Colourimetric Assay Research Articles

Efficacy of Reuterin and Bacteriocins Nisin and Pediocin in the Preservation of Raw Milk from Dairy Farms.

SUMMARYResearch backgroundIn the current scenario of milk production in developing and developed countries, several factors influence the shelf-life of raw milk and add significant numbers of microbial contaminants that drastically lower the initial microbial quality leading to milk spoilage by the time it reaches the processing units.Experimental approachThe present study was undertaken to investigate the biopreservative efficacy of reuterin system along with different combinations of bacteriocins in controlling the initial microflora of raw milk at farm level. Lactobacillus reuteri strain LR47, having effective antimicrobial activity, was shortlisted from our previous study and further characterized for reuterin production and tested in raw milk system.Results and conclusionsPreliminary testing of the cell-free supernatant from L. reuteri LR47 demonstrated significant growth inhibition of the majority of the tested bacterial indicators of milk spoilage. Further genetic analysis of the L. reuteri LR47 revealed the presence of two genes (pduC and dhaB) involved in the utilization of glycerol to produce reuterin via two different pathways. The strain LR47 was also found to possess comparatively higher capacity to convert glycerol into reuterin when checked through colorimetric assay. In the raw milk biopreservation experiment with reuterin alone or in combination with bacteriocins, the highest level of growth suppression in the total bacterial load and coliform counts was observed in the sample that was treated with a combination of reuterin, nisin and pediocin. The treatment combining these three natural biopreservatives at specific concentrations was able to maintain the initial microbial quality and extend the shelf-life of raw milk by 6 h at 37 °C based on the microbial counts and physicochemical properties, viz. pH and titratable acidity. In conclusion, the results confirm that the use of reuterin in combination with bacteriocins is a promising approach for temporary control of the raw milk microflora and extension of its shelf-life until further processing.Novelty and scientific contributionThis study demonstrates for the first time the use of reuterin for the extension of shelf-life of raw milk as an alternative treatment method.

In vitro anticancer activity of silver nanoparticle synthesized from Punica granatum dried peel against cancer cell lines

The main aim of the investigation was to screen the silver and gold nanoparticle synthesized from the methanol concentrate of Punica granatum for its in vitro anticancer activity against MCF 2, PC3, A-549, HeLa, and HepG2 cell lines. Silver and gold nanoparticles were prepared from methanol concentrate of P. granatum and nanoparticle synthesized was analyzed by UV and TEM analysis. The impact of nanoparticles synthesized on MCF-2, PC3, A-549, HeLa, and HepG2 disease cell lines was assessed by MTT colourimetric assay and the impact on cell cycle was assessed by flow cytometric method. After assessing the cytotoxicity effect, the impact of apoptosis was also analyzed. The TEM analysis showed the particle size of 25.56 and 22.02 nm for gold and silver nanoparticles respectively. The adequacy of silver nanoparticles synthesized from P. granatum against MCF-2, PC3, A-549, HeLa, and HepG2 cell line demonstrated that the hatching of malignancy cells decreased the suitability of PC-3 and A 549 cancer cells lines only with IC 50 values as 108.7 and 88.42 µg/mL respectively. Gold nanoparticles didn’t have any activity against all the cancer cell lines. Cell cycle analysis and apoptosis study showed that Silver nanoparticles are effective in controlling the cell cycle and blocking the apoptosis in all the cell lines used. P. granatum can be a very good anticancer drug for various cancer cell lines. All in all, P. granatum has a critical cell reinforcement movement and anticancer action.

