Heparin/heparan sulfate controls fibrillin-1, -2 and -3 self-interactions in microfibril assembly

Fibrillins form multifunctional microfibrils in most connective tissues. Deficiencies in fibrillin assembly can result in fibrillinopathies, such as Marfan syndrome. We demonstrate the presence of heparin/heparan sulfate binding sites in fibrillin-2 and -3. Multimerization of all three fibrillins drastically increased the apparent affinity of their interaction with heparin/heparan sulfate. Surprisingly, contrary to other reports heparin/heparan sulfate strongly inhibited homo- and heterotypic N-to-C-terminal fibrillin interactions. These data suggest that heparin/heparan sulfate controls the formation of microfibrils at the bead interaction stage.     

Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-based Approach

Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G⁺, miR-152^low cells, and their miR-152-overexpressing (miR^high) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein β/α/YWHAB (14-3-3β) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3β expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3β expression in miR-152^high cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients'' outcome, a link between 14-3-3β and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3β and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity.     

Salvia suspension cultures as production systems for oleanolic and ursolic acid

Oleanolic acid (OA) and ursolic acid (UA) are triterpenic acids with diverse biological activities that are of interest to the pharmaceutical industry. To investigate the scope for producing these compound using cell suspension cultures of Salvia species, calli from Salvia officinalis, S. virgata and S. fruticosa were induced using several plant growth regulator combinations. Eleven lines were selected for suspension induction from a pool of calli. Six suspension cultures were established successfully and cultivated in the respiration activity monitoring system (RAMOS^®) to obtain online data on their growth kinetics and to establish appropriate sampling schedules for the determination of their OA and UA production. Based on their observed growth behaviour, OA and UA contents, and aggregation properties, one suspension culture from each studied Salvia species was selected for further optimisation. The µ_max values for these suspension cultures ranged from 0.20 to 0.37 day^?1, their OA and UA contents were greater than 1.3 and 1.2 mg g^?1, respectively, and they afforded maximum volumetric yields of 21.0 mg l^?1 for OA and 32.8 mg l^?1 for UA. These results will be useful in the development of a refined Salvia suspension-based process for OA and UA production.     

Human‐facilitated metapopulation dynamics in an emerging pest species,Cimex lectularius

The number and demographic history of colonists can have dramatic consequences for the way in which genetic diversity is distributed and maintained in a metapopulation. The bed bug (Cimex lectularius) is a re‐emerging pest species whose close association with humans has led to frequent local extinction and colonization, that is, to metapopulation dynamics. Pest control limits the lifespan of subpopulations, causing frequent local extinctions, and human‐facilitated dispersal allows the colonization of empty patches. Founder events often result in drastic reductions in diversity and an increased influence of genetic drift. Coupled with restricted migration, this can lead to rapid population differentiation. We therefore predicted strong population structuring. Here, using 21 newly characterized microsatellite markers and approximate Bayesian computation (ABC), we investigate simplified versions of two classical models of metapopulation dynamics, in a coalescent framework, to estimate the number and genetic composition of founders in the common bed bug. We found very limited diversity within infestations but high degrees of structuring across the city of London, with extreme levels of genetic differentiation between infestations (F_ST = 0.59). ABC results suggest a common origin of all founders of a given subpopulation and that the numbers of colonists were low, implying that even a single mated female is enough to found a new infestation successfully. These patterns of colonization are close to the predictions of the propagule pool model, where all founders originate from the same parental infestation. These results show that aspects of metapopulation dynamics can be captured in simple models and provide insights that are valuable for the future targeted control of bed bug infestations.     

Complete genome determination and analysis of Acholeplasma oculi strain 19L,highlighting the loss of basic genetic features in the Acholeplasmataceae

Background

Acholeplasma oculi belongs to the Acholeplasmataceae family, comprising the genera Acholeplasma and ‘Candidatus Phytoplasma’. Acholeplasmas are ubiquitous saprophytic bacteria. Several isolates are derived from plants or animals, whereas phytoplasmas are characterised as intracellular parasitic pathogens of plant phloem and depend on insect vectors for their spread. The complete genome sequences for eight strains of this family have been resolved so far, all of which were determined depending on clone-based sequencing.

Results

The A. oculi strain 19L chromosome was sequenced using two independent approaches. The first approach comprised sequencing by synthesis (Illumina) in combination with Sanger sequencing, while single molecule real time sequencing (PacBio) was used in the second. The genome was determined to be 1,587,120 bp in size. Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence. High-quality sequences were obtained by both strategies differing in six positions, which are interpreted as reliable variations present in the culture population. Our genome analysis revealed 1,471 protein-coding genes and highlighted the absence of the F₁F_O-type Na⁺ ATPase system and GroEL/ES chaperone. Comparison of the four available Acholeplasma sequences revealed a core-genome encoding 703 proteins and a pan-genome of 2,867 proteins.

Conclusions

The application of two state-of-the-art sequencing technologies highlights the potential of single molecule real time sequencing for complete genome determination. Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi. The loss of the F₁F_O-type Na⁺ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-931) contains supplementary material, which is available to authorized users.     

