Induction of Autophagy by Cystatin C: A Mechanism That Protects Murine Primary Cortical Neurons and Neuronal Cell Lines

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimer''s disease, and other neurodegenerative disorders.     

Nature of sterols affects plasma membrane behavior and yeast survival during dehydration

The plasma membrane (PM) is a main site of injury during osmotic perturbation. Sterols, major lipids of the PM structure in eukaryotes, are thought to play a role in ensuring the stability of the lipid bilayer during physicochemical perturbations. Here, we investigated the relationship between the nature of PM sterols and resistance of the yeast Saccharomyces cerevisiae to hyperosmotic treatment. We compared the responses to osmotic dehydration (viability, sterol quantification, ultrastructure, cell volume, and membrane permeability) in the wild-type (WT) strain and the ergosterol mutant erg6Δ strain. Our main results suggest that the nature of membrane sterols governs the mechanical behavior of the PM during hyperosmotic perturbation. The mutant strain, which accumulates ergosterol precursors, was more sensitive to osmotic fluctuations than the WT, which accumulates ergosterol. The hypersensitivity of erg6Δ was linked to modifications of the membrane properties, such as stretching resistance and deformation, which led to PM permeabilization during the volume variation during the dehydration-rehydration cycles. Anaerobic growth of erg6Δ strain with ergosterol supplementation restored resistance to osmotic treatment. These results suggest a relationship between hydric stress resistance and the nature of PM sterols. We discuss this relationship in the context of the evolution of the ergosterol biosynthetic pathway.     

Dynamics of microbial planktonic food web components during a river flash flood in a Mediterranean coastal lagoon

Episodic river flash floods, characteristic of Mediterranean climates, are suspected to greatly affect the functioning of microbial food webs. For the first time, the abundance, biomass and diversities of microbial food web components were studied before and during 4 consecutive days after a flash flood that occurred in November 2008, in the surface waters of five stations along a salinity gradient from 20 to 36 in the Thau lagoon. Eukaryotic pico- and nanophytoplankton were discharged from the river into the lagoon and increased by 30- and 70-fold, respectively. Bacteria increased by only 2-fold in the lagoon, from around 4–8 × 10⁶ cells ml⁻¹, probably benefiting from river nutrient input. Chlorophyll a increased 4-fold, and pigment biomarkers showed that the dinophyceae, prasinophyceae and prymnesiophyceae were sensitive to the flood perturbation, whereas the bacillariophyceae, cryptophyceae and chlorophyceae were resistant and/or transported to the lagoon from the river. Predator responses were more complex as total heterotrophic flagellate abundance decreased slightly, whereas those of specific naked ciliates increased, particularly for Uronema sp. The flood also induced a specific change in diversity, from a community dominated by Strobilidium spiralis to a community dominated by Uronema sp. The tintinnid community was particularly sensitive to the flood event as the abundance of all species decreased greatly. The high increases in biomass, mainly brought by the river during the flood, could have eventually sedimented to the benthic layer and/or been transported further into the lagoon, supporting the pelagic food web, or have even been exported to the Mediterranean Sea.     

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.     

The role of spiking and bursting pacemakers in the neuronal control of breathing

Breathing is controlled by a distributed network involving areas in the neocortex, cerebellum, pons, medulla, spinal cord, and various other subcortical regions. However, only one area seems to be essential and sufficient for generating the respiratory rhythm: the preBötzinger complex (preBötC). Lesioning this area abolishes breathing and following isolation in a brain slice the preBötC continues to generate different forms of respiratory activities. The use of slice preparations led to a thorough understanding of the cellular mechanisms that underlie the generation of inspiratory activity within this network. Two types of inward currents, the persistent sodium current (I_NaP) and the calcium-activated non-specific cation current (I_CAN), play important roles in respiratory rhythm generation. These currents give rise to autonomous pacemaker activity within respiratory neurons, leading to the generation of intrinsic spiking and bursting activity. These membrane properties amplify as well as activate synaptic mechanisms that are critical for the initiation and maintenance of inspiratory activity. In this review, we describe the dynamic interplay between synaptic and intrinsic membrane properties in the generation of the respiratory rhythm and we relate these mechanisms to rhythm generating networks involved in other behaviors.     

