ABSTRACT
Human metapneumovirus (hMPV) is an important aetiological agent of respiratory tract infection (RTI) in infants. Based on genetic differences, hMPVs are separated into two main groups, A and B, and two subgroups 1 and 2. To better understand the genetic diversity of hMPV, we analyzed 435 bp fragments of the F gene from 49 isolates obtained from a paediatric clinical centre in Northern Italy from 2005 to 2009. The phylogenetic analysis showed that our hMPV sequences clustered into five main clades (A1, A2a, A2b, B1, B2) statistically supported. Most of the strains belong to A2 (49%) and to B1 (28.5%) lineages. The intermixing in the phylogenetic tree showed no seasonal distribution between samples collected over a 5 year period. Mean genetic distances showed that clade A2 was more heterogeneous than others. In our F glycoprotein dataset we observed only two positively selected sites with an w value of 1.408 and 1.429, respectively, and 27 negatively selected sites. The two positively selected sites could be considered evolutionary "hot spots" because they were under positive selection and/or relaxed selective constraints. The abundant negatively selected sites reflect a high degree of conservation of F protein, probably necessary for viral infection.
Subject(s)
Epidemics/statistics & numerical data , Metapneumovirus/genetics , Paramyxoviridae Infections/epidemiology , Paramyxoviridae Infections/virology , Phylogeny , Evolution, Molecular , Genes, Viral/genetics , Genetic Variation/genetics , Humans , Italy/epidemiology , Metapneumovirus/classification , Seasons , Viral Fusion Proteins/geneticsABSTRACT
PURPOSE: Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). METHODS: NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). RESULTS: Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. CONCLUSIONS: These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.
Subject(s)
Curcumin/pharmacology , Hippocampus/drug effects , N-Methylaspartate/toxicity , Neuroprotective Agents/pharmacology , Receptors, N-Methyl-D-Aspartate/metabolism , Retina/drug effects , Animals , Apoptosis/drug effects , Blotting, Western , Calcium/metabolism , Cells, Cultured , Dizocilpine Maleate/pharmacology , Dose-Response Relationship, Drug , Excitatory Amino Acid Agonists/toxicity , Excitatory Amino Acid Antagonists/pharmacology , Female , Hippocampus/embryology , Hippocampus/metabolism , Hippocampus/pathology , Immunohistochemistry , In Situ Nick-End Labeling , Kainic Acid/toxicity , Patch-Clamp Techniques , Pregnancy , Rats , Rats, Wistar , Retina/embryology , Retina/metabolism , Retina/pathology , Time Factors , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
OBJECTIVE: The goal of the OSCAR programme is to evaluate the performances of genotypic HIV-1 tropism testing in clinical practice using the enhanced sensitivity version of Trofile (ESTA) as reference-assay. METHODS: HIV-1 coreceptor-usage was assessed using plasma samples from 406 HIV-1 infected patients by ESTA and by gp120 V3 population-sequencing followed by Geno2pheno (set at a False Positive Rate [FPR] of 10% and 5%). RESULTS: ESTA was successful in 365 (89.9%) samples indicating R5 in 254 (69.6%), and DM/X4 in 111 (30.4% of samples (104 [28.5%] DM and 7 [1.9%] X4). Genotypic-testing successfully assessed viral tropism for all 406 samples, including the 41 with undetermined result by ESTA. Genotypic-tropism testing at a FPR of 5% and 10% was 81.1% and 78.4% concordant with ESTA, respectively. Despite a sensitivity of 48.7% and 55.9% at a FPR of 5% and 10%, respectively, a high concordance (specificity: 95.3% for FPR of 5% and 88.2% for FPR of 10%) between genotypic-tropism testing and ESTA was reached in the detection of R5-tropic viruses. CONCLUSION: Our results are in line with other European studies, and support the routine use of genotypic tropism testing in clinical-settings for monitoring of HIV-1 infected patients candidate to or failing CCR5-antagonists.
