Mapping regions containing binding residues within functional domains of Plasmodium vivax and Plasmodium knowlesi erythrocyte-binding proteins

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Whereas Plasmodium vivax and Plasmodium knowlesi use the Duffy blood group antigen, Plasmodium falciparum uses sialic acid residues of glycophorin A as receptors to invade human erythrocytes. P. knowlesi uses the Duffy antigen as well as other receptors to invade rhesus erythrocytes by multiple pathways. Parasite ligands that bind these receptors belong to a family of erythrocyte-binding proteins (EBP). The EBP family includes the P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi β and γ proteins, which bind alternate receptors on rhesus erythrocytes, and P. falciparum erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of human glycophorin A. Binding domains of each EBP lie in a conserved N-terminal cysteine-rich region, region II, which contains around 330 amino acids with 12 to 14 conserved cysteines. Regions containing binding residues have now been mapped within P. vivax and P. knowlesi β region II. Chimeric domains containing P. vivax region II sequences fused to P. knowlesi β region II sequences were expressed on the surface of COS cells and tested for binding to erythrocytes. Binding residues of P. vivax region II lie in a 170-aa stretch between cysteines 4 and 7, and binding residues of P. knowlesi β region II lie in a 53-aa stretch between cysteines 4 and 5. Mapping regions responsible for receptor recognition is an important step toward understanding the structural basis for the interaction of these parasite ligands with host receptors.

Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishing this by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (“seeding”) and support growth by lateral diffusion of protein molecules in the membrane (“feeding”). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 Å resolution, with a space group P63, and unit cell dimensions of a = b = 62 Å, c = 108 Å; α = β = 90° and γ = 120°.

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345.

Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: Use of a chemically preactivated reagent

Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at λ > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the Tα subunit. Crosslinking to Tα was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in Tα were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19–Arg-28 at the N-terminal region of Tα. The total results show that both the N and the C termini of Tα are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.

Development of a bovine X chromosome linkage group and painting probes to assess cattle, sheep, and goat X chromosome segment homologies.

The X chromosome linkage group is conserved in placental mammals. However, X chromosome morphological differences, due to internal chromosome rearrangements, exist among mammalian species. We have developed bovine chromosome painting probes for Xp and Xq to assess segment homologies between the submetacentric bovine X chromosome and the acrocentric sheep and goat X chromosomes. These painting probes and their corresponding DNA libraries were developed by chromosome micromanipulation, DNA micropurification, microcloning, and PCR amplification. The bovine Xp painting probe identified an interstitially located homologous segment in the sheep and goat Xq region, most probably resulting from chromosome inversion. Ten type II (microsatellite) markers obtained from the bovine Xq library and five other X chromosome assigned, but unlinked, markers were used to generate a linkage map for Xq spanning 89.4 centimorgans. The chromosome painting probes and molecular markers generated in this study would be useful for comparative mapping and tracing of internal X chromosome rearrangements in all ruminant species and would contribute to the understanding of mammalian sex chromosome evolution.

Mouse model for Usher syndrome: linkage mapping suggests homology to Usher type I reported at human chromosome 11p15.

Usher syndrome is a group of diseases with autosomal recessive inheritance, congenital hearing loss, and the development of retinitis pigmentosa, a progressive retinal degeneration characterized by night blindness and visual field loss over several decades. The causes of Usher syndrome are unknown and no animal models have been available for study. Four human gene sites have been reported, suggesting at least four separate forms of Usher syndrome. We report a mouse model of type I Usher syndrome, rd5, whose linkage on mouse chromosome 7 to Hbb and tub has homology to human Usher I reported on human chromosome 11p15. The electroretinogram in homozygous rd5/rd5 mouse is never normal with reduced amplitudes that extinguish by 6 months. Auditory-evoked response testing demonstrates increased hearing thresholds more than control at 3 weeks of about 30 decibels (dB) that worsen to about 45 dB by 6 months.

Estudos comparativos sobre a elaboração de mapas conceituais durante processo de avaliação da aprendizagem: identificando a importância das demandas e do efeito de preparação

Os mapas conceituais são ferramentas gráficas que possibilitam a representação dos modelos mentais do aluno. Devido a essa capacidade, o mapa conceitual pode ser utilizado como ferramenta avaliativa de conhecimento. O uso dessa ferramenta em sala de aula gera cargas na memória de trabalho que podem ser referentes ao conteúdo (carga intrínseca) ou à forma como esse recurso está sendo trabalhado na sala de aula (carga extrínseca). Este trabalho tem por objetivo investigar como os mapas conceituais auxiliam na avaliação da aprendizagem da disciplina Ciências da Natureza: Ciência, Cultura e Sociedade ofertada na Universidade de São Paulo para alunos ingressantes de diversos cursos de graduação (n = 64) durante o período de 2013.1 sobre o conteúdo de Mudanças Climáticas. Nas turmas avaliadas o mapa conceitual poderia ser utilizado como ferramenta de preparação para prova (MCPREP) como também era utilizado como parte da avaliação formal da disciplina (MC-AVAL). Essa pesquisa se constitui de três Estudos que investigam: 1. As diferenças de perfis de mapas conceituais obtidos na condição de MC-PREP x MCAVAL; 2. Se o grupo de alunos que fizeram MC-PREP tiveram MC-AVAL com perfil diferente daqueles que não fizeram; 3. A existência de uma correlação entre as características do MC-AVAL com o conhecimento declarativo dos estudantes. Os mapas conceituais foram avaliados considerando aspectos estruturais e semânticos. As metodologias das análises realizadas foram retiradas de trabalhos presente na literatura. Entre os aspectos semânticos estavam o uso de materiais instrucionais, a natureza das proposições, a presença de erros e a aderência a pergunta focal. Além dessas análises, foi verificada a presença de agrupamentos naturais que pudessem ser explicados com uso das categorias teóricas. A análise dos resultados indica que os mapas conceituais das condições MC-PREP e MC-AVAL são bastante distintas entre si, pois atendiam a objetivos educacionais diferentes. No Estudo 2 verificou-se que o MC-PREP não influenciou fortemente a elaboração do MC-AVAL, pois a diminuição da carga extrínseca provocada pela elaboração de MC-PREP não foi suficiente para mostrar diferenças entre os grupos, já que ambos estavam altamente treinados na técnica de mapeamento conceitual. Por fim, no Estudo 3, o principal fator correlacionando o conhecimento declarativo com a complexidade dos mapas conceituais foi o percentual de proposições apropriadas. As conclusões do trabalho são que a demanda de elaboração do mapa conceitual é realmente orientadora de produto, a elaboração de um mapa conceitual a mais em um grupo bem treinado na técnica não altera seu desempenho e o principal meio de avaliar os mapas conceituais deve ser a leitura de suas proposições constituintes.

