RESUMO
The widespread application of enzymes in industrial chemical synthesis requires efficient process control to maintain high yields and purity. Flow injection analysis-electrospray ionization-mass spectrometry (FIA-ESI-MS) offers a promising solution for real-time monitoring of these enzymatic processes, particularly when handling challenging compounds like sugars and glycans, which are difficult to quickly analyze using liquid chromatography-mass spectrometry due to their physical properties or the requirement for a derivatization step beforehand. This study compares the performance of FIA-MS with traditional hydrophilic interaction liquid chromatography (HILIC)-ultra high-performance liquid chromatography (UHPLC)-mass spectrometry (MS) setups for the monitoring of the enzymatic synthesis of N-acetyllactosamine (LacNAc) using beta-1,4-galactosyltransferase. Our results show that FIA-MS, without prior chromatographic separation or derivatization, can quickly generate accurate mass spectrometric data within minutes, contrasting with the lengthy separations required by LC-MS methods. The rapid data acquisition of FIA-MS enables effective real-time monitoring and adjustment of the enzymatic reactions. Furthermore, by eliminating the derivatization step, this method offers the possibility of being directly coupled to a continuously operated reactor, thus providing a rapid on-line methodology for glycan synthesis as well.
Assuntos
Análise de Injeção de Fluxo , Glicosiltransferases , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização por Electrospray/métodos , Análise de Injeção de Fluxo/métodos , Cromatografia Líquida de Alta Pressão/métodos , Glicosiltransferases/metabolismo , Amino Açúcares/análiseRESUMO
Continuous manufacturing is becoming increasingly important in the (bio-)pharmaceutical industry, as more product can be produced in less time and at lower costs. In this context, there is a need for powerful continuous analytical tools. Many established off-line analytical methods, such as mass spectrometry (MS), are hardly considered for process analytical technology (PAT) applications in biopharmaceutical processes, as they are limited to at-line analysis due to the required sample preparation and the associated complexity, although they would provide a suitable technique for the assessment of a wide range of quality attributes. In this study, we investigated the applicability of a recently developed micro simulated moving bed chromatography system (µSMB) for continuous on-line sample preparation for MS. As a test case, we demonstrate the continuous on-line MS measurement of a protein solution (myoglobin) containing Tris buffer, which interferes with ESI-MS measurements, by continuously exchanging this buffer with a volatile ammonium acetate buffer suitable for MS measurements. The integration of the µSMB significantly increases MS sensitivity by removing over 98% of the buffer substances. Thus, this study demonstrates the feasibility of on-line µSMB-MS, providing a versatile PAT tool by combining the detection power of MS for various product attributes with all the advantages of continuous on-line analytics.
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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.
Assuntos
Compostos de Sulfidrila , Tiadiazóis , Humanos , Cisteína/metabolismo , Tiadiazóis/farmacologia , Espectrometria de Massas em Tandem , Ácidos Sulfênicos , Oxirredução , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Leukemia patients undergo chemotherapy to combat the leukemic cells (LCs) in the bone marrow. During therapy not only the LCs, but also the blood-producing hematopoietic stem and progenitor cells (HSPCs) may be destroyed. Chemotherapeutics targeting only the LCs are urgently needed to overcome this problem and minimize life-threatening side-effects. Predictivein vitrodrug testing systems allowing simultaneous comparison of various experimental settings would enhance the efficiency of drug development. Here, we present a three-dimensional (3D) human leukemic bone marrow model perfused using a magnetic, parallelized culture system to ensure media exchange. Chemotherapeutic treatment of the acute myeloid leukemia cell line KG-1a in 3D magnetic hydrogels seeded with mesenchymal stem/stromal cells (MSCs) revealed a greater resistance of KG-1a compared to 2D culture. In 3D tricultures with HSPCs, MSCs and KG-1a, imitating leukemic bone marrow, HSPC proliferation decreased while KG-1a cells remained unaffected post treatment. Non-invasive metabolic profiling enabled continuous monitoring of the system. Our results highlight the importance of using biomimetic 3D platforms with proper media exchange and co-cultures for creatingin vivo-like conditions to enablein vitrodrug testing. This system is a step towards drug testing in biomimetic, parallelizedin vitroapproaches, facilitating the discovery of new anti-leukemic drugs.
