Cellular up-regulation of Nedd4 family interacting protein 1 (Ndfip1) using low levels of bioactive cobalt complexes

The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.     

Integrating the niche and neutral perspectives on community structure and dynamics

Elucidating the mechanisms underlying the assembly and dynamics of ecological communities is a fundamental goal of ecology. Two conceptual approaches have emerged in this respect: the niche-assembly view and the neutral perspective. The debate as to which approach best explains the biodiversity patterns observed in nature is becoming outdated, as ecologists increasingly agree on the existence of a niche-neutral continuum of community dynamical behaviors. However, attempts to make the continuum idea operational and measurable remain sparse. Here, we propose a model-based approach to achieving this. The proposed methodology consists of separating out fluctuations in species abundances into niche-mediated and stochastic factors, linking the niche configuration to community dynamics through competition, and adding demographic stochasticity. This results in a comprehensive framework including neutrality and strict niche segregation as extreme cases. We develop an index of departure from neutral drift as a surrogate for community position on the niche-neutral continuum. We evaluate the performance of our modeling approach with simulated data, and subsequently use the model to analyze rodent web-trapping data from a real-world system. The model fitting is carried out with a Bayesian approach using Markov chain Monte Carlo simulation methods.     

Glycoprotein structural genomics: solving the glycosylation problem

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.     

Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

Broadly neutralizing antibodies have been isolated that bind the glycan shield of the HIV-1 envelope spike. One such antibody, PGT135, contacts the intrinsic mannose patch of gp120 at the Asn332, Asn392, and Asn386 glycosylation sites. Here, site-specific glycosylation analysis of recombinant gp120 revealed glycan microheterogeneity sufficient to explain the existence of a minor population of virions resistant to PGT135 neutralization. Target microheterogeneity and antibody glycan specificity are therefore important parameters in HIV-1 vaccine design.     

Sexually antagonistic polymorphism in simultaneous hermaphrodites

In hermaphrodites, pleiotropic genetic trade‐offs between female and male reproductive functions can lead to sexually antagonistic (SA) selection, where individual alleles have conflicting fitness effects on each sex function. Although an extensive theory of SA selection exists for dioecious species, these results have not been generalized to hermaphrodites. We develop population genetic models of SA selection in simultaneous hermaphrodites, and evaluate effects of dominance, selection on each sex function, self‐fertilization, and population size on the maintenance of polymorphism. Under obligate outcrossing, hermaphrodite model predictions converge exactly with those of dioecious populations. Self‐fertilization in hermaphrodites generates three points of divergence with dioecious theory. First, opportunities for stable polymorphism decline sharply and become less sensitive to dominance with increased selfing. Second, selfing introduces an asymmetry in the relative importance of selection through male versus female reproductive functions, expands the parameter space favorable for the evolutionary invasion of female‐beneficial alleles, and restricts invasion criteria for male‐beneficial alleles. Finally, contrary to models of unconditionally beneficial alleles, selfing decreases genetic hitchhiking effects of invading SA alleles, and should therefore decrease these population genetic signals of SA polymorphisms. We discuss implications of SA selection in hermaphrodites, including its potential role in the evolution of “selfing syndromes.”     

The Folding of a Family of Three-Helix Bundle Proteins: Spectrin R15 Has a Robust Folding Nucleus,Unlike Its Homologous Neighbours

Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape.     

Uukuniemi Phlebovirus Assembly and Secretion Leave a Functional Imprint on the Virion Glycome

Uukuniemi virus (UUKV) is a model system for investigating the genus Phlebovirus of the Bunyaviridae. We report the UUKV glycome, revealing differential processing of the Gn and Gc virion glycoproteins. Both glycoproteins display poly-N-acetyllactosamines, consistent with virion assembly in the medial Golgi apparatus, whereas oligomannose-type glycans required for DC-SIGN-dependent cellular attachment are predominant on Gc. Local virion structure and the route of viral egress from the cell leave a functional imprint on the phleboviral glycome.     

