had rather high specific filtration resistance (SFR) of 2.49 × 1015 m-1·kg-1, and SFR of TEP solution firstly decreased and then increased rapidly with EDTA concentration increase (0-1 mM). A series of characterizations suggested that EDTA took roles in SFR of TEP solution by means of changing TEP microstructure. The rather high SFR of TEP layer can be attributed to the big chemical potential gap during filtration described by the extended Flory-Huggins lattice theory. Initial EDTA addition disintegrated TEP structure by EDTA chelating calcium in TEP, inducing reduced SFR. Continuous EDTA addition decreased solution pH, resulting into no effective chelating and accumulation of EDTA on membrane surface, increasing SFR. It was suggested that factors increasing homogeneity of TEP gel will increase SFR, and vice versa. This study revealed the thermodynamic mechanism of TEP fouling behaviors affected by EDTA, and also demonstrated the importance of EDTA dosage and pH adjustment for TEP-associated fouling control.

Radicular canal disinfection using photodynamic therapy and sodium hypochlorite following three chelating agents' green tea extract, grape extract, and EDTA on the push-out bond strength of epoxy resin-based sealer.

AIM: To appraise the efficacy of photodynamic therapy (PDT) and conventional regime (NaOCl) following three chelating agents ethylene diamine tetraacetic acid (EDTA), Green tea extract (GTE), grape extract (GE) on pushout bond strength (PBS) of epoxy resin-based sealer with root dentin. MATERIAL AND METHODS: 60 single-rooted human teeth were prepped using a ProTaper system and cleansed using photodynamic therapy (PDT) and 5% NaOCl (n = 30 each). Samples in each group PDT and 5% NaOCl were further divided into 6 sub-group (n = 10) based on the chelating agents used: 17% EDTA, GTE, and GE. Gutta-percha and AH Plus were used to obturate the canals. The push-out test was used to determine bond strength, and ANOVA was used to conduct statistical analysis while failure patterns were classified as adhesive, cohesive, or mixed. The Chi-squared test was used to examine the different failure modes at a significance level of 0.05. RESULTS: NaOCl disinfection, when applied with naturally derived reducing agents (17% EDTA, GTE, and GE) demonstrated significantly higher PBS compared to PDT when used with chelating agents (17% EDTA, GTE, and GE) (p < 0.05). The most predominant failure mode was an adhesive failure when root dentin was disinfected with PDT while NaOCl treatment showed a high percentage of cohesive failure. CONCLUSION: Radicular canal disinfection with sodium hypochlorite following three chelating agents (Green tea extract, Grape extract, and Ethylene diamine tetraacetic acid) exhibited better push-out bond strength bonded to radicular dentin with epoxy resin-based sealer.

Cryopreservation of undifferentiated and differentiated human neuronal cells.

The effective use of human-derived cells that are difficult to freeze, such as parenchymal cells and differentiated cells from stem cells, is crucial. A stable supply of damage-sensitive cells, such as differentiated neuronal cells, neurons, and glial cells can contribute considerably to cell therapy. We developed a serum-free freezing solution that is effective for the cryopreservation of differentiated neuronal cells. The quality of the differentiated and undifferentiated SK-N-SH cells was determined based on cell viability, live-cell recovery rate, and morphology of cultured cells, to assess the efficacy of the freezing solutions. The viability and recovery rate of the differentiated SK-N-SH neuronal cells were reduced by approximately 1.5-folds compared to that of the undifferentiated SK-N-SH cells. The viability and recovery rate of the differentiated SK-N-SH cells were remarkably different between the freezing solutions containing 10% DMSO and that containing 10% glycerol. Cryoprotectants such as fetal bovine serum (FBS), antifreeze proteins (sericin), and sugars (maltose), are essential for protecting against freeze damage in differentiated neuronal cells and parenchymal cells. Serum-free alternatives (sericin and maltose) could increase safety during cell transplantation and regenerative medicine. Considering these, we propose an effective freezing solution for the cryopreservation of neuronal cells.

Hexane fraction of Globimetula braunii induces mitochondria-mediated apoptosis in Plasmodium berghei-infected mice.

Background: Apoptosis is a common pathology in malaria and most antimalarial drugs induce apoptosis during chemotherapy. Globimetula braunii is an African mistletoe used for the treatment of malaria but its effect on mitochondria-mediated apoptosis is not known. Methods: Malarial infection was induced by the intraperitoneal injection of NK 65 strain Plasmodium berghei-infected erythrocytes into mice which were treated with graded doses (100-400 mg/kg) of methanol extract (ME), and fractions of n-hexane, dichloromethane, ethylacetate and methanol (HF, DF, EF and MF) for 9 days after the confirmation of parasitemia. Artequine (10 mg/kg) was used as control drug. The fraction with the highest antiplasmodial activity was used (same dose) to treat mice infected with chloroquine-resistant (ANKA) strain for 5 consecutive days after the confirmation of parasitemia. P-alaxin (10 mg/kg) was used as control drug. On the last day of the treatment, liver mitochondria were isolated and mitochondrial Permeability Transition (mPT) pore opening, mitochondrial F0F1 ATPase (mATPase) activity, lipid peroxidation (mLPO) and liver deoxyribonucleic acid (DNA) fragmentation were assessed spectrophotometrically. Caspases 3 and 9 were determined by Enzyme-Linked Immunosorbent Assay (ELISA) technique. Cytochrome c, P53, Bcl-2-associated X protein (Bax), and B-cell lymphoma-2 (Bcl2) were determined via immunohistochemistry. Phytochemical constituents of the crude methanol extract of Globimetula braunii were determined via the Gas Chromatography-Mass Spectrometry (GC-MS) analysis. Results: There was large amplitude mPT induction by malaria parasites, extract and fractions of Globimetula braunii. At 400 mg/kg, HF significantly (p < 0.01) downregulated mATPase activity, and mLPO in both (susceptible and resistant) models, caused DNA fragmentation (P < 0.0001), induced caspases activation, P53, bax and cytochrome c release but downregulated Bcl2 in both models. The GC-MS analysis of methanol extract of Globimetula braunii showed that α-amyrin is the most abundant phytochemical. Conclusion: The n-hexane fraction of Globimetula braunii induced mitochondrial-mediated apoptosis through the opening of the mitochondrial pore, fragmentation of genomic DNA, increase in the levels of P53, bax, caspase 3 and 9 activation and cytochrome c release with concomitant decrease in the level of Bcl2. α-Amyrin is a triterpene with apoptotic effects.

Capture and identification of bacteria from fish muscle based on immunomagnetic beads and MALDI-TOF MS.

In the present study, E. coli was taken as a model bacterium, anti-E. coli functionalized magnetic beads were constructed and used to capture E. coli from aqueous extracts of fish sarcoplasmic protein (FSP) and fish muscle protein of sablefish. The excellency of the reproducibility of the present protocol was demonstrated by capturing E. coli from sablefish FSP extracts. The presence of 10 CFU/mL E. coli is still detectable. A microbial safety test on the surface of fish muscle was successfully performed. The bacterial identification accuracy from samples with different matrices was found to be excellent with RSD = 3%. High specific detection of target bacteria in complex biological samples was testified by spiking Staphylococcus aureus and Klebsiella pneumoniae in samples as interference. Ten biomarker ions were discovered for E. coli's recognition. It is promising to apply the present protocol in bacterial analysis in muscle food samples to ensure their safety.

[Study on the correlation between the content of bone morphogenetic protein 2 in demineralized bone matrix and its osteogenic activity in vitro and in vivo].

