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Background. Red blood cells collected in citrate-phosphate-dextrose can be stored for up to 42 days at 4 degrees C in saline-adenine-glucose-mannitol additive solution. During this controlled, but nevertheless artificial, ex vivo ageing, red blood cells accumulate lesions that can be reversible or irreversible upon transfusion. The aim of the present study is to follow several parameters reflecting cell metabolism, antioxidant defences, morphology and membrane dynamics during storage. Materials and methods. Five erythrocyte concentrates were followed weekly during 71 days. Extracellular glucose and lactate concentrations, total antioxidant power, as well as reduced and oxidised intracellular glutathione levels were quantified. Microvesiculation, percentage of haemolysis and haematologic parameters were also evaluated. Finally, morphological changes and membrane fluctuations were recorded using label-free digital holographic microscopy. Results. The antioxidant power as well as the intracellular glutathione concentration first increased, reaching maximal values after one and two weeks, respectively. Irreversible morphological lesions appeared during week 5, where discocytes began to transform into transient echinocytes and finally spherocytes. At the same time, the microvesiculation and haemolysis started to rise exponentially. After six weeks (expiration date), intracellular glutathione was reduced by 25%, reflecting increasing oxidative stress. The membrane fluctuations showed decreased amplitudes during shape transition from discocytes to spherocytes. Discussion. Various types of lesions accumulated at different chemical and cellular levels during storage, which could impact their in vivo recovery after transfusion. A marked effect was observed after four weeks of storage, which corroborates recent clinical data. The prolonged follow-up period allowed the capture of deep storage lesions. Interestingly, and as previously described, the severity of the changes differed among donors.

Since the discovery and isolation of a synuclein (a syn) from human brains it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that a syn produced in E. Coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in a helical structure (12). However it remains unknown whether or not this putative tetramer is the main physiological form of a syn in the brain. In this study we investigated the oligomeric state of a syn in mouse rat and human brains. To assess the conformational and oligomeric state of native a syn in complex mixtures we generated a syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed a syn to these standards using native and denaturing gel electrophoresis techniques size exclusion chromatography and an oligomer specific ELISA. Our findings demonstrate that both human and rodent a syn expressed in the nervous system exist predominantly as an unfolded monomer. Similar results were observed when human a syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293 HeLa SH SY5Y). Furthermore we show that a syn expressed in E. Coli and purified under denaturing or non denaturing conditions whether as a free protein or as a fusion construct with GST is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that a syn becomes structured upon interaction with other proteins and/or biological membranes.

Electrochemical or photo-electrochemical reactions in both electrospray ionization and laser desorption ionization are discussed stressing the role of the electrode reaction in influencing the ionization process. In particular, upon application of a high voltage during electrospray ionization, the emitter includes a working electrode, where redox reactions are observed, such as electro-generation of benzoquinone and metal ions. In contrast, the target plate in laser-induced desorption ionization also acts as a photo-electrode, especially when modified with a mesoporous semiconductor. We illustrate here how these electrochemical reactions can be used for tagging purposes, and for oxidative or reductive dissociation reactions.

The present invention relates to an apparatus and a method of fabricating the apparatus for dispensing a sample for subsequent analysis by mass spectrometry. The apparatus comprises a polymer substrate (1), in which a covered microchannel (3) has been etched, a wall of the microchannel comprising an electrically conductive material (2) for applying a voltage allowing the sample to be sprayed into a mass spectrometer from the open end (4) of the microchannel. The present application also discloses various applications of the apparatus.

