MIND-BEST: Web server for drugs and target discovery; design, synthesis, and assay of MAO-B inhibitors and theoretical-experimental study of G3PDH protein from Trichomonas gallinae

Many drugs with very different affinity to a large number of receptors are described. Thus, in this work, we selected drug-target pairs (DTPs/nDTPs) of drugs with high affinity/nonaffinity for different targets. Quantitative structure-activity relationship (QSAR) models become a very useful tool in this context because they substantially reduce time and resource-consuming experiments. Unfortunately, most QSAR models predict activity against only one protein target and/or they have not been implemented on a public Web server yet, freely available online to the scientific community. To solve this problem, we developed a multitarget QSAR (mt-QSAR) classifier combining the MARCH-INSIDE software for the calculation of the structural parameters of drug and target with the linear discriminant analysis (LDA) method in order to seek the best model. The accuracy of the best LDA model was 94.4% (3,859/4,086 cases) for training and 94.9% (1,909/2,012 cases) for the external validation series. In addition, we implemented the model into the Web portal Bio-AIMS as an online server entitled MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (MIND-BEST), located at http://miaja.tic.udc.es/Bio-AIMS/MIND-BEST.php . This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally, we illustrated two practical uses of this server with two different experiments. In experiment 1, we report for the first time a MIND-BEST prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of eight rasagiline derivatives, promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF and -TOF/TOF MS, MASCOT search, 3D structure modeling with LOMETS, and MIND-BEST prediction for different peptides as new protein of the found in the proteome of the bird parasite Trichomonas gallinae, which is promising for antiparasite drug targets discovery.     

In silico two-hybrid system for the selection of physically interacting protein pairs

Deciphering the interaction links between proteins has become one of the main tasks of experimental and bioinformatic methodologies. Reconstruction of complex networks of interactions in simple cellular systems by integrating predicted interaction networks with available experimental data is becoming one of the most demanding needs in the postgenomic era. On the basis of the study of correlated mutations in multiple sequence alignments, we propose a new method (in silico two-hybrid, i2h) that directly addresses the detection of physically interacting protein pairs and identifies the most likely sequence regions involved in the interactions. We have applied the system to several test sets, showing that it can discriminate between true and false interactions in a significant number of cases. We have also analyzed a large collection of E. coli protein pairs as a first step toward the virtual reconstruction of its complete interaction network.     

Porous polyethylene implants in orbital floor reconstruction

The purpose of this article is to present the authors' experience with the use of porous polyethylene ultrathin sheets for orbital floor reconstruction. Thirty-two patients with orbital floor fractures were treated with porous polyethylene ultrathin sheets. Sixteen cases corresponded to orbitozygomatic fractures, 11 cases corresponded to pure orbital floor fractures, and five corresponded to panfacial fractures. The subciliary approach was used in 15 patients and the transconjunctival approach in nine; another three patients were operated on through a preexisting eyebrow wound, two were operated on with a subtarsal approach, two were operated on through an eyebrow extension of a facial wound, and one patient was operated on through the facial wound. Intraoperatively, all patients received a prophylactic dose of intravenous antibiotics. Postoperatively, 24 patients received amoxicillin clavulanate for 5 to 7 days, two patients received clindamycin, and six patients received no antibiotics. Enophthalmos was corrected in 15 of 24 patients (62.5 percent), and hypoglobus in nine of 11 (82 percent). Diplopia was resolved in 25 of 28 patients (89.3 percent) with preoperative impairment. Extrinsic eye movement impairment was resolved in 25 of 27 patients (92.6 percent). A preoperative visual acuity deficit was present in four patients (12.5 percent) and was resolved in one (from 20/100 to 20/20). Visual acuity improved in one patient (from 20/60 to 20/30). In the other two patients, visual acuity remained altered (from 20/30 to 20/30). One patient (3.1 percent) suffered blindness induced by surgery. Nine of 26 patients (34.6 percent) had residual infraorbital nerve hypesthesia and five (19.2 percent) had residual paresthesias. Postoperatively, epiphora was present in six patients (18.8 percent) and ectropion in five (15.6 percent). Although there was no statistical significance between the surgical approach and the presence of epiphora (p = 0.211) and ectropion (p = 0.422), patients who were treated using the transconjunctival approach suffered reduced ectropion (0 percent) compared with patients treated using the subciliary approach (20 percent). However, patients treated using the transconjunctival approach suffered increased epiphora (22.2 percent) compared with those treated with the subciliary approach (13.3 percent). There were four cases (12.5 percent) of postoperative facial infections. Two of these cases were resolved with systemic antibiotics, one was resolved with bone sequestrum resection, and one patient needed removal of the implant. Orbital infections were related in all cases to titanium osteosynthesis miniplates or skull bone graft. When comparing patients who were treated with and without antibiotics, no statistical differences (p = 0.958) were found relative to the presence of infections. Correction of hypoglobus is technically easier than enophthalmos, because enophthalmic correction requires a wide, deep subperiosteal dissection and implant positioning, posterior to the equator of the globe, with the inherent risk of orbital apex injury.     

Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens

Current methods for the serodiagnosis of sheep fascioliasis show suboptimal sensitivity, specificity, or both. With the aim of developing an improved method, we fractionated native Fasciola hepatica excretory-secretory antigens (ESAs) by size-exclusion FPLC (fast protein liquid chromatography) on a Superdex 75 HR 10/30 column and then tested the serodiagnostic value of the antigens contained in each one of the 4 peaks obtained (peaks I-IV). Serodiagnostic value was assessed using sera from sheep naturally infected with F. hepatica (group A); sera from the individuals of a fluke-free herd (most of which also had other intestinal nematodes, lung nematodes, Moniezia spp., and/or Cysticercus tenuicollis) sera from a fluke-free herd (group B); sera from lambs experimentally infected with 10-40 F. hepatica metacercariae (group C); and sera from uninfected control lambs (group D). Enzyme-linked immunosorbent assay (ELISA) with peak I or II as target antigens (and to a lesser extent with peak III as target) showed reactivity with negative sera, so that it was not possible to establish cutoff values discriminating infected and uninfected animals. In contrast, when peak IV was used as target, a low cutoff value of 0.235 optical density units (mean + 4 SD) discriminated infected and uninfected animals, with 100% sensitivity and 100% specificity. ELISA with peak IV as a target identified infected animals (even animals that had received only 10 metacercariae) within 3-5 wk of infection and subsequently throughout the rest of the 14-wk monitoring period. In Western blotting analysis, again only the antigens contained in peak IV (range 7-40 kDa, under reducing conditions) were specific for diagnosis of infected animals. These results indicate that molecular sieving of F. hepatica ESAs by this procedure is a fast, simple, reproducible way of obtaining antigens useful for serodiagnosis of sheep fascioliasis.     

Induction of a Crassulacean acid like metabolism in the C4 succulent plant,Portulaca oleracea L.: physiological and morphological changes are accompanied by specific modifications in phosphoenolpyruvate carboxylase

The induction of a Crassulacean acid like metabolism (CAM) was evidenced after 21–23 days of drought stress in the C₄ succulent plant Portulaca oleracea L. by changes in the CO₂ exchange pattern, in malic acid content and in titratable acidity during the day–night cycle. Light microscopy studies also revealed differences in the leaf structure after the drought treatment. Following the induction of the CAM-like metabolism, the regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the enzyme responsible for the diurnal fixation of CO₂ in C₄ plants but nocturnal in CAM plants, were studied. The enzyme from stressed plants showed different kinetic properties with respect to controls, notably its lack of cooperativity, higher sensitivity to L-malate inhibition, higher PEP affinity and lower enzyme content on a protein basis. In both conditions, PEPC's subunit mass was 110 kDa, although changes in the isoelectric point and electrophoretic mobility of the native enzyme were observed. In vivo phosphorylation and native isoelectrofocusing studies indicated variations in the phosphorylation status of the enzyme of samples collected during the night and day, which was clearly different for the control and stressed groups of plants. The results presented suggest that PEPC activity and regulation are modified upon drought stress treatment in a way that allows P. oleracea to perform a CAM-like metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

High Intensity Light Increases Olfactory Bulb Melatonin in Venezuelan Equine Encephalitis Virus Infection

In mice infected with the Venezuelan equine encephalomyelitis (VEE) virus and exposed to high intensity light (2500 lux) with a 12 h light: 12 h dark photoperiod, a significant increase in the levels of melatonin in the olfactory bulb was observed. The significance of these findings deserves further studies to understand the mechanisms involved in this effect since the olfactory bulbs have been proposed as first portal for VEE virus entry into the CNS. The increase in melatonin content could represent one of the mechanisms of defense against the viral attack.     

Phylogeny-independent detection of functional residues

MOTIVATION: Current projects for the massive characterization of proteomes are generating protein sequences and structures with unknown function. The difficulty of experimentally determining functionally important sites calls for the development of computational methods. The first techniques, based on the search for fully conserved positions in multiple sequence alignments (MSAs), were followed by methods for locating family-dependent conserved positions. These rely on the functional classification implicit in the alignment for locating these positions related with functional specificity. The next obvious step, still scarcely explored, is to detect these positions using a functional classification different from the one implicit in the sequence relationships between the proteins. Here, we present two new methods for locating functional positions which can incorporate an arbitrary external functional classification which may or may not coincide with the one implicit in the MSA. The Xdet method is able to use a functional classification with an associated hierarchy or similarity between functions to locate positions related to that classification. The MCdet method uses multivariate statistical analysis to locate positions responsible for each one of the functions within a multifunctional family. RESULTS: We applied the methods to different cases, illustrating scenarios where there is a disagreement between the functional and the phylogenetic relationships, and demonstrated their usefulness for the phylogeny-independent prediction of functional positions.