2D separation and profiling of complex oligosaccharide mixtures

Oligosaccharides present in various natural sources can contain different linkages, susbstitutions and building blocks with the same mass. Oligosaccharide mixtures often contain these isomeric and isobaric structures, which causes their complexity. These components are difficult to separate and analyze with conventional analytical methods. The technique described below was developed to obtain a higher resolution

Correction: Human and Drosophila Cryptochromes Are Light Activated by Flavin Photoreduction in Living Cells

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non–light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated

Human Cryptochrome-1 Confers Light Independent Biological Activity in Transgenic Drosophila Correlated with Flavin Radical Stability

Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism

Comparison of the hydrolytic capacity of Trichoderma reesei QM6a and RUT C30 enzyme mixtures on lignocellulosic substrates

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2D separation and profiling of complex oligosaccharide mixtures

The carbohydrate component of plant cell walls is made up of intricate polymeric structures which, in turn, comprise of a large number of different charged and uncharged monosaccharides. When this natural resource is used for biofuel production, saccharification is often the first step: this is the conversion of the polymeric cell wall polysaccharides to soluble oligo-and monosaccharides which can then be used as feedstock for biofuel-producing micro-organisms. However, to achieve complete saccharification of lignocellulosic biomass, complex cocktails of (mostly) fungal glycosidases have to be used, sofar with a suboptimal efficiency. Therefore, there is a keen interest in identifying the bottlenecks in this saccharification process, which requires comprehensive, high-resolution oligosaccharide mapping technology. These oligosaccharides can contain different isomeric linkages and building blocks with the same mass, which leads to isomeric and isobaric structures that are difficult to separate and analyze with conventional analytical methods. Over the past years, we developed an analytical fingerprinting method that is able to resolve many of these structures and provides adequate sensitivity and throughput to analyze samples from different natural sources. Using standard DNA sequencing equipment, fluorescently labeled carbohydrates are separated by CE and detected using laser-induced fluorescence. We describe here the off-line coupling of this electrophoretic technique to analytical high-performance anion-exchange chromatography (HPAEC). This first dimension separates unlabeled poly- and oligosaccharides based on their size and charge, and collection of these compounds in fractions. In the second dimension, these fractions are analyzed by carbohydrate electrophoresis on a capillary DNA sequencer to obtain a higher resolution, thus yielding a 2D map. To demonstrate the technique, the profiles of a xylan (hemicellulose) hydrolysate and a Belgian beer were analysed. Those fractions that contain oligosaccharides which accumulate over the course of a saccharification, are then available for structural analysis using MS/MS, compositional and linkage analysis

2D separation and profiling of complex oligosaccharide mixtures

Oligosaccharides present in various natural sources can contain different linkages, susbstitutions and building blocks with the same mass. Oligosaccharide mixtures often contain these isomeric and isobaric structures, which causes their complexity. These components are difficult to separate and analyze with conventional analytical methods. The technique described below was developed to obtain a higher resolution

Risks and Benefits of Prophylactic Transfusion before Cholecystectomy in Sickle Cell Disease

Preoperative transfusion (PT) reduces acute postoperative vaso-occlusive events (VOE) in sickle cell disease (SCD), but exposes patients to alloimmunization, encouraging a recent trend towards transfusion sparing. The aim of this study was to investigate the benefit–risk ratio of PT before cholecystectomy on the occurrence of postoperative VOE. Adult SCD patients who underwent cholecystectomy between 2008 and 2019 in our center were included. Patients’ characteristics, collected retrospectively, were compared according to PT. A total of 79 patients were included, 66% of whom received PT. Gallbladder histopathology found chronic cholecystitis (97%) and gallstones (66%). Transfused patients underwent more urgent surgeries and had experienced more painful vaso-occlusive crises (VOC) in the month before surgery (p = 0.05). Four (8.5%) post-transfusion alloimmunizations occurred, and two of them caused a delayed hemolytic transfusion reaction (DHTR) (4.3%). The occurrence of postoperative VOE was similar between the groups (19.2% vs. 29.6%, p = 0.45). Though not statistically significant, a history of hospitalized VOC within 6 months prior to surgery seemed to be associated to postoperative VOE among non-transfused patients (75% vs. 31.6%, p = 0.10). PT before cholecystectomy exposes to risks of alloimmunization and DHTR that could be avoided in some patients. Recent VOCs appear to be associated with a higher risk of postoperative VOE and prompt the preemptive transfusion of these patients