Iron-Chelating Potential of Novel Phytochemicals in Poplar and Cedar Trees

Introduction: Iron overload is not only a consequence of diseases such as thalassemia and hereditary hemochromatosis (HFE), but also of neurodegeneration with brain iron accumulation (NBIA). In HFE, iron increases the risk of developing hepatocellular and colorectal cancers. Excess iron resulting from disruptions in normal iron homeostasis can accumulate in major organs including liver, heart and brain, and has devastating effects if left untreated. Currently, treatment includes using iron chelators, which at higher concentrations can have significant adverse effects and require constant medical supervision. Therefore, the search for alternative unique or adjuvant iron chelators that have reduced toxicity could be of significant benefit. Plants that grow in alkaline soils may be a good source of chelators for this purpose. Since iron is generally unavailable in such soils, plant roots have evolved mechanisms to solubilize iron for uptake, such as soil acidification, but need additional strategies to overcome high alkalinity. This may include producing secondary metabolites that are exuded into the soil and can chelate iron directly, including phenolic acids that may chelate iron at physiological pH in humans. This project is focused on finding, isolating, and testing bioactivity of compounds from western red cedar (alkaline tolerant) and poplar (rich in phenolics). Methods: Plants are grown in iron-normal and -deficient conditions in an innovative aeroponic system to stimulate the production of secondary metabolites related to Fe deficiency. Plant tissue extracts and root washings are collected and concentrated with solid phase extraction chromatography to form plant-derived concentrates (PDC) that are analyzed by UPLC-MS and colourimetric assays to isolate, identify, and characterize compounds induced by iron-deficiency. For bio-activity testing, PDCs are introduced to cultures of THP-1 cells, a model human monocytic cell line, to study their effect on Fe homeostasis. Prior to treatment with chelators, cells are cultured under normal (Con) and Fe-overload (CrFe) conditions (produced by treatment with 10 and 20 µM Fe-citrate) for one week to model human chronic iron overload. Deferoxamine (DFO), a well-known clinical iron chelator, model phenolics like caffeic (CafA) and chlorogenic acid (CGA), and PDCs have been applied to cultures as potential chelators. Results: Leaf compared to root tissues from poplar vary greatly in their CGA and phenolic content. Leaf extracts contained 5 times more phenolics than root extracts, and root extracts from iron-deficient plants produced 66% more phenolic compounds than those from iron-normal plants. Compared to leaf extracts, root extracts showed a 4-fold increase in iron-binding activity in vitro. PDCs including these extracts were found to contain compounds responsive to iron deficiency, which are semi-polar and low in molecular weight (140 - 340 m/z). Distinct iron-responsive compounds were also identified from cedar. Following acute dosage with Fe-citrate, THP-1 cells showed a moderate reduction in iron content after treatment with CafA, CGA, and PDCs from roots, with no influence on cell viability. On-going work includes dose-dependency with CafA and PDCs and co-operative effects of PDCs with DFO. Iron-loading in THP-1 cells is time sensitive, with maximum iron uptake measured at 8 hours following delivery of 20 µM Fe-citrate. Detailed kinetics of cellular iron-loading in the presence of iron-chelators is currently being investigated. Conclusions: We found that low-molecular weight and water-soluble PDCs from iron-deficient plants had excellent iron-binding capacity in vitro, and inhibited iron uptake in THP-1 cells. Effects of chelators on cellular iron uptake is both dose and time dependent. Screening plants for novel chelators provides an abundance of opportunity to search for new chelators for human medicinal use. Disclosures No relevant conflicts of interest to declare.

A cost-effective colourimetric assay for quantifying hydrogen peroxide in honey.

Honey is a natural product with many beneficial properties including antimicrobial action. Production of hydrogen peroxide (H2O2) in diluted honey is central to this action. Here, we describe an optimized method for measuring levels of H2O2 in honey. This method is based on established methods, with the level of dilution, the time between dilution and reading the assay, and aeration of the samples during the assay identified as critical points for ensuring reliability and reproducibility. The method is cost-effective and easy to perform using common laboratory equipment. Using this method, we quantified the hydrogen peroxide content of five different, unprocessed polyfloral honeys collected in NC, USA. Our results show that H2O2 production by these honeys varies greatly, with some samples producing negligible levels of H2O2. We assessed the effect of colour on the assay by measuring the recovery of spiked H2O2 from light and dark honey and from serially diluted dark corn syrup, and found the amount of H2O2 that could be detected was lower in dark corn syrup and darker honey samples.

Substrate Flexibility of the Flavin-Dependent Dihydropyrrole Oxidases PigB and HapB Involved in Antibiotic Prodigiosin Biosynthesis.

