EDTA Treatment Research Articles

P032 Determining the minimum EDTA concentration necessary to eliminate prozone effect in the single antigen bead (SAB) assay

Aim A major limitation of the single antigen bead (SAB) assay is its susceptibility to interference by complement activation, which inhibits binding of anti-IgG-PE to HLA specific antibodies. This interference, known as prozone effect, can reduce MFI values leading to false negative reactions. Schnaidt et. al. (2011) described a method to eliminate prozone by treating sera with EDTA to chelate calcium, preventing complement activation. However, anecdotal evidence suggests that the method is not always effective. This may be due to differences in EDTA concentration used by different labs given the original description is ambiguous and lacks sufficient detail to follow the protocol. The goal of this study was to determine the minimum EDTA concentration required to eliminate prozone. Methods Six well characterized, prozone +ve sera (3 class I and 3 class II HLA) were selected. Prozone +ve specificities were defined as >5000 MFI increase in SAB assay reactivity upon titration. Sera were treated with several concentrations of dipotassium EDTA (1.2–6 mM) or PBS control and were tested in parallel using LABScreen SAB assay. MFI values of prozone +ve specificities were then compared. Results A total of 52 prozone +ve specificities (28 class I and 24 class II HLA) were identified in titration studies. Prozone was eliminated for all 52 specificities when sera were treated with either 6 mM or 3 mM EDTA (Fig. 1). In contrast, treatment with either 2 mM or 1.2 mM EDTA was ineffective (Fig. 1). Prozone −ve specificities were not affected by EDTA treatment at any concentrations used. Conclusion EDTA treatment of sera is effective at eliminating prozone if appropriate EDTA concentrations are used. Interestingly, our experiments demonstrate that the threshold at which EDTA is effective is very distinct (3 mM is effective while 2 mM is ineffective). Since EDTA forms a 1:1 chelate complex with calcium, the concentration threshold of EDTA required to eliminate prozone is consistent with physiologic serum calcium concentration (2.1–2.6 mM). Download : Download high-res image (369KB) Download : Download full-size image

P028 Uncovering the invisible; pre-treatment of sera with EDTA relieves prozone effect

Aim High titer HLA antibodies in patient sera can be falsely assessed due to interfering factors, also known as the prozone phenomenon, causing the masking of such antibodies. In this study we assessed the elimination of prozone by treatment of sera with DTT, EDTA and dilution. Methods Sera from 10 patients were treated with DTT, 6% EDTA, and 1:10 dilution. Antibody screening was performed by Luminex single antigen bead (SAB) assay. Effect of each treatment on the negative and positive controls, false-positive, and false-negative beads were compared to one another as well as to the untreated serum. Results Patient sera treated with DTT, EDTA and diluted at 1:10, all showed elimination of interfering factors and resolved the prozone effect. Comparison of the three treatment approaches showed that, DTT resolved the prozone, however it appeared to cause a slight increase in the mean fluorescent intensity (MFI) of the Luminex negative control (NC) bead. EDTA treatment eliminates prozone effect and has no effect on the NC bead. The 1:10 dilution also can resolve prozone, however the sensitivity can be compromised especially for the lesser strength antibodies. Conclusion In circumstances where prozone phenomenon is suspected, such as in the pre-transplant evaluation of highly sensitized patients, or during post-transplant monitoring for detection of donor specific antibodies, DTT, EDTA, and dilution are all effective strategies in elimination of interfering factors. However in addition to a higher NC, DTT-treatment, unlike EDTA, requires an additional incubation step with the sera (45 min) which affects the overall turnaround time. Dilution of serum can affect the sensitivity of the assay and in addition, often one dilution might not be sufficient for an accurate assessment of prozone, and performing multiple dilutions can prove to be costly. Routine treatment of sera with EDTA can accurately assess antibody specificities without affecting the sensitivity, turn around time and test cost.

EDTA Treatment of Serum Unmasks Complement-Mediated Prozone Inhibition in Human Leukocyte Antigen Antibody Testing.

