Monitoring intracellular calcium in response to GPCR activation using thin-film silicon photodiodes with integrated fluorescence filters.

G-protein coupled receptor (GPCRs) drug discovery is a thriving strategy in the pharmaceutical industry. The standard approach uses living cells to test millions of compounds in a high-throughput format. Typically, changes in the intracellular levels of key elements in the signaling cascade are monitored using fluorescence or luminescence read-out systems, which require external equipment for signal acquisition. In this work, thin-film amorphous silicon photodiodes with an integrated fluorescence filter were developed to capture the intracellular calcium dynamics in response to the activation of the endogenous muscarinic M1 GPCR of HEK 293T cells. Using the new device it was possible to characterize the potency of carbachol (EC50=10.5 µM) and pirenzepine (IC50=4.2 µM), with the same accuracy as standard microscopy optical systems. The smaller foot-print provided by the detection system makes it an ideal candidate for the future integration in microfluidic devices for drug discovery.

Protein-ligand docking in the new millennium--a retrospective of 10 years in the field.

Protein-ligand docking is currently an important tool in drug discovery efforts and an active area of research that has been the subject of important developments over the last decade. These are well portrayed in the rising number of available protein-ligand docking software programs, increasing level of sophistication of its most recent applications, and growing number of users. While starting by summarizing the key concepts in protein-ligand docking, this article presents an analysis of the evolution of this important field of research over the past decade. Particular attention is given to the massive range of alternatives, in terms of protein-ligand docking software programs currently available. The emerging trends in this field are the subject of special attention, while old established docking alternatives are critically revisited. Current challenges in the field of protein-ligand docking such as the treatment of protein flexibility, the presence of structural water molecules and its effect in docking, and the entropy of binding are dissected and discussed, trying to anticipate the next years in the field.

Spintronic platforms for biomedical applications.

Since the fundamental discovery of the giant magnetoresistance many spintronic devices have been developed and implemented in our daily life (e.g. information storage and automotive industry). Lately, advances in the sensors technology (higher sensitivity, smaller size) have potentiated other applications, namely in the biological area, leading to the emergence of novel biomedical platforms. In particular the investigation of spintronics and its application to the development of magnetoresistive (MR) biomolecular and biomedical platforms are giving rise to a new class of biomedical diagnostic devices, suitable for bench top bioassays as well as point-of-care and point-of-use devices. Herein, integrated spintronic biochip platforms for diagnostic and cytometric applications, hybrid systems incorporating magnetoresistive sensors applied to neuroelectronic studies and biomedical imaging, namely magneto-encephalography and magneto-cardiography, are reviewed. Also lab-on-a-chip MR-based platforms to perform biological studies at the single molecule level are discussed. Overall the potential and main characteristics of such MR-based biomedical devices, comparing to the existing technologies while giving particular examples of targeted applications, are addressed.

Quantitation of non-amplified genomic DNA by bead-based hybridization and template mediated extension coupled to alkaline phosphatase signal amplification.

Klenow I polymerase activity was combined with solid phase DNA hybridization to detect non-amplified genomic DNA (gDNA) sequences from Escherichia coli. Aminopropyl-controlled pore glass surface-bound oligonucleotides were hybridized to fragmented gDNA. The template-mediated extension at the 3'-terminus of the immobilized probe was then promoted in the presence of Klenow I polymerase and digoxigenin-labeled nucleotides. Detection of the extended probes was accomplished with an anti-digoxigenin alkaline phosphatase conjugate protocol coupled to colorimetric or fluorescent detection. Using the colorimetric protocol, the proof-of-concept was established. The fluorescence-based methodology, on the other hand, provided the basis for a quantitative interpretation of the data, affording a detection limit of 5 pM gDNA.

Application of central composite design for DNA hybridization onto magnetic microparticles.

Central composite face-centered (CCF) design and response surface methodologies were used to investigate the effect of probe and target concentration and particle number in immobilization and hybridization on a microparticle-based DNA/DNA hybridization assay. The factors under study were combined according to the CCF design matrix, and the intensity of the hybridization signal was quantified by flow cytometry. A second-order polynomial was fitted to data and validated by analysis of variance. The results showed a complex relationship between variables and response given that all factors as well as some interactions were significant, yet it could explain 95% of the data. Probe and target concentration had the strongest impact on hybridization signal intensity. Increments in initial probe concentration in solution positively affected the hybridization signal until a negative influence of a compact probe layer emerged. This trend was attributed to probe-probe interactions. By manipulating particle number on both immobilization and hybridization, enhancements on the assay sensitivity could be obtained. Under optimized conditions, the limit of detection (LOD) at the 95% confidence level was determined to be 2.3 nM of target solution concentration.

The role of probe-probe interactions on the hybridization of double-stranded DNA targets onto DNA-modified magnetic microparticles.

In this work, we have studied the effect of different probe lengths and surface densities on the hybridization of a 181-bp polymerase chain reaction product to probes tethered onto magnetic microparticles. Hybridization was shown to be favored by longer probes but only at probe surface densities where probe-to-probe interactions are absent. From these results, a simple rule was inferred for determining maximum surface densities above which hybridization signals decreased. According to this rule, if the average surface area occupied by an immobilized probe (Sigma) is larger than the projected surface area of each tethered probe molecule (S(ss)), hybridization efficiency increases with surface density, whereas the reverse occurs when Sigma-S(ss) < 0.

