Multi locus analysis of Pristionchus pacificus on La Réunion Island reveals an evolutionary history shaped by multiple introductions, constrained dispersal events and rare out-crossing

Pristionchus pacificus, recently established as a model organism in evolutionary biology, is a cosmopolitan nematode that has a necromenic association with scarab beetles. The diverse array of host beetle species and habitat types occupied by P. pacificus make it a good model for investigating local adaptation to novel environments. Presence of P. pacificus on La Réunion Island, a young volcanic island with a dynamic geological history and a wide variety of ecozones, facilitates such investigation in an island biogeographic setting. Microsatellite data from 20 markers and 223 strains and mitochondrial sequence data from 272 strains reveal rich genetic diversity among La Réunion P. pacificus isolates, shaped by differentially timed introductions from diverse sources and in association with different beetle species. Distinctions between volcanic zones and between arid western and wet eastern climatic zones have likely limited westward dispersal of recently colonized lineages and maintained a genetic distinction between eastern and western clades. The highly selfing lifestyle of P. pacificus contributes to the strong fine‐scale population structure detected, with each beetle host harbouring strongly differentiated assemblages of strains. Periodic out‐crossing generates admixture between genetically diverse lineages, creating a diverse array of allelic combinations likely to increase the evolutionary potential of the species and facilitate adaptation to local environments and beetle hosts.     

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH^•. In root cells, extracellular OH^• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH^•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH^•. An OH^•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.     

Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture

Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-alpha1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-alpha1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-alpha1I probe (apparent K(d) values of 0.5 and 50 microM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-alpha1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods.     