Luminex-based single-antigen bead human leukocyte antigen (HLA) antibody testing is widely used to define HLA antibodies for transplant compatibility. False-negative results can occur with complement-mediated prozone inhibition. This study assessed the effect of EDTA on the assay background reactivity and fluctuations in antibody mean fluorescent intensity. Serum specimens were retrospectively tested using Luminex-based single-antigen beads with and without EDTA. Treated and untreated serum samples were compared by two measures: changes in background reactivity and changes in HLA antibody strength after EDTA treatment. Ten pretransplant and 48 posttransplant specimens were identified: lung (22), heart (10), kidney (21), heart/lung (two), pancreas (one), small bowel (one), and liver (one). After EDTA treatment, weak antibodies (below 2,000 mean florescent intensity) demonstrated the largest fluctuations. Newly identified HLA antibodies were seen in 16% (8/49) of class I and 26% (15/57) of class II beads. EDTA treatment did not result in false-negative reactions compared with untreated serum. EDTA serum pretreatment mitigated complement-mediated prozone inhibition and improved accurate HLA antibody detection. The background reactivity and the false-negative rate of the assay appear unchanged.

Molecular and chemical characterization of a Sphagnum palustre clone: Key steps towards a standardized and sustainable moss bag technique

This work aimed to define the molecular and chemical signature of a S. palustre clone developed in the framework of the EU-FP7 Mossclone project to improve the standardization and reliability of the moss-bag technique. The molecular characterization was performed by a set of DNA molecular markers (RAPD, ISJ, PCR-RFLP, sequencing and microsatellites) to tag the clone produced within the project. Molecular characterization also provided new DNA markers that can be applied in systematic analyses of Sphagnum, and gave new insights to implement well established techniques. The elemental composition of the clone was measured by ICP-MS analysis of 54 major and trace elements, with and without commonly applied pre-exposure treatments (oven devitalization and EDTA washing). Concentrations of almost all analyzed elements were significantly lower (from 10 to 100 times) in the clone than in conspecific field moss, apart from some elements (K, Mo, P and Na) deriving from the culture medium or EDTA treatment. Oven devitalization and EDTA washing did not significantly affect the clone composition. A comparison between the elemental composition of the clone with that of naturally growing Sphagnum species proved the particularly low elemental content of the clone. Therefore, in view of a rigorously standardized moss-bag protocol for the monitoring of persistent atmospheric pollutants, the use of the S. palustre clone, a biomaterial with very low and constant element composition, and homogenous morphological characteristics is strongly recommended.

Influence of the post-synthesis method on the number and size of secondary mesoporous structure of NaY zeolite and its effect on catalyst loading. An efficient and eco-friendly catalyst for synthesis of xanthenes under conventional heating

Several identification techniques have been used to study the effect of the preparation method of dealuminated Y zeolite on catalyst loading. In this paper, NaY zeolite was dealuminated by chemical operation with ethylenediaminetetraacetic acid (H4EDTA) treatment. In this method, extra-framework aluminum species removed from the supercage of zeolite and therefore increases the Si/Al ratio and pore volume. Consequently, the loading of the molybdophosphoric acid (MPA) in the supercage of zeolite is increases (0.1 g/g equal 0.049 mmol/g support) in EDTA treatment (MPA-MDAZY) in comparison with (0.0875 g/g equal 0.043 mmol/g support) in hydrothermal method (MPA-DAZY). These results were also confirmed by XRF, AAS, FTIR, SEM, BET, XRD and WDX analysis. Reducing of the reaction time and increasing of the catalytic activity of EDTA treatment toward hydrothermal method can be related to the high catalyst loading based on removing of extra-framework aluminum. The catalytic activity of two catalysts has been compared in the xanthenes synthesis reactions.

