Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

The 5′-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bγ) and 2 (eIF2γ), respectively. The involvement of eIF2Bγ and eIF2γ in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

Loss of HMW1 and HMW3 in noncytadhering mutants of Mycoplasma pneumoniae occurs post-translationally

The genomic sequence of Mycoplasma pneumoniae establish this cell-wall-less prokaryote as among the smallest known microorganisms capable of self-replication. However, this genomic simplicity and corresponding biosynthetic austerity are sharply contrasted by the complex terminal structure found in this species. This tip structure (attachment organelle) directs colonization of the human respiratory mucosa, leading to bronchitis and atypical pneumonia. Furthermore, formation of a second tip structure appears to precede cell division, implying temporal regulation. However, the organization, regulation, and assembly of the attachment organelle in M. pneumoniae are poorly understood, and no counterparts have been identified among the walled bacteria. M. pneumoniae possesses a cytoskeleton-like structure required to localize adhesin proteins to the attachment organelle. The cytadherence-associated proteins HMW1, HMW2, and HMW3 are components of the mycoplasma cytoskeleton, with HMW1 localizing strictly along the filamentous extensions from the cell body and HMW3 being a key structural element of the terminal organelle. Disruptions in hmw2 result in the loss of HMW1 and HMW3. However, the hmw1 and hmw3 genes were transcribed and translated at wild-type levels in hmw2 mutants. HMW1 and HMW3 were relatively stable in the wild-type background over 8 h but disappeared in the mutants over this time period. Evaluation of recombinant HMW1 levels in mycoplasma mutants suggested a requirement for the C-terminal domain of HMW1 for turnover. Finally, an apparent defect in the processing of the precursor for the adhesin protein P1 was noted in the HMW− mutants.

Arrayed transposase-binding sequences on the ends of transposon Tn5090/Tn402

The transposon Tn5090/Tn402 encodes a 559 amino acid transposase, TniA, with a DDE motif. Gel mobility shifting and cleavage protection analysis with DNase I and hydroxyl radical probes revealed that TniA binds to multiple repeat sequences on either terminus of Tn5090/Tn402. Four of these TniA-binding 19mers occurred on the left-hand (t) end and two on the right-hand (i) end. Hydroxyl radical cleavage protection demonstrated the presence of 3–6 bp contact sequences on one face of the DNA helix. The binding pattern and organisation of repeats suggested parallels between Tn5090/Tn402 and Mu, which controls its transpositional activity in the assembly step of a higher order transpososome complex. The complex terminal structure and genes of transposase and nucleotide-binding proteins in tandem are hallmarks of the handful of Mu-like elements that are known to date.

Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

Estrutura da vegetação e sua relação com a diversidade, abundância e similaridade de coleópteros bioindicadores em diferentes sistemas vegetacionais, Piracicaba, SP