Coordination of aproximations in secondary school students’ understanding of limit concept

The aim of this research is to characterize the coordination of the processes of approximation related to the understanding of the limit of a function. We analyze the answers of 64 post-secondary school students to 7 problems considering the dynamic and metric conception of limit of a function. Results indicate that the metric understanding of the limit in terms of inequality supports that the student is capable of coordinating the approximations in the domain and in the range when lateral approximations coincide. However, the student is not capable of this coordination when lateral approximations do not coincide. This indicates that the metric understanding of the limit begins with the previous construction of the dynamic conception in case of coincidence of the lateral approximations in the range.

Mapping Binary Liquid-Vapor or Liquid-Liquid-Vapor Equilibria Regions, including the Different Azeotropic Behaviours, as a Function of the NRTL Binary Parameters

13th Mediterranean Congress of Chemical Engineering (Sociedad Española de Química Industrial e Ingeniería Química, Fira Barcelona, Expoquimia), Barcelona, September 30-October 3, 2014

Goal Orientation Profiles and Self-Concept of Secondary School Students

The objective of this study is to identify possible combinations of multiple goals that lead to different goal orientation profiles and to determine whether there are significant group differences in self-concept dimensions. The Achievement Goals Tendencies Questionnaire (AGTQ) and the Self-Description Questionnaire-II (SDQ-II) were administered to a sample of 2,022 students of Compulsory Secondary education, ranging in age from 12 to 16 years (M = 13.81, SD = 1.35). Cluster analysis identified four profiles of motivational goals: a group of students with a generalized high motivation profile, a group of students with generalized low motivation profile, a group of students with a predominance of learning goals and achievement goals, and a last group of students with a predominance of achievement goals and social reinforcement goals. Results reveal statistically significant differences among the profiles obtained regarding self-concept dimensions.

Mapping Emotion Terms into Affective Space: Further Evidence for a Four-Dimensional Structure

Recent empirical work on the semantics of emotion terms across many different cultures and languages, using a theoretical componential approach, suggested that four dimensions are needed to parsimoniously describe the semantic space of the emotion domain as reflected in emotion terms (Fontaine, Scherer, Roesch, & Ellsworth, 2007; Fontaine, Scherer, & Soriano, 2013). In addition to valence, power, and arousal, a novelty dimension was discovered that mostly differentiated surprise from other emotions. Here, we further explore the existence and nature of the fourth dimension in semantic emotion space using a much larger and much more representative set of emotion terms. A group of 156 participants each rated 10 out of a set of 80 French emotion terms with respect to semantic meaning. The meaning of an emotion term was evaluated with respect to 68 emotion features representing the appraisal, action tendency, bodily reaction, expression, and feeling components of the emotion process. A principal component analysis confirmed the four-dimensional valence, power, arousal, and novelty structure. Moreover, this larger and much more representative set of emotion terms revealed that the novelty dimension not only differentiates surprise terms from other emotion terms, but also identifies substantial variation within the fear and joy emotion families.

Radiomic Texture Analysis Mapping Predicts Areas of True Functional MRI Activity.

Individual analysis of functional Magnetic Resonance Imaging (fMRI) scans requires user-adjustment of the statistical threshold in order to maximize true functional activity and eliminate false positives. In this study, we propose a novel technique that uses radiomic texture analysis (TA) features associated with heterogeneity to predict areas of true functional activity. Scans of 15 right-handed healthy volunteers were analyzed using SPM8. The resulting functional maps were thresholded to optimize visualization of language areas, resulting in 116 regions of interests (ROIs). A board-certified neuroradiologist classified different ROIs into Expected (E) and Non-Expected (NE) based on their anatomical locations. TA was performed using the mean Echo-Planner Imaging (EPI) volume, and 20 rotation-invariant texture features were obtained for each ROI. Using forward stepwise logistic regression, we built a predictive model that discriminated between E and NE areas of functional activity, with a cross-validation AUC and success rate of 79.84% and 80.19% respectively (specificity/sensitivity of 78.34%/82.61%). This study found that radiomic TA of fMRI scans may allow for determination of areas of true functional activity, and thus eliminate clinician bias.

55 mph - Interstate Communications Project. Task 3: preliminary communications concept test. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Panoramic aerial photography for mapping gypsy moth defoliation.

"October 1982"--P. [1].