Chemiluminescent bead-based hybridization assay for the detection of genomic DNA from E. coli in purified plasmid samples.

A bead-based hybridization assay was developed for detection of traces of E. coli genomic DNA (gDNA) present in purified plasmid DNA (pDNA) samples. Standards of gDNA and pDNA samples were sheared by sonication and adsorbed onto aminopropyl controlled pore glass (CPG) particles (130 microm). A preliminary study was conducted to optimize the amount of DNA adsorbed on the particles. Results indicated that maximum attachment efficiency was obtained by adsorbing DNA for 2 h in 0.2 x SSC, pH 5.7. The DNA-bound particles were hybridized overnight with a 181-bp digoxigenin-labeled probe, specific for gDNA. Following a chemiluminescent detection protocol, signal intensities of the standards were plotted as a function of initial gDNA concentration. The calculated detection limit (LOD) was 1.4 pM of gDNA. The assay was able to detect gDNA in pure plasmid preparations at the 1% level even in the presence of 1,000-fold excess of noncomplementary target. Hybridization results were compared with a quantitative real-time PCR assay. Both methods afforded similar accurate results at the 95% confidence level.

Epithelial gill cells in the armored catfish, Hypostomus cf. plecostomus (Loricariidae).

Epithelial gill cell morphology and distribution were investigated in the armored catfish, Hypostomus cf. plecostomus, which lives in soft ion-poor Brazilian freshwaters. Pavement cells are the most abundant type of cell on both filament and lamellar epithelia and there are a great number of mucous and chloride cells between them. Mucous cells are almost covered by adjacent pavement cells and have large packed granules showing electrondense differences. No mucous cells were found on the lamellar epithelium. Chloride cell were distributed throughout both epithelia and usually have large apical surface facing the external medium and may exhibit short and sparsely distributed microvilli. The presence of chloride cells on the lamellar epithelium may be an adaptation to low ion concentrations in the water, allowing for improved ion-transport capacity of the gill. The large size of these cells increases the water-blood barrier and may affect the transference of respiratory gases. However, the negative effect on the respiratory process may be minimized by this species' ability to resort to atmospheric air to fulfill its oxygen requirements.

Resolution of microsporidial keratoconjunctivitis in an AIDS patient treated with highly active antiretroviral therapy.

PURPOSE: To report the outcome of microsporidial keratoconjunctivitis in a patient with acquired immunodeficiency syndrome (AIDS) after highly active antiretroviral therapy without any specific treatment for microsporidiosis. METHODS: Case report. A 42-year-old woman diagnosed with AIDS and severe immunodepression (CD4+ of 9 cells/mm(3) and viral load of 460,000/mm(3)), antiretroviral naive, presented with cerebral toxoplasmosis and microsporidial keratoconjunctivitis in the right eye documented by conjunctival scraping and electron microscopy. RESULTS: The patient was treated with a combination of indinavir, stavudine, and lamivudine, besides sulfadiazine and pyrimethamine. No specific treatment for the microsporidial keratoconjunctivitis was attempted. One month later, the keratoconjunctivitis had disappeared. CONCLUSION: This case suggests that microsporidial keratoconjunctivitis in the setting of AIDS and severe immunodepression can be effectively managed with highly active antiretroviral therapy alone.

Epithelial gill cells in the armored catfish, Hypostomus cf. plecostomus (Loricariidae)

Epithelial gill cell morphology and distribution were investigated in the armored catfish, Hypostomus cf. plecostomus, which lives in soft ion-poor Brazilian freshwaters. Pavement cells are the most abundant type of cell on both filament and lamellar epithelia and there are a great number of mucous and chloride cells between them. Mucous cells are almost covered by adjacent pavement cells and have large packed granules showing electrondense differences. No mucous cells were found on the lamellar epithelium. Chloride cell were distributed throughout both epithelia and usually have large apical surface facing the external medium and may exhibit short and sparsely distributed microvilli. The presence of chloride cells on the lamellar epithelium may be an adaptation to low ion concentrations in the water, allowing for improved ion-transport capacity of the gill. The large size of these cells increases the water-blood barrier and may affect the transference of respiratory gases. However, the negative effect on the respiratory process may be minimized by this species' ability to resort to atmospheric air to fulfill its oxygen requirements

Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography.

The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded plasmid DNA. RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC. RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.

Circadian rhythms in Neurospora crassa: effects of unsaturated fatty acids.

Employing a fatty acid-requiring strain (bd csp cel) of Neurospora crassa, the 21.5-h period of the circadian spore-forming rhythm was manipulated by fatty acid supplementation. The addition to the medium of an unsaturated fatty acid (oleic, linoleic, or linolenic acid) lengthened the period to 26, 40, or 33 h, respectively. Ther period-lengthening effect of linoleic acid was proportional to its concentration up to 1.3 X 10(-4) M, and also was reversed by the addition to the medium of a saturated fatty acid, palmitic acid. None of these period-lengthening effects was observed in the prototrophic strain (bd csp cel+).