Discovery of novel dengue virus entry inhibitors via a structure-based approach

Dengue is a mosquito-borne virus that has become a major public health concern worldwide in recent years. However, the current treatment for dengue disease is only supportive therapy, and no specific antivirals are available to control the infections. Therefore, the need for safe and effective antiviral drugs against this virus is of utmost importance. Entry of the dengue virus (DENV) into a host cell is mediated by its major envelope protein, E. The crystal structure of the E protein reveals a hydrophobic pocket occupied by the detergent n-octyl-β-d-glucoside (β-OG) lying at a hinge region between domains I and II, which is important for the low-pH-triggered conformational rearrangement required for fusion. Thus, the E protein is an attractive target for the development of antiviral agents. In this work, we performed prospective docking-based virtual screening to identify small molecules that likely bind to the β-OG binding site. Twenty-three structurally different compounds were identified and two of them had an EC₅₀ value in the low micromolar range. In particular, compound 2 (EC₅₀ = 3.1 μM) showed marked antiviral activity with a good therapeutic index. Molecular dynamics simulations were used in an attempt to characterize the interaction of 2 with protein E, thus paving the way for future ligand optimization endeavors. These studies highlight the possibility of using a new class of DENV inhibitors against dengue.     

Enteroparasitoses and Toxocarosis Affecting Children from Mar del Plata City,Argentina

This study evaluated the existence of enteroparasitoses and toxocarosis in children of peripheral (PC) and urban communities (UC) from Mar del Plata city (Argentina) and their associations with socio-environmental conditions. A Parasite Vulnerability Index (PVI) was elaborated using variables such as overcrowding, floor type, drinking water source, wastewater disposal, solid waste disposal, presence of animals and schooling level. The PC evidenced statistically significant higher frequencies of families with high (38.9%) and medium (55.5%) PVI, while in the UC low PVI (93%) was the most frequent. A statistically significant higher frequency of PC children was parasitized (30.2 vs. 14.5%; χ ² Pearson = 5.21; P < 0.05), presented higher parasite frequencies, specific richness, parasitic loads, and they also evidenced polyparasitism. The Multiple Correspondence Analysis (MCA) showed associations between PC-parasitized children, overcrowding and contact with pets and farm animals. The ELISA test to the specified determination of Toxocara canis IgG was reactive in a statistically significant higher proportion of PC children than the UC (55 vs. 8.5%; χ ² = 30.5; P < 0.01). The MCA associated PC reactive children, not adequate hand washing, moderate and hypereosinophilia and contact with pets and farm animals. Deficient socio-environmental conditions became children more vulnerable to get enteroparasitoses and toxocarosis in the PC than in the UC.     

The effect of temperature and irradiance on the growth and carotenogenic capacity of seven strains of Dunaliella salina (Chlorophyta) cultivated under laboratory conditions

The carotenogenic microalga Dunaliella salina is cultivated as a natural source of beta-carotene. The 9-cis isomer of beta-carotene is found only in natural sources having commercial advantages over the all-trans isomer due to its high liposolubility and antioxidant power. High irradiance appears to stimulate specifically all-trans beta-carotene accumulation in D. salina, whereas low temperature apparently elicits a-carotene and 9-cis betacarotene production. We studied the effect of temperature and irradiance on the growth and the carotenogenesis of three Chilean (CONC-001, CONC-006 and CONC-007) and four non-Chilean (from Mexico, China, Australia and Israel) strains of D. salina cultivated under two photon flux densities (40 and 110 micromol photons x m(-2) x s(-1)) and two temperatures (15 and 26 degrees C). The Chilean strain CONC-001 and all of the non-Chilean strains exhibited the highest growth rates and the maximum cell densities, whereas the Chilean strains CONC-006 and CONC-007 showed the lowest values in both parameters. The Australian strain showed the highest accumulation of total carotenoids per unit volume (40.7 mg x L(-1)), whereas the Chilean strains CONC-006 and CONC-007, the only ones isolated from Andean environments, yielded the highest amounts of carotenoids per cell (61.1 and 92.4 pg x cell(-1), respectively). Temperature was found to be more effective than irradiance in changing the qualitative and quantitative carotenoids composition. The Chilean strains accumulated 3.5-fold more alpha-carotene than the non-Chilean strains when exposed to 15 degrees C and, unlike the non-Chilean strains, also accumulated this pigment at 26 degrees C. The 9-cis/all-trans beta-carotene ratio was > 1.0 in all treatments for all strains, and the values were not greatly influenced by either temperature or photon flux density. Physiological and biotechnological implications of these results are discussed.     