Oxidative stability of chicken’s breast after vacuum packaging, EDTA, sage and rosemary essential oils treatment

In the present work, the effect of the sage and rosemary essential oils on oxidative stability of chicken breast muscles during chilled storage was investigated. In the experiment were chickens of hybrid combination Cobb 500 after 42 days of the fattening period slaughtered. All the broiler chickens were fed with the same feed mixtures and were kept under the same conditions. The feed mixtures were produced without any antibiotic preparations and coccidiostats. After slaughtering was dissection obtained fresh chicken breast with skin from left half-carcass, which were divided into five groups (n = 5): C - control air-packaged group; A1 - vacuum-packaged experimental group; A2 - vacuum-packaged experimental group with EDTA solution 1.50% w/w; A3 - vacuum-packaged experimental group with Salvia officinalis L. oil 2.0% v/w and A4 - vacuum-packaged experimental group with Rosmarinus officinalis L. essential oil 2.0% v/w. The sage and rosemary essential oils were applicate on surface chicken breasts and immediately after dipping, each sample was packaged using a vacuum packaging machine and storage in refrigerate at 4 ±0.5 °C. The value of thiobarbituric acid (TBA) expressed as amount of malondialdehyde (MDA) in 1 kg sample was measured during storage in 1st, 4th, 8th, 12th and 16th day. The treatments of chicken breasts with sage and rosemary essential oils show statistically significant differences between all testing groups and control group, where higher average value of MDA measured in breast muscle of broiler chickens was in samples of control group (0.396 mg.kg-1) compared to experimental groups A1 (0.060 mg.kg-1), A2 (0.052 mg.kg-1), A3 (0.042 mg.kg-1) and A4 (0.041 mg.kg-1) after 16-day of chilled storage. The results of experiment showed that the treatment of chicken breast with sage and rosemary essential oils had positive effect on the decrease of oxidative processes in breast muscles during chilling storage and use of plant essential oils is one of the possibilities increase shelf life of fresh chicken meat.

Isolation, characterization and valorizable applications of fish scale collagen in food and agriculture industries

Abstract Collagen has enormous applications in food, biomedical and pharmaceutical industries; but its high cost severely limits its use. Fish processing waste is a promising and cost efficacious source of collagen, which otherwise stated as an earnest environmental pollutant. Carp (Cyprinus carpio) is one of the main species of freshwater fish consumed in India. Acid-soluble collagen (ASC) was isolated from scales of Cyprinus carpio. Scales were demineralized by EDTA treatment and ASC was extracted from the demineralized scales. The yield of scale ASC was 9.79% (on the wet weight basis). SDS–PAGE and FTIR corroborated the isolated protein as collagen. Denaturation temperature (Td) of isolated collagen was found to be 37 °C. Considering bioactive properties of collagen, a milk based food product – paneer was developed by incorporation of the extracted collagen. Composed paneer was found to be acceptable with good sensorial and textural attributes. Same scales were further treated enzymatically and the released metabolites were tested for their competency to promote the plant growth. Released metabolites showed excellent plant growth promotion and hence could be successfully employed as an economic source of nitrogen fertilizers for plants.

Effects of Tetracycline, EDTA and Citric Acid Application on Nonfluorosed and Fluorosed Dentin: An In Vitro Study.

Fluorosis is one of the factors that may bring about mineralization changes in teeth. Routine treatment of root biomodification is commonly followed during Periodontal therapy.Background:The Purpose of the present study was to compare and evaluate the morphological changes in fluorosed and nonfluorosed root dentin subsequent to the application of Tetracycline, EDTA and Citric acid. Both fluorosed and nonfluorosed teeth comprising of periodontally healthy and diseased were included in this study.Method:Each of them was grouped into Tetracycline Hydrochloride, EDTA and Citric acid treatment groupes. Using scanning electron microscope (SEM), the photomicrographs of dentin specimens were obtained.Results:Showed that there was no significant difference in exposure of number of tubules in different groups, while significant increase in the tubular width and tubular surface area was seen in fluorosed healthy, followed by fluorosed diseased groups, nonfluorosed healthy and nonfluorosed diseased groups after root biomodification procedure using various root conditioning agents. The root biomodification procedure brings in definite difference between fluorosed and nonfluorosed dentin specimens.