NOVELTY - A plate comprises an electroconductive substrate (1) at least partially covered with sintered nanoparticles (2), deposited as an array of individual spots, for use as a sorbing phase for a sample, and for supporting ionization of sorbed sample molecules covered by or present in an overlayer or matrix. The overlayer or matrix comprises a light absorber and/or a charged carrier acid. USE - Plate is used for matrix-assisted laser desorption ionization mass spectrometry analysis of molecules or reaction products (oxidized or reduced) of molecules, such as oligomers, oligosaccharides and biomolecules e.g. peptides, oligonucleotides (all claimed), proteins, and DNA. Can also be used for tagging reactions, disulfide bridge reductions, and ion source decay reactions. ADVANTAGE - The plate provides a very efficient tool to analyze biomolecules by mass spectrometry and to study the products of photo-induced electron transfer reactions. DETAILED DESCRIPTION - A plate comprises an electroconductive substrate at least partially covered with sintered nanoparticles, deposited as an array of individual spots, for use as a sorbing phase for a sample, and for supporting ionization of sorbed sample molecules covered by or present in an overlayer or matrix. The overlayer or matrix comprises a light absorber and/or a charged carrier acid. The nanoparticles are made of titania, alumina, zinc oxide, silica, ferrous oxide, zirconia, and/or niobium oxide. Or, the nanoparticles are quantum dots, such as cadmium sulfide, cadmium selenide, zinc oxide, or materials which can be photosensitized. The sorbing nanoparticles are derivatized by hydrophobic molecules to specifically bind other hydrophobic molecules, such as peptides. The electroconductive substrate comprises stainless steel, aluminum, nickel, zinc, copper, silicon, tin-indium oxide on glass, or conductive/semi-conductive polymer. INDEPENDENT CLAIMS are included for the following: (1) preparation method of plate; and (2) usage method of plate. DESCRIPTION OF DRAWING(S) - The drawing shows a schematic view of the plate. Electroconductive substrate (1) Sintered nanoparticles (2) Laser (3) Material including molecules from sample (4)

Bloodstream infections rank among the most serious causes of morbidity and mortality in hospitalized patients, partly due to the long period (up to one week) required for clinical diagnosis. In this work, we have developed a sensitive method to quickly and accurately identify bacteria in human blood samples by combining optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and efficient immunoaffinity enrichment/separation. A library of bacteria reference mass spectra at different cell numbers was firstly built. Due to a reduced sample spot size, the reference spectra could be obtained from as few as 10 to 102 intact bacterial cells. Bacteria in human blood samples were then extracted using antibodies-modified magnetic beads for MS fingerprinting. By comparing the sample spectra with the reference spectra based on a cosine correlation, bacteria with concentrations as low as 500 cells per mL in blood serum and 8000 cells per mL in whole blood were identified. The proposed method was further applied to positive clinical blood cultures (BCs) provided by a local hospital, where Escherichia coli and Staphylococcus aureus were identified. Because of the method’s high sensitivity, the BC time required for diagnosis can be greatly reduced. As a proof of concept, whole blood spiked with a low initial concentration (102 or 103 cells per mL) of bacteria was cultured in commercial BC bottles and analysed by the developed method after different BC times. Bacteria were successfully identified after 4 hours of BC. Therefore, an entire diagnostic process could be accurately accomplished within half a day using the newly developed method, which could facilitate the timely determination of appropriate anti-bacterial therapy and decrease the risk of mortality from bloodstream infections.

NOVELTY - The plate has electrically conductive substrate (1) that is covered with light sensitive matrix (2). The matrix comprises light absorber, charge carrier, probe molecule and photo-sensitizer (3) arranged to oxidize probe molecule by irradiation of light from light source (4). The absorber and carrier comprise crystalline acid. The matrix comprises hybrid organic-inorganic gel, and xerogel. USE - Plate for matrix-assisted laser desorption ionization (MALDI) mass spectrometry for analyzing biomolecules such as peptide by structurally determining oxidation products of probe molecules (claimed) and the products of successive reactions involving the oxidation products of the probe molecules, and for screening and evaluation of anti-oxidants and drugs, and studying metabolic pathway in biological processes. ADVANTAGE - The structural determination of oxidation product of probe molecule can be enabled. DETAILED DESCRIPTION - An INDEPENDENT CLAIM is included for method for preparing plate. DESCRIPTION OF DRAWING(S) - The drawing shows the schematic view of photo-reactive MALDI plate. Substrate (1) Light sensitive matrix (2) Photo-sensitizer (3) Light source (4) Portion of matrix (5)