Melanin concentrating hormone increase hippocampal synaptic transmission in the rat

A retrograde facilitation has been demonstrated in the one trial step-down inhibitory avoidance of melanin-concentrating hormone (MCH), when it was infused into rat hippocampal formation. Considering the high density of specific binding sites for the MCH peptide on the hippocampus and the participation of this structure on learning and memory processes we have studied the effects of MCH on the hippocampal synaptic transmission. For this purpose, slices of rat hippocampus were perfused with different concentration of MCH. The main result of the present study was a long-lasting potentiation on the hippocampal evoked response on dentate gyrus induced by MCH (4-11 microM) at 30, 60 and 120 min with a maximum effect at 120 min. Previous perfusion of DL - 2- amino - 5 phosphonovaleric acid (APV, 20 microM) was unable to impair the increased hippocampal evoked response induced by MCH 4 microM. On the other hand, the channel blocker Dizocilpine (MK-801, 10 microM) completely impaired the increased hippocampal synaptic plasticity induced by MCH perfusion. We postulate the increased hippocampal synaptic efficacy induced by MCH as one of the mechanisms underlying the retrograde facilitation on the inhibitory avoidance paradigm, observed after MCH hippocampal microinjection. We cannot rule out other MCH neurochemical mechanism and other areas of the brain involved in the MCH effects.     

Pretranslational control of Menkes disease gene expression

The gene for Menkes disease codes for a Cu-transporting ATPase that regulates Cu homeostasis in all tissues with the exception of adult liver. The basis for developmental or tissue-specific regulation at present is not understood. To learn if the regulation is associated with the promoter, we cloned and sequenced a 2.2 kb genomic DNA fragment flanking exon 1. When ligated into a pGL2 luciferase reporter gene construct, the 2.2 kb showed promoter activity, but not nearly to the extent of a 1.3 kb fragment previously reporter by Levinson et al. Sequence analysis of the nucleotides spanning the region between 1.3 kb and 2.2 kb revealed a 13-nucleotide motif ACACAAAAAAATA 2059 bp upstream from the start site that duplicated the `hunchback' binding site, a key site controlling developmental gene expression in Drosophila. Eliminating 129 bp containing the hunchback site (Hb) from the 5 end of the 2.2 kb stimulated promoter activity, suggesting the Hb site was basically suppressive. When ligated upstream of an SV40 and tested in SY5Y cells, however, the SV40 promoter activity was strongly stimulated, which conflicts with the site being suppressive. Mutating the site in the 2.2 kb weakened the promoter activity in SY5Y and HepG2 cells and a fragment with mutated sequence ligated upstream of the SV40 cancelled the activation of SV40 promoter activity. All data suggested the Hb site was a positive controlling site for Cu-ATPase expression. Nuclear extracts from SY5Y and HepG2 cells were observed to bind to a 106 bp probe with the Hb site in a gel-shift assay. Only SY5Y proteins, however, showed a slower moving shift band indicative of a secondary interaction. A probe with mutated sequences displayed the same shift pattern, suggesting other sites in the 106 bp DNA strand were also recognizing the nuclear proteins. A Southwestern analysis suggested that proteins of 125 kD, 70 kD, 50 kD and 42 kD bound to the wild type probe; a 60 kD and all with the exception of the 42 kD bound to the mutant probe. The data support the conclusion that the distal promoter of the Menkes disease gene contains elements that interact in combinatorial fashion to regulate Cu-ATPase expression and that tissue specificity may lie with the quantity or types of distinct DNA binding proteins in the nucleus.