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin‐dependent oxidase responsible for the formation of 2‐methyl‐3‐amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5‐methyl‐4‐pentyl‐3,4‐dihydro‐2H‐pyrrole (5 a, resulting from transamination of the aldehyde of 3‐acetyloctanal) and 2‐methyl‐3‐pentyl‐3,4‐dihydro‐2H‐pyrrole (6, resulting from transamination of the ketone), were synthesised. Only 5 a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane‐associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colourimetric assays for PigB and HapB were developed, and the HapB‐catalysed reaction was kinetically characterised. Ten analogues of 5 a were synthesised, varying in the C2 and C3 side chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side chain tested at C3 were accepted, but only short side chains at C2 were accepted. The knowledge that 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis.

Abstract 1273: Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in EGFR-mutant lung cancer

Abstract Background Acquired drug resistance is the most challenging problem in treating nonsmall cell lung cancer (NSCLC) patients with EGFR tyrosine kinase inhibitor (TKI). We evaluated whether resistance cell subclones significantly increased shortly after treatment with EGFR-TKIs could determine the final resistance phenotype for EGFR-TKIs in the tumor with EGFR mutations. Methods We generated 2 EGFR-mutant lung cancer cell lines (PC9GR and HCC827GR) resistant to gefitinib and evaluated the resistance mechanisms in each cell line. In parent cell lines (PC9 and HCC827) the amounts of EGFR T790M mutation and C-MET and VIMENTIN mRNA expression were measured before and after 48 hours treatment with gefitinib or paclitaxel. Using a cell line derived directly from tumors of treatment-naïve NSCLC patient with EGFR mutation, the above tests were repeated. We prospectively collected the bloods from 4 NSCLC patients with EGFR mutations at baseline and eight weeks after EGFR-TKI treatment and measured the level of T790M mutation in circulating tumor DNAs (ctDNA) from their bloods by colourimetric assay. Results EGFR T790M mutation was detected in PC9GR cells whereas C-MET amplification in HCC827GR cells. The proportion of EGFR T790M mutation significantly increased in the PC9 cells treated with gefitinib whereas not in the same cells treated with paclitaxel or not in the HCC827 treated with gefitinib or paclitaxel. The mRNA expression of C-MET was significantly increased in the HCC827 treated with gefitinib. The VIMENTIN mRNA expression was not changed in both cell lines. When we treated the patient-derived cells with gefitnib, the frequency of T790M significantly increased whereas C-MET mRNA expression decreased and VIMENTIN mRNA expression was not changed. In the corresponding resistance tumor tissue, only T790M mutation was detected without C-MET amplification and VIMENTIN overexpression. Two cases whose T790M mutation level in their ctDNAs increased at 8 weeks by more than 50% revealed T790M mutation in their final resistance tumor tissue. But, in other cases without an early surge of T790M mutation in their ctDNAs, T790M mutation was not detected in posttreatment biopsy sample. Conclusions Early-on-treatment quantitative change of resistance-related gene alterations may predict the final mechanism of resistance to EGFR-TKI. Citation Format: Youngjoo Lee, Yu-Ra Choi. Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1273.

Cytotoxic and apoptotic properties of silver chloride nanoparticles synthesized using Escherichia coli cell-free supernatant on human breast cancer MCF 7 cell line

Synthesis of silver and silver based nanoparticles using microorganisms has received profound interest because of obtaining nanoparticles with unique physicochemical and biological properties. In the current study, for the first time, synthesis of silver chloride nanoparticles (AgClNPs) using cell-free supernatant of Escherichia coli culture is reported. Prepared AgClNPs were characterized by EDS, XRD and FESE. Data revealed the synthesized nanoparticles, mostly, have a spherical shape with an average size of 13 nm. Additionally, MTT assay elucidated a dose-dependent cytotoxicity of AgClNPs against MCF-7 cells (IC50 = 44 µg/mL). Quantitative real-time reverse transcription-PCR and colourimetric assays were employed to investigate the mechanism of cell toxicity in several cell death pathways. The results revealed the ability of AgClNPs to upregulate Bax/Bcl-2 ratio and p53 at mRNA level. Moreover, other apoptotic factors such as caspase-3, 8 and 9 were also upregulated at both mRNA and proteome levels. Finally, apoptosis induction was confirmed by Annexin-V/PI detection assay. Based on the obtained data, biosynthesized AgClNPs using E. coli cell-free supernatant exhibit a cytotoxic effect on human breast cancer cells through up-regulation of apoptotic factors, which suggest them as anti-tumour agents for further investigations.