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Leucemia Mieloide Aguda , Células-Tronco Mesenquimais , Células da Medula Óssea , Diferenciação Celular , Proliferação de Células , Técnicas de Cocultura , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismoRESUMO
Fully exploiting the potential of enzymes in cell-free biocatalysis requires stabilization of the catalytically active proteins and their integration into efficient reactor systems. Although in recent years initial steps towards the immobilization of such biomolecules in metal-organic frameworks (MOFs) have been taken, these demonstrations have been limited to batch experiments and to aqueous conditions. Here we demonstrate a MOF-based continuous flow enzyme reactor system, with high productivity and stability, which is also suitable for organic solvents. Under aqueous conditions, the stability of the enzyme was increased 30-fold, and the space-time yield exceeded that obtained with other enzyme immobilization strategies by an order of magnitude. Importantly, the infiltration of the proteins into the MOF did not require additional functionalization, thus allowing for time- and cost-efficient fabrication of the biocatalysts using label-free enzymes.
Assuntos
Enzimas Imobilizadas , Estruturas Metalorgânicas , Biocatálise , Catálise , Enzimas/metabolismo , Enzimas Imobilizadas/metabolismo , Estruturas Metalorgânicas/metabolismo , Proteínas/metabolismo , SolventesRESUMO
Metal-organic frameworks (MOFs) built from different building units offer functionalities going far beyond gas storage and separation. In connection with advanced applications, e.g., in optoelectronics, hierarchical MOF-on-MOF structures fabricated using sophisticated methodologies have recently become particularly attractive. Here, we demonstrate that the structural complexity of MOF-based architectures can be further increased by employing highly spatioselective photochemistry. Using a layer-by-layer, quasi-epitaxial synthesis method, we realized a photoactive MOF-on-MOF hetero-bilayer consisting of a porphyrinic bottom layer and a tetraphenylethylene (TPE)-based top layer. Illumination of the monolithic thin film with visible light in the presence of oxygen gas results in the generation of reactive oxygen species (1O2) in the porphyrinic bottom layer, which lead to a photocleavage of the TPE units at the internal interface. We demonstrate that this spatioselective photochemistry can be utilized to delaminate the top layers, yielding two-dimensional (2D) MOF sheets with well-defined thickness. Experiments using atomic force microscopy (AFM) demonstrate that these platelets can be transferred onto other substrates, thus opening up the possibility of fabricating planar MOF structures using photolithography.
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Salicylic acid is a phenolic phytohormone which controls plant growth and development. A methyl ester (MSA) derivative thereof is volatile and involved in plant-insect or plant-plant communication. Here we show that the nematode-trapping fungus Duddingtonia flagrans uses a methyl-salicylic acid isomer, 6-MSA as morphogen for spatiotemporal control of trap formation and as chemoattractant to lure Caenorhabditis elegans into fungal colonies. 6-MSA is the product of a polyketide synthase and an intermediate in the biosynthesis of arthrosporols. The polyketide synthase (ArtA), produces 6-MSA in hyphal tips, and is uncoupled from other enzymes required for the conversion of 6-MSA to arthrosporols, which are produced in older hyphae. 6-MSA and arthrosporols both block trap formation. The presence of nematodes inhibits 6-MSA and arthrosporol biosyntheses and thereby enables trap formation. 6-MSA and arthrosporols are thus morphogens with some functions similar to quorum-sensing molecules. We show that 6-MSA is important in interkingdom communication between fungi and nematodes.
