Functional Characterization of a Chimeric Soluble Fas Ligand Polymer with In Vivo Anti-Tumor Activity

Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas.     

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.     

Non-contiguous finished genome sequence and description of Oceanobacillus massiliensis sp. nov.

Oceanobacillus massiliensis strain N’Diop^T sp. nov. is the type strain of O. massiliensis sp. nov., a new species within the genus Oceanobacillus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. O. massiliensis is an aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,532,675 bp long genome contains 3,519 protein-coding genes and 72 RNA genes, including between 6 and 8 rRNA operons.     

Non-contiguous finished genome sequence of Phocaeicola abscessus type strain 7401987T

Phocaeicola abscessus strain 7401987^T is the sole member of the genus Phocaeicola. This bacterium is Gram-negative, non-spore-forming, coccoid to rod-shaped and motile by lophotrichous flagella. It was isolated from a human brain abscess sample. In this work, we describe a set of features of this organism, together with the complete genome sequence and annotation. The 2,530,616 bp long genome contains 2,090 protein-coding genes and 54 RNA genes, including 4 rRNA operons.     

Soil Environmental Conditions and Microbial Build-Up Mediate the Effect of Plant Diversity on Soil Nitrifying and Denitrifying Enzyme Activities in Temperate Grasslands

Random reductions in plant diversity can affect ecosystem functioning, but it is still unclear which components of plant diversity (species number – namely richness, presence of particular plant functional groups, or particular combinations of these) and associated biotic and abiotic drivers explain the observed relationships, particularly for soil processes. We assembled grassland communities including 1 to 16 plant species with a factorial separation of the effects of richness and functional group composition to analyze how plant diversity components influence soil nitrifying and denitrifying enzyme activities (NEA and DEA, respectively), the abundance of nitrifiers (bacterial and archaeal amoA gene number) and denitrifiers (nirK, nirS and nosZ gene number), and key soil environmental conditions. Plant diversity effects were largely due to differences in functional group composition between communities of identical richness (number of sown species), though richness also had an effect per se. NEA was positively related to the percentage of legumes in terms of sown species number, the additional effect of richness at any given legume percentage being negative. DEA was higher in plots with legumes, decreased with increasing percentage of grasses, and increased with richness. No correlation was observed between DEA and denitrifier abundance. NEA increased with the abundance of ammonia oxidizing bacteria. The effect of richness on NEA was entirely due to the build-up of nitrifying organisms, while legume effect was partly linked to modified ammonium availability and nitrifier abundance. Richness effect on DEA was entirely due to changes in soil moisture, while the effects of legumes and grasses were partly due to modified nitrate availability, which influenced the specific activity of denitrifiers. These results suggest that plant diversity-induced changes in microbial specific activity are important for facultative activities such as denitrification, whereas changes in microbial abundance play a major role for non-facultative activities such as nitrification.     

Functional Divergence in Shrimp Anti-Lipopolysaccharide Factors (ALFs): From Recognition of Cell Wall Components to Antimicrobial Activity

Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded β-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.     

Adapting the LCA framework to environmental assessment in land planning

Purpose

Since the implementation of the European directive (EC/2001/42) on strategic environmental assessment, an ex ante evaluation has become mandatory for plans and programs. This requirement could have significant consequences for the environment. Local authorities, who are in charge of land planning issues, must therefore conduct such assessments. However, they are faced with lack of uniform methodology. The aim of this paper is thus to propose a methodological framework for the required environmental assessment stages in land planning.

Methods

Life cycle assessment (LCA) has been identified as a promising tool to perform environmental assessment at a meso-level (i.e., territories). Yet, the standardized LCA framework has never been used for assessing the environmental impacts of a territory as such, which can be explained by the complexity that its application would involve. Four major methodological bottlenecks have been identified in this paper, i.e., (1) functional unit definition, (2) boundary selection, (3) data collecting, and (4) the refinement of the life cycle impact assessment phase in order to provide useful indicators for land planning. For each of these challenges, recommendations have been made to adapt the analytical framework of LCA.

Results and discussion

A revised framework is proposed to perform LCA of a territory. One of the major adaptations needed concerns the goal and scope definition phase. Henceforth, the association of a territory and the studied land planning scenario, defined by its geographical boundaries and its interactions with other territories, will be designated as the reference flow in LCA. Consequently, two kinds of indicators will be determined using this approach, i.e., (1) a vector of environmental impacts generated (conventional LCA) and (2) a vector of land use functions provided by the territory for different stakeholders (e.g., provision of work, recreation, culture, etc.). This revised framework has been applied to a theoretical case study in order to highlight its utility in land planning.