Development of Tubing-based Stationary Liquid-phase Enzyme-linked Immunosorbent Assay

Altzre-testing (POCT) applications, its commercialization has been hampered due to complex fabrication processes or the need for additional lab instruments. In this study, tubing-based colourimetric and volumetric enzyme-linked immunosorbent assay (ELISA) methods were developed for application in POCT of cortisol and Salmonella typhimurium, respectively. Different solutions required for each assay were contained in a piece of tubing, forming different aqueous layers that were separated by immiscible organic layers. The assay was carried out by moving antibody-functionalized, solid-phase magnetic particles through the layers with a hand-operated magnet. This method enabled detection of cortisol at a low detection limit of 0.1 ng/mL within 12 min and detection of S. typhimurium down to 102 cfu/sample within 1 h. The performance of the assay methods was comparable to or better than that of conventional ELISA despite the assay systems and methods being extremely simple, thus demonstrating its potential for POCT application.

The anti-carcinogenic activity of Hydrastis Canadensis on oral cancer cell lines

Naturals herbs are a topic commonly used for the research of drugs that could be used to treat cancer as it has become a leading cause of death in several developing countries. There is a constant demand for new therapies to treat and prevent this life-threatening disease. Scientific and research interest is drawing its attention towards naturally-derived compounds as they are considered to have less toxic side effects compared to current treatments such as chemotherapy. Goldenseal, commonly known as yellow puccoon has wide therapeutic activity. The bioactive components present are helpful in treating several problems in the human systems. They are capable of eliminating toxic substances due to the anti-cancer, anti-inflammatory and other properties. The study aims to do a study on the anti-carcinogenic activity of Hydrastis Canadensis on oral cancer cell lines. Through there researches it would be made available to all the people for an affordable cost. The sensitivity of KB (Oral squamous cell carcinoma) cells was determined individually by the MTT colourimetric assay. Higher concentrations of the herb induce increased cell inhibition, and it can give close and similar actions with tamoxifen. Studies indicate that goldenseal is biologically a very active plant which makes it very attention-able one.

Preparation of a highly stable drug carrier by efficient immobilization of human serum albumin (HSA) on drug-loaded magnetic iron oxide nanoparticles

Albumin immobilized nanoparticles are known to be biodegradable, easy to prepare and reproducible for drug delivery systems. In summary, we have synthesized a new drug carrier using modified iron oxide nanoparticles. The synthesized drug carrier was characterized by X-ray powder diffraction (XRD), field-emission scanning electron microscopy (FE-SEM), Fourier transform infrared (FT-IR), vibrating sample magnetometry (VSM) and energy-dispersive X-ray spectroscopy (EDX). Three different drugs were loaded on the modified iron oxide nanoparticles and then human serum albumin (HSA) immobilized on the iron oxide nanoparticles. In addition, the in-vitro antiproliferative activity of Fe3O4@SiO2@Nev@HSA nanoparticles against Hela cancer cell line using MTT colourimetric assay was compared with nevirapine. The results show that Fe3O4@SiO2@Nev@HSA nanoparticles in comparison to nevirapine itself have more effective antiproliferative activity on Hela cancer cell lines. The IC50 value for Fe3O4@SiO2@Nev@HSA nanoparticles was 59.20 μg/ml, which is close to the antiproliferative activity of anti-cancer gefitinib with IC50 value of 76.24 μg/ml. Moreover, in vitro calf thymus DNA (ct-DNA) binding studies were investigated by various spectroscopy techniques.

Evaluation of DNA damage in spinal cord and mutagenic effect of a Phα1β recombinant toxin with analgesic properties from the Phoneutria nigriventer spider

