Proteomic profiling of Rhizobium tropici PRF 81: Identification of conserved and specific responses to heat stress

ABSTRACT: BACKGROUND: Rhizobium tropici strain PRF 81 (= SEMIA 4080) has been used in commercial inoculants for application to common-bean crops in Brazil since 1998, due to its high efficiency in fixing nitrogen, competitiveness against indigenous rhizobial populations and capacity to adapt to stressful tropical conditions, representing a key alternative to application of N-fertilizers. The objective of our study was to obtain an overview of adaptive responses to heat stress of strain PRF 81, by analyzing differentially expressed proteins when the bacterium is grown at 28degreesC and 35degreesC. RESULTS: Two-dimensional gel electrophoresis (2DE) revealed up-regulation of fifty-nine spots that were identified by MALDI-TOF/TOF-TOF. Differentially expressed proteins were associated with the functional COG categories of metabolism, cellular processes and signaling, information storage and processing. Among the up-regulated proteins, we found some related to conserved heat responses, such as molecular chaperones DnaK and GroEL, and other related proteins, such as translation factors EF-Tu, EF-G, EF-Ts and IF2. Interestingly, several oxidative stress-responsive proteins were also up-regulated, and these results reveal the diversity of adaptation mechanisms presented by this thermotolerant strain, suggesting a cross-talk between heat and oxidative stresses. CONCLUSIONS: Our data provide valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81, and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species.     

Rapid de novo evolution of X chromosome dosage compensation in Silene latifolia, a plant with young sex chromosomes

Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ～10 million years ago and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems. Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin of sex chromosomes.     

Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.     

Vaccination of a melanoma patient with mature dendritic cells pulsed with MAGE-3 peptides triggers the activity of nonvaccine anti-tumor cells

We previously characterized the CTL response of a melanoma patient who experienced tumor regression following vaccination with an ALVAC virus coding for a MAGE-A3 Ag. Whereas anti-vaccine CTL were rare in the blood and inside metastases of this patient, anti-tumor CTL recognizing other tumor Ags, mainly MAGE-C2, were 100 times more frequent in the blood and considerably enriched in metastases following vaccination. In this study we report the analysis of the CTL response of a second melanoma patient who showed a mixed tumor response after vaccination with dendritic cells pulsed with two MAGE-A3 antigenic peptides presented, respectively, by HLA-A1 and HLA-DP4. Anti-MAGE-3.A1 CD8 and anti-MAGE-3.DP4 CD4 T cells became detectable in the blood after vaccination at a frequency of approximately 10(-5) among the CD8 or CD4 T cells, respectively, and they were slightly enriched in slowly progressing metastases. Additional anti-tumor CTL were present in the blood at a frequency of 2x10(-4) among the CD8 T cells and, among these, an anti-MAGE-C2 CTL clone was detected only following vaccination and was enriched by >1,000-fold in metastases relative to the blood. The striking similarity of these results with our previous observations further supports the hypothesis that the induction of a few anti-vaccine T cells may prime or restimulate additional anti-tumor T cell clones that are mainly responsible for the tumor regression.     

Dissolution of Dead Corals by Euendolithic Microorganisms Across the Northern Great Barrier Reef (Australia)

Spatial and temporal variabilities in species composition, abundance, distribution, and bioeroding activity of euendolithic microorganisms were investigated in experimental blocks of the massive coral Porites along an inshore–offshore transect across the northern Great Barrier Reef (Australia) over a 3-year period. Inshore reefs showed turbid and eutrophic waters, whereas the offshore reefs were characterized by oligotrophic waters. The euendolithic microorganisms and their ecological characteristics were studied using techniques of microscopy, petrographic sections, and image analysis. Results showed that euendolithic communities found in blocks of coral were mature. These communities were dominated by the chlorophyte Ostreobium quekettii, the cyanobacterium Plectonema terebrans, and fungi. O. quekettii was found to be the principal agent of microbioerosion, responsible for 70–90% of carbonate removal. In the offshore reefs, this oligophotic chlorophyte showed extensive systems of filaments that penetrated deep inside coral skeletons (up to 4.1 mm) eroding as much as 1 kg CaCO₃ eroded m⁻² year⁻¹. The percentage of colonization by euendolithic filaments at the surface of blocks did not vary significantly among sites, while their depths of penetration, especially that of O. quekettii (0.6–4.1 mm), increased significantly and gradually with the distance from the shore. Rates of microbioerosion (0.1–1.4 kg m⁻² after 1 year and 0.2–1.3 kg m⁻² after 3 years of exposure) showed a pattern similar to the one found for the depth of penetration of O. quekettii filaments. Accordingly, oligotrophic reefs had the highest rates of microbioerosion of up to 1.3 kg m⁻² year⁻¹, whereas the development of euendolithic communities in inshore reefs appeared to be limited by turbidity, high sedimentation rates, and low grazing pressure (rates <0.5 kg m⁻² after 3 years). Those results suggest that boring microorganisms, including O. quekettii, have a significant impact on the overall calcium carbonate budget of coral reef ecosystems, which varies according to environmental conditions.     

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.     

Isolation and characterization of polymorphic microsatellite loci for the horn fly, Haematobia irritans (L.) (Diptera: Muscidae)

The horn fly, Haematobia irritans (L.) (Diptera: Muscidae), is a cosmopolitan livestock pest that has caused a great negative impact on the animal production sector throughout the world. Here, we describe 10 polymorphic microsatellite loci isolated from H. irritans. The number of alleles found ranged from two to eight per locus and the expected heterozygosity from 0.1421 to 0.7702. These loci are potentially useful for the fine-scale genetic characterization of horn fly populations and provide fundamental information for pest management and planning of control programs.     

Pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to NAD

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFalpha levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.