Enrichment of phosphoproteins and phosphopeptide derivatization identify universal stress proteins in elicitor-treated Arabidopsis

Protein phosphorylation is a key biological process that regulates reactions involved in plant-microbe interactions. The phosphorylated form of a protein often represents only a small fraction of the total population and can be problematic to analyze in a mass spectrometer. We demonstrate how a titanium dioxide (TiO(2)) resin can be employed for the enrichment of phosphoproteins, as well as a method to derivatize TiO(2)-purified phosphopeptides to facilitate determination of the exact site of phosphorylation. The use of these methods was exemplified by the identification of two plant proteins that were shown to be phosphorylated after the elicitation of Arabidopsis cells with Phytophthora infestans zoospores and xylanase. Both of the proteins that were identified, At5g54430.1 and At4g27320.1, were found to contain a universal stress protein domain with conserved residues for ATP binding.     

Determination of the Diversity of Rhodopirellula Isolates from European Seas by Multilocus Sequence Analysis

In the biogeography of microorganisms, the habitat size of an attached-living bacterium has never been investigated. We approached this theme with a multilocus sequence analysis (MLSA) study of new strains of Rhodopirellula sp., an attached-living planctomycete. The development of an MLSA for Rhodopirellula baltica enabled the characterization of the genetic diversity at the species level, beyond the resolution of the 16S rRNA gene. The alleles of the nine housekeeping genes acsA, guaA, trpE, purH, glpF, fumC, icd, glyA, and mdh indicated the presence of 13 genetically defined operational taxonomic units (OTUs) in our culture collection. The MLSA-based OTUs coincided with the taxonomic units defined by DNA-DNA hybridization experiments. BOX-PCR supported the MLSA-based differentiation of two closely related OTUs. This study established a taxon-area relationship of cultivable Rhodopirellula species. In European seas, three closely related species covered the Baltic Sea and the eastern North Sea, the North Atlantic region, and the southern North Sea to the Mediterranean. The last had regional genotypes, as revealed by BOX-PCR. This suggests a limited habitat size of attached-living Rhodopirellula species.The biogeography of microorganisms describes the habitat size of the species and the distribution of microorganisms on Earth. The experimental approaches depend on the focus of the studies. Habitats are often analyzed by environmental microbiologists with genetic-fingerprinting techniques, with up to 200 bands or fragments representing the whole community. Although the taxonomic resolution of these operational taxonomic units (OTUs) is limited, the studies revealed a community biogeography (22). Medical microbiologists analyze the alleles of housekeeping genes of microorganisms to gain insight into the epidemiology of pathogens, the population biogeography (2). This strain-specific, fine-scale taxonomic resolution within a species is well suited to observance of recent dispersal events. At the species level, multilocus sequence typing (MLST) and analysis (MLSA), which were developed for intraspecies and intragenus specific studies, respectively, consist of the sequences of several (at least seven) housekeeping gene fragments concatenated to an approximately 5-kilobase alignment (17). Recent MLSA studies revealed its applicability to marine isolates and the analysis of biogeographic patterns: Alteromonas macleodii isolates could be grouped in an epipelagic and an abyssal clade (6), and strains of Pseudomonas aeruginosa were genetically well separated into groups of coastal and oceanic origin (8). However, for Salinibacter ruber strains, biogeographical distinctness was not resolved in an MLSA study but showed allopatry in a metabolic analysis (31). Several studies used MLSA together with DNA-DNA hybridization (DDH) for the delineation of new species, e.g., for Vibrio and Ensifer spp. (20, 36).In the biogeography of microorganisms, the experimental proof of a local genetic evolution was first revealed at sample sites that were physically separated by over 18,000 km (39). Large populations and the small size of microbes have been considered as facilitators for dispersal over long distances, eventually establishing cosmopolitan microbial populations. On the other hand, the smallest spatial scale of a microbial species in an open system has not been investigated. Attached-living bacteria disperse only during a distinct, short time span in their lives. This limitation of the dispersal time stimulated this study of the biogeography of Rhodopirellula baltica in European seas.R. baltica is a planctomycete with typical morphological features. The peptidoglycanless bacteria have an intracellular compartmentation: the riboplasm with the nucleoid is separated by a membrane from the surrounding paryphoplasm. Cells attach with a holdfast substance to surfaces or, in culture, to themselves, forming typical rosettes. Proliferation occurs by budding, and offspring cells live free in the water column: they are motile with a flagellum until they settle on the sediment (4).Seventy recently isolated strains affiliated according to the 16S rRNA gene analysis with R. baltica SH1^T as the closest validly described species (40). The 16S rRNA gene sequences do not offer sufficient information at the species level. A dissimilarity of the 16S rRNA genes of more than 3%, recently reduced to 1.3% (34, 35), indicates that the strains under consideration belong to two species. These thresholds yielded in our strain collection, according to an ARB-based calculation, five or eight operational taxonomic units besides the species R. baltica (40). For strains with highly identical sequences, whole-genome DDH experiments have to be performed to identify the affiliation to established species. Recently, multilocus sequence analyses have emerged as a possible alternative method. Our strain collection comprised many strains with a 16S rRNA gene sequence very closely related to that of R. baltica SH1^T. To gain insight into the genetic identity of the isolates on the species level and the habitat sizes of the species, we developed a multilocus sequence analysis and applied it to the strain collection. The MLSA results were calibrated with a DDH study. The closely related strains were additionally characterized by BOX-PCR, a fingerprinting method (15). Transmission electron microscopy (EM) was performed on some isolates to support the identification as Planctomycetes and to visualize morphological differences between strains.     