Suitability of three Ostrinia species as hosts for Macrocentrus cingulum： A comparison of their encapsulation abilities

Two cornborer species, Ostrinia furnacalis （Lepidoptera： Crambidae） and O. nubilalis, are major corn pests in Asia and Europe, respectively. In both continents, the larval endoparasitoid Macrocentrus cingulum （Hymenoptera： Braconidae） develops on another, closely related stemborer, O. scapulalis, which feeds on mugwort and other dicotyledons. M. cingulum also emerges from O. furnacalis in Asia and （9. nubilalis in North America, but not from O. nubilalis in Europe. We assessed the ability of three populations of each of the three Ostrinia species to encapsulate foreign bodies of a size similar to that of a M. cingulum egg. We conclude that variations in encapsulation ability alone cannot account for the differences observed in the field between parasite emergence rates in these different host species and geographic areas.     

Functional diversity of leaf nitrogen concentrations drives grassland carbon fluxes

Little is known about the role of plant functional diversity for ecosystem‐level carbon (C) fluxes. To fill this knowledge gap, we translocated monoliths hosting communities with four and 16 sown species from a long‐term grassland biodiversity experiment (‘The Jena Experiment’) into a controlled environment facility for ecosystem research (Ecotron). This allowed quantifying the effects of plant diversity on ecosystem C fluxes as well as three parameters of C uptake efficiency (water and nitrogen use efficiencies and apparent quantum yield). By combining data on ecosystem C fluxes with vegetation structure and functional trait‐based predictors, we found that increasing plant species and functional diversity led to higher gross and net ecosystem C uptake rates. Path analyses and light response curves unravelled the diversity of leaf nitrogen concentration in the canopy as a key functional predictor of C fluxes, either directly or indirectly via LAI and aboveground biomass.     

High resolution assembly and characterization of genomes of Canadian isolates of Salmonella Enteritidis

Background

There is a need to characterize genomes of the foodborne pathogen, Salmonella enterica serovar Enteritidis (SE) and identify genetic information that could be ultimately deployed for differentiating strains of the organism, a need that is yet to be addressed mainly because of the high degree of clonality of the organism. In an effort to achieve the first characterization of the genomes of SE of Canadian origin, we carried out massively parallel sequencing of the nucleotide sequence of 11 SE isolates obtained from poultry production environments (n = 9), a clam and a chicken, assembled finished genomes and investigated diversity of the SE genome.

Results

The median genome size was 4,678,683 bp. A total of 4,833 chromosomal genes defined the pan genome of our field SE isolates consisting of 4,600 genes present in all the genomes, i.e., core genome, and 233 genes absent in at least one genome (accessory genome). Genome diversity was demonstrable by the presence of 1,360 loci showing single nucleotide polymorphism (SNP) in the core genome which was used to portray the genetic distances by means of a phylogenetic tree for the SE isolates. The accessory genome consisted mostly of previously identified SE prophage sequences as well as two, apparently full- sized, novel prophages namely a 28 kb sequence provisionally designated as SE-OLF-10058 (3) prophage and a 43 kb sequence provisionally designated as SE-OLF-10012 prophage.

Conclusions

The number of SNPs identified in the relatively large core genome of SE is a reflection of substantial diversity that could be exploited for strain differentiation as shown by the development of an informative phylogenetic tree. Prophage sequences can also be exploited for SE strain differentiation and lineage tracking. This work has laid the ground work for further studies to develop a readily adoptable laboratory test for the subtyping of SE.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-713) contains supplementary material, which is available to authorized users.