Phα1β peptide isolated from the venom of the Phoneutria nigriventer spider has shown higher analgesic action in pre-clinical studies than ω-conotoxin MVIIA peptide used to treat severe chronic pain. In view of the great potential for the development of a new Phα1β-based drug, a Phα1β recombinant form (CTK 01512-2) has been studied for efficacy and safety. The aim of this study was to evaluate cytotoxic, genotoxic and mutagenic effects of a Phα1β recombinant form and compare it with native Phα1β and ω-conotoxin MVIIA. Cytotoxicity was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colourimetric assay in L929 mouse fibroblast cells (0.5-10.0μmol/L). Genotoxic and mutagenic activities were analysed using the alkaline comet assay in peripheral blood and spinal cord, and the micronucleus test in bone marrow from Wistar rats treated by intrathecal injection of CTK 01512-2 (200, 500 and 1000pmol/site), native Phα1β (500pmol/site) and ω-conotoxin MVIIA (200pmol/site). CTK 01512-2 decreased the cell viability of the L929, showing IC50 of 3.3±0.1µmol/L, while the Phα1β and ω-conotoxin MVIIA did not show cytotoxicity (IC50 >5.0µmol/L). Native and recombinant Phα1β forms induced DNA damage in the spinal cord, but not in peripheral blood. CTK 01512-2 at 1000pmol/site increased the micronucleus frequency suggesting mutagenic effects. In conclusion, the recombinant form has cytotoxic, genotoxic and mutagenic effects, evidenced in doses five times above the therapeutic dose.

95 N-Acetyl cysteine as a potential treatment for equine persistent breeding-induced endometritis

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Transient uterine inflammation is a normal response to breeding; however, PBIE-susceptible mares do not clear this inflammation in a timely fashion. Uterine inflammation at the time of embryonic descent from the oviducts ultimately results in early embryonic death and is seen clinically as infertility. Several risk factors for PBIE have been identified and include age, parity, anatomical conformation, such as poor perineal conformation and cervical fibrosis, as well as other reproductive tract abnormalities. N-Acetyl cysteine (NAC), a mucolytic used to treat endometritis in mares, has anti-inflammatory properties, affects inflammatory cytokines, and is a mild inhibitor of nitric oxide synthase. Increased nitric oxide, causing smooth muscle relaxation and alterations in inflammatory cytokines, has been documented in PBIE mares. The objective of our study was to determine if NAC treatment would lower nitric oxide and inflammatory cytokine levels, thereby resolving PBIE. A randomised, blinded, crossover design clinical trial was performed utilising PBIE-susceptible mares (n=10). Mares were screened for bacterial endometritis before enrolment in the study and only mares that had negative bacterial cultures were used. No other treatments were given to mares while they were enrolled in the study. Intrauterine infusion of 180mL of 3.3% NAC was performed 12h before insemination, when a follicle >35mm was present. Mares were sampled for endometrial cytology and endometrial fluid to determine inflammatory cytokine (ELISA) and nitric oxide (colourimetric assay) levels at 12 and 72h post-insemination. Endometrial biopsies were taken at the same time points post-insemination to determine gene expression of inflammatory cytokines by qPCR. Clinical signs of endometrial oedema and intrauterine fluid volumes were assessed at 12 and then every 24h post-breeding. Statistical assessment of the data was performed using a repeated-measures ANOVA. Uterine inflammation, as determined by percent number of neutrophils on endometrial cytology (P=0.0006), and interleukin 6 gene expression (P=0.003) were highest at 12h. Uterine oedema was greatest at 12 and 24h (P=0.02) and uterine fluid volumes were highest at 24h (P=0.02). Interestingly, interleukin-6 protein levels were not in accordance with gene expression, and were highest at 72h. In this clinical trial, pre-breeding intrauterine treatment with NAC did not affect nitric oxide levels, cytokine gene expression, or clinical signs of PBIE. However, the pattern of inflammation noted in this study is consistent with that described in PBIE mares. Nevertheless, the assessment of inflammatory cytokines, both at the gene and protein level at different time points post-AI, in combination with clinical signs will add to the understanding of the alterations in inflammatory cytokine levels in mares susceptible to PBIE.

Increased NEFA levels reduce blood Mg2+ in hypertriacylglycerolaemic states via direct binding of NEFA to Mg2+

