Revealing protein-protein interactions at the transcriptome scale by sequencing

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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.     

Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk

The soluble form of penicillin-binding protein 3 (sPBP3^∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3^∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3^∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.     

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.     

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.     

Japonica cultivars' susceptibility to the rice water weevil Lissorhoptrus oryzophilus (Coleoptera: Curculionoidea: Brachyceridae)

Italy is the largest rice‐producing country in the European Union. In Italy, only japonica cultivars are listed in the Italian National Register. Almost all of the rice production is concentrated in the Po Valley, where the rice water weevil Lissorhoptrus oryzophilus Kuschel was first detected and settled. This study investigated the performance of this pest in terms of feeding, reproduction and plant injury on 10 rice cultivars chosen among the most widely grown in Italy. No‐choice experiments were conducted to evaluate the plant susceptibility to larval attack and to find out how cultivars can influence the adult leaf area consumption. The results gave evidence of different types of attack depending on the density of the insect (0.6 adults/plant vs. 0.9 adults/plant), the cultivar type and climatic conditions. Different cultivars with the same level of infestation gave different results in terms of productivity. Production was significantly affected by the larval presence in 4 of the 10 cultivars tested. A different population structure reflected a different damage severity. Statistically different values for total adult leaf area consumption were found according to adult female age and to the cultivar.     

Evaluation of oxidative DNA damage and antioxidant defense in patients with nasal polyps

Abstract

Objectives

The presence of inflammatory cells indicates the development of epithelial cell injury in nasal polyposis (NP) and the potential for production of high levels of reactive oxygen and nitrogen species. The aim of our study was to clarify the role of oxidative stress and antioxidant status in the deterioration accompanying NP.

Methods

Twenty patients (11 men) aged 47.2 ± 17.0 years with nasal polyps were included in the study. Twenty healthy subjects (7 men) aged 48.2 ± 15.3 years formed the control group. The erythrocyte activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and plasma nitric oxide (NO) concentrations were measured. An alkaline comet assay was used to determine the extent of blood lymphocyte DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites.

Results

A significant increase of NO (P < 0.05) and non-significant decreases of SOD (P > 0.05), CAT (P > 0.05), and GPx (P > 0.05) were seen in NP patients compared to healthy controls. The level of blood lymphocyte oxidative DNA damage in NP patients was significantly higher compared to the control group (P = 0.01).

Discussion

The blood lymphocyte DNA damage level increased in patients with NP. Elevated DNA damage may be related to overproduction of reactive oxygen and nitrogen species and/or decreased antioxidant protection.     

Coming soon to a journal near you—The updated guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first guidelines paper for monitoring autophagy and interpreting the data resulting from the various assays used in our field. The guidelines paper was substantially expanded and updated in 2012. Based on the number of citations, and on comments from many users, I think it is accurate to say that the guidelines have been very useful for many researchers. Because the field continues to undergo rapid development, it is necessary to update the guidelines once again.     

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.     

Methodological evaluation of a new chemiluminescence immunoassay for thyrotropin using acridinium ester-labelled antibody

A recently available immunochemiluminometric assay (ICMA) for TSH developed by Ciba Corning Corp. has been evaluated. This system (Magic Lite) uses an acridinium-ester-labelled antibody and magnetizable particle for bound-free separation. In each assay only two calibrators are carried out and used to re-scale a manufacturer-generated curve stored in the memory of the luminometer. The precision of the response (RLU) estimated by all duplicates of 14 runs was about 4% for responses >12,000 RLU (corresponding to a concentration interval 0.7–113 μlU/ml) and worsened in the lower range (up to 10% CV); the sensitivity, computed from the mean within-assay precision profile, was 0.028 μlU/ml; the between-assay precision ranged from 4.6 to 13.1 CV%. Regression analysis of ICMA results (y) against consensus values of Behring IRMA (x) on 15 QC sera assayed in an inter-laboratory survey (concentration range 1–30 μlU/ml) gave y = ?0.003 + 0.98x indicating a good agreement of the two techniques. Similar conclusions have been derived from the comparison of the ICMA results (y) in the low TSH concentration range (< 1 μlU/ml) against the IRMA Boots Celltech (x) on 80 patient samples (y = 0.04 + 1.04x).