Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer’s disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and ...behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.
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•Astrocytes have impaired glycolytic flux in a mouse model of Alzheimer’s disease•Consequently, astrocytes produce less glycolysis-derived l-serine•Low NMDAR occupancy by d-serine leads to impairment of synaptic plasticity and memory•Dietary supplementation of l-serine restores synaptic plasticity and memory
Le Douce et al. show that glycolysis is impaired in astrocytes in the early stages of disease in a mouse model of Alzheimer’s. This leads to the reduction of both l- and d-serine synthesis and to the alteration of synaptic plasticity and memory. Dietary supplementation with l-serine restores both deficits, suggesting it to be a potential therapy.
Human serum albumin (HSA) has been used for a long time as a resuscitation fluid in critically ill patients. It is known to exert several important physiological and pharmacological functions. Among ...them, the antioxidant properties seem to be of paramount importance as they may be implied in the potential beneficial effects that have been observed in the critical care and hepatological settings. The specific antioxidant functions of the protein are closely related to its structure. Indeed, they are due to its multiple ligand-binding capacities and free radical-trapping properties. The HSA molecule can undergo various structural changes modifying its conformation and hence its binding properties and redox state. Such chemical modifications can occur during bioprocesses and storage conditions of the commercial HSA solutions, resulting in heterogeneous solutions for infusion. In this review, we explore the mechanisms that are responsible for the specific antioxidant properties of HSA in its native form, chemically modified forms, and commercial formulations. To conclude, we discuss the implication of this recent literature for future clinical trials using albumin as a drug and for elucidating the effects of HSA infusion in critically ill patients.
The use of magnetic beads bio-functionalized by antibodies (Ab) is constantly increasing with a wide range of biomedical applications. However, despite an urgent need for current methods to monitor ...Ab’s grafting process and orientation, existing methods are still either cumbersome and/or limited. In this work, we propose a new simple and rapid analytical approach to evaluate antibody orientation and density on magnetic beads. This approach relies on the cleavage by
IdeS
, a highly specific protease for human immunoglobulin G (hIgG), of immobilized antibodies. The F(ab)
2
and Fc fragments could be then accurately quantified by size exclusion chromatography (SEC)-coupled to fluorescent detection (FLD), and the ratio of these fragments was used to give insight on the IgG orientation at the bead surface. Four different commercially available magnetic beads, bearing carboxyl groups, tosyl groups, streptavidin, or protein G on their surface have been used in this study. Results obtained showed that this approach ensures reliable information on hIgG orientation and bead surface coverage. Protein G magnetic beads demonstrated an optimal orientation of antibodies for antigen capture (75% of accessible F(ab)
2
fragment) compared to tosylactivated, carboxylated, and streptavidin ones. Capture efficiency of the different functionalized beads towards human TNF-α immunocapture, a biomarker of inflammation, has been also compared. Protein G beads provided a more efficient capture compared to other beads. In the future, this approach could be applied to any type of surface and beads to assess hIgG coverage and orientation after any type of immobilization.
Graphical abstract
A rapid and simple approach to evaluate orientation and density of antibodies immobilized on magnetic beads
It is reported herein a new approach to study the orientation and density of mouse antibody 27 grafting on magnetic beads, serving for immunoassays and biosensors with fluorescent 28 detection of ...biomolecules. This approach is based on selective enzymatic digestion of target 29 grafted antibodies at a specific site below the hinge position to provide F(ab’)2 and Fc 30 fragments, followed by separation and determination of these fragments with size exclusion 31 chromatography (SEC) coupled with fluorescence detection (FLD). The developed method was 32 applied for evaluation of immunoglobulin (IgG2a) grafting capacity on three different 33 biofunctionalized magnetic beads (i.e., Tosyl-activated, carboxylic, protein G). Tosyl-activated 34 and protein G beads at different optimal grafting IgG: bead ratios (i.e.,110 μg: 1000 μg and 240 35 μg: 1000 μg, respectively) exhibited superior grafting capacity than carboxylic counterparts. 36 Under the optimized conditions, more than 70 % of antibodies were grafted on tosyl-activated 37 and protein G beads in the right orientation. This approach was then demonstrated with different 38 commercially available antibodies specific to amyloid-beta peptide 1-42 (Aβ1-42) for magneto-39 immunoassays and fluorescent detection of this peptide that is an established biomarker for 40 molecular diagnosis of Alzheimer’s disease.