Aims/hypothesisThe blood triacylglycerol level is one of the main determinants of blood Mg2+ concentration in individuals with type 2 diabetes. Hypomagnesaemia (blood Mg2+ concentration <0.7 mmol/l) has serious consequences as it increases the risk of developing type 2 diabetes and accelerates progression of the disease. This study aimed to determine the mechanism by which triacylglycerol levels affect blood Mg2+ concentrations.MethodsUsing samples from 285 overweight individuals (BMI >27 kg/m2) who participated in the 300-Obesity study (an observational cross-sectional cohort study, as part of the Human Functional Genetics Projects), we investigated the association between serum Mg2+ with laboratory variables, including an extensive lipid profile. In a separate set of studies, hyperlipidaemia was induced in mice and in healthy humans via an oral lipid load, and blood Mg2+, triacylglycerol and NEFA concentrations were measured using colourimetric assays. In vitro, NEFAs harvested from albumin were added in increasing concentrations to several Mg2+-containing solutions to study the direct interaction between Mg2+ and NEFAs.ResultsIn the cohort of overweight individuals, serum Mg2+ levels were inversely correlated with triacylglycerols incorporated in large VLDL particles (r = −0.159, p ≤ 0.01). After lipid loading, we observed a postprandial increase in plasma triacylglycerol and NEFA levels and a reciprocal reduction in blood Mg2+ concentration both in mice (Δ plasma Mg2+ −0.31 mmol/l at 4 h post oral gavage) and in healthy humans (Δ plasma Mg2+ −0.07 mmol/l at 6 h post lipid intake). Further, in vitro experiments revealed that the decrease in plasma Mg2+ may be explained by direct binding of Mg2+ to NEFAs. Moreover, Mg2+ was found to bind to albumin in a NEFA-dependent manner, evidenced by the fact that Mg2+ did not bind to fatty-acid-free albumin. The NEFA-dependent reduction in the free Mg2+ concentration was not affected by the presence of physiological concentrations of other cations.Conclusions/interpretationThis study shows that elevated NEFA and triacylglycerol levels directly reduce blood Mg2+ levels, in part explaining the high prevalence of hypomagnesaemia in metabolic disorders. We show that blood NEFA level affects the free Mg2+ concentration, and therefore, our data challenge how the fractional excretion of Mg2+ is calculated and interpreted in the clinic.

Bacillus amyloliquefaciens levan and its silver nanoparticles with antimicrobial properties

In this study, the exopolysaccharide from Bacillus amyloliquefaciens PB6 was purified and identified as levan. To explore the application of this levan in nanotechnology, its silver nanoparticles (AgNPs) were prepared in a simple and green method. Transmission electron microscopy analysis revealed that the size of the synthesised AgNPs was in the range of 19.47 ± 2.45 nm. X-ray powder diffraction analysis confirmed the face-centred cubic crystalline structure of metallic silver. Moreover, the AgNPs at the concentration of 0–12 μg/mL exhibited a significant inhibitive effect on Staphylococcus aureus and Escherichia coli. Tested by tetrazolium-based colourimetric assay, the viability of RAW 264.7 cells was higher than 75% in the presence of 20 μg/mL AgNPs. The results not only proved the feasibility of producing levan from B. amyloliquefaciens with high safety and a high yield, but also demonstrated the simple and cost-efficient approach of AgNPs synthesis as potential antibacterial agents from levan.

Effective detection of biocatalysts with specified activity by using a hydrogel-based colourimetric assay - β-galactosidase case study.

The main aim of this study was to prepare gelatine-based hydrogels containing entrapped substrate and to examine the applicability of these matrices for detection of enzymes with a specified catalytic activity. The general research concept assumed the use of a substrate that, in the presence of a particular enzyme, will quickly undergo conversion to a coloured product. ortho-Nitrophenyl-β-D-galactopyranoside (ONPG) was used as the immobilized substrate and β-galactosidase from Kluyveromyces lactis as the biocatalyst to be determined. Among other factors, the range of detectable concentrations of galactosidase, the operational pH range, the time necessary to achieve a visible response and the preferred storage conditions for the test were determined. As a result, an effective colourimetric test for β-galactosidase detection was obtained. Its main advantages include (i) the effective detection of the enzyme at concentrations greater than or equal to 0.6 mg.L-1, (ii) the ability to perform initial quantification of the enzyme on the basis of the intensity of the obtained colour (iii) applicability in a wide pH range (from 4.0 to 9.0), (iv) a relatively short response time (from 1 to a maximum of 30 minutes) and (v) stability in long-term storage at 4°C (90 days without loss of specific properties).

Tiron is Protective Against ROS-Induced Damage in Human Bronchial Epithelial Cells

Background Reactive oxygen species (ROS)-induced damage and resultant oxidative stress have been documented within the lung and have been implicated in the development of pulmonary diseases such as chronic obstructive pulmonary disorder and asthma (1). Antioxidant supplementation has been used to inhibit accumulation of oxidative damage such as DNA strand breaks and lipid peroxidation in inflammation (1,2). Tiron, a superoxide scavenger, has previously been shown to reduce murine airway remodelling and associated inflammation as well as offer protection against UV and H2O2-induced mitochondrial DNA (mtDNA) damage in human dermal fibroblasts (2,3). Aim This study aimed to investigate the use of the Tiron to prevent ROS-induced damage in human bronchial epithelial cells (BEAS-2B). Methods BEAS-2B cells were pre-treated with 3mM Tiron in BEGM (Lonza, UK) for 24 hours prior to induction of ROS damage (1 hour H2O2 (0.25mM) or exposure to 120 hour hypoxic (1% O2) conditions). Simultaneous Tiron treatment and ROS-induction was also assessed. The level of ROS production was assessed via flow cytometric analysis using the DCFDA assay (Abcam, UK). MtDNA strand breaks were assessed using a qPCR 1kb amplification following 83bp alignment (4). Concentration of the lipid peroxidation biomarker malondialdehyde (MDA) was quantified using a colourimetric assay (Sigma, UK). All data was normalised to the cell controls where appropriate and summarised as mean ±SEM, analysed by one-way ANOVA and Dunnett’s posthoc test (p≤0.05). Results Both pre-treatment and the addition of Tiron at the time of ROS-induction demonstrated 100% protection (±0.18 vs. H2O2; ±0.06 vs. hypoxia pre-treatment and ±0.30 co-treatment) against both H2O2 and hypoxia induced mtDNA strand breaks. This was supported by a significant (p≤0.001) reduction in the production of ROS and diminished formation of MDA with both H2O2 and hypoxic conditions compared to untreated controls. Conclusion This study demonstrates a promising use for Tiron as a supplement against both H2O2 and hypoxia-induced ROS in BEAS-2B cells. However, the precise mechanism of Tiron’s action requires further elucidation. The findings of this study indicate a potential use for Tiron in the treatment and prevention of pulmonary diseases associated with oxidative stress.

Improving antiproliferative effect of the nevirapine on Hela cells by loading onto chitosan coated magnetic nanoparticles as a fully biocompatible nano drug carrier

The freshly prepared magnetic iron oxide nanoparticles (MIONPs) were coated with SiO2 and then modified with a Si-based linker (SiL) having chlorine atom at the end of its chain. The resulting chlorine functionalized MIONPs were bonded to chitosan (CT) in trimethylamine solution. Then nevirapine (Nev) drug was loaded into above CT–SiL–MIONPs system and resulting Nev-loaded magnetic nanoparticles, Nev@CT–SiL–MIONPs, studied using different techniques. Furthermore, the value of Nev loading efficiency and also controlled delivery effect of Nev@CT–SiL–MIONP particles was determined by UV–vis spectrometer. Interestingly, the above nanomaterial showed a superparamagnetic property with a saturation magnetization value of 35.66 emu/g, indicating that it has an excellent potential application in the treatment of cancer using magnetic targeting drug delivery technology. Furthermore, the in-vitro antiproliferative activity of Nev@CT–SiL–MIONPs against cancer cell line (Hela) was compared with nevirapine using MTT colourimetric assay. The Nev-loaded magnetic nanoparticles were shown to be more effective antiproliferative on Hela cancer cell lines than nevirapine itself. Moreover, in vitro ct-DNA binding studies were investigated by UV–Vis and competitive fluorescence spectroscopies. The results showed that DNA aggregated on Nev-loaded nanoparticles via groove binding mode.