Characterization of Phospholipase C�� Enzymes with Gain-of-Function Mutations

Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.Phosphoinositide-specific phospholipase C (PLC)² enzymes, comprising several families (PLCβ, γ, δ, ϵ, η, and ζ), have been established as crucial signaling molecules involved in regulation of a variety of cellular functions (1–4). PLC-catalyzed formation of the second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol, from phosphatidylinositol 4,5-bisphosphate (PIP₂), constitutes one of the major cell signaling responses. These second messengers provide a common link from highly specific receptors for hormones, neurotransmitters, antigens, and growth factors to downstream, intracellular targets; thus, they contribute to regulation of biological functions as diverse as cell motility, fertilization, and sensory transduction. Despite this central role for PLC enzymes in signaling networks, the molecular details of their regulation and possible subversion of these regulatory mechanisms in disease remain poorly understood.Of two PLCγ enzymes, PLCγ1 is ubiquitously expressed and appears to regulate a multitude of cellular functions in many tissues. Plcg1-null mice die by embryonic day 9, highlighting the widespread importance of this enzyme (5). PLCγ1 is activated in response to growth factor stimulation; in addition, its function in T-cell responses has been extensively documented (1). PLCγ2, in contrast, is most highly expressed in cells of the hematopoietic system and plays a key role in regulation of the immune response. Consistent with this, Plcg2-null mice display defects in the functioning of B cells, platelets, mast cells, and natural killer cells (6).Both PLCγ enzymes have also been implicated in signaling events underlying aberrant cellular responses. PLCγ1 is critically involved in the regulation of cancer cell motility (7–11) while PLCγ2 has been implicated in deregulation of the immune responses resembling Btk-dependent X-linked agammaglobulinaemia and SLE disease in humans (12–14). It has been suggested that, in cancer cells, PLCγ1 could function as a key, rate-limiting, common component involved in cell motility triggered by several growth factors and integrins (7). In some cancer cells, this increased motility could result from deregulation i.e. higher levels of expression of PLCγ1 (15, 16). The possibility that the activity of PLCγ could be up-regulated due to mutation has not yet been fully investigated in cancer. Previous studies of PLCγ2, however, have demonstrated the first gain-of-function mutation in a PLC molecule in the context of an organism, and shown that, in principle, PLC activity can be greatly enhanced by point mutations (13). Furthermore, this work has demonstrated that such a mutation is linked to a dramatic phenotypic disorder. By using a large scale ENU mutagenesis to discover new immune regulators, several mouse strains were generated with spontaneous autoimmune and inflammatory symptoms; two of these strains harbor a mutation in PLCγ2. In addition to the previously described ALI5 mutation (13) the ALI14 mutation has been identified very recently.³ Strikingly, the well-characterized ALI5 phenotype has shown that the mutation affects many cellular functions deregulated in Plcg2-null mice. Notably, while in Plcg2-null mice such responses are lacking, the ALI5 mutation resulted in their enhancement. In particular, further analyses of the ALI5 mutation in the context of signaling in B-cells have demonstrated that calcium responses to the crosslinking of the B-cell receptor were enhanced and prolonged resulting in enhanced deletion of B cells and autoreactivity (13).The domain organization of PLCγ enzymes is characterized by the insertion of a highly structured region (PLCγ-specific array, γSA) between the two halves of the TIM-barrel catalytic domain common to all PLCs. The γSA comprises a split PH (spPH) domain flanking two tandem SH2 domains and a SH3 domain (1). A distinct regulatory feature of PLCγ enzymes is that their activation is linked to an increase in phosphorylation of specific tyrosine residues (most notably within the γSA) by receptor and non-receptor tyrosine kinases (17, 18). Furthermore, multiple protein-protein interactions (mainly mediated by SH2 domains) also contribute to activation and have an important role in localizing PLCγ into protein complexes with different binding partners, depending on cell type and specific cellular compartments. One mode of activation that is specific for the PLCγ2 isozyme is direct binding to and activation by Rac. The interaction involves the spPH domain, and this activation mechanism does not require tyrosine phosphorylation (19, 20). In molecular terms, changes that lead to PLC activation in response to different input signals, or due to point mutations, are not well understood and require further studies.Here we describe further analysis of the two gain-of-function mutations, ALI5 and ALI14, obtained using ENU mutagenesis. These mutations map to different regions in PLCγ2, and we performed detailed analysis of these regions in both PLCγ isozymes. To characterize the molecular mechanism of gain-of-function, we combined studies in vitro and in different cellular signaling contexts. We have found that ALI5- and ALI14-type point mutations lead, by distinct mechanisms, to an enhancement of responses to a variety of input signals while their combination results in a constitutively active PLC enzyme.     

Investigating the Targets of MIR-15a and MIR-16-1 in Patients with Chronic Lymphocytic Leukemia (CLL)

Background

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate the expression of multiple target genes. Deregulation of miRNAs is common in human tumorigenesis. The miRNAs, MIR-15a/16-1, at chromosome band 13q14 are down-regulated in the majority of patients with chronic lymphocytic leukaemia (CLL).

Methodology/Principal Findings

We have measured the expression of MIR-15a/16-1, and 92 computationally-predicted MIR-15a/16-1 target genes in CLL patients and in normal controls. We identified 35 genes that are deregulated in CLL patients, 5 of which appear to be specific targets of the MIR-15a/16-1 cluster. These targets included 2 genes (BAZ2A and RNF41) that were significantly up-regulated (p<0.05) and 3 genes (RASSF5, MKK3 and LRIG1) that were significantly down-regulated (p<0.05) in CLL patients with down-regulated MIR-15a/16-1 expression.

Significance

The genes identified here as being subject to MIR-15a/16-1 regulation could represent direct or indirect targets of these miRNAs. Many of these are good biological candidates for involvement in tumorigenesis and as such, may be important in the aetiology of CLL.     