Run-Off Replication of Host-Adaptability Genes Is Associated with Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella grahamii

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella.     

Notch-signalling is required for head regeneration and tentacle patterning in Hydra

Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals.     

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset.

Objective

To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism.

Methods

25-OH vitamin D and 1,25-(OH)₂ vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia.

Results

In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)₂ vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001).

Summary

Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.     

Furan in heat-treated foods: Formation, exposure, toxicity, and aspects of risk assessment

Furan is formed in a variety of heat-treated foods through thermal degradation of natural food constituents. Relatively high levels of furan contamination are found in ground roasted coffee, instant coffee, and processed baby foods. European exposure estimates suggest that mean dietary exposure to furan may be as high as 1.23 and 1.01 μg/kg bw/day for adults and 3- to 12-month-old infants, respectively. Furan is a potent hepatotoxin and hepatocarcinogen in rodents, causing hepatocellular adenomas and carcinomas in rats and mice, and high incidences of cholangiocarcinomas in rats at doses ≥2 mg/kg bw. There is therefore a relatively low margin of exposure between estimated human exposure and doses that cause a high tumor incidence in rodents. Since a genotoxic mode of action cannot be excluded for furan-induced tumor formation, the present exposures may indicate a risk to human health and need for mitigation. This review summarizes the current knowledge on mechanisms of furan formation in food, human dietary exposure to furan, and furan toxicity, and highlights the need to establish the risk resulting from the genotoxic and carcinogenic properties of furan at doses lower than 2 mg/kg bw.     

Prehospital Thrombolysis: A Manual from Berlin

In acute ischemic stroke, time from symptom onset to intervention is a decisive prognostic factor. In order to reduce this time, prehospital thrombolysis at the emergency site would be preferable. However, apart from neurological expertise and laboratory investigations a computed tomography (CT) scan is necessary to exclude hemorrhagic stroke prior to thrombolysis. Therefore, a specialized ambulance equipped with a CT scanner and point-of-care laboratory was designed and constructed. Further, a new stroke identifying interview algorithm was developed and implemented in the Berlin emergency medical services. Since February 2011 the identification of suspected stroke in the dispatch center of the Berlin Fire Brigade prompts the deployment of this ambulance, a stroke emergency mobile (STEMO). On arrival, a neurologist, experienced in stroke care and with additional training in emergency medicine, takes a neurological examination. If stroke is suspected a CT scan excludes intracranial hemorrhage. The CT-scans are telemetrically transmitted to the neuroradiologist on-call. If coagulation status of the patient is normal and patient''s medical history reveals no contraindication, prehospital thrombolysis is applied according to current guidelines (intravenous recombinant tissue plasminogen activator, iv rtPA, alteplase, Actilyse).Thereafter patients are transported to the nearest hospital with a certified stroke unit for further treatment and assessment of strokeaetiology. After a pilot-phase, weeks were randomized into blocks either with or without STEMO care. Primary end-point of this study is time from alarm to the initiation of thrombolysis. We hypothesized that alarm-to-treatment time can be reduced by at least 20 min compared to regular care.     

Immediate and Heterogeneous Response of the LiaFSR Two-Component System of Bacillus subtilis to the Peptide Antibiotic Bacitracin

Background

Two-component signal transduction systems are one means of bacteria to respond to external stimuli. The LiaFSR two-component system of Bacillus subtilis consists of a regular two-component system LiaRS comprising the core Histidine Kinase (HK) LiaS and the Response Regulator (RR) LiaR and additionally the accessory protein LiaF, which acts as a negative regulator of LiaRS-dependent signal transduction. The complete LiaFSR system was shown to respond to various peptide antibiotics interfering with cell wall biosynthesis, including bacitracin.

Methodology and Principal Findings

Here we study the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. Using quantitative fluorescence microscopy, we performed a whole population study analyzed on the single cell level. We investigated switching from the non-induced ‘OFF’ state into the bacitracin-induced ‘ON’ state by monitoring gene expression of a fluorescent reporter from the RR-regulated liaI promoter. We found that switching into the ‘ON’ state occurred within less than 20 min in a well-defined switching window, independent of the bacitracin concentration. The switching rate and the basal expression rate decreased at low bacitracin concentrations, establishing clear heterogeneity 60 min after bacitracin induction. Finally, we performed time-lapse microscopy of single cells confirming the quantitative response as obtained in the whole population analysis for high bacitracin concentrations.

Conclusion

The LiaFSR system exhibits an immediate, heterogeneous and graded response to the inducer bacitracin in the exponential growth phase.