Spermidine, but not spermine, is essential for pigment pattern formation in zebrafish

Polyamines are small poly-cations essential for all cellular life. The main polyamines present in metazoans are putrescine, spermidine and spermine. Their exact functions are still largely unclear; however, they are involved in a wide variety of processes affecting cell growth, proliferation, apoptosis and aging. Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry. We show that spermidine, but not spermine, is essential for early development, organogenesis and colour pattern formation. Whereas in other vertebrates spermidine deficiency leads to very early embryonic lethality, maternally provided spermidine synthase in zebrafish is sufficient to rescue the early developmental defects. This allows us to uncouple them from events occurring later during colour patterning. Factors involved in the cellular interactions essential for colour patterning, likely targets for spermidine, are the gap junction components Cx41.8, Cx39.4, and Kir7.1, an inwardly rectifying potassium channel, all known to be regulated by polyamines. Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines

Title: Synthesis of UDP-activated Oligosaccharides with Commercial ␤-galactosidase from Bacillus circulans under Microwave Irradiation Synthesis of UDP-activated Oligosaccharides with Commercial β- galactosidase from Bacillus circulans under Microwave Irr

Abstract We report here on the synthesis of nucleotide activated oligosaccharides by transglycosylation with β-galactosidase from Bacillus circulans applying microwave irradiation (MWI) 1 and conventional heating. The presented products could serve as novel inhibitors or donor substrates of Leloir-glycosyltransferases. Some of them have been isolated from human milk but the biological role remains unclear due to limited access to the nucleotide oligosaccharides. The synthesis with β-galactosidases is challenging, because of competing hydrolysis of the product by the same enzyme. Effects of MWI and thermal heating on the hydrolytic and synthetic performance of the enzyme were systematically analysed and described here. We demonstrate that under both conditions similar product yields are obtained, however, the enzymatic hydrolysis of the product is significantly decreased under MWI leading to stable product formation. The obtained product yields and absence of product hydrolysis under MWI can be rationalized by time-dependent activation and inactivation of the β-galactosidase. Keywords Bacillus circulans; β-galactosidase; microwave; transglycosylation; nucleotide oligosaccharides Graphical abstract 1 MWI : microwave irradiatio

Statically Adsorbed Coatings for High Separation Efficiency and Resolution in CE–MS Peptide Analysis: Strategies and Implementation

Coatings are necessary to prevent protein and peptide adsorption to the capillary surface and obtain high intermediate precision. In this protocol, we first present our basic strategy to address peptide separation using three different coatings: one neutral and two cationic coatings, the latter largely differing in their induced electroosmotic mobility. In detail, we will describe how we apply the statically adsorbed coatings to obtain very high plate numbers and high repeatability.With some model examples, we clearly describe the scope of the method for the analysis of peptide samples: tryptic digests are addressed as well as small glycoproteins and glycopeptides largely differing in their effective electrophoretic mobility. We also show that the method is suitable for a fast screening of peptide samples despite a high matrix load comprising of up to 500 mmol/L sodium chloride. We demonstrate that this basic CE-MS method is rather independent of the polarity of the analytes with a very fast near-baseline separation of very hydrophobic Aβ peptides related to the onset of Alzheimer's disease. These examples will give an impression, which coating is most suitable for a specific analytical application.Special attention is paid to difficult aspects of the coating procedure and the CE-MS method, e.g., the potential of cross-contamination when changing the coatings

Column coupling isotachophoresis-capillary electrophoresis with mass spectrometric detection: Characterization and optimization of microfluidic interfaces

Two-dimensional electrophoretic separations are one of the most promising tools for the continuously growing needs of different bioanalytical fields such as proteomics and metabolomics. In this work we present the design and the implementation of a two-dimensional electrophoretic separation coupled to mass spectrometry. We started our work studying the sample transfer characteristics of different microfluidic interfaces compatible with capillary coupling for two-dimensional electrophoretic separations. These junctions are aimed at method decoupling and sample transfer in a modular two-dimensional electrophoretic separation system. In order to perform the characterization of the interfaces, we carried out capillary electrophoresis experiments and numerical simulations using three cationic compounds under different flow conditions. The comparison of the experimental and simulation results enables us to clearly define the desirable characteristics of interfaces in order to achieve method orthogonality with lossless sample transfer in a two-dimensional separation system. Finally, we present a glass microfluidic chip as interface for the implementation of a novel hybrid modular system for performing two-dimensional electrophoretic separations involving isotachophoresis and capillary electrophoresis. In this setup we include mass spectrometric and contactless capacitively coupled conductivity detection to monitor the separation process. We demonstrate the ability of the setup to be used as a flexible analysis tool by performing preconcentration, separation, detection and identification of four different human angiotensin peptides

Novel RP-HPLC based assay for selective and sensitive endotoxin quantification

The paper presents a novel instrumental analytical endotoxin quantification assay. It uses common analytical laboratory equipment (HPLC-FLD) and allows quantifying endotoxins (ETs) in different matrices from about 109 EU / mL down to about 40 EU / mL (RSE based). Test results are obtained in concentration units (e.g. ng ET / mL), which can then be converted to commonly used endotoxin units (EU / mL) in case of known pyrogenic activity. During endotoxin hydrolysis, the endotoxin specific rare sugar acid KDO is obtained quantitatively. After that, KDO is stoichiometrically reacted with DMB, which results in a highly fluorescent derivative. The mixture is separated using RP-HPLC followed by KDO-DMB quantification with a fluorescence detector. Based on the KDO content, the endotoxin content in the sample is calculated. The developed assay is economic and has a small error. Its applicability was demonstrated in applied research. ETs were quantified in purified bacterial biopolymers, which were produced by Gram-negative bacteria. Results were compared to LAL results obtained for the same samples. A high correlation was found between the results of both methods. Further, the new assay was utilized with high success during the development of novel endotoxin specific depth filters, which allow efficient, economic and sustainable ET removal during DSP. Those examples demonstrate that the new assay has the potential to complement the animal-based biological LAL pyrogenic quantification tests, which are accepted today by the major health authorities worldwide for the release of commercial pharmaceutical products