Inhibitors of Trypanosoma brucei trypanothione reductase: comparative molecular field analysis modeling and structural basis for selective inhibition

Background: Sleeping sickness is a major cause of death in Africa. Since no secure treatment is available, the development of novel therapeutic agents is urgent. In this context, the enzyme trypanothione reductase (TR) is a prominent molecular target that has been investigated in drug design for sleeping sickness. Results: In this study, comparative molecular field analysis models were generated for a series of Trypanosoma brucei TR inhibitors. Statistically significant results were obtained and the models were applied to predict the activity of external test sets, with good correlation between predicted and experimental results. We have also investigated the structural requirements for the selective inhibition of the parasite's enzyme over the human glutathione reductase. Conclusion: The quantitative structure-activity relationship models provided valuable information regarding the essential molecular requirements for the inhibitory activity upon the target protein, providing important insights into the design of more potent and selective TR inhibitors.

Structure- and ligand-based drug design approaches for neglected tropical diseases

Drug discovery has moved toward more rational strategies based on our increasing understanding of the fundamental principles of protein-ligand interactions. Structure( SBDD) and ligand-based drug design (LBDD) approaches bring together the most powerful concepts in modern chemistry and biology, linking medicinal chemistry with structural biology. The definition and assessment of both chemical and biological space have revitalized the importance of exploring the intrinsic complementary nature of experimental and computational methods in drug design. Major challenges in this field include the identification of promising hits and the development of high-quality leads for further development into clinical candidates. It becomes particularly important in the case of neglected tropical diseases (NTDs) that affect disproportionately poor people living in rural and remote regions worldwide, and for which there is an insufficient number of new chemical entities being evaluated owing to the lack of innovation and R&D investment by the pharmaceutical industry. This perspective paper outlines the utility and applications of SBDD and LBDD approaches for the identification and design of new small-molecule agents for NTDs.

Structure-based drug design studies on a series of aldolase inhibitors

Human African trypanosomiasis, also known as sleeping sickness, is a major cause of death in Africa, and for which there are no safe and effective treatments available. The enzyme aldolase from Trypanosoma brucei is an attractive, validated target for drug development. A series of alkyl‑glycolamido and alkyl-monoglycolate derivatives was studied employing a combination of drug design approaches. Three-dimensional quantitative structure-activity relationships (3D QSAR) models were generated using the comparative molecular field analysis (CoMFA). Significant results were obtained for the best QSAR model (r2 = 0.95, non-cross-validated correlation coefficient, and q2 = 0.80, cross-validated correlation coefficient), indicating its predictive ability for untested compounds. The model was then used to predict values of the dependent variables (pKi) of an external test set,the predicted values were in good agreement with the experimental results. The integration of 3D QSAR, molecular docking and molecular dynamics simulations provided further insight into the structural basis for selective inhibition of the target enzyme.

Molecular barcode and morphological analyses reveal the taxonomic and biogeographical status of the striped-legged hermit crab species Clibanarius sclopetarius (Herbst, 1796) and Clibanarius vittatus (Bosc, 1802) (Decapoda: Diogenidae)

The taxonomic status of the species Clibanarius sclopetarius (Herbst, 1796) and Clibanarius vittatus (Bosc, 1802), which have sympatric biogeographical distributions restricted to the western Atlantic Ocean, is based only on differences in the colour pattern of the walking legs of adults. Their morphological similarity led to the suggestion that they be synonymised. In order to investigate this hypothesis, we included species of Clibanarius Dana, 1892 in a molecular phylogenetic analysis of partial sequences of the mitochondrial 16S rDNA gene and the COI barcode region. In addition, we combined the molecular results with morphological observations obtained from several samples of these two species. The genetic divergences of the 16S rDNA and COI sequences between C. sclopetarius and C. vittatus ranged from 4.5 to 5.9% and 9.4 to 11.9%, which did not justify their synonymisation. Differences in the telson morphology, chela ornamentation, and coloration of the eyestalks and antennal peduncle provided support for the separation of the two species. Another interesting result was a considerable genetic difference found between populations of C. vittatus from Brazil and the Gulf of Mexico, which may indicate the existence of two homonymous species.

