Lipid ratios as a marker for red blood cell storage quality and as a possible explanation for donor gender differences in storage quality

Background and objectives Red blood cells that are stored for transfusions as red cell concentrates (RCCs) undergo changes during the storage period, culminating in the lysis of the cells. The goal of this work is to find markers that are linked to high haemolysis, in order to explain the inter‐donor variability that is known to occur in storage quality, and also the known differences between RCCs from male and female donors. Materials and methods The relative amounts of lipids at the end of the storage period were compared for one group of low haemolysis samples (24 units, all ≤0·15% haemolysis), and one group of high haemolysis samples (26 units, all ≥0·5% haemolysis). Representative lipids were analysed from different lipid classes, including cholesterol, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and ceramide. Whole membrane preparations were analysed with one mass spectrometry technique, and lipid extracts were analysed with a second mass spectrometry technique. Results The ratio of palmitoyl‐oleoyl phosphatidylcholine (POPC) to sphingomyelin was different for the high and low haemolysis groups (P = 0·0001) and for the RCCs from male and female donors (P = 0·0009). The ratio of cholesterol to phospholipids showed only minimal links to haemolysis. Higher relative amounts of sphingomyelin were associated with lower haemolysis, and higher relative amounts of ceramides were associated with increased haemolysis. Conclusion The level of sphingomyelinase activity and the resulting ratio of sphingomyelin to POPC is proposed as a possible marker for RCC storage quality

The oxidative stress agent hypochlorite stimulates c-di-GMP synthesis and biofilm formation in Pseudomonas aeruginosa

The opportunistic human pathogen Pseudomonas aeruginosa is able to survive under a variety of often harmful environmental conditions due to a multitude of intrinsic and adaptive resistance mechanisms, including biofilm formation as one important survival strategy. Here, we investigated the adaptation of P. aeruginosa PAO1 to hypochlorite (HClO), a phagocyte-derived host defense compound and frequently used disinfectant. In static biofilm assays, we observed a significant enhancement in initial cell attachment in the presence of sublethal HClO concentrations. Subsequent LC-MS analyses revealed a strong increase in cyclic-di-GMP (c-di-GMP) levels suggesting a key role of this second messenger in HClO-induced biofilm development. Using DNA microarrays, we identified a 26-fold upregulation of ORF PA3177 coding for a putative diguanylate cyclase (DGC), which catalyzes the synthesis of the second messenger c-di-GMP – an important regulator of bacterial motility, sessility and persistence. This DGC PA3177 was further characterized in more detail demonstrating its impact on P. aeruginosa motility and biofilm formation. In addition, cell culture assays attested a role for PA3177 in the response of P. aeruginosa to human phagocytes. Using a subset of different mutants, we were able to show that both Pel and Psl exopolysaccharides are effectors in the PA3177-dependent c-di-GMP network

Switching the Switch: Ligand Induced Disulfide Formation in HDAC8

Human histone deacetylase 8 is a well‐recognized target for T‐cell lymphoma and particularly childhood neuroblastoma. PD‐404,182 was shown to be a selective covalent inhibitor of HDAC8 that forms mixed disulfides with several cysteine residues and is also able to transform thiol groups to thiocyanates. Moreover, HDAC8 was shown to be regulated by a redox switch based on the reversible formation of a disulfide bond between cysteines Cys$_{102}$ and Cys$_{153}$. This study on the distinct effects of PD‐404,182 on HDAC8 reveals that this compound induces the dose‐dependent formation of intramolecular disulfide bridges. Therefore, the inhibition mechanism of HDAC8 by PD‐404,182 involves both, covalent modification of thiols as well as ligand mediated disulfide formation. Moreover, this study provides a deep molecular insight into the regulation mechanism of HDAC8 involving several cysteines with graduated capability to form reversible disulfide bridges

Glycolipids produced by Rouxiella sp. DSM 100043 and isolation of the biosurfactants via foam-fractionation

Additional file 1. Table S1, Figure S1–Figure S3: Mass spectrometry data and plots of purified foam extracts of Rouxiella sp. DSM 100043. Figure S4: Full NMR spectra of Rouxiella sp. DMS 100043 glycolipids present in fractions 64-65

Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS) Promotes Biofilm Formation of Pseudomonas aeruginosa by Increasing Iron Availability