Efficient Agrobacterium transformation of elite wheat germplasm without selection

A previously reported Agrobacterium tumefaciens transformation system that transformed wheat cultivar Fielder at high efficiency was shown to also transform eight out of nine Triticum aestivum (hexaploid wheat) cultivars tested and two Triticum turgidum (durum wheat) cultivars. Transformation efficiencies of these wheat lines ranged from 1.5 to 51 %. Included amongst this germplasm were elite Australian hexaploid wheat cultivars that are currently in commercial cultivation and two of these cultivars, Gladius and Westonia, were transformed at 32 and 45 % efficiency, respectively. Similar high transformation efficiencies were observed for durum wheat cultivars Kronos (51 %) and Stewart (26 %). This highly efficient transformation system was used to generate transgenic plants in the absence of selection and high heritability of unselected transgenes was observed. Selectable marker free transgenic wheat plants were produced at 3 % efficiency. These data demonstrate highly efficient Agrobacterium transformation of diverse wheat germplasm, including elite cultivars, which enables routine production of selectable marker free transgenics.     

Rab5 and Ndfip1 Are Involved in Pten Ubiquitination and Nuclear Trafficking

The spatial regulation of Pten is critical for its role as a tumour suppressor with both nuclear and cytoplasmic locations being implicated with distinct functions. In the cytoplasm, Pten plays a central role in opposing PI3K/Akt cell signalling, whereas in the nucleus, Pten is important for maintaining genome stability and enhancing the tumour suppressor activity of APC‐CDH1. Despite this diversity in protein function at different subcellular locations, there is limited knowledge on how Pten is able to find different cellular niches. Here, we report that Rab5 GTPase is required for efficient trafficking and ubiquitination of Pten on endosomes inside the cytosol. Using bimolecular fluorescence complementation (BiFC) for imaging protein interactions, we observed that ubiquitinated Pten is localized to peri‐nuclear and nuclear regions of the cell. Nuclear trafficking of Pten required both Rab5 as well as the E3 ligase adaptor protein Ndfip1. Rab5 colocalization with Pten was observed on endosomes and expression of a dominant negative form of Rab5 significantly reduced Pten ubiquitination and nuclear trafficking. Genomic deletion of Ndfip1 abrogated nuclear trafficking of ubiquitinated Pten, even in the presence of Rab5. Our findings show that endosomal trafficking and ubiquitination are important mechanisms for the subcellular distribution of Pten.      

Should I Get Screened for Sleeping Sickness? A Qualitative Study in Kasai Province,Democratic Republic of Congo

Background

Control of human African trypanosomiasis (sleeping sickness) in the Democratic Republic of Congo is based on mass population active screening by mobile teams. Although generally considered a successful strategy, the community participation rates in these screening activities and ensuing treatment remain low in the Kasai-Oriental province. A better understanding of the reasons behind this observation is necessary to improve regional control activities.

Methods

Thirteen focus group discussions were held in five health zones of the Kasai-Oriental province to gain insights in the regional perceptions regarding sleeping sickness and the national control programme''s activities.

Principal Findings

Sleeping sickness is well known among the population and is considered a serious and life-threatening disease. The disease is acknowledged to have severe implications for the individual (e.g., persistence of manic periods and trembling hands, even after treatment), at the family level (e.g., income loss, conflicts, separations) and for communities (e.g., disruption of community life and activities). Several important barriers to screening and treatment were identified. Fear of drug toxicity, lack of confidentiality during screening procedures, financial barriers and a lack of communication between the mobile teams and local communities were described. Additionally, a number of regionally accepted prohibitions related to sleeping sickness treatment were described that were found to be a strong impediment to disease screening and treatment. These prohibitions, which do not seem to have a rational basis, have far-reaching socio-economic repercussions and severely restrict the participation in day-to-day life.

Conclusions/Significance

A mobile screening calendar more adapted to the local conditions with more respect for privacy, the use of less toxic drugs, and a better understanding of the origin as well as better communication about the prohibitions related to treatment would facilitate higher participation rates among the Kasai-Oriental population in sleeping sickness screening and treatment activities organized by the national HAT control programme.