Biological Effects of a Root Conditioning Treatment on Periodontally Affected Teeth--An In Vitro Analysis.

The aim of this study was to evaluated the root surfaces modifications resulted by application of different chemicals agents, and their influence on the fibrin network and fibroblasts attachment. From 96 anterior mandibular human extracted incisor teeth, 192 dentin blocks of buccal and lingual surface were obtained and randomly divided into 6 groups: Cont- control group, which received no treatment; Root surface scaling and root planing (Srp); Citric acid-Srp; EDTA-Srp; Tetracycline capsule-Srp; Tetracycline gel-Srp. After dentin treatments the specimens were analyzed as follows: 1) demineralization level and residues of the product by scanning electron microscopy (SEM); 2) adhesion of blood components after 20 min of surface treatment by SEM; 3) fibroblast attachment after 24 h by SEM; 4) cell metabolism after 24 h by MTT assay. Data were analyzed using Fisher Exact, One-way ANOVA test followed by Dunn's test, Tukey test and Dunnett test (α=0.05). Citric acid, EDTA and Tetracycline gel resulted in adequate demineralization with no completely smear layer and smear plug removal on root dentin surface. Tetracycline capsule produced great tetracycline residues with several demineralization areas. Tetracycline gel and EDTA groups presented more fibroblast fixation than other experimental groups. The highest mean blood clot adhesion score was observed in roots treated with tetracycline gel. EDTA and Tetracycline gel surface treatment removed the smear layer over dentin surface and promoted adhesion of fibrin network and fibroblast cells attachment.

Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A

Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed β-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,β-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins.

Elimination of complement interference can improve the diagnostic performance of the VIDAS CMV IgG assay in acute cytomegalovirus infections

In this study we showed that complement factors are responsible for assay interference in the VIDAS cytomegalovirus (CMV) immunoglobulin G (IgG) assay. Three different serum treatments were applied to show the cause of interference: heat treatment (56 °C), adding cobra venom factor, and adding EDTA. Elimination of complement interference by EDTA treatment of serum was prospectively evaluated on 215 CMV IgM positive samples and a sensitivity increase of the VIDAS CMV IgG assay was noticed. On average the CMV IgG level increased 100% after EDTA treatment of the serum. In paired serum samples from 38 patients we could show that serum treatment with EDTA can make the CMV IgG level changes more obvious in recent CMV infections. Since the CMV IgG avidity II assay on VIDAS depends on the determination of CMV IgG, the CMV IgG avidity was also evaluated in this study but only a limited effect of the complement interference was observed.

Enhanced isolation of lymphoid cells from human skin.

SummaryStudying skin immune cells under various pathophysiological conditions is vital for understanding the nature of cutaneous inflammatory responses. Available methods of isolating cells from the skin have relatively low yield or require in vitro culture. To increase the effective isolation of skin immune cells, we used collagenase P treatment. The number of T cells obtained ex vivo using this technique was dramatically greater than that obtained with conventional methods, without the need for long‐term culture. The phenotype and function of isolated cells were comparable with those of cells isolated by EDTA treatment. Collagenase P‐based methods will enhance the ability to investigate lymphoid cell function in both healthy and diseased skin.

New Insights into EDTA In Vitro Effects on Endothelial Cells and on In Vivo Labeled EDTA Biodistribution

EDTA chelation therapy has recently been proposed for the treatment of patients affected by neurodegenerative (ND) or cardiovascular (CVD) diseases due to its efficacy in removing toxic metals that affect the functions of neurons and endothelial cells. We examined the effects of EDTA in modulating human umbilical vein endothelial cell (HUVEC) activation induced by tumor necrosis factor alpha (TNFα). Moreover, we studied the biodistribution of technetium [99 mTc] EDTA injected intravenously into healthy adult rats. Our results show that EDTA treatment reverses the HUVEC activation induced by TNFα, as highlighted by normal tubulin distribution, in keeping with a well spread, quiescent endothelial phenotype. In addition, the biodistribution of labeled EDTA reaches the central nervous system (CNS), as assessed by scintigraphy (5853.6 cpm/pixel measured at 1 hour following intravenous labeled EDTA injection) and autopsy data (3.67% at 3 hours following labeled EDTA intravenous injection). Such evidence highlights new insights into the usefulness of EDTA chelation therapy in the treatment of ND and CVD.