Assuntos
Ascomicetos/fisiologia , Caenorhabditis elegans/fisiologia , Hifas/fisiologia , Comportamento Predatório/fisiologia , Ácido Salicílico/metabolismo , Animais , Ascomicetos/genética , Ascomicetos/metabolismo , Quimiotaxia/fisiologia , Proteínas Fúngicas/metabolismo , Hifas/genética , Hifas/metabolismo , Policetídeo Sintases/metabolismo , Ácido Salicílico/química , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
BACKGROUND: Red blood cells (RBCs) stored for transfusions can lyse over the course of the storage period. The lysis is traditionally assumed to occur via the formation of spiculated echinocyte forms, so that cells that appear smoother are assumed to have better storage quality. We investigate this hypothesis by comparing the morphological distribution to the hemolysis for samples from different donors. METHODS: Red cell concentrates were obtained from a regional blood bank quality control laboratory. Out of 636 units processed by the laboratory, we obtained 26 high hemolysis units and 24 low hemolysis units for assessment of RBC morphology. The association between the morphology and the hemolysis was tested with the Wilcoxon-Mann-Whitney U test. RESULTS: Samples with high stomatocyte counts (p = 0.0012) were associated with increased hemolysis, implying that cells can lyse via the formation of stomatocytes. CONCLUSION: RBCs can lyse without significant echinocyte formation. Lower degrees of spiculation are not a good indicator of low hemolysis when RBCs from different donors are compared.
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We herein present a case study on the templated, Pd-catalyzed polymerization reaction of methyl propiolate in the confined pore space of three different surface anchored metal-organic framework (SURMOF) systems in order to introduce electrical conductivity to MOF thin films and provide predictions for potential device integrations. To gain comprehensive insight into the influence of the template on polymerization, we chose Cu(bpdc), Cu2(bdc)2(dabco) and HKUST-1 because of their different types of pore channels, 1D, quasi-1D and 3D, and their free pore volumes. Well-defined MOF thin films were prepared using layer-by-layer deposition, which allows for the application of several characterization techniques not applicable for conventional powder MOFs. With SEM, AFM, XRD, MALDI-ToF/MS, ToF-SIMS and QCM, we were able to investigate the behaviour of the polymer formation. For lower dimensional pore channels, we find a depot-like release of monomeric units leading to top-layer formation determined by desorption kinetics, whereas for the 3D channels, quick release of an excess amount of monomers was observed and polymerization proceeds perfectly. Despite polymerization issues, control over the maximum chain lengths and the molecular weight distribution was achieved depending on the dimensionality of the pore systems. For the HKUST-1 system, polymerization was optimized and we were able to measure the electrical conductivity introduced by the conjugated polymer inside the channels.
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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.
RESUMO
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.
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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.
Assuntos
Preservação de Sangue/normas , Eritrócitos/metabolismo , Lipídeos/análise , Caracteres Sexuais , Colesterol/análise , Feminino , Hemólise , Humanos , Masculino , Fosfolipídeos/análiseRESUMO
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 Cys102 and Cys153 . 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.
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Iron is one of the most abundant elements on earth and essential for life. However, Fe3+ ions are rather insoluble and microorganisms such as fungi may use siderophores as strong chelators for uptake. In addition, free cytoplasmic iron is rather toxic and intracellular siderophores are used to control the toxicity. Siderophores are also important for iron storage. We studied two siderophore systems in the plant necrotrophic fungus Alternaria alternata and show that the non-ribosomal peptide synthase, Nps2, is required for the biosynthesis of intracellular ferricrocin, whereas Nps6 is needed for the formation of extracellular coprogen and coprogen B. Whereas nps2 was dispensable for growth on iron-depleted medium, nps6 was essential under those conditions. nps2 deletion caused an increase in spore formation and reduced pathogenicity on tomato. Our results suggest that A. alternata employs an external and an internal siderophore system to adapt to low iron conditions.
Assuntos
Alternaria/metabolismo , Ferro/metabolismo , Sideróforos/biossíntese , Alternaria/enzimologia , Alternaria/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismoRESUMO
Pseudomonas aeruginosa rapidly adapts to altered conditions by quorum sensing (QS), a communication system that it uses to collectively modify its behavior through the production, release, and detection of signaling molecules. QS molecules can also be sensed by hosts, although the respective receptors and signaling pathways are poorly understood. We describe a pattern of regulation in the host by the aryl hydrocarbon receptor (AhR) that is critically dependent on qualitative and quantitative sensing of P. aeruginosa quorum. QS molecules bind to AhR and distinctly modulate its activity. This is mirrored upon infection with P. aeruginosa collected from diverse growth stages and with QS mutants. We propose that by spying on bacterial quorum, AhR acts as a major sensor of infection dynamics, capable of orchestrating host defense according to the status quo of infection.