A Mata Atlântica é considerada um dos biomas mais importantes do mundo devido à sua alta biodiversidade e funções ecossistêmicas. Entretanto, encontra-se fragmentada em porções de pequenas dimensões esparsas em uma matriz predominantemente agrícola, composta principalmente por extensas pastagens e monoculturas. Desse modo, os sistemas agroflorestais por apresentarem uma estrutura diferenciada dos monocultivos e similar às condições naturais, podem ser utilizados como uma alternativa para o manejo e a conservação da biodiversidade nos remanescentes florestais. A fragmentação provoca modificações no ambiente que irão refletir na perda e no deslocamento da biodiversidade, estando os insetos entre os grupos mais afetados. Uma das formas de se avaliar o estado de conservação dos fragmentos e o impacto antrópico nos sistemas vegetacionais, é estudar a presença e distribuição de organismos bioindicadores. Dentre esses, os insetos ocupam posição de destaque. Os insetos da família Scarabaeidae e da subfamília Scolytinae são bons indicadores de distúrbios, pois são muito sensíveis ás mudanças ambientais. Neste trabalho hipotetisou-se que a presença desses insetos está relacionada com a estrutura da vegetação e as condições de vida proporcionadas pelas diferentes formas de uso-da-terra. O objetivo desta pesquisa foi avaliar a diversidade de espécies, o padrão de abundância e a similaridade entre as populações de coleópteros (Scarabaeidae e Scolytinae) em diferentes sistemas vegetacionais de diferentes estruturas: i) Fragmento de floresta estacional semidecidual dividido em três áreas: beira do rio, centro e borda; ii) Sistema Agroflorestal (SAF) (interface entre o fragmento e o pasto); iii) Pasto composto de Brachiaria decumbens (L.); iv) Monocultivo de café (Coffea arábica L.); v) Monocultivo de seringueira (Hevea brasiliensis Müell. Arg.); vi) SAF de café e seringueira - todos situados numa região de domínio anterior de floresta estacional semidecidual em Piracicaba-SP. Os sistemas foram caracterizados quanto à sua estrutura e condições micrometeorológicas. Os insetos foram coletados mensalmente entre agosto/2013 e julho/2014 utilizando-se dois tipos de armadilhas: Pitfall e etanol modelo ESALQ-84. Foram coletados 1.047 espécimes distribuídos em 21 espécies da família Scarabaeidae e 1.833 indivíduos de 38 espécies da subfamília Scolytinae. A maior quantidade de espécies de Scarabaeidae foi encontrada na borda do fragmento florestal, enquanto que a maior abundância ocorreu no fragmento florestal perto do rio. A subfamília Scolytinae apresentou a maior riqueza de espécies no sistema agroflorestal misto (borda) e a maior abundância no sistema agroflorestal café-seringueira. A abundância e riqueza de espécies da família Scarabaeidae foram correlacionadas positivamente com a temperatura do ar, temperatura e umidade do solo e a precipitação. Por outro lado, a abundância e a riqueza de espécies da subfamília Scolytinae apresentaram correlação negativa com a temperatura do ar e a temperatura e umidade do solo. Ambos os grupos de insetos apresentaram a maior abundância e riqueza de espécies nas áreas com estrutura vegetacional mais complexa, sendo influenciadas pelas condições microclimáticas dentro de cada local.

RetinaStudio: A bioinspired framework to encode visual information

The retina is a very complex neural structure, which performs spatial, temporal, and chromatic processing on visual information and converts it into a compact ‘digital’ format composed of neural impulses. This paper presents a new compiler-based framework able to describe, simulate and validate custom retina models. The framework is compatible with the most usual neural recording and analysis tools, taking advantage of the interoperability with these kinds of applications. Furthermore it is possible to compile the code to generate accelerated versions of the visual processing models compatible with COTS microprocessors, FPGAs or GPUs. The whole system represents an ongoing work to design and develop a functional visual neuroprosthesis. Several case studies are described to assess the effectiveness and usefulness of the framework.

Ras plasma membrane signalling platforms

The plasma membrane is a complex, dynamic structure that provides platforms for the assembly of many signal transduction pathways. These platforms have the capacity to impose an additional level of regulation on cell signalling networks. In this review, we will consider specifically how Ras proteins interact with the plasma membrane. The focus will be on recent studies that provide novel spatial and dynamic insights into the micro-environments that different Ras proteins utilize for signal transduction. We will correlate these recent studies suggesting Ras proteins might operate within a heterogeneous plasma membrane with earlier biochemical work on Ras signal transduction.

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.

Macrocycles and medicine - facile synthesis of a bis(macrocycle) with pendent functionality

The crystal structure of the Cu(II) perchlorate complex of a functionalised bis(rnacrocycle) ligand, where the individual macrocycle units are of the cyclam type and adopt the trans-III configuration, is analysed in terms of its possible relationship to those of bis(macrocycle) complexes possessing anti-viral activity. To cite this article: P V Bernhardt et al., C. R. Chimie 8 (2005). (C) 2004 Academie des sciences. Published by Elsevier SAS. All rights reserved.

Knots in rings - The circular knotted protein momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys(1)-Cys(18) disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.

Multi-level zero-inflated Poisson regression modelling of correlated count data with excess zeros

Count data with excess zeros relative to a Poisson distribution are common in many biomedical applications. A popular approach to the analysis of such data is to use a zero-inflated Poisson (ZIP) regression model. Often, because of the hierarchical Study design or the data collection procedure, zero-inflation and lack of independence may occur simultaneously, which tender the standard ZIP model inadequate. To account for the preponderance of zero counts and the inherent correlation of observations, a class of multi-level ZIP regression model with random effects is presented. Model fitting is facilitated using an expectation-maximization algorithm, whereas variance components are estimated via residual maximum likelihood estimating equations. A score test for zero-inflation is also presented. The multi-level ZIP model is then generalized to cope with a more complex correlation structure. Application to the analysis of correlated count data from a longitudinal infant feeding study illustrates the usefulness of the approach.