Role of alpha- and beta-adrenoreceptors in rat monocyte/macrophage function at rest and acute exercise

Previous studies from our laboratory have demonstrated that a single bout of moderate exercise stimulates macrophage function, increasing phagocytic capacity, and production of hydrogen peroxide and nitric oxide (NO˙) through nuclear factor kappa B activation. In this work, we investigated the role of α- and β-adrenoreceptors on the function of monocyte/macrophages during rest and exercise. Adult male Wistar rats were i.p. administered (100 μL/100 g) with specific adrenergic antagonists before an acute moderate exercise bout: prazosin (α₁-specific antagonist 2 mg/kg), propranolol (unspecific β_1/β₂ antagonist 10 mg/kg), double blockade (α₁ and β_1/β₂), or phosphate-buffered saline (control). Acute exercise consisted in a single swimming session of moderate intensity (5 % body weight overload on the chest) for 60 min. Control groups (rest) received the same antagonists and were killed 60 min after drug administration. Exercise increased phagocytic capacity (1.7-fold, p?<?0.05), NO˙ production (5.24 fold, p?<?0.001), and inducible nitric oxide synthase (NOS2) expression (by 58.1 %), thus suggesting macrophage activation. The β-adrenoreceptor blockade did not change this behavior. In resting animals, α₁ antagonist, as well as the double (α₁/β) blockade, however, further increased phagocytic capacity (by up to 261 %, p?<?0.001), NO˙ production (by up to 328 %, p?<?0.001), and the expressions of NOS2 (by 182 %, p?<?0.001) and HSP70 (by 42.5 %, p?<?0.01) suggesting a tonic inhibitory effect of α₁ stimulation on macrophage activation. In exercised animals, α₁-blockade showed similar enhancing effect on phagocytic indices and expressions of NOS and HSP70, particularly in double-blocked groups, although NO˙ production was found to be reduced in exercised animals submitted to both α- and β-blockade. Redox (glutathione) status and lipoperoxidation were evaluated in all test groups and approximately paralleled macrophage NO˙ production. We suggest the prevalence of a peripheral α₁-adrenoreceptor inhibitory tonus that limits macrophage responsiveness but operates differently after physical exercise.     

Blood cis-eQTL Analysis Fails to Identify Novel Association Signals among Sub-Threshold Candidates from Genome-Wide Association Studies in Restless Legs Syndrome

Restless legs syndrome (RLS) is a common neurologic disorder characterized by nightly dysesthesias affecting the legs primarily during periods of rest and relieved by movement. RLS is a complex genetic disease and susceptibility factors in six genomic regions have been identified by means of genome-wide association studies (GWAS). For some complex genetic traits, expression quantitative trait loci (eQTLs) are enriched among trait-associated single nucleotide polymorphisms (SNPs). With the aim of identifying new genetic susceptibility factors for RLS, we assessed the 332 best-associated SNPs from the genome-wide phase of the to date largest RLS GWAS for cis-eQTL effects in peripheral blood from individuals of European descent. In 740 individuals belonging to the KORA general population cohort, 52 cis-eQTLs with p_nominal<10⁻³ were identified, while in 976 individuals belonging to the SHIP-TREND general population study 53 cis-eQTLs with p_nominal<10⁻³ were present. 23 of these cis-eQTLs overlapped between the two cohorts. Subsequently, the twelve of the 23 cis-eQTL SNPs, which were not located at an already published RLS-associated locus, were tested for association in 2449 RLS cases and 1462 controls. The top SNP, located in the DET1 gene, was nominally significant (p<0.05) but did not withstand correction for multiple testing (p = 0.42). Although a similar approach has been used successfully with regard to other complex diseases, we were unable to identify new genetic susceptibility factor for RLS by adding this novel level of functional assessment to RLS GWAS data.     

Dietary Enterococcus faecium NCIMB 10415 and Zinc Oxide Stimulate Immune Reactions to Trivalent Influenza Vaccination in Pigs but Do Not Affect Virological Response upon Challenge Infection

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy-two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Zn^high; 50 ppm, Zn^low). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn^high and E. faecium groups gained weight after infection while those in the control group (Zn^low) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn^high+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Zn^high and E. faecium groups at single time points after infection compared to the Zn^low control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology.     

S-Nitroso-Proteome in Poplar Leaves in Response to Acute Ozone Stress

Protein S-nitrosylation, the covalent binding of nitric oxide (NO) to protein cysteine residues, is one of the main mechanisms of NO signaling in plant and animal cells. Using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome of the woody model plant Populus x canescens. Under normal conditions, constitutively S-nitrosylated proteins in poplar leaves and calli comprise all aspects of primary and secondary metabolism. Acute ozone fumigation was applied to elicit ROS-mediated changes of the S-nitroso-proteome. This treatment changed the total nitrite and nitrosothiol contents of poplar leaves and affected the homeostasis of 32 S-nitrosylated proteins. Multivariate data analysis revealed that ozone exposure negatively affected the S-nitrosylation status of leaf proteins: 23 proteins were de-nitrosylated and 9 proteins had increased S-nitrosylation content compared to the control. Phenylalanine ammonia-lyase 2 (log2[ozone/control] = −3.6) and caffeic acid O-methyltransferase (−3.4), key enzymes catalyzing important steps in the phenylpropanoid and subsequent lignin biosynthetic pathways, respectively, were de-nitrosylated upon ozone stress. Measuring the in vivo and in vitro phenylalanine ammonia-lyase activity indicated that the increase of the phenylalanine ammonia-lyase activity in response to acute ozone is partly regulated by de-nitrosylation, which might favor a higher metabolic flux through the phenylpropanoid pathway within minutes after ozone exposure.