Potassium‐ion‐selective fluorescent sensors to detect cereulide, the emetic toxin of B. cereus, in food samples and HeLa cells

We report the development of new chemical probes for cereulide, a toxic metabolite produced by specific strains of Bacillus cereus, through displacement of potassium cations from a preformed specific complex and a subsequent change in the fluorescence emission. For this purpose, we designed fluorescent probes for potassium cations that were suitable for displacement assays with cereulide from organic extracts. The fluorescence detection of natural cereulide in rice samples was achieved by using synthetic cereulide as a reference and a potassium fluorescent reporter, and this was found to be useful as a portable and fast method for the in situ detection of cereulide in food extracts. To study the fate of cereulide in live cells, we designed a procedure that was suitable for live‐cell microscopy imaging of HeLa cells by comparing the cellular location of the potassium fluorogenic probe, which stained intracellular endolysosomes, in the absence and presence of cereulide; we concluded that in the presence of cereulide, the fluorescence of the probe was decreased because of complexation of the potassium ions by cereulide.Ministerio de Econom&a y Competitividad, Spain (Projects CTQ2015-71353-R and AES-PI16/000496), Junta de Castilla y Lejn, Consejer&a de Educaci jn y Cultura y Fondo Social Europeo (Project BU232U13), and the European Commission, Seventh Framework Programme (Project SNIFFER FP7-SEC-2012–312411

Monitoring Insulin Aggregation via Capillary Electrophoresis

Early stages of insulin aggregation, which involve the transient formation of oligomeric aggregates, are an important aspect in the progression of Type II diabetes and in the quality control of pharmaceutical insulin production. This study is the first to utilize capillary electrophoresis (CE) with ultraviolet (UV) detection to monitor insulin oligomer formation at pH 8.0 and physiological ionic strength. The lag time to formation of the first detected species in the aggregation process was evaluated by UV-CE and thioflavin T (ThT) binding for salt concentrations from 100 mM to 250 mM. UV-CE had a significantly shorter (5–8 h) lag time than ThT binding (15–19 h). In addition, the lag time to detection of the first aggregated species via UV-CE was unaffected by salt concentration, while a trend toward an increased lag time with increased salt concentration was observed with ThT binding. This result indicates that solution ionic strength impacts early stages of aggregation and β-sheet aggregate formation differently. To observe whether CE may be applied for the analysis of biological samples containing low insulin concentrations, the limit of detection using UV and laser induced fluorescence (LIF) detection modes was determined. The limit of detection using LIF-CE, 48.4 pM, was lower than the physiological insulin concentration, verifying the utility of this technique for monitoring biological samples. LIF-CE was subsequently used to analyze the time course for fluorescein isothiocyanate (FITC)-labeled insulin oligomer formation. This study is the first to report that the FITC label prevented incorporation of insulin into oligomers, cautioning against the use of this fluorescent label as a tag for following early stages of insulin aggregation

Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size

The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-β protein (Aβ) associated with Alzheimer’s disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aβ species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting

Surface Modified Capillaries in Capillary Electrophoresis Coupled to Mass Spectrometry : Method Development and Exploration of the Potential of Capillary Electrophoresis as a Proteomic Tool

The increased knowledge about the complexity of the physiological processes increases the demand on the analytical techniques employed to explore them. A comprehensive analysis of the entire sample content is today the most common approach to investigate the molecular interplay behind a physiological deviation. For this purpose a method that offers a number of important properties, such as speed and simplicity, high resolution and sensitivity, minimal sample volume requirements, cost efficiency and robustness, possibility of automation, high-throughput and wide application range of analysis is requested. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has a great potential and fulfils many of these criteria. However, further developments and improvements of these techniques and their combination are required to meet the challenges of complex biological samples. Protein analysis using CE is a challenging task due to protein adsorption to the negatively charged fused-silica capillary wall. This is especially emphasised with increased basicity and size of proteins and peptides. In this thesis, the adsorption problem was addressed by using an in-house developed physically adsorbed polyamine coating, named PolyE-323. The coating procedure is fast and simple that generates a coating stable over a wide pH range, 2-11. By coupling PolyE-323 modified capillaries to MS, either using electrospray ionisation (ESI) or matrix-assisted laser desorption/ionisation (MALDI), successful analysis of peptides, proteins and complex samples, such as protein digests and crude human body fluids were obtained. The possibilities of using CE-MALDI-MS/MS as a proteomic tool, combined with a proper sample preparation, are further demonstrated by applying high-abundant protein depletion in combination with a peptide derivatisation step or isoelectric focusing (IEF). These approaches were applied in profiling of the proteomes of human cerebrospinal fluid (CSF) and human follicular fluid (hFF), respectively. Finally, a multiplexed quantitative proteomic analysis was performed on a set of ventricular cerebrospinal fluid (vCSF) samples from a patient with traumatic brain injury (TBI) to follow relative changes in protein patterns during the recovery process. The results presented in this thesis confirm the potential of CE, in combination with MS, as a valuable choice in the analysis of complex biological samples and clinical applications