Lobe growth in foliose lichen thalli

The margins of foliose lichen thalli comprise individual lobes which grow radially and divide. This results in a complex marginal structure in which lobes differing in morphology, state of division, and growth pattern are crowded together. Various aspects of the biology of these lobes are reviewed including their carbohydrate supply, morphology, pattern of division and branching, the effect of lobe overcrowding and interactions between neighbouring lobes. As the thalus grows, lobes become increasingly crowded together and this restricts further lateral growth. Restriction of lobe width may be reponsible for the changes in radial growth rate (RGR) with size observed in foliose species. Various aspects of lobe biology may be responsible for lobe growth variation including the physical independence of lobes from their neighbours, the genetic origin of the lobes, and the pattern of lobe branching. Overall symmetry of a thallus is maintained by a fluctuating pattern of growth of individual lobes in successive months together with competition for space at the margin

Generalization by Computation Through Memory

Usually, generalization is considered as a function of learning from a set of examples. In present work on the basis of recent neural network assembly memory model (NNAMM), a biologically plausible 'grandmother' model for vision, where each separate memory unit itself can generalize, has been proposed. For such a generalization by computation through memory, analytical formulae and numerical procedure are found to calculate exactly the perfectly learned memory unit's generalization ability. The model's memory has complex hierarchical structure, can be learned from one example by a one-step process, and may be considered as a semi-representational one. A simple binary neural network for bell-shaped tuning is described.

Thermalized polarization dynamics of a discrete optical-waveguide system with four-wave mixing

Statistical mechanics of two coupled vector fields is studied in the tight-binding model that describes propagation of polarized light in discrete waveguides in the presence of the four-wave mixing. The energy and power conservation laws enable the formulation of the equilibrium properties of the polarization state in terms of the Gibbs measure with positive temperature. The transition line T=∞ is established beyond which the discrete vector solitons are created. Also in the limit of the large nonlinearity an analytical expression for the distribution of Stokes parameters is obtained, which is found to be dependent only on the statistical properties of the initial polarization state and not on the strength of nonlinearity. The evolution of the system to the final equilibrium state is shown to pass through the intermediate stage when the energy exchange between the waveguides is still negligible. The distribution of the Stokes parameters in this regime has a complex multimodal structure strongly dependent on the nonlinear coupling coefficients and the initial conditions.

Gato-do-mato-pequeno (Leopardus tigrinus) na Caatinga: ocupação e padrão de atividade de um felídeo ameaçado e pouco conhecido na floresta tropical seca do nordeste do Brasil

While the carnivores are considered regulators and structuring of natural communities are also extremely threatened by human activities. Endangered little-spotted-cat (Leopardus tigrinus) is one of the lesser known species from the Neotropical cats. In this work we investigate the occupancy and the activity pattern of L. tigrinus in Caatinga of Rio Grande do Norte testing: 1) how environmental and anthropogenic factors influence their occupation and 2) how biotic and abiotic factors influence their activity pattern. For this we raised occurrence data of species in 10 priority areas for conservation. We built hierarchical models of occupancy based on maximum likelihood to represent biological hypotheses which were ranked using the Akaike Information Criterion (AIC). According to the results the feline occupancy is more likely away from rural settlements and in areas with a higher proportion of woody vegetation. The opportunistic killing of L. tigrinus and in retaliation for poultry predation close to residential areas can explain this result; as well as more complex vegetation structure can better serve as refuge and ensure more food. Analyzing the records of the species through circular statistics we conclude that the activity pattern is mostly nocturnal, although considerable crepuscular and a small diurnal activity. L. tigrinus activity was directly affected by the availability of small terrestrial mammals, which are essentially nocturnal. In addition, the temperatures recorded in the environment directly and indirectly affect the activity of the little-spotted-cat, as also influence the activity of their potential prey. Generally, the cats were more active when possible prey were active, and this happened at night when lower temperatures are recorded. Moreover, the different lunar phases did not affect the activity pattern. The results improve the understanding of an endangered feline inhabiting the Caatinga biome, and thus can help develop conservation and management strategies, as well as in planning future research in this semi-arid ecosystem.