Lectotypification of the name Helianthus dentatus Cav., basionym of Viguiera dentata (Cav.) Spreng. (Asteraceae: Heliantheae)

A recent molecular phylogenetic analysis (Schilling & Panero 2011) proposed to narrow the concept of the paraphyletic genus Viguiera Kunth (1818: 176; Asteraceae: Heliantheae) to embrace only the type, V. dentata (Cavanilles 1794: 10) Sprengel (1826: 615). Robinson (1977) emphasized the peculiarities of Viguiera dentata as the "distinctive and unique" presence of hairs on the filaments of the anther, the disk corollas throat less than twice as long as the lobes and densely scabrous below, a combination of features that differs from most member of the genus. During taxonomic studies of Viguiera and related genera, it was discovered that V. dentata has never been typified. The basionym, Helianthus dentatus, was described by Antonio Jose Cavanilles in Icones et Descriptiones Plantarum based on plants grown in the Real Jardin Botanico from seed sent by Martin Sesse from Mexico (Blanco 2000). Previous workers including Blake (1918) and Robinson (1977) have accepted the plate in Cavanilles as the type for Helianthus dentatus Cav.

Two versions of the threshold contact model in two dimensions

Two versions of the threshold contact process ordinary and conservative - are studied on a square lattice. In the first, particles are created on active sites, those having at least two nearest neighbor sites occupied, and are annihilated spontaneously. In the conservative version, a particle jumps from its site to an active site. Mean-field analysis suggests the existence of a first-order phase transition, which is confirmed by Monte Carlo simulations. In the thermodynamic limit, the two versions are found to give the same results. (C) 2012 Elsevier B.V. All rights reserved.

Is Drosera meristocaulis a pygmy sundew? Evidence of a long-distance dispersal between Western Australia and northern South America

South America and Oceania possess numerous floristic similarities, often confirmed by morphological and molecular data. The carnivorous Drosera meristocaulis (Droseraceae), endemic to the Neblina highlands of northern South America, was known to share morphological characters with the pygmy sundews of Drosera sect. Bryastrum, which are endemic to Australia and New Zealand. The inclusion of D. meristocaulis in a molecular phylogenetic analysis may clarify its systematic position and offer an opportunity to investigate character evolution in Droseraceae and phylogeographic patterns between South America and Oceania. was included in a molecular phylogenetic analysis of Droseraceae, using nuclear internal transcribed spacer (ITS) and plastid rbcL and rps16 sequence data. Pollen of D. meristocaulis was studied using light microscopy and scanning electron microscopy techniques, and the karyotype was inferred from root tip meristem. The phylogenetic inferences (maximum parsimony, maximum likelihood and Bayesian approaches) substantiate with high statistical support the inclusion of sect. Meristocaulis and its single species, D. meristocaulis, within the Australian Drosera clade, sister to a group comprising species of sect. Bryastrum. A chromosome number of 2n approx. 3236 supports the phylogenetic position within the Australian clade. The undivided styles, conspicuous large setuous stipules, a cryptocotylar (hypogaeous) germination pattern and pollen tetrads with aperture of intermediate type 78 are key morphological traits shared between D. meristocaulis and pygmy sundews of sect. Bryastrum from Australia and New Zealand. The multidisciplinary approach adopted in this study (using morphological, palynological, cytotaxonomic and molecular phylogenetic data) enabled us to elucidate the relationships of the thus far unplaced taxon D. meristocaulis. Long-distance dispersal between southwestern Oceania and northern South America is the most likely scenario to explain the phylogeographic pattern revealed.

Expanding the Clinical and Genetic Spectrum of Human CD40L Deficiency: The Occurrence of Paracoccidioidomycosis and Other Unusual Infections in Brazilian Patients

CD40 ligand (CD40L) deficiency or X-linked hyper-IgM syndrome (X-HIGM) is a well-described primary immunodeficiency in which Pneumocystis jiroveci pneumonia is a common clinical feature. We have identified an unusual high incidence of fungal infections and other not yet described infections in a cohort of 11 X-HIGM patients from nine unrelated Brazilian families. Among these, we describe the first case of paracoccidioidomycosis (PCM) in X-HIGM. The molecular genetic analysis of CD40L was performed by gene sequencing and evaluation of CD40L protein expression. Nine of these 11 patients (82%) had fungal infections. These included fungal species common to CD40L deficiency (P. jiroveci and Candida albicans) as well as Paracoccidioides brasiliensis. One patient presented with PCM at age 11 years and is now doing well at 18 years of age. Additionally, one patient presented with a simultaneous infection with Klebsiella and Acinetobacter, and one with condyloma caused by human papilloma virus. Molecular analysis revealed four previously described CD40L mutations, two novel missense mutations (c.433 T>G and c.476 G>C) resulting in the absence of CD40L protein expression by activated CD4(+) cells and one novel insertion (c.484_485insAA) within the TNFH domain leading to a frame shift and premature stop codon. These observations demonstrated that the susceptibility to fungal infections in X-HIGM extends beyond those typically associated with X-HIGM (P. jiroveci and C. albicans) and that these patients need to be monitored for those pathogens.