Uptake of halloysite clay nanotubes by human cells: Colourimetric viability tests and microscopy study

This study is a systemic investigation of the uptake and toxicity of halloysite nanotubes using human adenocarcinoma epithelial cells (A549). A549 cells were chosen as a popular model of cancer cells extensively studied in nanotoxicity and drug delivery research. The adverse effects of a range of halloysite concentrations were evaluated. The viability of A549 cells was determined using several colourimetric assays. Dark-field microscopy was used to visualize the uptake and distribution of halloysite nanotubes in cells. The morphology of the cells was evaluated using dark-field, transmission electron and atomic force microscopies. The results showed that halloysite had a dose-dependent effect on human cells at concentrations of 5–900 μg per 105 cells in the MTT assay. The reduced toxicity of halloysite nanotubes at lower concentrations (5–75 μg per 105 cells) was additionally supported by the results of other colorimetric assays. Microscopy assays have demonstrated that the nanotubes, though affecting the biochemical processes, do not alter the morphology of the cells and do not penetrate into the nuclei.

Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid.

Vitamin C (ascorbate) is the major water-soluble antioxidant in plasma and its oxidation to dehydroascorbic acid (DHA) has been proposed as a marker of oxidative stress in vivo. However, controversy exists in the literature around the amount of DHA detected in blood samples collected from various patient cohorts. In this study, we report on DHA concentrations in a selection of different clinical cohorts (diabetes, pneumonia, cancer, and critically ill). All clinical samples were collected into EDTA anticoagulant tubes and processed at 4 °C prior to storage at −80 °C for subsequent analysis by HPLC with electrochemical detection. We also investigated the effects of different handling and processing conditions on short-term and long-term ascorbate and DHA stability in vitro and in whole blood and plasma samples. These conditions included metal chelation, anticoagulants (EDTA and heparin), and processing temperatures (ice, 4 °C and room temperature). Analysis of our clinical cohorts indicated very low to negligible DHA concentrations. Samples exhibiting haemolysis contained significantly higher concentrations of DHA. Metal chelation inhibited oxidation of vitamin C in vitro, confirming the involvement of contaminating metal ions. Although EDTA is an effective metal chelator, complexes with transition metal ions are still redox active, thus its use as an anticoagulant can facilitate metal ion-dependent oxidation of vitamin C in whole blood and plasma. Handling and processing blood samples on ice (or at 4 °C) delayed oxidation of vitamin C by a number of hours. A review of the literature regarding DHA concentrations in clinical cohorts highlighted the fact that studies using colourimetric or fluorometric assays reported significantly higher concentrations of DHA compared to those using HPLC with electrochemical detection. In conclusion, careful handling and processing of samples, combined with appropriate analysis, is crucial for accurate determination of ascorbate and DHA in clinical samples.

Association Study of the ATP - Binding Cassette Transporter A1 (ABCA1) Rs2230806 Genetic Variation with Lipid Profile and Coronary Artery Disease Risk in an Iranian Population.

BACKGROUND:ATP - binding cassette transporter A1 (ABCA1) plays essential roles in the biogenesis of high -density lipoprotein - cholesterol. Variations in the ABCA1 gene may influence the risk of coronary artery disease (CAD).AIM:Present study aimed to investigate the association of rs2230806 (R219K) polymorphism of ABCA1 gene with the development and severity of CAD in an Iranian population.MATERIALS AND METHODS:Our study population consisted of 100 patients with angiographically confirmed CAD and 100 controls. The genotyping of R219K mutation of ABCA1 gene was determined by PCR - RFLP method. Lipid profile was determined using routine colourimetric assays. Statistical analysis was done by SPSS - 16.RESULTS:The genotypic (P = 0.024) and allelic (P = 0.001) distribution of the ABCA1 R219K polymorphism were significantly different between the two groups. In a univariate analysis (with genotype RR as the reference), the RK genotype (OR = 0.46, 95%CI = 0.25-0.86, P = 0.020) and KK genotype (OR = 0.27, 95%CI = 0.11 – 0.66, P = 0.005) was significantly associated with a decreased risk of CAD. A multiple logistic regression analysis revealed that smoking (0.008), diabetes (P = 0.023), triglyceride (P = 0.001), HDL - cholesterol (P = 0.002) and ABCA1 KK genotype (P = 0.009) were significantly and independently associated with the risk of CAD. The association between different genotypes of R219K polymorphism with lipid profile was not significant in both groups (P > 0.05). The R219K polymorphism was significantly associated with severity of CAD (P < 0.05).CONCLUSION:The carriage of K allele of ABCA1 R219K polymorphism has a protective effect on CAD risk and correlates with a decreased severity of CAD. This protective effect seems to be mediated independently of plasma lipid levels.