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N-linked glycosylation is a primary source of heterogeneity associated with recombinant monoclonal antibodies and plays a key role in a myriad of drug properties associated with biological function. The glycosylation profile of recombinant monoclonal antibodies is influenced by an array of cell culture inputs which must be carefully controlled in order to engineer the desired glycan distribution. A platform reduced intact mass method applied to monoclonal antibodies has been validated as a quantitative method to monitor the relative mannose-5 level as a surrogate for overall high mannose content in cell culture as a control strategy to ensure product quality and process consistency. The method was shown to be linear, accurate, specific, and precise for an IgG1 and IgG4 mAb allowing relative quantitation of mannose-5 in the range 0.8-11.0% and 1.0-6.2%, respectively. The method can be applied at several stages of the production process from cell culture harvest to drug substance/drug product and is amenable to routine GMP batch testing in a quality control laboratory. Testing upstream during cell culture rather than for product release allows for an earlier assessment of product quality as the glycosylation profile remains unchanged during downstream purification.
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Anticorpos Monoclonais/metabolismo , Proteínas Recombinantes/metabolismo , Glicosilação , Imunoglobulina G/metabolismo , Manose/metabolismo , Modelos BiológicosRESUMO
This work presents a comprehensive, multi-analytical scientific approach for determining the type of lacquer and artistic materials used by Jean Dunand on his work "The Return of the Hunters" (1935). For this purpose, thermally assisted hydrolysis and methylation - gas chromatography/mass spectrometry (THM-GC/MS), optical microscopy (OM) in visible (Vis) and ultraviolet light (UV), and scanning electron microscopy - energy-dispersive X-ray spectroscopy (SEM-EDX) were selected. Furthermore, a novel application of micro attenuated total reflection Fourier transform infrared (µATR-FTIR) spectroscopic mapping by univariate and multivariate analysis was applied for studying the complex lacquer paint stratigraphy. The results show that Vietnamese lacquer was used as a binder, mixed together with linseed oil and pine resins as additives in combination with inorganic pigments, and that shellac was included on the top of the paint; they document an important step in the story of the transfer of Vietnamese lacquer painting techniques to Europe.
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Dielectric barrier discharges (DBDs) have been used as soft ionization sources (DBDI) for organic mass spectrometry (DBDI-MS) for approximately ten years. Helium-based DBDI is often used because of its good ionization efficiency, low ignition voltage, and homogeneous plasma conditions. Argon needs much higher ignition voltages than helium when the same discharge geometry is used. A filamentary plasma, which is not suitable for soft ionization, may be produced instead of a homogeneous plasma. This difference results in N2, present in helium and argon as an impurity, being Penning-ionized by helium but not by metastable argon atoms. In this study, a mixture of argon and propane (C3H8) was used as an ignition aid to decrease the ignition and working voltages, because propane can be Penning-ionized by argon metastables. This approach leads to homogeneous argon-based DBDI. Furthermore, operating DBDI in an open environment assumes that many uncharged analyte molecules do not interact with the reactant ions. To overcome this disadvantage, we present a novel approach, where the analyte is introduced in an enclosed system through the discharge capillary itself. This nonambient DBDI-MS arrangement is presented and characterized and could advance the novel connection of DBDI with analytical separation techniques such as gas chromatography (GC) and high-pressure liquid chromatography (HPLC) in the near future.
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Transcytosis across the blood-brain barrier (BBB) regulates key processes of the brain, but the intracellular sorting mechanisms that determine successful receptor-mediated transcytosis in brain endothelial cells (BECs) remain unidentified. Here, we used Transferrin receptor-based Brain Shuttle constructs to investigate intracellular transport in BECs, and we uncovered a pathway for the regulation of receptor-mediated transcytosis. By combining live-cell imaging and mathematical modeling in vitro with super-resolution microscopy of the BBB, we show that intracellular tubules promote transcytosis across the BBB. A monovalent construct (sFab) sorted for transcytosis was localized to intracellular tubules, whereas a bivalent construct (dFab) sorted for degradation formed clusters with impaired transport along tubules. Manipulating tubule biogenesis by overexpressing the small GTPase Rab17 increased dFab transport into tubules and induced its transcytosis in BECs. We propose that sorting tubules regulate transcytosis in BECs and may be a general mechanism for receptor-mediated transport across the BBB.
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Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Estruturas Citoplasmáticas/metabolismo , Células Endoteliais/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Barreira Hematoencefálica/ultraestrutura , Encéfalo/ultraestrutura , Estruturas Citoplasmáticas/ultraestrutura , Células Endoteliais/ultraestrutura , Feminino , Corantes Fluorescentes/química , Expressão Gênica , Genes Reporter , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transcitose , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusive. Autophagy is an essential catabolic process frequently failing in neurodegeneration. Selective autophagy routes have recently emerged, including nucleophagy, defined as degradation of nuclear components by autophagy. Here, we show that, in a mouse model for the polyglutamine disease dentatorubral-pallidoluysian atrophy (DRPLA), progressive acquirement of an ataxic phenotype is linked to severe cerebellar cellular pathology, characterized by nuclear degeneration through nucleophagy-based LaminB1 degradation and excretion. We find that canonical autophagy is stalled in DRPLA mice and in human fibroblasts from patients of DRPLA. This is evidenced by accumulation of p62 and downregulation of LC3-I/II conversion as well as reduced Tfeb expression. Chronic autophagy blockage in several conditions, including DRPLA and Vici syndrome, an early-onset autolysosomal pathology, leads to the activation of alternative clearance pathways including Golgi membrane-associated and nucleophagy-based LaminB1 degradation and excretion. The combination of these alternative pathways and canonical autophagy blockade, results in dramatic nuclear pathology with disruption of the nuclear organization, bringing about terminal cell atrophy and degeneration. Thus, our findings identify a novel progressive mechanism for the terminal phases of neuronal cell degeneration and death in human neurodegenerative diseases and provide a link between autophagy block, activation of alternative pathways for degradation, and excretion of cellular components.