Recoupled separated-local-field experiments and applications to study intermediate-regime molecular motions

A specific separated-local-field NMR experiment, dubbed Dipolar-Chemical-Shift Correlation (DIPSHIFT) is frequently used to study molecular motions by probing reorientations through the changes in XH dipolar coupling and T-2. In systems where the coupling is weak or the reorientation angle is small, a recoupled variant of the DIPSHIFT experiment is applied, where the effective dipolar coupling is amplified by a REDOR-like pi-pulse train. However, a previously described constant-time variant of this experiment is not sensitive to the motion-induced T-2 effect, which precludes the observation of motions over a large range of rates ranging from hundreds of Hz to around a MHz. We present a DIPSHIFT implementation which amplifies the dipolar couplings and is still sensitive to T-2 effects. Spin dynamics simulations, analytical calculations and experiments demonstrate the sensitivity of the technique to molecular motions, and suggest the best experimental conditions to avoid imperfections. Furthermore, an in-depth theoretical analysis of the interplay of REDOR-like recoupling and proton decoupling based on Average-Hamiltonian Theory was performed, which allowed explaining the origin of many artifacts found in literature data. (C) 2012 Elsevier Inc. All rights reserved.

Genetic characterization of fig tree mutants with molecular markers

The fig (Ficus carica L.) is a fruit tree of great world importance and, therefore, the genetic improvement becomes an important field of research for better crops, being necessary to gather information on this species, mainly regarding its genetic variability so that appropriate propagation projects and management are made. The improvement programs of fig trees using conventional procedures in order to obtain new cultivars are rare in many countries, such as Brazil, especially due to the little genetic variability and to the difficulties in obtaining plants from gamete fusion once the wasp Blastophaga psenes, responsible for the natural pollinating, is not found in Brazil. In this way, the mutagenic genetic improvement becomes a solution of it. For this reason, in an experiment conducted earlier, fig plants formed by cuttings treated with gamma ray were selected based on their agronomic characteristics of interest. We determined the genetic variability in these fig tree selections, using RAPD and AFLP molecular markers, comparing them to each other and to the Roxo-de-Valinhos, used as the standard. For the reactions of DNA amplification, 140 RAPD primers and 12 primer combinations for AFLP analysis were used. The selections did not differ genetically between themselves and between them and the Roxo-de-Valinhos cultivar. Techniques that can detect polymorphism between treatments, such as DNA sequencing, must be tested. The phenotypic variation of plants may be due to epigenetic variation, necessitating the use of techniques with methylation-sensitive restriction enzymes.

Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from L-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019331, 1245-1262, 2012.

Large-Scale Population Analysis Challenges the Current Criteria for the Molecular Diagnosis of Fascioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (<= 8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4-8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns

Background: Sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs) are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out. Results: Sixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. FISH analysis resulted in the expected pattern of Gypsy/heterochromatin, Copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. Analysis of related ESTs and RT-PCR showed transcriptional variation between tissues and families. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements. Conclusions: Individual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNA's that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome.

The first modern solitary Agariciidae (Anthozoa, Scleractinia) revealed by molecular and microstructural analysis

Dactylotrochus cervicornis (= Tridacophyllia cervicornis Moseley, 1881), which occurs in Indo-Pacific waters between 73 and 852 m, was originally described as an astraeid but was later transferred to the Caryophylliidae. Assumed to be solitary, this species has no stolons and only one elongated fossa, and is unique among azooxanthellate scleractinians in often displaying extremely long thecal extensions that are septate and digitiform. Based on both molecular phylogenetic analyses (partial mitochondrial CO1 and 16S rDNA, and partial nuclear 28S rDNA) and morphological characteristics, we propose the transfer of D. cervicornis from the Caryophylliidae to the Agariciidae, making it the first extant representative of the latter family that is solitary and from deep water (azooxanthellate). The basal position of D. cervicornis within the agariciids implied by our analyses strengthens the case for inclusion of fossil species that were solitary, such as Trochoseris, in this family and suggests that the ancestor of this scleractinian family, extant members of which are predominantly colonial and zooxanthellate, may have been solitary and azooxanthellate.

Molecular and phylogenetic analysis of bovine coronavirus based on the spike glycoprotein gene

Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract infections in calves and feedlot cattle. In Cuba, the presence of BCoV was first reported in 2006. Since then, sporadic outbreaks have continued to occur. This study was aimed at deepening the knowledge of the evolution, molecular markers of virulence and epidemiology of BCoV in Cuba. A total of 30 samples collected between 2009 and 2011 were used for PCR amplification and direct sequencing of partial or full S gene. Sequence comparison and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100% bootstrap and 1.00 posterior probability values. The Cuban bovine coronavirus sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique. (C) 2012 Elsevier B.V. All rights reserved.