Assuntos
Interações Hospedeiro-Patógeno , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/fisiologia , Receptores de Hidrocarboneto Arílico/fisiologia , Células A549 , Animais , Humanos , Larva , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Receptores de Hidrocarboneto Arílico/genética , Peixe-ZebraRESUMO
Strontium, calcium, and magnesium silicate hydrate phases are synthesized by the reaction between silica and solution of metal hydroxides. The kinetics of the reaction is recorded using a quartz crystal microbalance (QCM), continuously monitoring the change in frequency and dissipation energy. Based on QCM results, it is shown that properties of solutions like the pH-value or the type of ions play a pivotal function on the rate-determining stage of the reaction, the thickness of the diffuse layer, the formation of carbonates, as well as the kinetics of the formed phases. Further properties of the reaction products are investigated using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and infrared spectroscopy (IR). With the help of thermogravimetric analysis (TGA) and temperature-dependent X-ray diffraction (XRD), we investigate how our synthesized phases can be turned into MSiO3 structures. Finally, the Goldschmidt rules for perovskites structures show that this might be an attractive way for new and nontoxic phases in the future.
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Owing to the hierarchical organization of biology, from genomes over transcriptomes and proteomes down to metabolomes, there is continuous debate about the extent to which data and interpretations derived from one level, e.g. the transcriptome, are in agreement with other levels, e.g. the metabolome. Here, we tested the effect of ocean acidification (OA; 400 vs. 1000 µatm CO2) and its modulation by light intensity (50 vs. 300 µmol photons m-2 s-1) on the biomass composition (represented by 75 key metabolites) of diploid and haploid life-cycle stages of the coccolithophore Emiliania huxleyi (RCC1216 and RCC1217) and compared these data with interpretations from previous physiological and gene expression screenings. The metabolite patterns showed minor responses to OA in both life-cycle stages. Whereas previous gene expression analyses suggested that the observed increased biomass buildup derived from lipid and carbohydrate storage, this dataset suggests that OA slightly increases overall biomass of cells, but does not significantly alter their metabolite composition. Generally, light was shown to be a more dominant driver of metabolite composition than OA, increasing the relative abundances of amino acids, mannitol and storage lipids, and shifting pigment contents to accommodate increased irradiance levels. The diploid stage was shown to contain vastly more osmolytes and mannitol than the haploid stage, which in turn had a higher relative content of amino acids, especially aromatic ones. Besides the differences between the investigated cell types and the general effects on biomass buildup, our analyses indicate that OA imposes only negligible effects on E. huxleyi´s biomass composition.
Assuntos
Haptófitas/crescimento & desenvolvimento , Água do Mar/química , Biomassa , Dióxido de Carbono/análise , Diploide , Haploidia , Haptófitas/genética , Haptófitas/metabolismo , Concentração de Íons de Hidrogênio , Estágios do Ciclo de Vida , Metaboloma , Oceanos e Mares , TranscriptomaRESUMO
Distinct streptomycetes such as Streptomyces mobaraensis produce the protein cross-linking enzyme transglutaminase. Bioinformatic analysis predicted the occurrence of seven sortases exerting transpeptidation reactions similarly to transglutaminase. Here, we report the production and characterization of sortase E2 (Sm-SrtE2) solubilized by removal of its membrane anchor domain. Sm-SrtE2 activity was measured using pentapeptides predicted to be cell wall sorting signals of putative sortase substrate proteins. Preferred linkage to Gly3 by Sm-SrtE2 was in the order LAETG>>LAHTG>>LAQTG~LANTG>LARTG. Chaplin 1 from S. mobaraensis was further demonstrated to be an excellent substrate of both the intrinsic Sm-SrtE2 and transglutaminase. The unexpected discovery showing Gln-62 and Gln-65 of Δ1-50 -Sm-SrtE2 as transglutaminase cross-linking sites suggests that low enzyme stability might be due to anchor domain truncation and a disordered N terminus.