Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.     

Characterization of postdive recovery using sound recordings and its relationship to dive duration,exertion, and foraging effort of southern elephant seals (Mirounga leonina)

It is notoriously difficult to measure physiological parameters in cryptic free‐ranging marine mammals. However, it is critical to understand how marine mammals manage their energy expenditure and their diving behavior in environments where the predation risks are low and where survival is mainly linked to capacities to maintain physiological homeostasis and energy budget balance. Elephant seals are top marine predators that dive deeply and continuously when at sea. Using acoustic recorders deployed on two postbreeding southern elephant seals (SES) females, we developed methods to automatically estimate breathing frequency at the surface. Using this method, we found that seals took successive identical breaths at high frequency (0.29 Hz) when recovering at the surface and that breath count was strongly related to postdive surfacing time. In addition, dive depth was the main factor explaining surfacing time through the effects of dive duration and total underwater swimming effort exerted. Finally, we found that recovery does not only occur over one dive timescale, but over a multidive time scale for one individual. The way these predators manage their recovery will determine how they respond to the change in oceanic water column structure in the future.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

Plant resistance to phytopathogenic microorganisms mainly relies on the activation of an innate immune response usually launched after recognition by the plant cells of microbe-associated molecular patterns. The plant hormones, salicylic acid (SA), jasmonic acid, and ethylene have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids (RLs) are glycolipids produced by bacteria and are involved in surface motility and biofilm development. Here we report that RLs trigger an immune response in Arabidopsis (Arabidopsis thaliana) characterized by signaling molecules accumulation and defense gene activation. This immune response participates to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora arabidopsidis, and the necrotrophic fungus Botrytis cinerea. We show that RL-mediated resistance involves different signaling pathways that depend on the type of pathogen. Ethylene is involved in RL-induced resistance to H. arabidopsidis and to P. syringae pv tomato whereas jasmonic acid is essential for the resistance to B. cinerea. SA participates to the restriction of all pathogens. We also show evidence that SA-dependent plant defenses are potentiated by RLs following challenge by B. cinerea or P. syringae pv tomato. These results highlight a central role for SA in RL-mediated resistance. In addition to the activation of plant defense responses, antimicrobial properties of RLs are thought to participate in the protection against the fungus and the oomycete. Our data highlight the intricate mechanisms involved in plant protection triggered by a new type of molecule that can be perceived by plant cells and that can also act directly onto pathogens.In their environment, plants are challenged by potentially pathogenic microorganisms. In response, they express a set of defense mechanisms including preformed structural and chemical barriers, as well as an innate immune response quickly activated after microorganism perception (Boller and Felix, 2009). Plant innate immunity is triggered after recognition by pattern recognition receptors of conserved pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively) or by plant endogenous molecules released by pathogen invasion and called danger-associated molecular patterns (Boller and Felix, 2009; Dodds and Rathjen, 2010). This first step of recognition leads to the activation of MAMP-triggered immunity (MTI). Successful pathogens can secrete effectors that interfere or suppress MTI, resulting in effector-triggered susceptibility. A second level of perception involves the direct or indirect recognition by specific receptors of pathogen effectors leading to effector-triggered immunity (ETI; Boller and Felix, 2009; Dodds and Rathjen, 2010). Whereas MTI and ETI are thought to involve common signaling network, ETI is usually quantitatively stronger than MTI and associated with more sustained and robust immune responses (Katagiri and Tsuda, 2010; Tsuda and Katagiri, 2010).The plant hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) have emerged as key players in the signaling networks involved in MTI and ETI (Robert-Seilaniantz et al., 2007; Tsuda et al., 2009; Katagiri and Tsuda, 2010; Mersmann et al., 2010; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate spectrum of responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). It is assumed that JA and ET signaling pathways are important for resistance to necrotrophic fungi including Botrytis cinerea and Alternaria brassicicola (Thomma et al., 2001; Ferrari et al., 2003; Glazebrook, 2005). Infection of Arabidopsis (Arabidopsis thaliana) with B. cinerea causes the induction of the JA/ET responsive gene PLANT DEFENSIN1.2 (PDF1.2; Penninckx et al., 1996; Zimmerli et al., 2001). Induction of PDF1.2 by B. cinerea is blocked in ethylene-insensitive2 (ein2) and coronatine-insensitive1 (coi1) mutants that are respectively defective in ET and JA signal transduction pathways. Moreover, ein2 and coi1 plants are highly susceptible to B. cinerea infection (Thomma et al., 1998; Thomma et al., 1999). JA/ET-dependent responses do not seem to be usually induced during resistance to biotrophs, but they can be effective if they are stimulated prior to pathogen challenge (Glazebrook, 2005). Plants impaired in SA signaling are highly susceptible to biotrophic and hemibiotrophic pathogens. Following pathogen infection, SA hydroxylase (NahG), enhanced disease susceptibility5 (eds5), or SA induction-deficient2 (sid2) plants are unable to accumulate high SA levels and they display heightened susceptibility to Pseudomonas syringae pv tomato (Pst), Hyaloperonospora arabidopsidis, or Erysiphe orontii (Delaney et al., 1994; Lawton et al., 1995; Wildermuth et al., 2001; Nawrath et al., 2002; Vlot et al., 2009). Mutants that are insensitive to SA, such as nonexpressor of PATHOGENESIS-RELATED (PR) genes1 (npr1), have enhanced susceptibility to these pathogens (Cao et al., 1994; Glazebrook et al., 1996; Shah et al., 1997; Dong, 2004). According to some reports, plant defense against necrotrophs also involves SA. Arabidopsis plants expressing the nahG gene and infected with B. cinerea show larger lesions compared with wild-type plants (Govrin and Levine, 2002). In tobacco (Nicotiana tabacum), acidic isoforms of PR3 and PR5 gene that are specifically induced by SA (Ménard et al., 2004) are up-regulated after challenge by B. cinerea (El Oirdi et al., 2010). Resistance to some necrotrophs like Fusarium graminearum involves both SA and JA signaling pathways (Makandar et al., 2010). It is assumed that SA and JA signaling can be antagonistic (Bostock, 2005; Koornneef and Pieterse, 2008; Pieterse et al., 2009; Thaler et al., 2012). In Arabidopsis, SA inhibits JA-dependent resistance against A. brassicicola or B. cinerea (Spoel et al., 2007; Koornneef et al., 2008). Recent studies demonstrated that ET modulates the NPR1-mediated antagonism between SA and JA (Leon-Reyes et al., 2009; Leon-Reyes et al., 2010a) and suppression by SA of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway (Leon-Reyes et al., 2010b). Synergistic effects of SA- and JA-dependent signaling are also well documented (Schenk et al., 2000; van Wees et al., 2000; Mur et al., 2006) and induction of some defense responses after pathogen challenge requires intact JA, ET, and SA signaling pathways (Campbell et al., 2003).Isolated MAMPs trigger defense responses that also require the activation of SA, JA, and ET signaling pathways (Tsuda et al., 2009; Katagiri and Tsuda, 2010). For instance, treatment with the flagellin peptide flg22 induces many SA-related genes including SID2, EDS5, NPR1, and PR1 (Ferrari et al., 2007; Denoux et al., 2008), causes SA accumulation (Tsuda et al., 2008; Wang et al., 2009), and activates ET signaling (Bethke et al., 2009; Mersmann et al., 2010). Local application of lipopolysaccharides elevates the level of SA (Mishina and Zeier, 2007). The oomycete Pep13 peptide induces defense responses in potato (Solanum tuberosum) that require both SA and JA (Halim et al., 2009). Although signaling networks induced by isolated MAMPs are well documented, the contribution of SA, JA, and ET in MAMP- or PAMP-induced resistance to biotrophs and necrotrophs is poorly understood.Rhamnolipids (RLs) are glycolipids produced by various bacteria species including some Pseudomonas and Burkholderia species. They are essential for bacterial surface motility and biofilm development (Vatsa et al., 2010; Chrzanowski et al., 2012). RLs are potent stimulators of animal immunity (Vatsa et al., 2010). They have recently been shown to elicit plant defense responses and to induce resistance against B. cinerea in grapevine (Vitis vinifera; Varnier et al., 2009). They also participate to biocontrol activity of the plant beneficial bacteria Pseudomonas aeruginosa PNA1 against oomycetes (Perneel et al., 2008). However, the signaling pathways used by RLs to stimulate plant innate immunity are not known. To gain more insights into RL-induced MTI, we investigated RL-triggered defense responses and resistance to the necrotrophic fungus B. cinerea, the biotroph oomycete H. arabidopsidis, and the hemibiotroph bacterium Pst in Arabidopsis. Our results show that RLs trigger an innate immune response in Arabidopsis that protects the plant against these different lifestyle pathogens. We demonstrate that RL-mediated resistance involves separated signaling sectors that depend on the type of pathogen. In plants challenged by RLs, SA has a central role and participates to the restriction of the three pathogens. ET is fully involved in RL-induced resistance to the biotrophic oomycete and to the hemibiotrophic bacterium whereas JA is essential for the resistance to the necrotrophic fungus.     

Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.     

Sperm production and mating potential of males after a cold shock on pupae of the parasitoid wasp Dinarmus basalis (Hymenoptera: Pteromalidae)

For ectothermic species, temperature is a key environmental factor influencing several aspects of their physiology and ecology, acting particularly on reproduction. To measure the consequences of a severe thermal stress during development on male reproduction, a cold shock (1h at -18 degrees C) was tested on Dinarmus basalis pupae. D. basalis (Hymenoptera: Pteromalidae) is a parasitoid wasp in which sperm management in both male and female is of prime importance. After a cold shock, developmental success was reduced, with a quarter of cold-shocked males not emerging correctly. The stress effects were estimated at the level of sperm stock in seminal vesicles of males at different ages and on the ability of 2-day-old males to access females in single and multiple mating and in male-male competition. Cold-shocked males had a reduced sperm stock compared to control males and this difference persisted with age. The rate of sperm production was similar in both groups. The consequences of a cold shock on male reproductive ability were perceptible in multiple mating and male-male competition but not in single mating. Cold-shocked males were at a disadvantage, inseminating fewer females and copulating less frequently. Finally, male pupae of D. basalis were able to withstand severe temperature stresses and their reproductive functions were partially preserved.     

The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity.     

Mycobacterium tuberculosis Phylogeny Reconstruction Based on Combined Numerical Analysis with IS1081, IS6110, VNTR, and DR-Based Spoligotyping Suggests the Existence of Two New Phylogeographical Clades

This paper deals with phylogenetic relationships among a set of 90 clinical strains representative of the worldwide diversity of the Mycobacterium tuberculosis complex (Kremer et al. 1999) using eight independent genetic markers: IS6110, IS1081, the direct repeat (DR) locus, and five variable number of tandem DNA repeat loci (VNTR). In a preliminary experiment, phylogenetic trees based on single markers were constructed that led to the detection of some similarities between the VNTR-based and the spoligotyping-based phylogenetic trees. In the second step, a more global phenetic approach based on pairwise comparison of strains within each typing system was used, followed by calculations of mean genetic distances based on all the eight loci and the use of the neighbor-joining algorithm for tree reconstruction. This analysis confirmed our preliminary observations and suggested the existence of at least two new phylogeographical clades of M. tuberculosis, one defined as the ``East African–Indian family' (EA-I), which may find its origin on the African or Asian continents, and the other as the ``Latin American and Mediterranean' (LA-M) family. The existence of these two families was also validated by an independent phylogenetic analysis of spoligotyping on a larger set of shared types (n= 252) and further corroborated by VNTR and katG–gyrA results. The potential origin of these families of bacilli is discussed based on cattle domestication and human migration history. In conclusion, the information contained in insertion sequence and repetitive DNAs may serve as a model for the phylogenetic reconstruction of the M. tuberculosis complex.     

Unbalanced biparental care during colony foundation in two subterranean termites

Parental care is a major component of reproduction in social organisms, particularly during the foundation steps. Because investment into parental care is often costly, each parent is predicted to maximize its fitness by providing less care than its partner. However, this sexual conflict is expected to be low in species with lifelong monogamy, because the fitness of each parent is typically tied to the other's input. Somewhat surprisingly, the outcomes of this tug‐of‐war between maternal and paternal investments have received important attention in vertebrate species, but remain less known in invertebrates. In this study, we investigated how queens and kings share their investment into parental care and other social interactions during colony foundation in two termites with lifelong monogamy: the invasive species Reticulitermes flavipes and the native species R. grassei. Behaviors of royal pairs were recorded during six months using a non‐invasive approach. Our results showed that queens and kings exhibit unbalanced investment in terms of grooming, antennation, trophallaxis, and vibration behavior. Moreover, both parents show behavioral differences toward their partner or their descendants. Our results also revealed differences among species, with R. flavipes exhibiting shorter periods of grooming and antennation toward eggs or partners. They also did more stomodeal trophallaxis and less vibration behavior. Overall, this study emphasizes that despite lifelong monogamy, the two parents are not equally involved in the measured forms of parental care and suggests that kings might be specialized in other tasks. It also indicates that males could play a central, yet poorly studied role in the evolution and maintenance of the eusocial organization.     

Soil–plant–atmosphere conditions regulating convective cloud formation above southeastern US pine plantations

Loblolly pine trees (Pinus taeda L.) occupy more than 20% of the forested area in the southern United States, represent more than 50% of the standing pine volume in this region, and remove from the atmosphere about 500 g C m per year through net ecosystem exchange. Hence, their significance as a major regional carbon sink can hardly be disputed. What is disputed is whether the proliferation of young plantations replacing old forest in the southern United States will alter key aspects of the hydrologic cycle, including convective rainfall, which is the focus of the present work. Ecosystem fluxes of sensible () and latent heat (LE) and large‐scale, slowly evolving free atmospheric temperature and water vapor content are known to be first‐order controls on the formation of convective clouds in the atmospheric boundary layer. These controlling processes are here described by a zero‐order analytical model aimed at assessing how plantations of different ages may regulate the persistence and transition of the atmospheric system between cloudy and cloudless conditions. Using the analytical model together with field observations, the roles of ecosystem and LE on convective cloud formation are explored relative to the entrainment of heat and moisture from the free atmosphere. Our results demonstrate that cloudy–cloudless regimes at the land surface are regulated by a nonlinear relation between the Bowen ratio and root‐zone soil water content, suggesting that young/mature pines ecosystems have the ability to recirculate available water (through rainfall predisposition mechanisms). Such nonlinearity was not detected in a much older pine stand, suggesting a higher tolerance to drought but a limited control on boundary layer dynamics. These results enable the generation of hypotheses about the impacts on convective cloud formation driven by afforestation/deforestation and groundwater depletion projected to increase following increased human population in the southeastern United States.