The protein tyrosine phosphatase Pez regulates TGFbeta, epithelial-mesenchymal transition, and organ development

Epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT.     

Characterization of human immunodeficiency virus type 1 monomeric and trimeric gp120 glycoproteins stabilized in the CD4-bound state: antigenicity, biophysics, and immunogenicity

The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S+S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S+S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.     

Endogenous retroviral insertion in Cryge in the mouse No3 cataract mutant

No3 (nuclear opacity 3) is a novel congenital nuclear cataract in mice. Microsatellite mapping placed the No3 locus on chromosome 1 between D1Mit480 (32cM) and D1Mit7 (41cM), a region containing seven crystallin genes; Cryba2 and the Cryga-Crygf cluster. Although polymorphic variants were observed, no candidate mutations were found for six of the genes. However, DNA walking identified a murine endogenous retrovirus (IAPLTR1: ERVK) insertion in exon 3 of Cryge, disrupting the coding sequence for gammaE-crystallin. Recombinant protein for the mutant gammaE was completely insoluble. The No3 cataract is mild compared with the effects of similar mutations of gammaE. Quantitative RT-PCR showed that gammaE/F mRNA levels are reduced in No3, suggesting that the relatively mild phenotype results from suppression of gammaE levels due to ERVK insertion. However, the severity of cataract is also strain dependent suggesting that genetic background modifiers also play a role in the development of opacity.     

Target assessment for antiparasitic drug discovery

Drug discovery is a high-risk, expensive and lengthy process taking at least 12 years and costing upwards of US$500 million per drug to reach the clinic. For neglected diseases, the drug discovery process is driven by medical need and guided by pre-defined target product profiles. Assessment and prioritisation of the most promising targets for entry into screening programmes is crucial for maximising the chances of success. Here, we describe criteria used in our drug discovery unit for target assessment and introduce the 'traffic-light' system as a prioritisation and management tool. We hope this brief review will stimulate basic scientists to acquire additional information necessary for drug discovery.     

Phylogeographical patterns in Coenosia attenuata (Diptera: Muscidae): a widespread predator of insect species associated with greenhouse crops

The tiger‐fly Coenosia attenuata is a globally widespread predatory fly which is not only associated with greenhouse crops, but also occurs in open fields. It is a potential control agent against some of the more common pests in these crops. Assessing the genetic structure and gene flow patterns may be important for planning crop protection strategies and for understanding the historical processes that led to the present distribution of genetic lineages within this species. In the present study, the phylogeographical patterns of this species, based on mitochondrial cytochrome oxidase I and nuclear white and elongation factor‐1α genes, are described, revealing relatively low genetic diversity and weak genetic structure associated with a recent and sudden population expansion of the species. The geographical distribution of mitochondrial haplotypes indicates the Mediterranean as the most likely region of origin of the species. Some dispersal patterns of the species are also revaled, including at least three independent colonizations of North and South America: one from Middle East to North America with a strong bottleneck event, another from Europe to South America (Chile), with both likely to be a result of unintentional introduction, and a third one of still undetermined origin to South America (Ecuador). © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 308–326.     

Interactions between temperature and nutrients across levels of ecological organization

Temperature and nutrient availability play key roles in controlling the pathways and rates at which energy and materials move through ecosystems. These factors have also changed dramatically on Earth over the past century as human activities have intensified. Although significant effort has been devoted to understanding the role of temperature and nutrients in isolation, less is known about how these two factors interact to influence ecological processes. Recent advances in ecological stoichiometry and metabolic ecology provide a useful framework for making progress in this area, but conceptual synthesis and review are needed to help catalyze additional research. Here, we examine known and potential interactions between temperature and nutrients from a variety of physiological, community, and ecosystem perspectives. We first review patterns at the level of the individual, focusing on four traits – growth, respiration, body size, and elemental content – that should theoretically govern how temperature and nutrients interact to influence higher levels of biological organization. We next explore the interactive effects of temperature and nutrients on populations, communities, and food webs by synthesizing information related to community size spectra, biomass distributions, and elemental composition. We use metabolic theory to make predictions about how population‐level secondary production should respond to interactions between temperature and resource supply, setting up qualitative predictions about the flows of energy and materials through metazoan food webs. Last, we examine how temperature–nutrient interactions influence processes at the whole‐ecosystem level, focusing on apparent vs. intrinsic activation energies of ecosystem processes, how to represent temperature–nutrient interactions in ecosystem models, and patterns with respect to nutrient uptake and organic matter decomposition. We conclude that a better understanding of interactions between temperature and nutrients will be critical for developing realistic predictions about ecological responses to multiple, simultaneous drivers of global change, including climate warming and elevated nutrient supply.     

Organic dust augments nucleotide-binding oligomerization domain expression via an NF-{kappa}B pathway to negatively regulate inflammatory responses

Nucleotide-binding oligomerization domain 2 (NOD2) is involved in innate immune responses to peptidoglycan degradation products. Peptidoglycans are important mediators of organic dust-induced airway diseases in exposed agriculture workers; however, the role of NOD2 in response to complex organic dust is unknown. Monocytes/macrophages were exposed to swine facility organic dust extract (ODE), whereupon NOD2 expression was evaluated by real-time PCR and Western blot. ODE induced significant NOD2 mRNA and protein expression at 24 and 48 h, respectively, which was mediated via a NF-κB signaling pathway as opposed to a TNF-α autocrine/paracrine mechanism. Specifically, NF-κB translocation increased rapidly following ODE stimulation as demonstrated by EMSA, and inhibition of the NF-κB pathway significantly reduced ODE-induced NOD2 expression. However, there was no significant reduction in ODE-induced NOD2 gene expression when TNF-α was inhibited or absent. Next, it was determined whether NOD2 regulated ODE-induced inflammatory cytokine production. Knockdown of NOD2 expression by small interfering RNA resulted in increased CXCL8 and IL-6, but not TNF-α production in response to ODE. Similarly, primary lung macrophages from NOD2 knockout mice demonstrated increased IL-6, CXCL1, and CXCL1, but not TNF-α, expression. Lastly, a higher degree of airway inflammation occurred in the absence of NOD2 following acute (single) and repetitive (3 wk) ODE exposure in an established in vivo murine model. In summary, ODE-induced NOD2 expression is directly dependent on NF-κB signaling, and NOD2 is a negative regulator of complex, organic dust-induced inflammatory cytokine/chemokine production in mononuclear phagocytes.     

REPRODUCTIVE BIOLOGY OF ASCLEPIAS EXALTATA

An analysis of the relationships between plant size and survivorship and reproductive success was carried out by sampling four populations of the herbaceous perennial milkweed Asclepias exaltata in Virginia from 1980 to 1982. The annual survivorship rate (about 65%) is the lowest measured for any species of Asclepias. Survivorship was strongly size-dependent but showed no clear relationship with previous history of fruit production. Non-flowering plants were significantly smaller than flowering plants and showed very strong (r > 0.87) correlations between root dry weight and stem or leaf dry weight. Flowering plants were similar to nonflowering plants in root: shoot ratio (approximately 1:1) but differed in that root dry weight was not strongly correlated with stem or leaf dry weight. Components of inflorescence size were strongly correlated within a given level of comparison (e.g., stems per plant with flowers per plant) but less strongly correlated between levels (e.g., stems per plant with flowers per stem). Number of fruits per plant and percentage fruit-set were positively correlated with every component of inflorescence size. Although overall fruit-set was low (about 2%), fruits that were initiated had a high probability of surviving to maturity. There was no evidence of an early period of high fruit abortion: a relatively constant proportion of fruits aborted between each age class.     

THE EVOLUTION OF SELF-POLLINATION IN GRANITE OUTCROP SPECIES OF ARENARIA (CARYOPHYLLACEAE). IV. CORRELATED CHANGES IN THE GYNOECIUM

Intraspecific variation in gynoecial characters was analyzed by SEM for nine populations of Arenaria uniflora, a winter annual endemic to rock outcrops in the southeastern United States. Style lengths in self-pollinating populations, formerly called A. alabamensis, were significantly shorter than in outcrossing populations, and style length showed a very strong correlation (r = 0.97) with outcrossing potential as assessed by degree of protandry. Selfers were also characterized by the production of fewer but longer stigmatic papillae. Papillae extended to the base of the style and diverged from all sides. Outcrossers had longer styles with dense but short papillae, mostly restricted to the upper surface of the style and lacking at its base. Styles also were observed to curl downward in outcrossers. These fine-scale changes appear to enhance the effectiveness of plants as either outcross-pollen or self-pollen receivers.     

Biochemical and immunogenic characterization of soluble human immunodeficiency virus type 1 envelope glycoprotein trimers expressed by semliki forest virus

The current lack of envelope glycoprotein immunogens that elicit broadly neutralizing antibody responses remains a major challenge for human immunodeficiency virus type 1 (HIV-1) vaccine development. However, the recent design and construction of stable soluble gp140 trimers have shown that some neutralization breadth can be achieved by using immunogens that better mimic the functional viral spike complex. The use of genetic delivery systems to drive the in vivo expression of such immunogens for the stimulation of neutralizing antibodies against HIV-1 may offer advantages by maintaining the quaternary structure of the trimeric envelope glycoproteins. Here, we describe the biochemical and immunogenic properties of soluble HIV-1 envelope glycoprotein trimers expressed by recombinant Semliki Forest virus (rSFV). The results presented here demonstrate that rSFV supports the expression of stable soluble gp140 trimers that retain recognition by conformationally sensitive antibodies. Further, we show that rSFV particle immunizations efficiently primed immune responses as measured after a single boost with purified trimeric gp140 protein, resulting in a Th1-biased antibody response. This differed from the Th2-biased antibody response obtained after repeated immunizations with purified gp140 protein trimers. Despite this difference, both regimens stimulated neutralizing antibody responses of similar potency. This suggests that rSFV may be a useful component of a viral vector prime-protein boost regimen aimed at stimulating both cell-mediated immune responses and neutralizing antibodies against HIV-1.