The 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase HodC was previously described to cleave the Pseudomonas quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS) system of Pseudomonas aeruginosa, an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of P.aeruginosa attenuated production of virulence factors, and reduced virulence inplanta. However, proteolytic cleavager educed the efficacy of HodC. Here, we identified the secreted protease LasB of P.aeruginosa to be responsible for HodC degradation. In static biofilms of the P.aeruginosa PA14 lasB::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as pvdS, pvdL, pvdA, and pvdQ. This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite ist ability to quench the production of virulence factors, is contraindicated for combating P.aeruginosa biofilms

3‐Chloro‐5‐Substituted‐1,2,4‐Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers

The study of cysteine modifications has gained much attention in recent years. This includes detailed investigations in the field of redox biology with focus on numerous redox derivatives like nitrosothiols, sulfenic acids, sulfinic acids and sulfonic acids resulting from increasing oxidation, S-lipidation, and perthiols. For these studies selective and rapid blocking of free protein thiols is required to prevent disulfide rearrangement. In our attempt to find new inhibitors of human histone deacetylase 8 (HDAC8) we discovered 5-sulfonyl and 5-sulfinyl substituted 1,2,4-thiadiazoles (TDZ), which surprisingly show an outstanding reactivity against thiols in aqueous solution. Encouraged by these observations we investigated the mechanism of action in detail and show that these compounds react more specifically and faster than commonly used N-ethyl maleimide, making them superior alternatives for efficient blocking of free thiols in proteins. We show that 5-sulfonyl-TDZ can be readily applied in commonly used biotin switch assays. Using the example of human HDAC8, we demonstrate that cysteine modification by a 5-sulfonyl-TDZ is easily measurable using quantitative HPLC/ESI-QTOF-MS/MS, and allows for the simultaneous measurement of the modification kinetics of seven solvent-accessible cysteines in HDAC8

Extracellular DNA release, quorum sensing, and PrrF1/F2 small RNAs are key players in Pseudomonas aeruginosa tobramycin-enhanced biofilm formation

Biofilms are structured microbial communities that are the leading cause of numerous chronic infections which are difficult to eradicate. Within the lungs of individuals with cystic fibrosis (CF), Pseudomonas aeruginosa causes persistent biofilm infection that is commonly treated with aminoglycoside antibiotics such as tobramycin. However, sublethal concentrations of this aminoglycoside were previously shown to increase biofilm formation by P. aeruginosa, but the underlying adaptive mechanisms still remain elusive. Herein, we combined confocal laser scanning microscope analyses, proteomics profiling, gene expression assays and phenotypic studies to unravel P. aeruginosa potential adaptive mechanisms in response to tobramycin exposure during biofilm growth. Under this condition, we show that the modified biofilm architecture is related at least in part to increased extracellular DNA (eDNA) release, most likely as a result of biofilm cell death. Furthermore, the activity of quorum sensing (QS) systems was increased, leading to higher production of QS signaling molecules. We also demonstrate upon tobramycin exposure an increase in expression of the PrrF small regulatory RNAs, as well as expression of iron uptake systems. Remarkably, biofilm biovolumes and eDNA relative abundances in pqs and prrF mutant strains decrease in the presence of tobramycin. Overall, our findings offer experimental evidences for a potential adaptive mechanism linking PrrF sRNAs, QS signaling, biofilm cell death, eDNA release, and tobramycin-enhanced biofilm formation in P. aeruginosa. These specific adaptive mechanisms should be considered to improve treatment strategies against P. aeruginosa biofilm establishment in CF patients’ lungs

Activation of the cell wall stress response in pseudomonas aeruginosa infected by a pf4 phage variant

Pseudomonas aeruginosa PAO1 has an integrated Pf4 prophage in its genome, encoding a relatively well-characterized filamentous phage, which contributes to the bacterial biofilm organization and maturation. Pf4 variants are considered as superinfectives when they can re-infect and kill the prophage-carrying host. Herein, the response of P. aeruginosa H103 to Pf4 variant infection was investigated. This phage variant caused partial lysis of the bacterial population and modulated H103 physiology. We show by confocal laser scanning microscopy that a Pf4 variant-infection altered P. aeruginosa H103 biofilm architecture either in static or dynamic conditions. Interestingly, in the latter condition, numerous cells displayed a filamentous morphology, suggesting a link between this phenotype and flow-related forces. In addition, Pf4 variant-infection resulted in cell envelope stress response, mostly mediated by the AlgU and SigX extracytoplasmic function sigma factors (ECFσ). AlgU and SigX involvement may account, at least partly, for the enhanced expression level of genes involved in the biosynthesis pathways of two matrix exopolysaccharides (Pel and alginates) and bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) metabolism

The Temperature-Regulation of Pseudomonas aeruginosa cmaX-cfrX-cmpX Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX

Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECFσ SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis