Sustained neurochemical plasticity in central terminals of mouse DRG neurons following colitis

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.     

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.     

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.     

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.     

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.     

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.     

Sulfite inhibition of photochemical activity of intact pea leaves

Sulfite treatment of pea leaf disks in light caused a significant decrease in the relative quantum yield of photosynthetic oxygen evolution and energy storage (ES) as measured by photoacoustic (PA) spectroscopy. The inhibition was concentration dependent and was less in darkness than in light, indicating light-dependent inhibitory site(s) on the photosynthetic electron transport chain. Further, in darksulfite-treated leaves, the energy storage was more affected than the relative quantum yield of oxygen evolution, suggesting that photophosphorylation and/or cyclic electron transport around PS I are sites of sulfite action in darkness. The R_fd values, the ratio of fluorescence decrease (fd) to the steady-state fluorescence (fs), decreased significantly in leaves treated with sulfite in light but were not affected in dark-treated ones, confirming the photoacoustic observations. Similarly, the ratio of variable fluorescence (F_v) to maximum fluorescence (F_m), a measure of PS II photochemical efficiency, was affected by sulfite treatment in light and not changed by treatment in darkness. An attempt was made to explain the mechanism of sulfite action on photosynthetic electron transport in light and in darkness.Abbreviations A_PT amplitude of photothermal signal - A_ox amplitude of oxygen signal - ES energy storage - fd fluorescence decrease - fs steady-state fluorescence - F_m maximum fluorescence - F_v variable fluorescence - PA photoacoustic(s)     

Biochemical and proton NMR characterization of the isolated functional beta-subunit of coupling factor one from spinach chloroplasts

Beta subunits have been dissociated from CF1 of spinach chloroplasts, purified by HPLC and characterized by two-dimensional electrophoresis and fluorescence emission. The solutions of isolated beta subunits are able to hydrolyze MgATP; this ATPase activity is an intrinsic property of the beta molecule. From proton NMR at 300 and 500 MHz, it is shown that the preparations are fully reproducible and that beta subunits remain monomeric with 75% aliphatic protons associated with rigid parts of the molecule. The other 25% give rise to separate resonances and belong to mobile side-chains and/or to flexible regions. The measurement of the transverse relaxation times T2 has permitted a detailed characterization of the molecular dynamics of the isolated beta subunits.     

Self-specific MHC class II-restricted CD4-CD8- T cells that escape deletion and lack regulatory activity

In the presence of the I-Ealpha protein, transgenic (Tg) mice expressing the 1H3.1 alphabeta TCR that is specific for the Ealpha52-68:I-A(b) complex display drastic intrathymic deletion. Although peripheral T cells from these mice remained unresponsive to the Ealpha52-68:I-A(b) complex, they contained a subpopulation able to specifically react to this complex in the presence of exogenous IL-2, indicating that some 1H3.1 alphabeta TCR Tg T cells have escaped clonal deletion and efficiently populated the periphery. IL-2-dependent, Ealpha52-68:I-A(b) complex-responsive T cells were CD4-CD8- and expressed the 1H3.1 alphabeta TCR. Such T cells could develop intrathymically, did not show sign of regulatory/suppressor activity, displayed a typical naive phenotype, and seemed to persist in vivo over time. CD4-CD8- TCR Tg T cells were also detected when the surface density of the deleting ligand was increased on MHC class II+ cells. In addition, the development of CD4-CD8- 1H3.1 alphabeta TCR Tg T cells could be supported by I-A(b) molecules. These observations indicate that CD4 surface expression neither specifies, nor is required for, the thymic export of mature thymocytes expressing a MHC class II-restricted alphabeta TCR. The data also show that, although the avidity of the interaction involved in intrathymic deletion is significantly lower than that involved in mature T cell activation, its range can be large enough to be influenced by the presence or absence of coreceptors. Finally, the margin created by the absence of CD4 coreceptor was substantial because it could accommodate various amounts of the deleting ligand on thymic stromal cells.