It´s also a part of the profession, I think : A phenomenographic study of preschool teachers´ profession

Denna studie har som syfte att synliggöra de uppfattningar förskollärare har om sin egen profession. För att undersöka detta har vi förhållit oss till kvalitativa metoder med fenomenografi som perspektiv och ansats, övergripande genom hela studien. Fenomenet och begreppet som är i fokus är profession och med hjälp av kvalitativa intervjuer och tidigare forskning bearbetar vi vad detta kan innebära för förskollärarna. I vårt resultat får vi fram fem olika huvudkategorier som synliggör variationen i förskollärarnas uppfattningar. Det komplexa uppdraget och förskollärarnas kompetens kommer fram som viktiga delar, men även det förhållningssätt som förskollärare har till professionen. Förskollärarna har också varierande uppfattningar av hur professionen framställs i övriga samhället och de har olika uppfattningar över vilka insatser som krävs för att stärka och synliggöra professionens betydelse i syfte att höja dess status

It´s also a part of the profession, I think : A phenomenographic study of preschool teachers´ profession

Denna studie har som syfte att synliggöra de uppfattningar förskollärare har om sin egen profession. För att undersöka detta har vi förhållit oss till kvalitativa metoder med fenomenografi som perspektiv och ansats, övergripande genom hela studien. Fenomenet och begreppet som är i fokus är profession och med hjälp av kvalitativa intervjuer och tidigare forskning bearbetar vi vad detta kan innebära för förskollärarna. I vårt resultat får vi fram fem olika huvudkategorier som synliggör variationen i förskollärarnas uppfattningar. Det komplexa uppdraget och förskollärarnas kompetens kommer fram som viktiga delar, men även det förhållningssätt som förskollärare har till professionen. Förskollärarna har också varierande uppfattningar av hur professionen framställs i övriga samhället och de har olika uppfattningar över vilka insatser som krävs för att stärka och synliggöra professionens betydelse i syfte att höja dess status

Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: a gel-free multidimensional electrophoresis approach for proteomic profiling--exemplified on human follicular fluid.

Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development

Quantitative Analysis of Cereulide Toxin from Bacillus cereus in Rice and Pasta Using Synthetic Cereulide Standard and 13C6-Cereulide Standard—A Short Validation Study

A single laboratory validation study of a rapid and sensitive quantitative method for the analysis of cereulide toxin produced by Bacillus cereus using ultra high performance liquid chromatography-electrospray-tandem mass spectrometry is presented. The analysis of this cyclic peptide toxin was validated for pasta and rice samples using a newly presented synthetic cereulide peptide standard, together with 13C6-cereulide that previously have not been commercially available. The use of cereulide standard was also compared to the most frequently used surrogate standard, the antibiotic valinomycin. The performance of the method was evaluated by analyzing spiked sample pools from different types of rice and pasta, as well as 21 individual rice and pasta samples from differently prepared meals. Inoculation of samples with three cereulide toxin-producing strains of Bacillus cereus was finally used to mimic naturally contaminated foods. The quantification range of the method was 1–500 ng/g (R2 = 0.999) and the limits of detection and quantification were 0.1 and 1 ng/g, respectively. The precision varied from 3% to 7% relative standard deviation and the trueness from −2% to +6% relative bias at different concentration levels in cooked rice and pasta

CE MALDI-TOF/TOF MS for multiplexed quantification of proteins in human ventricular cerebrospinal fluid.

CE, interfaced off-line to MALDI-TOF/TOF MS, was for the first time used to quantitatively monitor the protein content in complex biological and clinical samples with iTRAQ labeling. The usefulness and advantage of iTRAQ labeling, in combination, with CE MALDI-TOF/TOF MS is demonstrated on mixtures of protein standards and by a case study on human ventricular cerebrospinal fluid samples collected from a patient with traumatic brain injury during patient recovery. Mixtures of five standard proteins were initially analyzed to optimize the experimental conditions for the CE MALDI-MS and MS/MS analysis. The interactions of proteins and peptides with the capillary inner wall during CE separation were minimized using PolyE-323 modified capillaries. The analysis of the ventricular cerebrospinal fluid samples yielded 43 significantly (