Conclusions

This work is a first step in the adaptation of the LCA framework to environmental assessment in land planning. We believe that this revised framework has the potential to provide relevant information in decision-making processes. Nonetheless, further work is still needed to broaden and deepen this approach (i.e., normalization of impacts and functions, coupled application with GIS, uncertainties, etc.).     

Involvement of kinase PKC-zeta in the p62/p62P392L-driven activation of NF-κB in human osteoclasts

Mutations of the gene encoding sequestosome1 (SQSTM1/p62), clustering in or near the UBA domain, have been described in Paget's disease of bone (PDB); among these the P392L substitution is the most prevalent. Protein p62 mediates several cell functions, including the control of NF-κB signaling, and autophagy. This scaffolding protein interacts with atypical PKCζ in the RANKL-induced signaling complex. We have previously shown that osteoclasts (OCs) overexpressing the p62^P392L variant were in a constitutively activated state, presenting activated kinase p-PKCζ/λ and activated NF-κB prior to RANKL stimulation. In the present study, we investigated the relationships between PKCζ and NF-κB activation in human OCs transfected with p62 variants. We showed that PKCζ and p-PKCζ/λ co-localize with p62, and that PKCζ is involved in the RANKL-induced NF-κB activation and in the RANKL-independent activation of NF-κB observed in p62^P392L-transfected cells. We also observed a basal and RANKL-induced increase in IκBα levels in the presence of the p62^P392L mutation that contrasted with the NF-κB activation. In this study we propose that PKCζ plays a role in the activation of NF-κB by acting as a p65 (RelA) kinase at Ser⁵³⁶, independently of IκBα; this alternative pathway could be used preferentially in the presence of the p62^P392L mutation, which may hinder the ubiquitin–proteasome pathway. Overall, our results highlight the importance of p62-associated PKCζ in the overactive state of pagetic OCs and in the activation of NF-κB, particularly in the presence of the p62^P392L mutation.     

Population dynamics of calanoid copepods in the subtropical mesohaline Danshuei Estuary (Taiwan) and typhoon effects

We studied the populations of the dominant calanoid copepods Pseudodiaptomus annandalei and Acartia spp. in the mesohaline Danshuei Estuary for two successive springs and summers. We analyzed environmental factors (i.e., temperature, salinity, chlorophyll a, light attenuation coefficient, and suspended particulate matter) and the population densities and structures at the surface and near the estuarine bed. The population of P. annandalei dominated the zooplankton community from surface to bottom, except during a medusa bloom in 2009 and during the post-typhoon period, when Acartia spinicauda dominated. The relationships between environmental factors and densities differed between and within populations of P. annandalei and Acartia spp. We concluded that the primary determinants of the P. annandalei population are predation and the typhoon-related freshwater runoff, which can strongly influence the copepod succession in this part of the estuary.     

Management history determines gene flow in a prominent invader

Invasive plants pose substantial threats to protected areas globally. Although management can limit impacts, spread and reinvasion from neighbouring areas into protected areas are a major and an on‐going problem for land managers. However, identifying the main sources of propagules and the dimensions of invasion pathways is challenging. This study used population genetic markers [inter simple sequence repeats (ISSRs) and amplified fragment length polymorphisms (AFLPs)] to infer the source(s) of re‐colonization and dispersal patterns for a typical invader of riparian and terrestrial habitats (Lantana camara) along the Sabie‐Sand catchment, one of the most important river systems flowing into and across South Africa’s flagship protected area, the Kruger National Park (KNP). Results indicate that populations located along the lower reaches of the Sabie and Sand tributaries harboured substantially higher genetic diversity than those in the upper Sabie catchment. Bayesian assignments indicated that the upper Sabie tributary contributed far fewer propagules than the Sand tributary to the lower Sabie River. Current invasion patterns are due to a combination of a major flood event in 2000 and differences in the degree to which the upstream reaches were managed after the flooding. The major flood of 2000 effectively cleared lantana from the riparian areas. However, whereas on‐going management efforts against riparian species in the KNP have been effective, rendering the upper Sabie relatively clear of lantana, only a small part of the Sand tributary falls under jurisdiction of the KNP and has received consistent management attention. The reinvasion of the lower Sabie in the KNP was therefore almost entirely by propagules from the Sand tributary. The study highlights the important role that molecular tools can play in determining dispersal dynamics and directing invasive species management. For invasive plant species that invade both riparian habitats and landscapes away from rivers in protected areas, such as lantana, management must focus on all major sources of propagules to limit reinvasion.