Biosensor for dengue virus detection: sensitive, rapid, and serotype specific

A serotype-specific RNA biosensor was developed for the rapid detection of Dengue virus (serotypes 1-4) in blood samples. After RNA amplification, the biosensor allows the rapid detection of Dengue virus RNA in only 15 min. In addition, the biosensor is portable, inexpensive, and very easy to use, making it an ideal detection system for point-of-care and field applications. The biosensor is coupled to the isothermal nucleic acid sequence-based amplification (NASBA) technique with which small amounts of virus RNA are amplified using a simple water bath. During the NASBA reaction, a generic sequence is attached to all RNA molecules as described earlier (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtliff, R. N.; Porter, K R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). It has been shown earlier that Dengue virus can be detected specifically using two DNA probes: a first probe hybridized with the attached generic sequence and, therefore, bound to every amplified RNA molecule; and a second probe either bound to all four Dengue virus serotypes or chosen to be specific for only one serotype. These probes were utilized in the biosensor described in this publication. For a generic Dengue virus biosensor, the second probe is complementary to a conserved region found in all Dengue serotypes. For identification of the individual Dengue virus serotypes, four serotype-specific probes were developed (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtiff, R. N.; Porter, K. R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The generic DNA probe (reporter probe) is coupled to the outside of dye-encapsulating liposomes. The conserved or Dengue serotype specific probes (capture probes) are immobilized on a polyethersulfone membrane strip. Liposomes are mixed with amplified target sequence and are then applied to the membrane. The mixture is allowed to migrate along the test strip, and the liposome-target sequence complexes are immobilized in the capture zone via hybridization of the capture probe with target sequence. The amount of liposomes present in the immobilized complex is directly proportional to the amount of target sequence present in the sample and can be quantified using a portable reflectometer. The different biosensor components have been optimized with respect to sensitivity and, foremost, specificity toward the different serotypes. An excellent correlation to a laboratory-based detection system was demonstrated. Finally, the assay was tested using a limited number of clinical human serum samples. Although Dengue serotypes 1, 2 and 4 were identified correctly, serotype 3 displayed low cross-reactivity with biosensors designed for detection of serotypes 1 and 4.     

Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.     

Spontaneous Assembly in Organic Thin Films Spread on Aqueous Subphase: A Scanning Force Microscope (SFM) Study

Structure, morphology, and mechanical properties of mono- and several-layer structures of amphiphiles or pure n-alkane crystallites generated by spontaneous self-assembly on aqueous subphase have been analyzed by scanning force microscopy (SFM). Pure-component and heterogeneous mixtures of molecules were allowed to spread and self-assemble without compression on an aqueous subphase. The self-assembled films were transferred to an atomically smooth mica substrate by drainage for measurement using SFM. Results were compared with a variety of techniques including cryo-transmission electron microscopy, grazing incidence X-ray diffraction, X-ray reflectivity, and reflectance/absorption infrared spectroscopy. Whereas the collaborative techniques provide spatially-averaged information, we find that the SFM accesses both individual crystallites and amorphous material, thus providing unique information on the morphology, number of layers, and complementary structural features.     

Use of the confocal laser scanning microscope in studies on the developmental biology of marine crustaceans

Confocal Laser Scanning Microscope techniques have been applied to study the developmental biology of marine copepods and decapod larvae. The lipophylic probes DiI and DiOC(6) were used to study both the external and internal morphology of these crustaceans, whereas the same DiOC(6) and the specific nuclear probe Hoechst 33342 were used to study embryonic development of copepods in vivo. To distinguish viable from non-viable copepod embryos, the vital dye dichlorodihydrofluorescein diacetate (H(2)DCFDA) was used. Major advantages and difficulties in the use of these non-invasive techniques in studies of the reproductive biology of marine crustaceans are discussed.     

Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification

A reliable method using nucleic acid sequence based amplification (NASBA) with subsequent electrochemiluminescent detection for the specific and sensitive detection of viable oocysts of Cryptosporidium parvum in environmental samples was developed. The target molecule was a 121-nt sequence from the C. parvum heat shock protein hsp70 mRNA. Oocysts of C. parvum were isolated from environmental water via vortex flow filtration and immunomagnetic separation. A brief heat shock was applied to the oocysts and the nucleic acid purified using an optimized very simple but efficient nucleic acid extraction method. The nucleic acid was amplified in a water bath for 60-90 min with NASBA, an isothermal technique that specifically amplifies RNA molecules. Amplified RNA was hybridized with specific DNA probes and quantified with an electrochemiluminescence (ECL) detection system. We optimized the nucleic acid extraction and purification, the NASBA reaction, amplification, and detection probes. We were able to amplify and detect as few as 10 mRNA molecules. The NASBA primers as well as the ECL probes were highly specific for C. parvum in buffer and in environmental samples. Our detection limit was approximately 5 viable oocysts/sample for the assay procedure, including nucleic acid extraction, NASBA, and ECL detection. Nonviable oocysts were not detected.     

Experimental Study of the NO Oxidation to NO2 Over Metal Promoted Zeolites Aimed at the Identification of the Standard SCR Rate Determining Step

Steady state and transient kinetic runs devoted to the comparative analysis of NO oxidation and standard SCR reactions over commercial Cu- and Fe-promoted zeolite catalysts are herein presented with the aim to clarify whether NO oxidation to NO₂ is the rate determining step (rds) of the standard SCR reaction. It is found that this statement seems questionable in the light both of the herein collected experimental results and of scattered evidence from the literature.     

Study of the Expression and Function of Calcium-Sensing Receptor in Human Skeletal Muscle

Skeletal muscle has an outstanding capacity for regeneration in response to injuries, but there are disorders in which this process is seriously impaired, such as sarcopenia. Pharmacological treatments to restore muscle trophism are not available, therefore, the identification of suitable therapeutic targets that could be useful for the treatment of skeletal reduced myogenesis is highly desirable. In this in vitro study, we explored the expression and function of the calcium-sensing receptor (CaSR) in human skeletal muscle tissues and their derived satellite cells. The results obtained from analyses with various techniques of gene and protein CaSR expression and of its secondary messengers in response to calcium (Ca²⁺) and CaSR drugs have demonstrated that this receptor is not present in human skeletal muscle tissues, neither in the established satellite cells, nor during in vitro myogenic differentiation. Taken together, our data suggest that, although CaSR is a very important drug target in physiology and pathology, this receptor probably does not have any physiological role in skeletal muscle in normal conditions.     

A glucose derivative as natural alternative to the cyclohexane‐1,2‐diamine ligand in the anticancer drug oxaliplatin?

Having oxaliplatin as archetype, several platinum complexes with a carbohydrate moiety resembling the cyclohexane-1,2-diamine ligand of oxaliplatin have been prepared. As leaving groups, the anionic ligands iodide, oxalate, and malonate were utilized, and for comparison purposes the chloro complex was employed. All compounds were characterized by elemental analysis, nuclear magnetic resonance spectroscopy, and electrospray mass spectrometry. The crystal structure of (SP-4-3)-diiodo(2,3-diamino-2,3-dideoxy-D-glucose-kappa(2)N,N')platinum(II) was determined by X-ray diffraction. The affinity toward dGMP was assayed by capillary electrophoresis, revealing that the chloro complex shows the highest reactivity, followed by the iodo complex. In contrast, the binding kinetics of the dicarboxylato complexes are slower, with the malonato complex being the least reactive. Reactivity to dGMP in the cell-free system correlates with cytotoxicity in two of four human cancer cell lines as determined by means of the MTT assay. In three of the four cell lines, the chloro and the malonato complex are the most and the least active of the carbohydrate-Pt complexes, respectively, with IC(50) values differing only by factors of up to 3.2. Cytotoxicity of the chloro complex is one to two orders of magnitude lower than that of oxaliplatin, but still comparable to that of carboplatin in two of the four cell lines.