The use of duplex-specific crab nuclease for rapid analysis of single-nucleotide polymorphisms and the detection of DNA targets in complex PCR products

A rapid method for single-nucleotide polymorphism analysis and the detection of target clones after cloning of complex PCR products based on the use of duplex-specific crab nuclease and a universal fluorescent probe has been developed. The method is an alternative to the labor-intensive procedures of clone screening employing radioactively labeled probes, gel-based restriction analysis, and costly sequencing. The efficiency of the novel method has been demonstrated in a range of model systems.     

ABC transporters affect the detection of intracellular oxidants by fluorescent probes

Intracellular production of reactive oxygen species (ROS) plays an important role in the control of cell physiology. For the assessment of intracellular ROS production, a plethora of fluorescent probes is commonly used. Interestingly, chemical structures of these probes imply they could be substrates of plasma membrane efflux pumps, called ABC transporters. This study tested whether the determination of intracellular ROS production and mitochondrial membrane potential by selected fluorescent probes is modulated by the expression and activity of ABC transporters. The sub-clones of the HL-60 cell line over-expressing MDR1, MRP1 and BCRP transporters were employed. ROS production measured by luminol- and L-012-enhaced chemiluminescence and cytochrome c reduction assay showed similar levels of ROS production in all the employed cell lines. It was proved that dihydrorhodamine 123, dihexiloxocarbocyanine iodide, hydroethidine, tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide and tetramethylrhodamine ethyl ester perchlorate are substrates for MDR1; dichlorodihydrofluoresceine, hydroethidine and tetramethylrhodamine ethyl ester perchlorate are substrates for MRP1; dichlorodihydrofluoresceine, dihydrorhodamine 123, hydroethidine and tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide are substrates for BCRP. Thus, the determination of intracellular ROS and mitochondrial potential by the selected probes is significantly altered by ABC transporter activities. The activity of these transporters must be considered when employing fluorescent probes for the assessment of ROS production or mitochondrial membrane potential.     

Glutathione-S-transferase as a molecular biomarker of the state of marine organisms influenced by anthropogenic pressure

The activity of glutathione-S-transferase (GST), a key phase II enzyme catalyzing the biotrans-formation of organic electrophilic compounds, was studied in the bivalve Crenomytilus grayanus, the mysid shrimp Neomysis mirabilis, and the flounder Liopsetta pinnifasciata from Peter the Great Bay, Sea of Japan. GST activity was increased in fish and mollusks from polluted areas of the bay, compared to background areas. In mysid shrimps, pollution caused inhibition of the activity of this enzyme. The role of GST in adaptation processes of different species to the current level of pollution is discussed.     

A systematic review of re-identification attacks on health data

BackgroundPrivacy legislation in most jurisdictions allows the disclosure of health data for secondary purposes without patient consent if it is de-identified. Some recent articles in the medical, legal, and computer science literature have argued that de-identification methods do not provide sufficient protection because they are easy to reverse. Should this be the case, it would have significant and important implications on how health information is disclosed, including: (a) potentially limiting its availability for secondary purposes such as research, and (b) resulting in more identifiable health information being disclosed. Our objectives in this systematic review were to: (a) characterize known re-identification attacks on health data and contrast that to re-identification attacks on other kinds of data, (b) compute the overall proportion of records that have been correctly re-identified in these attacks, and (c) assess whether these demonstrate weaknesses in current de-identification methods.ConclusionsThe current evidence shows a high re-identification rate but is dominated by small-scale studies on data that was not de-identified according to existing standards. This evidence is insufficient to draw conclusions about the efficacy of de-identification methods.     

Oncological principles of surgery in the periorbital region

The aim of this study is to describe the most common malignant periorbital tumours and principles of their surgical treatment. The most common malignant tumours of the eyelids are: basocellular carcinoma, squamocellular carcinoma, sebaceous carcinoma and malignant melanoma. The primary treatment of periorbital tumours is surgery, other methods are cryotherapy or radiotherapy. Malignant tumours of the eyelids are fairly rare. Diagnosis in some of the cases is difficult, since the first signs of the tumours are small changes on the eyelid margin or eyelid skin. Focal loss of the eyelashes could be the first sign of a malignant tumour. General oncological principles are valid also in the surgery of periorbital region. Eyelids have special anatomical structure and their main role is protection of the eyeball. Therefore, early diagnosis and total tumour removal--regardless of the anatomical borders--as well as immediate reconstruction are important in the treatment of eyelid tumours.     

A live cell hormone-binding assay on transgenic bacteria expressing a eukaryotic receptor protein

The investigation of hormone-receptor interaction normally needs isolation and extensive purification of the receptor protein or a particular receptor-containing fraction. To bypass these time- and resource-consuming procedures, we have established a live cell-based assay using transgenic bacteria expressing single eukaryotic receptors. Here we describe some biochemical features of the Arabidopsis cytokinin receptor CRE1/AHK4 expressed in Escherichia coli. The data show that the main characteristics of the ligand-receptor interaction, including binding affinity and ligand specificity, can be determined using intact bacteria expressing a functional receptor.     

Clades of Adeno-associated viruses are widely disseminated in human tissues

The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors.