Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

Working at the nexus between public health policy, practice and research : dynamics of knowledge sharing in the Netherlands

Background
Joining the domains of practice, research and policy is an important aspect of boosting the quality performance required to tackle complex public health problems. “Joining domains” implies a departure from the linear and technocratic knowledge-translation approach. Integrating the practice, research and policy triangle means knowing its elements, appreciating the barriers, identifying possible cooperation strategies and studying strategy effectiveness under specified conditions.
This article examines the dynamic process of developing an Academic Collaborative Centre for Public Health in the Netherlands, with the objective of achieving that the three domains of policy, practice and research become working partners on an equal footing.
Method
An interpretative hermeneutic approach was used to interpret the phenomenon of collaboration at the nexus between the three domains. The project was explicitly grounded in current organizational culture and routines, applied to nexus action. In the process of examination, we used both quantitative (e.g. records) and qualitative data (e.g., interviews and observations). The data were interpreted using the Actor-Network, Institutional Re-Design and Blurring the Boundaries theories.
Results
Results show commitment at strategic level. At the tactical level, however, managers were inclined to prioritize daily routine, while the policy domain remained absent. At the operational level, practitioners learned to do PhD research in real-life practice and researchers became acquainted with problems of practice and policy, resulting in new research initiatives.
Conclusion
We conclude that working at the nexus is an ongoing process of formation and reformation. Strategies based on Institutional Re-Design theories in particular might help to more actively stimulate managers’ involvement to establish mutually supportive networks.

The P-Loop domain of yeast Clp1 mediates interactions between CF IA and CPF factors in Pre-mRNA 3′ end formation

 Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3' end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5' RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3' end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3' end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3' endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3' end formation.

Literature as activism: interrogating the 21st century demise of the Arendtian political public sphere

This article explores Don DeLillo's literary activism through Arendtian perspectives to investigate what the demise of literature's relevance, specifically in a political context, may mean in the current era of an increasingly complex and conflicting 'web of human relationships'.  In that it is accepted that narrative has a particular ability to reveal insights as prelinguistic elements that are distinct from all we are able to access through our limited human perceptions, it remarks the Lacanian paradox that if being is in excess of language, then language is the medium by which this is accessed in the world. For DeLillo, writers may be under threat in a dynamic but destabilizing era, their art superceded by technology and fundamentalist terrorism, however, as suggested in Mao II, this renders the writer all the more necessary.  It is at the point at which the writer has nothing to say or is under duress to say noting, that a human crisis is reached. I ask, do current forms of political pressure to censure literature constitute a further diminishing of the Arendtian political public domain in which speech as action has primacy?

An online transfer learning RBF neural network for cross domain data classification

In this paper, a Radial Basis Function Network (RBFN) trained with the Dynamic Decay Adjustment (DDA) algorithm (i.e., RBFNDDA) is deployed as an incremental learning model for tackling transfer learning problems. An online learning strategy is exploited to allow the RBFNDDA model to transfer knowledge from one domain and applied to classification tasks in a different yet related domain. An experimental study is carried out to evaluate the effectiveness of the online RBFNDDA model using a benchmark data set obtained from a public domain. The results are analyzed and compared with those from other methods. The outcomes positively reveal the potentials of the online RBFNDDA model in handling transfer learning tasks. © 2014 The authors and IOS Press. All rights reserved.

Information society and public ICT policies in the Caribbean: a review of advances and challenges, policy instruments and country experiences

Includes bibliography

Anemia and hemoglobinopathies in pregnant women attended in a public hospital

Objectives: to identify the demographic profile and frequency of anemia and hemoglobinopathies, as a basis for future implementation of actions aimed at pregnant women in the public health domain. Method: this is a cross-sectional study developed with pregnant women attended in a university hospital at Mato Grosso do Sul, Brazil. Blood samples were collected for the erythrogram analysis for detection of anemia and selective and specific tests for abnormal hemoglobin. The patients regarded as indigenous and mentally ill, as well as inmates, were excluded from the research, as they represent a vulnerable population which needs a cohort different from that of the sample. For data collection, a particular questionnaire was used. The research was approved by the Ethics Committee of Universidade Federal de Mato Grosso do Sul (UFMS), under the Protocol 873/2006. Results: of the 215 pregnant women under study, 20% were adolescents; 36.3% had incomplete primary education; 53.0% were non-Caucasian; 43.3% were from Campo Grande, Mato Grosso do Sul, Brazil; and 21.1% were of European descent. 17.7% had some type of anemia and, in the evaluation of hemoglobinopathies, 4.7% of patients were detected with some abnormal hemoglobin, with the following frequencies: 3.3% with HbAS; 0.9% with HbAC; and 0.5% with intermediate β-thalassemia. Conclusion: the frequencies of anemia and hemoglobinopathy found in these pregnant women showed the importance of early diagnosis, revealing indicators able to provide a basis for preventive and assistance actions for adequate clinical monitoring, reducing maternal and neonatal morbimortality in the public health services. Descriptors: pregnant women; anemia; hemoglobinopathies; public health; nursing.