Molecular phylogenetics and biogeography of the Neocellia Series of Anopheles mosquitoes in the Oriental Region

Molecular studies of population divergence and speciation across the Oriental Region are sparse, despite the region’s high biodiversity and extensive Pliocene and Pleistocene environmental change. A molecular phylogenetic study of the Neocellia Series of Anopheles mosquitoes was undertaken to identify patterns of diversification across the Oriental Region and to infer the role of Pleistocene and Pliocene climatic change. A robust phylogeny was constructed using CO2 and ND5 mitochondrial genes and ITS2 and D3 nuclear ribosomal markers. Bayesian analysis of mitochondrial genes was used to date divergence events. The repeated contraction and expansion of forest habitat resulting from Pleistocene climatic fluctuations appears to have had a substantial impact on intraspecific diversification, but has not driven speciation within this group. Primarily early to mid Pliocene speciation was detected within the Annularis Group, whereas speciation within the Maculatus and Jamesii Groups occurred during the mid and late Pliocene. Both allopatric divergence driven by late Pliocene environmental changes and ecological adaptation, involving altitudinal replacement and seasonality, are likely to have influenced speciation in the Maculatus Group.     

Thermodynamics of binding of regulatory ligands to tissue transglutaminase

Amino Acids - The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the...     

Leaf miner and plant galler species richness on Acacia: relative importance of plant traits and climate

Diversity patterns of herbivores have been related to climate, host plant traits, host plant distribution and evolutionary relationships individually. However, few studies have assessed the relative contributions of a range of variables to explain these diversity patterns across large geographical and host plant species gradients. Here we assess the relative influence that climate and host plant traits have on endophagous species (leaf miners and plant gallers) diversity across a suite of host species from a genus that is widely distributed and morphologically variable. Forty-six species of Acacia were sampled to encapsulate the diversity of species across four taxonomic sections and a range of habitats along a 950 km climatic gradient: from subtropical forest habitats to semi-arid habitats. Plant traits, climatic variables, leaf miner and plant galler diversity were all quantified on each plant species. In total, 97 leaf mining species and 84 plant galling species were recorded from all host plants. Factors that best explained leaf miner richness across the climatic gradient (using AIC model selection) included specific leaf area (SLA), foliage thickness and mean annual rainfall. The factor that best explained plant galler richness across the climatic gradient was C:N ratio. In terms of the influence of plant and climatic traits on species composition, leaf miner assemblages were best explained by SLA, foliage thickness, mean minimum temperature and mean annual rainfall, whilst plant gall assemblages were explained by C:N ratio, %P, foliage thickness, mean minimum temperature and mean annual rainfall. This work is the first to assess diversity and structure across a broad environmental gradient and a wide range of potential key climatic and plant trait determinants simultaneously. Such methods provide key insights into endophage diversity and provide a solid basis for assessing their responses to a changing climate.     

Reporting the implementation of the Three Rs in European primate and mouse research papers: are we making progress?

It is now more than 20 years since both Council of Europe Convention ETS123 and EU Directive 86/609/EEC were introduced, to promote the implementation of the Three Rs in animal experimentation and to provide guidance on animal housing and care. It might therefore be expected that reports of the implementation of the Three Rs in animal research papers would have increased during this period. In order to test this hypothesis, a literature survey of animal-based research was conducted. A randomly-selected sample from 16 high-profile medical journals, of original research papers arising from European institutions that featured experiments which involved either mice or primates, were identified for the years 1986 and 2006 (Total sample = 250 papers). Each paper was scored out of 10 for the incidence of reporting on the implementation of Three Rs-related factors corresponding to Replacement (justification of non-use of non-animal methods), Reduction (statistical analysis of the number of animals needed) and Refinement (housing aspects, i.e. increased cage size, social housing, enrichment of cage environment and food; and procedural aspects, i.e. the use of anaesthesia, analgesia, humane endpoints, and training for procedures with positive reinforcement). There was no significant increase in overall reporting score over time, for either mouse or primate research. By 2006, mouse research papers scored an average of 0 out of a possible 10, and primate research papers scored an average of 1.5. This review provides systematic evidence that animal research is still not properly reported, and supports the call within the scientific community for action to be taken by journals to update their policies.