Sensitive and rapid detection of microcystin synthetase E Gene (mcyE) by loop-mediated isothermal amplification: A new assay for detecting the potential microcystin-producing Microcystis in the aquatic ecosystem

In order to develop a new molecular technique that has the potential to assist with monitoring and management of water bodies for potential microcystin producing cyanobacterial species that occur in mixed populations in many regions of the world, we designed a new loop-mediated isothermal amplification (LAMP) assay based on microcystin biosynthesis genes. Four sets of primers were designed to recognize six distinct sequences on target the mcyE gene that encodes a protein (McyE) being responsible to catalyze the addition of d-glutamate to Adda. One set (MCYE2) was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for mcyE detection were determined. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 40 min at an isothermal temperature of 61 °C. For the sensitivity of LAMP, the detection limit was 8.5 pg/μl (approximately 17 pg) DNA. The eleven microcystin producing and four non-toxic cyanobacterial strains were selected for testing of specificity. The results of the amplification were positive with all microcystin-producing strains tested and not with four non-toxic strains, which showed that the primers had good levels of specificity. For testing the application of LAMP assay in the aquatic ecosystem, seven environmental samples from ponds and lakes in Ningbo City were also analyzed using the LAMP targeting the mcyE gene as well as an ELISA assay. Compared with these results of ELISA assay, LAMP assay is satisfied. All of these validated LAMP method being fast, simple and low in cost is a potentially valuable means for potential toxic of cyanobacterial blooms detection, especially for routine monitoring purposes in future.     

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC₅₀ values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC₅₀ values.     

An Enzymatic Fluorometric Assay for Pyridoxal with High Specificity and Sensitivity

An enzymatic fluorometric assay for pyridoxal with pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of pyridoxal showed high linearity (r=0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for pyridoxal, and thus animal samples could be directly analyzed without separation of pyridoxal 5′-phosphate by column chromatography.     

Indoleacetic acid enhancement of 14C methionine uptake by tomato cell cultures aids assay of pathogen extracts

Incorporation of one micromolar IAA in the assay system increased the rate of ¹⁴C methionine uptake by tomato cells in suspension and effectively differentiated the rates of uptake such that cells treated with pathogenic and non-pathogenic Fusarium extracts could be easily distinguished in a rapid 3 h assay.     

Oxidative Damage and Motor Neurone Disease Difficulties in the Measurement of Protein Carbonyls in Human Brain Tissue

It has been suggested in the literature that elevated oxidative protein damage, measured as protein carbonyls, is present in the nervous system of patients with sporadic motor neurone disease (MND). However, the actual reported levels of brain protein carbonyls vary over a wide range. We show here that this is probably due to the use of different protocols for the carbonyl assay; results differ depending on when the dinitrophenylhydrazine reagent is added and at what stage in the procedure protein is assayed for the calculation of carbonyls on a unit protein basis. Using a range of different procedures, we were unable to confirm reports of elevated protein carbonyls in motor cortex from brains of patients with MND. We also measured thiobarbituric acid-reactive material in the brain samples using an HPLC-based TBA test in the presence of butylated hydroxytoluene. In general, there was no significant elevation of TBARS in MND motor cortex. However, four patients showed values higher than any of the control patients (both 'normal' control and 'disease control'). There was no correlation of TBARS with protein carbonyl values. We suggest that oxidative damage in motor cortex in sporadic MND, if it occurs, may be confined to a small group of patients and may affect different molecular targets in each patient.     

Rate transition and regulatory coupling in endocytosis of interferon-alpha and tumor necrosis factor-alpha in human epithelial tumor cells.

The time-dependent concentrations of interferon-alpha and tumor necrosis factor-alpha associated with the membrane and internalized by cells contain information on the kinetics of endocytosis and their cellular processing. This information can be reduced quantitatively by application of the respective compartmental models. In our studies of human epithelial tumor cells interacting with human interferon-alpha and human tumor necrosis factor-alpha, we accounted only for actual endocytosis and elimination of the tracer from cells by a novel method sensitive to changes in the rate of endocytosis, to the delay in tracer elimination, and to the nonlinear regulatory coupling between endocytosis and the internalized ligand. Data reduced by this method resulted in best-fit parameter values statistically superior to values obtained by previous methods (Bajzer et al., 1989). The results indicate a change with time in the rate of endocytosis of tumor necrosis factor-alpha and the inhibition of endocytosis by the endocytosed ligand-receptor complex. We conclude that sorting and processing of interferon-alpha and tumor necrosis factor-alpha are restricted by the type of both the receptor and the cell.     

Clinical implications of microRNAs in cancer

Abstract

MicroRNAs (miRNAs) are endogenously produced non-coding RNAs that serve as micromanagers by negatively regulating gene expression. MiRNAs are implicated in several biological pathways including development of neoplasia. Because altered miRNA expression is implicated in the pathobiology of various cancers, these molecules serve as potential therapeutic targets. Using miRNA mimics to restore levels of aberrantly down-regulated miRNAs or miRNA inhibitors to inactivate over-expressed miRNAs shows promise as the next generation of therapeutic strategies. Manipulation of miRNAs offers an alternative therapeutic approach for chemo- and radiation-resistant tumors. Similarly, miRNA expression patterns can be used for diagnosis and to predict prognosis and efficacy of therapy. We present here an overview of how miRNAs affect cancers, how they may be used as biomarkers, and the clinical implications of miRNAs in cancer.     

A human exposure study to investigate biological monitoring methods for 2-butoxyethanol

The enzymatic activity of acetylcholinesterase (AChE) has been shown to be altered by environmental contaminants such as metals. However, the available literature illustrates a background of contradictory results regarding these effects. Therefore, the main purpose of this study was to investigate the potential of five metal ions (nickel, copper, zinc, cadmium and mercury) to inhibit AChE activity in vitro. First, to accomplish this objective, the possible interference of metals as test toxicants in the Ellman's assay, which is widely used to assess AChE activity, was studied. The potential influence of two different reaction buffers (phosphate and Tris) was also determined. The results suggest that the selected metals react with the products of this photometric technique. It is impossible to ascertain the artefactual contribution of the interaction of the metals with the technique when measuring AChE inhibition. This constitutes a major obstacle in obtaining accurate data. The presence of phosphate ions also makes enzymatic inhibition difficult to analyse. Attending to this evidence, an assay using the substrate o-nitrophenyl acetate and Tris buffer was used to investigate the effects of metals on AChE activity. O-nitrophenyl acetate is also a substrate for esterases other than cholinesterases. It is therefore only possible to use it for the measurement of cholinesterase activity with purified enzymes or after a previous verification of the absence of other esterases in the sample tissue. Under these conditions, the results indicate that with the exception of nickel, all tested metals significantly inhibit AChE activity.     

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes

Importin β1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin β1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin β1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.