Exercise tolerance can be enhanced through a change in work rate within the severe intensity domain: work above critical power is not constant

The characterization of the hyperbolic power-time (P-tlim) relationship using a two-parameter model implies that exercise tolerance above the asymptote (Critical Power; CP), i.e. within the severe intensity domain, is determined by the curvature (W') of the relationship. The purposes of this study were (1) to test whether the amount of work above CP (W>CP) remains constant for varied work rate experiments of high volatility change and (2) to ascertain whether W' determines exercise tolerance within the severe intensity domain. Following estimation of CP (208 ± 19 W) and W' (21.4 ± 4.2 kJ), 14 male participants (age: 26 ± 3; peak [Formula: see text]: 3708 ± 389 ml.min-1) performed two experimental trials where the work rate was initially set to exhaust 70% of W' in 3 ('THREE') or 10 minutes ('TEN') before being subsequently dropped to CP plus 10 W. W>CP for TEN (104 ± 22% W') and W' were not significantly different (P>0.05) but lower than W>CP for THREE (119 ± 17% W', P<0.05). For both THREE (r = 0.71, P<0.01) and TEN (r = 0.64, P<0.01), a significant bivariate correlation was found between W' and tlim. W>CP and tlim can be greater than predicted by the P-tlim relationship when a decrement in the work rate of high-volatility is applied. Exercise tolerance can be enhanced through a change in work rate within the severe intensity domain. W>CP is not constant.

Fairness, Justice and an Individual Basis for Public Policy

Prior models of the policy process have examined how human characteristics can affect policy decision-making in such a way that it leads to aggregate effects on policy outcomes as a whole. I develop a model of the policy process which suggests that emotions related to fair and unfair experiences in the same policy domain are utilized by decision-makers as policy criteria. In the lab, I empirically tested this, and find that emotions and experience related to fairness do influence the policy decision to move away from the status quo alternative. Based upon this result, I simulated the evolution of a society of agents engaged in decision-making using similar criteria. The simulation suggests that incentives have an important role in leading to cooperation and social success. The external validity of the simulation also implies that it can act as a platform for future evolutionary policy experimentation.

Systematic Analysis of FKBP Inducible Degradation Domain Tagging Strategies for the Human Malaria Parasite Plasmodium falciparum

Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression.

Optic Disc Margin Anatomy in Patients with Glaucoma and Normal Controls with Spectral Domain Optical Coherence Tomography

Objective: To characterize optic nerve head (ONH) anatomy related to the clinical optic disc margin with spectral domain-optical coherence tomography (SD-OCT). Design: Cross-sectional study. Participants: Patients with open-angle glaucoma with focal, diffuse, and sclerotic optic disc damage, and age-matched normal controls. Methods: High-resolution radial SD-OCT B-scans centered on the ONH were analyzed at each clock hour. For each scan, the border tissue of Elschnig was classified for obliqueness (internally oblique, externally oblique, or nonoblique) and the presence of Bruch's membrane overhanging the border tissue. Optic disc stereophotographs were co-localized to SD-OCT data with customized software. The frequency with which the disc margin identified in stereophotographs coincided with (1) Bruch's membrane opening (BMO), defined as the innermost edge of Bruch's membrane; (2) Bruch's membrane/border tissue, defined as any aspect of either outside BMO or border tissue; or (3) border tissue, defined as any aspect of border tissue alone, in the B-scans was computed at each clock hour. Main Outcome Measures: The SD-OCT structures coinciding with the disc margin in stereophotographs. Results: There were 30 patients (10 with each type of disc damage) and 10 controls, with a median (range) age of 68.1 (42-86) years and 63.5 (42-77) years, respectively. Although 28 patients (93%) had 2 or more border tissue configurations, the most predominant one was internally oblique, primarily superiorly and nasally, frequently with Bruch's membrane overhang. Externally oblique border tissue was less frequent, observed mostly inferiorly and temporally. In controls, there was predominantly internally oblique configuration around the disc. Although the configurations were not statistically different between patients and controls, they were among the 3 glaucoma groups. At most locations, the SD-OCT structure most frequently identified as the disc margin was some aspect of Bruch's membrane and border tissue external to BMO. Bruch's membrane overhang was regionally present in the majority of patients with glaucoma and controls; however, in most cases it was not visible as the disc margin. Conclusions: The clinically perceived disc margin is most likely not the innermost edge of Bruch's membrane detected by SD-OCT. These findings have important implications for the automated detection of the disc margin and estimates of the neuroretinal rim. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references. Ophthalmology 2012;119:738-747 (C) 2012 by the American Academy of Ophthalmology.