OBJECTIVE: To investigate the correlation between the content of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) and its osteogenic activity in vitro and in vivo, in order to choose a simple and convenient method to evaluate the osteogenic activity of DBM. METHODS: The left mid-femoral tissues of 9 donors were taken, and DBMs (S1-S9) were prepared by dynamic decalcification process, and inactivated DBM (control group) was prepared at the same time. Protease inhibitor method, collagenase method, guanidine hydrochloride/ethylene diamine tetraacetic acid (EDTA) method, and RIPA lysate method were used to extract BMP-2 in S1-S9 and inactivated DBMs. The BMP-2 content was measured and the differences between DBMs were compared. Then the S1-S9 and inactivated DBMs were co-cultured with mouse embryonic osteoblasts MC3T3-E1, respectively. The cell proliferation was detected by MTT method and fluorescence staining, and alkaline phosphatase (ALP) activity was detected at the same time. Thirty BALB/c male nude mice were divided into 10 groups, namely S1-S9 DBM groups (S1-S9 groups) and inactivated DBM group (control group), with 3 mice in each group. Muscle pockets of the middle thighs were prepared on both hindlimbs of mice in each group, and implanted corresponding DBM materials. At 4 weeks after operation, the samples were taken for HE staining observation and semi-quantitative evaluation, and the new bone formation score was calculated. RESULTS: The BMP-2 content of DBM derived from different donor bones was distinct. The BMP-2 content obtained by different extraction methods for DBM prepared from the same donor bone was also different, and the extraction efficiency of the guanidine hydrochloride/EDTA method was the highest. In vitro cell experiments, MTT test displayed that cell proliferations and ALP activity were significantly higher in S4 and S6 groups than in other groups at each time point after co-cultivation ( P<0.05). Moreover, the cell proliferation of S4 group was the most significant at 7 days ( P<0.05); fluorescence staining demonstrated that the osteoblasts of each group was in good condition, but the osteoblasts of S1, S2, S3, S4, and S6 groups were significantly more than other groups. In vivo ectopic osteogenesis experiments, the cartilage and new bone formation could be seen in the bone graft area of S1-S6 groups at 4 weeks after operation, and with the increase of BMP-2 content, the more new bone formation induced by the material, the higher the score of new bone formation of the material ( P<0.05). Among them, S4 and S6 groups contained a large number of chondrocytes and osteoblasts in the osteogenesis area. CONCLUSION: The osteogenic activity of DBM can be evaluated through BMP-2 quantitative detection combined with in vitro osteoblast proliferation and differentiation experiments.

Utility of Ethylene-Diamine-Tetraacetic Acid Buffer Solution With Boric Acid for Immunostaining of Specimens Stored for an Extended Period.

Antigen modification and denaturation are recognized causes of false negatives in immunostaining. Specimens that have been stored for an extended period at room temperature show decreased immunoreactivity and may mislead the diagnosis. Studies of the molecular targeting of drugs often involve immunostaining of previous samples and, in some situations, only unstained specimens can be used. The present study aimed to develop an effective staining method to recover antigen activation in unstained specimens stored for an extended period by using ethylene-diamine-tetraacetic acid (EDTA) buffer solution with boric acid. We compared several commonly used antigen retrieval solutions and found that Tris-borate-EDTA (TBE) buffer solution with a pH ≥8.3 provided sufficient antigen retrieval. However, pH values higher than 8.3 (9.0, 10.0, and 11.0) frequently caused severe tissue damage. Thus, TBE with pH 8.3 was the most suitable antigen retrieval solution for recovering the antigenicity of specimens stored for an extended period. This procedure may allow useful immunohistochemical information, even from sections that have been stored for an extended period.

Cyanobacterium Synechocystis sp. PCC 6803 lacking adc1 gene produces higher polyhydroxybutyrate accumulation under modified nutrients of acetate supplementation and nitrogen-phosphorus starvation.

Increased polyhydroxybutyrate production in cyanobacterium Synechocystis sp. PCC 6803 lacking adc1 gene (Δadc1) is first-timely reported in this study. We constructed the mutant by disrupting adc1 gene encoding arginine decarboxylase, thereby exhibiting a partial blockade of polyamine synthesis. This Δadc1 mutant had a proliferative growth and certain contents of intracellular pigments including chlorophyll a and carotenoids as similar as those of wild type (WT). Highest PHB production was certainly induced by BG11-N-P+A condition in both WT and Δadc1 mutant of about 24.9 %w/DCW at day 9 and 36.1 %w/DCW at day 7 of adaptation time, respectively. Abundant PHB granules were also visualized under both BG11-N-P and BG11-N-P+A conditions. All pha transcript amounts of Δadc1 mutant grown at 7 days-adaptation time were clearly upregulated corresponding to its PHB content under BG11-N-P+A condition. Our finding indicated that this adc1 perturbation is alternatively achieved for PHB production in Synechocystis sp. PCC 6803.