Assembly and characterization of pandemic influenza A H1N1 genome in nasopharyngeal swabs using high-throughput pyrosequencing

De novo high-throughput pyrosequencing was used to detect and characterize 2009 pandemic influenza A (H1N1) virus directly in nasopharyngeal swabs in the context of the microbial community. Data were generated with a prior sequence independent amplification by 454 pyrosequencing on GS-FLX platform (Roche). Influenza A assembled reads allowed near full-length genome reconstruction with the simultaneous analysis of site-specific heterogeneity. The molecular approach applied proved to be a powerful tool to characterize the new pandemic H1N1 influenza virus in clinical samples. This approach could be of great value in identifying possibly new reassortants that may occur in the near future.     

Type-1 cannabinoid receptors reduce membrane fluidity of capacitated boar sperm by impairing their activation by bicarbonate

Background

Mammalian spermatozoa acquire their full fertilizing ability (so called capacitation) within the female genital tract, where they are progressively exposed to inverse gradients of inhibiting and stimulating molecules.

Methodology/Principal Findings

In the present research, the effect on this process of anandamide, an endocannabinoid that can either activate or inhibit cannabinoid receptors depending on its concentration, and bicarbonate, an oviductal activatory molecule, was assessed, in order to study the role exerted by the type 1 cannabinoid receptor (CB1R) in the process of lipid membrane remodeling crucial to complete capacitation. To this aim, boar sperm were incubated in vitro under capacitating conditions (stimulated by bicarbonate) in the presence or in the absence of methanandamide (Met-AEA), a non-hydrolysable analogue of anandamide. The CB1R involvement was studied by using the specific inhibitor (SR141716) or mimicking its activation by adding a permeable cAMP analogue (8Br-cAMP). By an immunocytochemistry approach it was shown that the Met-AEA inhibits the bicarbonate-dependent translocation of CB1R from the post-equatorial to equatorial region of sperm head. In addition it was found that Met-AEA is able to prevent the bicarbonate-induced increase in membrane disorder and the cholesterol extraction, both preliminary to capacitation, acting through a CB1R-cAMP mediated pathway, as indicated by MC540 and filipin staining, EPR spectroscopy and biochemical analysis on whole membranes (CB1R activity) and on membrane enriched fraction (C/P content and anisotropy).

Conclusions/Significance

Altogether, these data demonstrate that the endocannabinoid system strongly inhibits the process of sperm capacitation, acting as membrane stabilizing agent, thus increasing the basic knowledge on capacitation-related signaling and potentially opening new perspectives in diagnostics and therapeutics of male infertility.     

Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide (CO), and biliverdin. We investigated subcellular localization of HO-1 using confocal laser scanning microscopy (CLSM) and the expression by Western blot in primary astroglial cells during differentiation and after exposure to glutamate (100microM). CLSM analysis of immunostained HO-1 in cultured astroglial cells during differentiation showed an increase of fluorescence between 7 and 14 days and a decrease between 14 and 21, although HO-1 peaked at 14 days it remained at high levels. The distribution of HO-1 protein undergoes modification in the various cellular compartments. Furthermore, localization of the protein in untreated astrocytes at 7 days appeared prevalently localized in the cytosol and in the perinuclear region. In contrast, at 14 and 21 days, fluorescence detection suggests that HO-1 was present also in the nucleus, and in the nucleoli. Fluorescence intensity significantly increased in glutamate-treated astrocytes during all development stages and the protein appeared in the cytosol, in the nucleus and in the nucleoli. The involvement of AMPA/Ka receptors was studied in glutamate-treated astroglial cells at 14 days by the preincubation of the cells with GYKI 52466, a specific receptor inhibitor, of AMPA/Ka receptor demonstrating the involvement of these receptors. Western blot analysis of HO-1 confirmed the CLSM results. Our results demonstrate that changes in HO-1 protein expression and localization in primary cultured astroglial cells may be part of the underlying mechanisms involved in brain development as well as in neurodegenerative diseases.     

Synthesis, solution equilibria and antiproliferative activity of copper(II) aminomethyltriazole and aminomethylthioxotriazoline complexes

The aim of the present study was the synthesis, the determination of formation constants, and the evaluation of the antiproliferative activity of two copper(II) complexes formed with triazole-type ligands. The synthesis of the unsymmetrical triazole ligand 4-amino-3-aminomethyl-5-methyl-1,2,4-triazole (L1), and its copper(II) complex is reported. The ligand was prepared by functionalization of the carboxylate function of tert-butyloxycarbonyl (BOC) protected glycine O-methyl ester. All intermediates and final products were isolated and characterized with IR, 1H NMR, and elemental analysis. X-ray structures of the ligand as a sulfate salt ((H2L1)2SO4.H2O) and the copper(II) complex [CuCl2(L1)(2)] are described. The ligand forms a (N,N) bidentate chelate with the amino group and one triazole nitrogen atom. The tetragonally distorted octahedral coordination of Cu(II) results from two axially coordinated chloride ions. Protonation constants for L1 and speciation of the Cu(II)/L1 system were determined in 0.1 M aqueous KCl solution at 25 degrees C. Complexes formed in solution were also characterized by visible spectrophotometry. Ligand substitution competition between L1 and glycine has also been studied using potentiometric titrations. Antiproliferative activities of ([CuCl2(L1)2]) and [CuCl2(H2L2)]Cl, where HL2 is the 5-thioxo analog of L1, against human tumor cell lines HT1080 and HT29 as well as normal human fibroblasts (HF) are presented along with the antiproliferative activities of L1, CuCl2, and cisplatin. Activity of these two complexes are discussed and compared with the activity of analogous compounds reported in the literature which contain pyridyl groups in place of the aminomethyl group. In particular, it is suggested that a lypophilic residue such as a pyridyl group is important for antiproliferative activity of this class of compounds.     

Dientamoeba fragilis is more prevalent than Giardia duodenalis in children and adults attending a day care centre in Central Italy

Giardia duodenalis is a well recognised enteropathogen, while Dientamoeba fragilis is rarely detected and consequently it is not recognised as an important human pathogen. In 2002-2003, a survey has been carried out on enteroparasites in faecal samples of outpatients attending a day care centre in the town of Perugia (Central Italy). To improve the detection level, at least three samples from each patient were collected at different days and within two hours from defecation. The coproparasitological examination has been carried out by direct microscopic examination, faecal concentration, and Giemsa and modified Ziehl-Nielsen stainings of faecal smears. The genotypes of Giardia duodenalis isolates were determined by PCR of the beta-giardin gene. Of 1,989 enrolled people (966 children, 1,023 adults), 165 persons (8.3%; 153 adults, 15.0%; 12 children, 1.2%), were positive for parasites, but only 1 12 adults (73.2% of those infected) and eight children (66.7% of those infected) harboured D. fragilis and G. duodenalis. Both the Assemblages A and B were detected in 18 G. duodenalis isolates examined at the beta-giardin gene. The higher prevalence of D. fragilis infections than that of G. duodenalis is probably related to the method used, a procedure, which is rarely followed in laboratories for the diagnosis of enteric parasites. These epidemiological data suggest that when faecal samples are examined after a period of time and without Giemsa staining, most D. fragilis infections goes undetected.     

Tissue arrays as fiducial markers for section alignment in 3-D reconstruction technology

Combination of conventional histology and the three-dimensional spatial view of tissue structures offers new prospects for understanding and diagnosing nature and development of human diseases. The essential technical problem related to three-dimensional reconstruction in histopathology is represented by the correct alignment of serial sections. During the past years several methods have been proposed but failed to become popular because of their limits in terms of time consume and restricted applicability. We aimed to overcome this problem by applying the technology of Tissue Array, thus by positioning adequate fiducial markers from specific "donor" blocks into the "recipient" paraffin block of interest. Digitized pictures of serially cut sections were aligned according to the tissue markers embedded by Tissue Array, and then processed with specific softwares for three-dimensional reconstruction. Thirteen models, including fetal hearts, breast and thyroid carcinomas, were elaborated. We found the procedure to be easy, fast and reproducible. Moreover, by selectively embedding the fiducial markers according to specific angles, the Tissue Arrays can be exploited in order to establish the distance between sections. This original methodology of incorporating Tissue Arrays into paraffin blocks as fiducial markers for three-dimensional reconstruction has a potential impact on histology for research purposes and diagnostic applications.     

Mitochondrial sequence variation in the Guahibo Amerindian population from Venezuela

New data were obtained on mitochondrial DNA (mtDNA) from Guahibo from Venezuela, a group so far not studied using molecular data. A population sample (n = 59) was analyzed for mtDNA variation in two control-region hypervariable segments (HV1 and HV2) by sequencing. The presence or absence of a 9-bp polymorphism in the COII/tRNA(Lys) region was studied by direct amplification and electrophoretic identification. Thirty-eight variable sites were detected in regions HV1 and HV2, defining 26 mtDNA lineages; 23.7% of these were present in a single individual. The 9-bp deletion was found in 3.39% of individuals. Nucleotide and haplotype diversities were relatively high compared with other New World populations. The identified sequence haplotypes were classified into four major haplogroups (A-D) according to previous studies, with high frequencies for A (47.46%) and C (49.15%), low frequency for B (3.39%), and an absence of D.     

The endocannabinoid system in neurodegeneration

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed.