Molecular Diversity of New Thermococcales Isolates from a Single Area of Hydrothermal Deep-Sea Vents as Revealed by Randomly Amplified Polymorphic DNA Fingerprinting and 16S rRNA Gene Sequence Analysis

Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13°N 104°W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales. About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.     

MCM8- and MCM9-Deficient Mice Reveal Gametogenesis Defects and Genome Instability Due to Impaired Homologous Recombination

We generated knockout mice for MCM8 and MCM9 and show that deficiency for these genes impairs homologous recombination (HR)-mediated DNA repair during gametogenesis and somatic cells cycles. MCM8(-/-) mice are sterile because spermatocytes are blocked in meiotic prophase I, and females have only arrested primary follicles and frequently develop ovarian tumors. MCM9(-/-) females also are sterile as ovaries are completely devoid of oocytes. In contrast, MCM9(-/-) testes produce spermatozoa, albeit in much reduced quantity. Mcm8(-/-) and Mcm9(-/-) embryonic fibroblasts show growth defects and chromosomal damage and cannot overcome a transient inhibition of replication fork progression. In these cells, chromatin recruitment of HR factors like Rad51 and RPA is impaired and HR strongly reduced. We further demonstrate that MCM8 and MCM9 form a complex and that they coregulate their stability. Our work uncovers essential functions of MCM8 and MCM9 in HR-mediated DSB repair during gametogenesis, replication fork maintenance, and DNA repair.     

Phospholipases C and D modulate proline accumulation in Thellungiella halophila/salsuginea differently according to the severity of salt or hyperosmotic stress

Proline accumulation is one of the most common responses of plants to environmental constraints. Thellungiella halophila/salsuginea, a model halophyte, accumulates high levels of proline in response to abiotic stress and in the absence of stress. Recently, lipid signaling pathways have been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. Here we investigated the relationship between lipid signaling enzymes and the level of proline in T. salsuginea. Inhibition of phospholipase C (PLC) enzymes by the specific inhibitor U73122 demonstrated that proline accumulation is negatively controlled by PLCs in the absence of stress and under moderate salt stress (200 mM NaCl). The use of 1-butanol to divert some of the phospholipase D (PLD)-derived phosphatidic acid by transphosphatidylation revealed that PLDs exert a positive control on proline accumulation under severe stress (400 mM NaCl or 400 mM mannitol) but have no effect on its accumulation in non-stress conditions. This experimental evidence shows that positive and negative lipid regulatory components are involved in the fine regulation of proline metabolism. These signaling pathways in T. salsuginea are regulated in the opposite sense to those previously described in A. thaliana, revealing that common signaling components affect the physiology of closely related glycophyte and salt-tolerant plants differently.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

Monitoring Strategy for Eight Amphibian Species in French Guiana,South America

Although dramatic amphibian declines have been documented worldwide, only few of such events have been quantitatively documented for the tropical forests of South America. This is due partly to the fact that tropical amphibians are patchily distributed and difficult to detect. We tested three methods often used to monitor population trends in amphibian species in a remote lowland tropical forest of French Guiana. These methods are capture-mark-recapture (CMR), estimation of the number of calling males with repeated counts data and distance sampling, and rates of occupancy inferred by presence/absence data. We monitored eight diurnal, terrestrial amphibian species including five Dendrobatidae and three Bufonidae. We found that CMR, the most precise way of estimating population size, can be used only with two species in high density patches where the recapture rate is high enough. Only for one of the species (Dendrobates tinctorius), a low coefficient of variation (CV = 0.19) can be achieved with 15 to 20 capture events. For dendrobatid species with day-calling males, audio surveys yield a better probability of detection with only 8 audio surveys needed; quantitative estimates can be achieved by computing the number of calling males inferred from audio counts or distance sampling analysis. We therefore suggest that an efficient monitoring protocol for Neotropical amphibian species should include a combination of sighting and audio techniques, and we discuss the need of implementing a large-scale monitoring in order to provide a baseline for comparison with future changes.     

Possible Links Between Stress Defense and the Tricarboxylic Acid (TCA) Cycle in Francisella Pathogenesis

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. In vivo, this facultative intracellular bacterium survives and replicates mainly in the cytoplasm of infected cells. We have recently identified a genetic locus, designated moxR that is important for stress resistance and intramacrophage survival of F. tularensis. In the present work, we used tandem affinity purification coupled to mass spectrometry to identify in vivo interacting partners of three proteins encoded by this locus: the MoxR-like ATPase (FTL_0200), and two proteins containing motifs predicted to be involved in protein–protein interactions, bearing von Willebrand A (FTL_0201) and tetratricopeptide (FTL_0205) motifs. The three proteins were designated here for simplification, MoxR, VWA1, and TPR1, respectively. MoxR interacted with 31 proteins, including various enzymes. VWA1 interacted with fewer proteins, but these included the E2 component of 2-oxoglutarate dehydrogenase and TPR1. The protein TPR1 interacted with one hundred proteins, including the E1 and E2 subunits of both oxoglutarate and pyruvate dehydrogenase enzyme complexes, and their common E3 subunit. Remarkably, chromosomal deletion of either moxR or tpr1 impaired pyruvate dehydrogenase and oxoglutarate dehydrogenase activities, supporting the hypothesis of a functional role for the interaction of MoxR and TPR1 with these complexes. Altogether, this work highlights possible links between stress resistance and metabolism in F. tularensis virulence.Francisella tularensis is responsible for the disease tularamia in a large number of animal species. This highly infectious bacterial pathogen can be transmitted to humans in numerous ways (1, 2, 3), including direct contact with sick animals, inhalation, ingestion of contaminated water or food, or by bites from ticks, mosquitoes, or flies. Four different subspecies (subsp.) of F. tularensis that differ in virulence and geographic distribution exist, designated subsp. tularensis (type A), subsp. holarctica (type B), subsp. Novicida, and subsp. mediasiatica, respectively. F. tularensis subsp. tularensis is the most virulent subspecies causing a severe disease in humans, whereas F. tularensis subsp. holarctica causes a similar disease but of less severity (4). Because of its high infectivity and lethality, F. tularensis is considered a potential bioterrorism agent (5).F. tularensis is able to survive and to replicate in the cytoplasm of a variety of infected cells, including macrophages. To resist this stressful environment, the bacterium must have developed stress resistance mechanisms, most of which are not yet well characterized. We recently reported the identification of a novel genetic locus that is important for stress resistance and intracellular survival of F. tularensis (6). This locus was designated moxR because the first gene FTL_0200, encodes a protein belonging to the AAA+ ATPase of the MoxR family ((7) and references therein). The data obtained in that first study had led us to suggest that the F. tularensis MoxR-like protein might constitute, in combination with other proteins of the locus, a chaperone complex contributing to F. tularensis pathogenesis.To further validate this hypothesis and expand our initial observations, we here decided to perform tandem affinity purification (TAP),¹ using a dual affinity tag approach coupled to mass spectroscopy analyses (8), to identify proteins interacting in vivo with three proteins encoded by the proximal portion of the moxR locus. For this, we chose as baits: the MoxR-like protein (FTL_0200) and two proteins bearing distinct motifs possibly involved in protein–protein interactions, FTL_0201 (Von Willebrand Factor Type A domain, or VWA) and FTL_0205 (tetratrichopeptide repeat or TPR). The three proteins were designated here for simplification, MoxR, VWA1, and TPR1; and the corresponding genes moxR, vwa1, and tpr1, respectively.VWA domains are present in all three kingdoms of life. They consist of a β-sheet sandwiched by multiple α helices. Frequently, VWA domain-containing proteins function in multiprotein complexes (9). TPR typically contain 34 amino acids. Many three-dimensional structures of TPR domains have been solved, revealing amphipathic helical structures (10). TPR-containing proteins are also found in all kingdoms of life. They can be involved in a variety of functions, and generally mediate protein–protein interactions. In the past few years, several TPR-related proteins have been shown to be involved in virulence mechanisms in pathogenic bacteria ((11) and references therein).Our proteomic approach allowed us to identify a series of protein interactants for each of the three moxR-encoded proteins. Remarkably, the protein TPR1 interacted with all the subunits of the pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (OGDH) complexes. Furthermore, inactivation of tpr1 also severely impaired the activities of these two enzymes. Inactivation of tpr1 affected bacterial resistance to several stresses (and in particular oxidative stress), intramacrophagic bacterial multiplication and bacterial virulence in the mouse model. Functional implications and possible relationship between bacterial metabolism, stress defense, and bacterial virulence are discussed.     

Curcuma longa Extract Associated with White Pepper Lessens High Fat Diet-Induced Inflammation in Subcutaneous Adipose Tissue

Background

Supra-nutritional doses of curcumin, derived from the spice Curcuma longa, have been proposed as a potential treatment of inflammation and metabolic disorders related to obesity. The aim of the present study was to test whether Curcuma longa extract rich in curcumin and associated with white pepper (Curcuma-P®), at doses compatible with human use, could modulate systemic inflammation in diet-induced obese mice. We questioned the potential relevance of changes in adiposity and gut microbiota in the effect of Curcuma-P® in obesity.

Methodology/Principal Findings

Mice were fed either a control diet (CT), a high fat (HF) diet or a HF diet containing Curcuma longa extract (0.1 % of curcumin in the HF diet) associated with white pepper (0.01 %) for four weeks. Curcumin has been usually combined with white pepper, which contain piperine, in order to improve its bioavailability. This combination did not significantly modify body weight gain, glycemia, insulinemia, serum lipids and intestinal inflammatory markers. Tetrahydrocurcumin, but not curcumin accumulated in the subcutaneous adipose tissue. Importantly, the co-supplementation in curcuma extract and white pepper decreased HF-induced pro-inflammatory cytokines expression in the subcutaneous adipose tissue, an effect independent of adiposity, immune cells recruitment, angiogenesis, or modulation of gut bacteria controlling inflammation.

Conclusions/Significance

These findings support that nutritional doses of Curcuma longa, associated with white pepper, is able to decrease inflammatory cytokines expression in the adipose tissue and this effect could be rather linked to a direct effect of bioactive metabolites reaching the adipose tissue, than from changes in the gut microbiota composition.     

A cycle ergometer mounted on a standard force platform for three-dimensional pedal forces measurement during cycling

This report describes a new method allowing to measure the three-dimensional forces applied on right and left pedals during cycling. This method is based on a cycle ergometer mounted on a force platform. By recording the forces applied on the force platform and applying the fundamental mechanical equations, it was possible to calculate the instantaneous three-dimensional forces applied on pedals. It was validated by static and dynamic tests. The accuracy of the present system was -7.61 N, -3.37 N and -2.81 N, respectively, for the vertical, the horizontal and the lateral direction when applying a mono-directional force and -4.52 N when applying combined forces. In pedaling condition, the orientation and magnitude of the pedal forces were comparable to the literature. Moreover, this method did not modify the mechanical properties of the pedals and offered the possibility for pedal force measurement with materials often accessible in laboratories. Measurements obtained showed that this method has an interesting potential for biomechanical analyses in cycling.