Metal-dependent thermal stability of recombinant endo-mannanase (ManB-1601) belonging to family GH 26 from Bacillus sp. CFR1601

A GH 26 endo-mannanase from Bacillus sp. CFR1601 was purified to homogeneity (Mw ∼39kDa, specific activity 10,461.5±100IU/mg). Endo-mannanase gene (manb-1601, 1083bp, accession No. KM404299) was expressed in Escherichia coli BL21 (DE3) and showed typical fingerprints of α/β proteins in the far-UV CD. A high degree of conservation among amino acid residues involved in metal chelation (His-1, 23 and Glu-336) and internal repeats (122–152 and 181–212) was observed in endo-mannanases reported from various Bacillus sp. Thermal inactivation kinetics suggested that metal ions are quintessential for stabilization of ManB-1601 structure as holoenzyme (Ea 87.4kcal/mol, ΔH 86.7kcal/mol, ΔS 186.6cal/k/mol) displayed better values of thermodynamic parameters compared to metal-depleted ManB-1601 (Ea 47kcal/mol, ΔH 45.7kcal/mol, ΔS 64.7cal/k/mol). EDTA treatment of ManB-1601 not only lead to transitions in both secondary and tertiary structure but also promulgated the population of conformational state that unfolds at lower temperature. ManB-1601 followed a three-state process for thermal inactivation wherein loss of tertiary structure preceded the concurrent loss of secondary structure and activity.

Towards Clinical Applications of Blood-Borne miRNA Signatures: The Influence of the Anticoagulant EDTA on miRNA Abundance.

BackgroundCirculating microRNAs (miRNAs) from blood are increasingly recognized as biomarker candidates for human diseases. Clinical routine settings frequently include blood sampling in tubes with EDTA as anticoagulant without considering the influence of phlebotomy on the overall miRNA expression pattern. We collected blood samples from six healthy individuals each in an EDTA blood collection tube. Subsequently, the blood was transferred into PAXgeneTM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgeneTM blood RNA tubes that contain a reagent to directly lyse blood cells and stabilize their content. For all six blood donors at the four conditions (24 samples) we analyzed the abundance of 1,205 miRNAs by human Agilent miRNA V16 microarrays.ResultsWhile we found generally a homogenous pattern of the miRNA abundance in all 24 samples, the duration of the EDTA treatment appears to influence the miRNA abundance of specific miRNAs. The most significant changes are observed after longer EDTA exposition. Overall, the impact of the different blood sample conditions on the miRNA pattern was substantially lower than intra-individual variations. While samples belonging to one of the six individuals mostly cluster together, there was no comparable clustering for any of the four tested blood sampling conditions. The most affected miRNA was miR-769-3p that was not detected in any of the six PAXgene blood samples, but in all EDTA 2h samples. Accordingly, hsa-miR-769-3p was also the only miRNA that showed a significantly different abundance between the 4 blood sample conditions by an ANOVA analysis (Benjamini-Hochberg adjusted p-value of 0.003). Validation by qRT-PCR confirmed this finding.ConclusionThe pattern of blood-borne miRNA abundance is rather homogenous between the four tested blood sample conditions of six blood donors. There was a clustering between the miRNA profiles that belong to a specific blood donor, but not between any of the four tested blood sampling conditions. The results show a limited overall impact of the blood sampling conditions on the miRNA pattern. Notwithstanding, the abundance of single miRNAs can be significantly altered by different blood sampling conditions.