It is reported in this study a new approach for modulation and even suppression of the electroosmotic flow (EOF) to achieve better electrokinetic preconcentration in capillary electrophoresis. This ...is based on the augmentation of the buffer’s concentrations to very high levels (more than a thousand of mM) without recourse to any dynamic/permanent coating nor viscous gel. The use of large weakly charged molecules as background electrolyte’s constituents allows working at extreme concentration ranges without penalty of high electric currents and Joule heating. By this way, the electroosmotic mobility could be modulated over a wide range (2–60 × 10−5 cm2 V−1 s−1 under alkaline conditions), and suppressed to levels equivalent to those obtained with several neutral coatings. The highest buffer concentrations, and the lowest EOF magnitudes, accordingly, were achieved with diethanolamine/3-(Cyclohexylamino)-1-propanesulfonic acid (ionic strength (IS) of 250 mM, pH 9.5), Tris(hydroxymethyl)aminomethane (Tris)/2-(Cyclohexylamino)ethanesulfonic acid (CHES) (IS of 280 mM, pH 8.7) and triethanolamine/2-(Cyclohexylamino)ethanesulfonic acid (IS of 250 mM, pH 8.5). For demonstration, this new approach was applied for sensitive determination of core-shell magnetic nanoparticles (CSMNPs) having high potential for healthcare applications such as imaging agents for diagnostics and controllable cargos for nanomedicine. Different profiles were achieved for purpose-made and commercial magnetic nanoparticles using CE coupled with light-emitting-diode induced fluorescence (LEDIF) detection. The best performance for EOF-assisted preconcentration and CE-LEDIF of CSMNPs was achieved with these nanoparticles prepared in TRIS/CHES (IS 10 mM, pH 8.4) for preconcentration, and separation under BGE of TRIS/CHES (IS 100 mM, pH 8.4). Compared to the conventional capillary electrophoresis (CE-UV) method for characterization of magnetic nanoparticles, our proposed approach with fluorescent detection and EOF-assisted preconcentration offers almost 350-fold sensitivity improvement. Furthermore, our scheme can be used for monitoring the interaction between CSMNPs and target pharmaceutical molecules, serving for drug delivery development. A preliminary study with two antibiotics using this approach revealed that kanamycin interacts better with the target nanoparticles than amikacin.
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•EOF suppression and modulation in silica capillaries without recourse to coating or gel is reported.•A new principle of EOF-assisted electrokinetic preconcentration for CE-LIF is proposed.•The first method for CE-LIF of core-shell magnetic nanoparticles (CSMNPs) was developed.•Detection and interaction study of CSMNPs could be realized with a 350-fold sensitivity improvement.
Determination of proteins and peptides is among the main challenges of today's bioanalytical chemistry. The application of microchip technology in this field is an exhaustively developed concept that ...aims to create integrated and fully automated analytical devices able to quantify or detect one or several proteins from a complex matrix. Selective extraction and preconcentration of targeted proteins and peptides especially from biological fluids is of the highest importance for a successful realization of these microsystems. Incorporation of solid structures or supports is a convenient solution employed to face these demands. This review presents a critical view on the latest achievements in sample processing techniques for protein determination using solid supports in microfluidics. The study covers the period from 2006 to 2015 and focuses mainly on the strategies based on microbeads, monolithic materials and membranes. Less common approaches are also briefly discussed. The reviewed literature suggests future trends which are discussed in the concluding remarks.
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•Miniaturization offers unique opportunities for extraction and preconcentration.•Solid supports include microbeads, monoliths and membranes.•High performances are achieved via the synergy of microfluidics and solid supports.•Integrated microsystems enable analysis of complex samples.
Type 2 diabetes is characterized by the aggregation of human islet amyloid polypeptide (hIAPP), from monomer to amyloid deposits that are made of insoluble fibrils. Discrepancies concerning the ...nature of formed species or oligomerization kinetics among reported in vitro studies on hIAPP aggregation process have been highlighted. In this work, we investigated if the sample itself could be at the origin of those observed differences. To this aim, four hIAPP samples obtained from three different sources or suppliers have been analyzed and compared by ThT fluorescence spectroscopy and by two recently developed techniques, capillary electrophoresis (CE), and ESI-IMS-QToF-MS. Lots provided by the same supplier were shown to be very similar whatever the analytical technique used to characterize them. In contrast, several critical differences could be pointed out for hIAPP provided by different suppliers. We demonstrated that in several samples, some oligomerized peptides (e.g., dimer) were already present upon reception. Purity was also different, and the proneness of the peptide solution to form fibrils in vitro within 24 h could vary considerably from one sample source to another but not from lot to lot of the same source. All those results demonstrate that the initial state of conformation, oligomerization, and quality of the hIAPP can greatly impact the aggregation kinetics, and thus the information provided by these in vitro tests. Finally, a careful selection of the peptide batch and source is mandatory to perform relevant in vitro studies on hIAPP oligomerization and to screen new molecules modulating this pathological process.
