A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Quinacrine reactivity with prion proteins and prion-derived peptides

Quinacrine is a drug that is known to heal neuronal cell culture infected with prions, which are the causative agents of neurodegenerative diseases called transmissible spongiform encephalopathies. However, the drug fails when it is applied in vivo. In this work, we analyzed the reason for this failure. The drug was suggested to “covalently” modify the prion protein via an acridinyl exchange reaction. To investigate this hypothesis more closely, the acridine moiety of quinacrine was covalently attached to the thiol groups of cysteines belonging to prion-derived peptides and to the full-length prion protein. The labeled compounds were conveniently monitored by fluorescence and absorption spectroscopy in the ultraviolet and visible spectral regions. The acridine moiety demonstrated characteristic UV–vis spectrum, depending on the substituent at the C-9 position of the acridine ring. These results confirm that quinacrine almost exclusively reacts with the thiol groups present in proteins and peptides. The chemical reaction alters the prion properties and increases the concentration of the acridine moiety in the prion protein.     

Identification of a new genotype of Torque Teno Mini virus

Background

Since we were able to isolate viable virus from brain and lung of H7N1 low pathogenic avian influenza virus (LPAIV) infected chickens, we here examined the distribution of different LPAIV strains in chickens by measuring the viral AI RNA load in multiple organs. Subtypes of H5 (H5N1, H5N2), H7 (H7N1, H7N7) and H9 (H9N2), of chicken (H5N2, H7N1, H7N7, H9N2), or mallard (H5N1) origin were tested. The actual presence of viable virus was evaluated with virus isolation in organs of H7N7 inoculated chickens.

Findings

Viral RNA was found by PCR in lung, brain, intestine, peripheral blood mononuclear cells, heart, liver, kidney and spleen from chickens infected with chicken isolated LPAIV H5N2, H7N1, H7N7 or H9N2. H7N7 virus could be isolated from lung, ileum, heart, liver, kidney and spleen, but not from brain, which was in agreement with the data from the PCR. Infection with mallard isolated H5N1 LPAIV resulted in viral RNA detection in lung and peripheral blood mononuclear cells only.

Conclusion

We speculate that chicken isolated LPAI viruses are spreading systemically in chicken, independently of the strain.     

Exploring the Chemical Space around 8-Mercaptoguanine as a Route to New Inhibitors of the Folate Biosynthesis Enzyme HPPK

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg^2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (K_D ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC₅₀ ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N ⁹ substitution, or tautomerization arising from N ⁷ substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N ⁹-H···Val46 hydrogen bond, combined with the limited space available around the N ⁹ position. The water-filled pocket under the N ⁷ position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N ⁷ (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (K_D ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N ⁷ position towards the Mg²⁺-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg²⁺ centres or Mg²⁺ -binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.     

Unique Gene Expression and MR T2 Relaxometry Patterns Define Chronic Murine Dextran Sodium Sulphate Colitis as a Model for Connective Tissue Changes in Human Crohn’s Disease

Introduction

Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS) ingestion, to mimic the relapsing nature of the human disease.

Materials and Methods

C57BL/6 mice were exposed to DSS in drinking water for 1 week, followed by a recovery phase of 2 weeks. This cycle of exposure was repeated for up to 3 times (9 weeks in total). Colonic inflammation, fibrosis, extracellular matrix proteins and colonic gene expression were studied. In vivo MRI T ₂ relaxometry was studied as a potential non-invasive imaging tool to evaluate bowel wall inflammation and fibrosis.

Results

Repeated cycles of DSS resulted in a relapsing and remitting disease course, which induced a chronic segmental, transmural colitis after 2 and 3 cycles of DSS with clear induction of fibrosis and remodeling of the muscular layer. Tenascin expression mirrored its expression in Crohn’s colitis. Microarray data identified a gene expression profile different in chronic colitis from that in acute colitis. Additional recovery was associated with upregulation of unique genes, in particular keratins, pointing to activation of molecular pathways for healing and repair. In vivo MRI T₂ relaxometry of the colon showed a clear shift towards higher T₂ values in the acute stage and a gradual regression of T₂ values with increasing cycles of DSS.

Conclusions

Repeated cycles of DSS exposure induce fibrosis and connective tissue changes with typical features, as occurring in Crohn’s disease. Colonic gene expression analysis revealed unique expression profiles in chronic colitis compared to acute colitis and after additional recovery, pointing to potential new targets to intervene with the induction of fibrosis. In vivo T₂ relaxometry is a promising non-invasive assessment of inflammation and fibrosis.     

Hospital Standardized Mortality Ratio: Consequences of Adjusting Hospital Mortality with Indirect Standardization

Background

The hospital standardized mortality ratio (HSMR) is developed to evaluate and improve hospital quality. Different methods can be used to standardize the hospital mortality ratio. Our aim was to assess the validity and applicability of directly and indirectly standardized hospital mortality ratios.

Methods

Retrospective scenario analysis using routinely collected hospital data to compare deaths predicted by the indirectly standardized case-mix adjustment method with observed deaths. Discharges from Dutch hospitals in the period 2003–2009 were used to estimate the underlying prediction models. We analysed variation in indirectly standardized hospital mortality ratios (HSMRs) when changing the case-mix distributions using different scenarios. Sixty-one Dutch hospitals were included in our scenario analysis.

Results

A numerical example showed that when interaction between hospital and case-mix is present and case-mix differs between hospitals, indirectly standardized HSMRs vary between hospitals providing the same quality of care. In empirical data analysis, the differences between directly and indirectly standardized HSMRs for individual hospitals were limited.

Conclusion

Direct standardization is not affected by the presence of interaction between hospital and case-mix and is therefore theoretically preferable over indirect standardization. Since direct standardization is practically impossible when multiple predictors are included in the case-mix adjustment model, indirect standardization is the only available method to compute the HSMR. Before interpreting such indirectly standardized HSMRs the case-mix distributions of individual hospitals and the presence of interactions between hospital and case-mix should be assessed.     

Anti-cancer activities of cytokinin ribosides

Phytochemistry Reviews - Cytokinins are plant hormones and play essential roles in regulating plant growth and development. They also have diverse pharmacological effects in animals and humans....     

Effects of Bark Beetle Disturbance on Soil Nutrient Retention and Lake Chemistry in Glacial Catchment

Ecosystems - Forest ecosystems worldwide are subjected to human-induced stressors, including eutrophication and acidification, and to natural disturbances (for example, insect infestation,...