Discovery of the Cyclic Lipopeptide Gacamide A by Genome Mining and Repair of the Defective GacA Regulator in Pseudomonas fluorescens Pf0-1.

Genome mining of the Gram-negative bacterium Pseudomonas fluorescens Pf0-1 showed that the strain possesses a silent NRPS-based biosynthetic gene cluster encoding a new lipopeptide; its activation required the repair of the global regulator system. In this paper, we describe the genomics-driven discovery and characterization of the associated secondary metabolite gacamide A, a lipodepsipeptide that forms a new family of Pseudomonas lipopeptides. The compound has a moderate, narrow-spectrum antibiotic activity and facilitates bacterial surface motility.

Differences in healthcare costs in youths with conduct disorders in rural vs. urban regions: an analysis of German health insurance data.

BACKGROUND: For children and adolescents with mental health problems, there is a lack of data as to whether the type of residential area (urban vs. rural) influences healthcare costs for affected individuals. The aim of this study was therefore to explore potential urban vs. rural healthcare cost differences in children and adolescents with conduct disorder (CD), one of the most frequent and cost-intensive child and adolescent psychiatric disorders. Additionally, we aimed to compare healthcare costs of youths with CD, and of youths without this diagnosis. METHODS: We analysed data from a German health insurance company, extracting all youths with a CD diagnosis in 2011 (CD group; N = 6337), and an age- and sex-matched group without this diagnosis (control group). For both groups, annual costs per person for outpatient and inpatient healthcare were aggregated, stratified by area of residence (urban vs. rural). RESULTS: While mean annual overall costs in the CD group did not differ significantly between urban and rural areas of residence (2785 EUR vs. 3557 EUR, p = 0.253), inpatient treatment costs were significantly higher in rural areas (2166 EUR (60.9% of overall costs) vs. 1199 EUR (43.1% of overall costs), p < 0.0005). For outpatient healthcare costs, the reverse effect was found, with significantly higher costs in individuals from urban areas of residence (901 EUR (32.3% of overall costs) vs. 581 EUR (16.3% of overall costs), p < 0.0005). In the control group, no significant rural vs. urban difference was found for either overall health costs, inpatient or outpatient costs. Mean overall costs in the CD group were four times higher than in the control group (3162 (±5934) EUR vs. 795 (±4425) EUR). CONCLUSIONS: This study is the first to demonstrate urban vs. rural differences in healthcare costs among youths with CD. The higher costs of inpatient treatment in rural regions may indicate a need for alternative forms of service provision and delivery in rural settings.

Insect Adhesion Secretions: Similarities and Dissimilarities in Hydrocarbon Profiles of Tarsi and Corresponding Tibiae.

Spatially controlled in vivo sampling by contact solid phase microextraction with a non-coated silica fiber combined with gas chromatography-mass spectrometry (GC-MS) was utilized for hydrocarbon profiling in tarsal adhesion secretions of four insect species (Nicrophorus vespilloides, Nicrophorus nepalensis, Sagra femorata, and Gromphadorhina portentosa) by using distinct adhesion systems, viz. hairy or smooth tarsi. For comparison, corresponding samples from tibiae, representing the general cuticular hydrocarbon profile, were analyzed to enable the statistical inference of active molecular adhesion principles in tarsal secretions possibly contributed by specific hydrocarbons. n-Alkanes, monomethyl and dimethyl alkanes, alkenes, alkadienes, and one aldehyde were detected. Multivariate statistical analysis (principal component and orthogonal partial least square discriminant analyses) gave insights into distinctive molecular features among the various insect species and between tarsus and tibia samples. In general, corresponding hydrocarbon profiles in tarsus and tibia samples largely resembled each other, both qualitatively and in relative abundances as well. However, several specific hydrocarbons showed significantly different relative abundances between corresponding tarsus and tibia samples, thus indicating that such differences of specific hydrocarbons in the complex mixtures might constitute a delicate mechanism for fine-tuning the reversible attachment performances in tarsal adhesive fluids that are composed of substances originating from the same pool as cuticular hydrocarbons. Caused by melting point depression, the multicomponent tarsal adhesion secretion, made up of straight chain alkanes, methyl alkanes, and alkenes will have a semi-solid, grease-like consistency, which might provide the basis for a good reversible attachment performance.

Methods for the comprehensive structural elucidation of constitution and stereochemistry of lipopeptides.