Graphical abstract
•For the first time, a CZE method was developed to analyze ApoC-III glycoforms.•Carbamylated ApoC-III forms were highlighted and carbamylation sites were identified.•Carbamylation was evidenced in ...ApoC-III batches from 3 distinct commercial sources.•Carbamylated and sialylated forms of ApoC-III can be efficiently separated by CZE.
ApolipoproteinC-III (ApoC-III) is a human plasma glycoprotein whose O-glycosylation can be altered as a result of congenital disorders of glycosylation (CDG). ApoC-III exhibits three major glycoforms whose relative quantification is of utmost importance for the diagnosis of CDG patients. Considering the very close structures of these glycoforms and their tendency to adsorb on the capillary, a thorough optimization of capillary electrophoresis (CE) parameters including preconditioning and in-between rinsing procedures was required to efficiently separate all the ApoC-III glycoforms. Permanent coatings did not contribute to high resolution separations. A fast and reliable method based on a bare-silica capillary combining the effect of urea and diamine additives allowed to separate up to six different ApoC-III forms. We demonstrated by a combination of MALDI-TOF mass spectrometry (MS) analyses and CE of intact and neuraminidase-treated samples that this method well resolved glycoforms differing not only by their sialylation degree but also by carbamylation state, an undesired chemical modification of primary amines. This method allowed to demonstrate the carbamylation of ApoC-III glycoforms for the first time. Our CZE method proved robust and accurate with excellent intermediate precision regarding migration times (RSDs < 0.7%) while RSDs for peak areas were less than 5%. Finally, the quality of three distinct batches of commercial ApoC-III obtained from different suppliers was assessed and compared. Quite similar but highly structurally heterogeneous ApoC-III profiles were observed for these samples.
We report an improved CE method to monitor in vitro the self‐assembly of monomeric amyloid β‐peptide (42 amino acids amyloid β‐peptide, Aβ1–42) and in particular the crucial early steps involved in ...the formation of the neurotoxic oligomers. In order to start the kinetics from the beginning, sample preparation was optimized to provide samples containing exclusively the monomeric form. The CE method was also improved using a dynamic coating and by reducing the separation distance. Using this method, the disappearance of the monomer as well as the progressive formation of four species during the self‐assembly process can now be monitored and quantified over time. The hydrodynamic radius of the species present at the initial kinetics step was estimated around 1.8 nm by Taylor dispersion analysis while SDS‐PAGE analyses showed the predominance of the monomer. These results confirmed that the Aβ1–42 species present at this initial time was the monomer. Methylene blue, an anti‐Alzheimer disease candidate, was then evaluated. In spite of an oligomerization inhibition, the enhanced disappearance of the Aβ1–42 monomer provoked by methylene blue was demonstrated for the first time. This method, allowing the monomeric and smallest oligomeric species to be monitored, represents a new accurate and precise way to evaluate compounds for drug discovery.
A microdevice combining online preconcentration and separation of phosphopeptides was developed in a glass microchip. An ethylene glycol methacrylate phosphate (EGMP), acrylamide (AM) and ...bisacrylamide (BAA) based monolith was synthesized within microchannels through a photo-driven process. Morphological investigations revealed a homogeneous monolithic structure composed of uniform nodules (∼0.8 μm), with a large pore volume (0.62 cm
g
) and sufficiently high specific surface area (34.1 m
g
). These features make the monolith particularly interesting for preconcentration purposes. Immobilization of Zr
ions on the phosphate groups present at the poly(EGMP-co-AM-co-BAA) monolith surface leads to immobilized metal affinity chromatography support. This monolith-Zr
showed a great capacity to capture phosphopeptides. Successful preconcentration and separation of a mixture of ERK2 derived peptides differing only by their phosphorylation degree and sites could be achieved with signal enhancement factors between 340 and 910 after only 7 min of preconcentration. This integrated microdevice represents a novel approach for phosphoproteomic applications.
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