The complement interference phenomenon as a cause of underestimation of high titer anti-HLA antibodies in a renal transplant recipient

Single antigen bead assay is the most sensitive laboratory tool for the anti-HLA antibody (Ab) identification in renal transplant recipients. However, this assay was shown to be prone to the prozone effect (PrE), giving false negative results in sera with high titer anti-HLA Ab. The ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement was described to eliminate the PrE allowing the anti-HLA Ab identification. A mean fluorescence intensity (MFI) ⩾ 2 fold increase in neat versus EDTA treated sera was considered relevant to PrE. We report a case of chronic Ab mediated rejection in a renal transplant recipient with anti-HLA-DQA1∗05:05,DQB1∗03:01 donor specific Ab (DSA), revealed after treatment of the sera with EDTA. Female patient, 36 years old, diagnosed with juvenile nephronophthisis, received a first graft from a living related donor in 1991. During the first year post transplantation the patient experienced two biopsy proven reversible rejection episodes and until 2010 she had no other evidence of graft malfunction. De novo DSA were detected in 2010, as shown on the table. In 2015, due to inconsistency between the sudden elevation of serum creatinine levels and absence of DSA, the patient’s sera were further analyzed after EDTA treatment. DSA with high MFI were detected post-EDTA treatment. The presence of DSA was confirmed with positive B-Flow crossmatch with the donor. Also, a kidney biopsy on 03/26/2015 showed chronic antibody mediated rejection with C4d deposition. Taking into account that the follow up post-transplantation is based on HLA Ab detection and identification, these results highlight the importance of using patients’ sera with EDTA treatment to abolish the PrE before the performance of single antigen bead assay, in order to detect the presence of HLA antibodies. Download : Download full-size image

Treating sera with ethylenediaminetetraacetic acid: A promising technical solution for the complement-mediated prozone effect in anti-hla antibody detection by single antigen bead assay

Aim The Luminex-single antigen bead (SAB) assay is considered the most sensitive method for the identification of anti-HLA antibodies (Ab). However, false negative results can occur due to the prozone effect (PrE) leading to underestimation of some high titer anti-HLA Ab. The aim of this study was to evaluate the ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement in eliminating the PrE in anti-HLA Ab identification by SAB. Method Ten patients with PRAs > 65% were included in the study. Neat and EDTA treated samples were tested using SAB for either HLA class I (n=14) or class II (n = 10) specificities. A mean fluorescence intensity (MFI) ⩾ 2 fold increase in EDTA treated versus neat sera was considered relevant to PrE. The PrE was confirmed with 1:10 dilution and with dithiothreitol treatment of the sera. To confirm the clinical significance of the new identified Ab, sera with defined PrE and MFI value 1000 in neat sera were further tested with AHG-CDC crossmatch against donor cells expressing the relevant HLA. Results PrE was found in 7/10 patients concerning either HLA class I (n = 4) or class II (n = 3) Ab specificities. Using an MFI = 1000 as a cut off, six ‘new’ HLA specificities were identified either HLA class I (HLA-A∗01:01, A∗34:02, A∗11:02) or class II (DQB1∗03:01, DQB1∗03:02, DQB1∗03:03). However, a significant shift of MFI value of HLA Ab class I and II was observed. More precisely, 38 HLA-class I Ab directed against HLA-A (n = 31) and HLA-B (n = 7) showed a significant mean MFI value shift from 5162 (266 lower value) to 20207. Regarding HLA class II Ab, 26 directed against HLA-DQ (n = 23) and HLA-DR (n = 3) showed a significant shift from MFI 4419 (113 lower value) to 23795. New HLA Ab specificities detected after EDTA treatment were confirmed with a strong positive AHG-CDC crossmatch. Conclusion Taking into account that selection of potential recipients pre-Tx as well as the follow up post-Tx is based to HLA Ab identification, these preliminary results highlight the importance of using EDTA treatment of the patient’s sera prior to SAB assay, in order to abolish the prozone effect in HLA antibody detection.

Attenuation of Oxidative Damage by Boerhaavia diffusa L. Against Different Neurotoxic Agents in Rat Brain Homogenate

ABSTRACTDue to a high rate of oxidative metabolic activity in the brain, intense production of reactive oxygen metabolite occurs, and the subsequent generation of free radicals is implicated in the pathogenesis of traumatic brain injury, epilepsy, and ischemia as well as chronic neurodegenerative diseases. In the present study, protective effects of polyphenol rich ethanolic extract of Boerhaavia diffusa (BDE), a neuroprotective edible medicinal plant against oxidative stress induced by different neurotoxic agents, were evaluated. BDE was tested against quinolinic acid (QA), 3-nitropropionic acid (NPA), sodium nitroprusside (SNP), and Fe (II)/EDTA complex induced oxidative stress in rat brain homogenates. QA, NPA, SNP, and Fe (II)/EDTA treatment caused an increased level of thiobarbituric acid reactive substances (TBARS) in brain homogenates along with a decline in the activities of antioxidant enzymes. BDE treatment significantly decreased the production of TBARS (p < .05) and increased the activities of antioxidant enzymes like catalase and superoxide dismutase along with increased concentration of non-enzymatic antioxidant, reduced glutathione (GSH). Similarly, BDE caused a significant decrease in the lipid peroxidation (LPO) in the cerebral cortex. Inhibitory potential of BDE against deoxyribose degradation (IC50 value 38.91 ± 0.12 μg/ml) shows that BDE can protect hydroxyl radical induced DNA damage in the tissues. Therefore, B. diffusa had high antioxidant potential that could inhibit the oxidative stress induced by different neurotoxic agents in brain. Since many of the neurological disorders are associated with free radical injury, these data may imply that B. diffusa, functioning as an antioxidant agent, may be beneficial for reducing various neurodegenerative complications.

Insight on the Feasibility of Producing Durable Paper from Spruce Pulp using the Sulfate Process

Bleached spruce sulfate pulp was used in this study to produce paper handsheets. Ethylene diamine tetra acetic acid (EDTA( was introduced as a chelating agent in concentrations of 0, 0.25, 0.5, and 0.75%. The handsheets were exposed to UV light at wavelengths ranging from 330 to 440 nm, with time intervals of 0, 10, 20, 30, 40, and 50 h. Finally, the strength properties were measured based on ISO standards. The strength indices of the handsheets were improved by adding the proper concentration of EDTA chelating agent, in comparison with the control sample. Furthermore, increasing the aging time reduced the breaking length, tear strength, folding endurance, burst strength, and tensile strength. Tear index, tensile strength, tearing strength, bursting strength, and folding endurance were decreased respectively by 41.9, 3.1, 28.2, 29.7, and 8.6 percent without EDTA treatment by increasing the aging time.

Co-aggregation of Pseudomonas fluorescens and Bacillus subtilis in culture and co-colonization in black gram (Vigna mungo L.) roots

Abstract Many plant growth promoting rhizobacteria (PGPR), biocontrol agents and biofertilizer bacteria are used in agriculture. They are applied to crops by seed or soil application which colonizes in the rhizosphere. Although there are extensive reports on the output (growth, yield, disease resistance, phenotypes, etc) of the combined inoculations of these bacteria the companionship among these bacteria and the relative contribution to overall output is largely unknown. The starting point to unravel this would be to study the bacterial companionship in terms of ability to co-aggregate, stability of co-aggregation and developing molecular evidences for co-colonization in crop roots. Our work mainly aimed at understanding the extent of co-aggregation between Pseudomonas fluorescens and Bacillus subtilis and their co-colonization abilities in plant roots. This was verified by conducting several experiments of co-aggregation and effect of various factors like pH and growth phase, effect of various treatments like urea, protease K, EDTA, heat, thermal stability, sonication and desiccation on the extent of the co-aggregation. Co-aggregation between these two bacteria was found to be pH and growth phase dependent. Protease and EDTA treatment reduced the co-aggregation whereas sonication treatment improved the same. Talc based formulation of the bacteria was prepared separately and used for seed treatment in black gram. The seeds were germinated under laboratory conditions and growth parameters were observed. DNA from roots of germinated seedlings was used as template to amplify bacterial DNA with species specific primers. PCR analysis results indicated that P. fluorescens and B. subtilis are compatible in colonizing black gram.