A panel of methods of general suitability for complete structural elucidation of the stereochemistry of cyclopeptides, depsipeptides and lipopeptides is presented and described in detail. The suitability of the proposed methods was exemplified on the lipopeptide poaeamide from Pseudomonas poae. Amino acid configurations have been assigned by direct LC enantiomer separation with Chiralpak ZWIX(+) and were confirmed by GC enantiomer separation on Chirasil L-Val. 3-Hydroxydecanoic acid absolute configuration was analyzed on Chiralpak ZWIX(+) and confirmed by injection on ZWIX(-) which showed opposite elution order. Plenty of d-amino acids have been found in this lipopeptide. It contained in total 5 Leu residues of which one had d-configuration. The position of the d-Leu in the peptide sequence was determined by pepsin and chemical digestions in combination with isolation of diagnostic peptide-fragments and subsequent identification of absolute configurations of the Leu residues. This allowed pinpointing the position of the d-amino acid. The complementarity of the peptide retention profiles on Chiralpak ZWIX column as compared to both RPLC and HILIC suggests its great utility as an alternative peptide separation tool.

Contact solid-phase microextraction with uncoated glass and polydimethylsiloxane-coated fibers versus solvent sampling for the determination of hydrocarbons in adhesion secretions of Madagascar hissing cockroaches Gromphadorrhina portentosa (Blattodea) by gas chromatography-mass spectrometry.

Molecular profiles of adhesion secretions of Gromphadorrhina portentosa (Madagascar hissing cockroach, Blattodea) were investigated by gas chromatography mass spectrometry with particular focus on a comprehensive analysis of linear and branched hydrocarbons. For this purpose, secretions from the tarsi (feet), possibly contributing to adhesion on smooth surfaces, and control samples taken from the tibiae (lower legs), which contain general cuticular hydrocarbons that are supposed to be not involved in the biological adhesion function, were analyzed and their molecular fingerprints compared. A major analytical difficulty in such a study constitutes the representative, spatially controlled, precise and reproducible sampling from a living insect as well as the minute quantities of insect secretions on both tarsi and tibiae. Thus, three different in vivo sampling methods were compared in terms of sampling reproducibility and extraction efficiency by replicate measurement of samples from tarsi and tibiae. While contact solid-phase microextraction (SPME) with a polydimethylsiloxane (PDMS) fiber showed higher peak intensities, a self-made uncoated glass fiber had the best repeatability in contact-SPME sampling. Chromatographic profiles of these two contact-SPME sampling methods were statistically not significantly different. Inter-individual variances were larger than potentially existing minor differences in molecular patterns of distinct sampling methods. Sampling by solvent extraction was time consuming, showed lower sensitivities and was less reproducible. In general, sampling by contact-SPME with a cheap glass fiber turned out to be a viable alternative to PDMS-SPME sampling. Hydrocarbon patterns of the tarsal adhesion secretions were qualitatively similar to those of epicuticular hydrocarbon profiles of the tibiae. However, hydrocarbons were in general less abundant in tarsal secretions than secretions from tibiae.

The Novel Lipopeptide Poaeamide of the Endophyte Pseudomonas poae RE*1-1-14 Is Involved in Pathogen Suppression and Root Colonization.

Endophytic Pseudomonas poae strain RE*1-1-14 was originally isolated from internal root tissue of sugar beet plants and shown to suppress growth of the fungal pathogen Rhizoctonia solani both in vitro and in the field. To identify genes involved in its biocontrol activity, RE*1-1-14 random mutagenesis and sequencing led to the identification of a nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode a lipopeptide (LP) with a 10-amino-acid peptide moiety. The two unlinked gene clusters consisted of three NRPS genes, designated poaA (cluster 1) and poaB and poaC (cluster 2), spanning approximately 33.7 kb. In silico analysis followed by chemical analyses revealed that the encoded LP, designated poaeamide, is a structurally new member of the orfamide family. Poaeamide inhibited mycelial growth of R. solani and different oomycetes, including Phytophthora capsici, P. infestans, and Pythium ultimum. The novel LP was shown to be essential for swarming motility of strain RE*1-1-14 and had an impact on root colonization of sugar beet seedlings The poaeamide-deficient mutant colonized the rhizosphere and upper plant cortex at higher densities and with more scattered colonization patterns than the wild type. Collectively, these results indicate that Pseudomonas poae RE*1-1-14 produces a structurally new LP that is relevant for its antagonistic activity against soilborne plant pathogens and for colonization of sugar beet roots.

New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential.

Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme required for hydroxylation of the aminobutyl side chain of deoxyhypusine in eukaryotic translation initiation factor 5A (eIF-5A), the second step in hypusine biosynthesis. DOHH has been recently identified in P. falciparum and P. vivax. Both enzymes have very peculiar features including E-Z type HEAT-like repeats and a diiron centre in their active site. Both proteins share only 26 % amino acid identity to the human paralogue. Hitherto, no X-ray structure exists from either enzyme. However, structural predictions based on the amino acid sequence of the active site in comparison to the human enzyme show that four conserved histidine and glutamate residues provide the coordination sites for chelating the ferrous iron ions. Recently, we showed that P. vivax DOHH is inhibited by zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea), a drug that is known for inhibiting human 5-lipoygenase (5-LOX) by the complexation of ferrous iron. A novel discovery program was launched to identify inhibitors of the P. falciparum DOHH from the Malaria Box, consisting of 400 chemical compounds, which are highly active in the erythrocytic stages of Malaria infections. In a first visual selection for potential ligands of ferrous iron, three compounds from different scaffold classes namely the diazonapthyl benzimidazole MMV666023 (Malaria Box plate A, position A03), the bis-benzimidazole MMV007384 (plate A, position B08), and a 1,2,5,-oxadiazole MMV665805 (plate A, position C03) were selected and subsequently evaluated in silico for their potential to complex iron ions. As a proof of principle, a bioanalytical assay was performed and the inhibition of hypusine biosynthesis was determined by GC-MS. All tested compounds proved to be active in this assay and MMV665805 exhibited the strongest inhibitory effect. Notably, the results were in accordance with the preliminary quantum-mechanical calculations suggesting the strongest iron complexation capacity for MMV665805. This compound might be a useful tool as well as a novel lead structure for inhibitors of P. falciparum DOHH.

Analysis of chemical profiles of insect adhesion secretions by gas chromatography-mass spectrometry.

This article reports on the chemical analysis of molecular profiles of tarsal secretions of the desert locust Schistocerca gregaria (Forsskål, 1775) by gas chromatography hyphenated with quadrupol mass spectrometry (GC-MS) as well as (1)H-nuclear magnetic resonance ((1)H NMR) spectroscopy. Special focus of this study was to elaborate on sampling methods which enable selective microscale extraction of insect secretions in a spatially controlled manner, in particular tarsal adhesive secretions and secretions located on cuticle surfaces at the tibia. Various solvent sampling procedures and contact solid-phase microextraction (SPME) methods were compared in terms of comprehensiveness and extraction efficiencies as measured by signal intensities in GC-MS. Solvent sampling with water as extraction solvent gave access to the elucidation of chemical profiles of polar compound classes such as amino acids and carbohydrates, but is extremely tedious. Contact SPME on the other hand can be regarded as a simplified and more elegant alternative, in particular for the lipophilic compound fraction. Many proteinogenic amino acids and ornithine as well as carbohydrate monomers arabinose, xylose, glucose, and galactose were detected in tarsal secretions after acid hydrolysis of aqueous extracts. Qualitatively similar but quantitatively significantly different molecular profiles were found for the lipid fraction which contained mainly n-alkanes and internally branched monomethyl-, dimethyl-, and trimethyl-alkanes in the C23-C49 range as well as long chain fatty acids and aldehydes. Especially, hydrocarbons with >C40 carbon numbers have previously been rarely reported for insect secretions. The results suggest that the investigated insect secretions are complex emulsions which allow the attachment of tarsi on various otherwise incompatible materials of smooth and rough surfaces. The solid consistence of the established alkanes at ambient temperatures might contribute to a semi-solid consistence of the adhesive, amalgamating partly opposing functions such as slip resistance, tarsal release, desiccation resistance, and mechanical compliance. The methods developed can be extended to other similar applications of studying compositions of insect secretions of other species.

Target evaluation of deoxyhypusine synthase from Theileria parva the neglected animal parasite and its relationship to Plasmodium.

East Coast fever (ECF) is a tick-borne disease caused by the parasite Theileria parva which infects cattle. In Sub-Saharan Africa it leads to enormous economic costs. After a bite of a tick, sporozoites invade the host lymphocytes and develop into schizonts. At this stage the parasite transforms host lymphocytes resulting in the clonal expansion of infected lymphocytes. Animals develop a lymphoma like disorder after infection which is rapidly fatal. Hitherto, a few drugs of the quinone type can cure the disease. However, therapy can only be successful after early diagnosis. The genera Theileria and Plasmodium, which includes the causative agent of human malaria, are closely related apicomplexan parasites. Enzymes of the hypusine pathway, a posttranslational modification in eukaryotic initiation factor EIF-5A, have shown to be druggable targets in Plasmodium. We identified the first enzyme of the hypusine pathway from T. parva, the deoxyhypusine synthase (DHS), which is located on chromosome 2 of the Muguga strain. Transcription is significantly increased in schizonts. The expressed T. parva DHS reveals an open reading frame (ORF) of 370 amino acids after expression in Escherichia coli Rosetta cells with a molecular size of 41.26 kDa and a theoretical pI of 5.26. Screening of the Malaria Box which consists of 400 active compounds resulted in a novel heterocyclic compound with a guanyl spacer which reduced the activity of T. parva DHS to 45%. In sum, the guanyl residue seems to be an important lead structure for inhibition of Theileria DHS. Currently, more different guanyl analogues from the Malaria Box are tested in inhibitor experiments to determine their efficacy.