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Autofagia , Cerebelo/patologia , Lisossomos/metabolismo , Epilepsias Mioclônicas Progressivas/fisiopatologia , Adolescente , Animais , Ataxia , Pré-Escolar , Feminino , Fibroblastos , Humanos , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Epilepsias Mioclônicas Progressivas/genética , FenótipoRESUMO
Although giving bad news at work is a stressful experience, managers are often underprepared for this challenging task. As a solution, we introduce organizational bad news training that integrates (a) principles of delivering bad news from the context of health care (i.e., bad news delivery component), and (b) principles of organizational justice theory (i.e., fairness component). We argue that both the formal and fair delivery of bad news at work can be enhanced with the help of training to mitigate distress both for the messenger and the recipient. We tested the effectiveness of training for the delivery of a layoff as a typical bad news event at work. In 2 studies, we compared the performance of a training group (receiving both components of training) with that of a control group (Study 1, Study 2) and a basics group (receiving the bad news delivery component only; Study 2) during a simulated dismissal notification meeting. In general, the results supported our hypotheses: Training improved the formal delivery of bad news and predicted indicators of procedural fairness during the conversation in both studies. In Study 2, we also considered layoff victims' negativity after the layoff and found that training significantly reduced negative responses. This relationship was fully mediated by layoff victims' fairness perceptions. Despite preparation, however, giving bad news remained a challenging task in both studies. In summary, we recommend that organizations provide managers with organizational bad news training in order to promote professional and fair bad news conversations at work. (PsycINFO Database Record
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Emprego/psicologia , Relações Interpessoais , Gestão de Recursos Humanos/métodos , Ensino , Revelação da Verdade , Adulto , Humanos , Adulto JovemRESUMO
A multi-dielectric-barrier-nano-electrospray ionization (multi-DB-nESI) emitter setup is presented where the emitters are quasi-simultaneously switched to ignite the respective spray solving the problem of coulombic interferences. Since the switching is done electronically, the sprays can be synchronized to the mass spectrometry (MS) ion trap and the resulting mass spectra can be assigned to the corresponding nESI emitter. Graphical Abstract Multi-dielectric-barrier-nano-electrospray in front of a mass spectrometer inlet.
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Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia LíquidaRESUMO
From a tomb in Upper Egypt we isolated a strain of Penicillium chrysogenum that was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some micro-organisms in adverse environments. We tested type strains of Pe. chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. Fourier transform IR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of Pe. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription.
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Melaninas/biossíntese , Penicillium chrysogenum/metabolismo , Tirosina/metabolismo , Cromatografia Líquida , Meios de Cultura/química , DNA Fúngico/química , DNA Fúngico/genética , Egito , Microbiologia Ambiental , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de Massas , Dados de Sequência Molecular , Penicillium chrysogenum/classificação , Penicillium chrysogenum/genética , Penicillium chrysogenum/isolamento & purificação , Análise de Sequência de DNA , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
A collection of 76 synthetic organic pigments was analysed using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). The purpose of this work was to expand the knowledge on synthetic pigments and to assess characteristic pyrolysis products that could help in the identification of these pigments in paint samples. We analysed several classes of synthetic pigments not previously reported as being analysed by this technique: some metal complexes, ß-naphthol pigment lakes, BONA pigment lakes, disazopyrazolone, triarylcarbonium, dioxazine, anthraquinone, indanthrone, isoindoline and thioindigo classes. We also report for the first time the Py-GC/MS analysis of a number of naphthol AS, benzimidazolone, phthalocyanine and perylene pigments and other miscellaneous pigments including pigments with unpublished chemical structure. We successfully used the Py-GC/MS technique for the analysis of paints by artists Clyfford Still and Jackson Pollock to identify the synthetic organic pigments and the binding media.
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The parameters influencing the combination of the dielectric barrier electrospray (DB-ES) with an ion trap mass spectrometer are investigated. Two approaches are presented: the application of different polarity cycles in the DB electrospray high voltage signal and the triggering of it to an output signal received by the mass spectrometer. Both approaches are addressed to improve the detection sensitivity over the sensitivity of conventional nano ES.
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In this work, the combined use of desorption by a continuous wave near-infrared diode laser and ionization by a dielectric barrier discharge-based probe (laser desorption dielectric barrier discharge ionization mass spectrometry (LD-DBDI-MS)) is presented as an ambient ionization method for the mass spectrometric detection of nonvolatile chemicals on surfaces. A separation of desorption and ionization processes could be verified. The use of the diode laser is motivated by its low cost, ease of use, and small size. To achieve an efficient desorption, the glass substrates are coated at the back side with a black point (target point, where the sample is deposited) in order to absorb the energy offered by the diode laser radiation. Subsequent ionization is accomplished by a helium plasmajet generated in the dielectric barrier discharge source. Examples on the application of this approach are shown in both positive and negative ionization modes. A wide variety of multiclass species with low vapor pressure were tested including pesticides, pharmaceuticals and explosives (reserpine, roxithromycin, propazine, prochloraz, spinosad, ampicillin, dicloxacillin, enrofloxacin, tetracycline, oxytetracycline, erythromycin, spinosad, cyclo-1,3,5,7-tetramethylene tetranitrate (HMX), and cyclo-1,3,5-trimethylene trinitramine (RDX)). A comparative evaluation revealed that the use of the laser is advantageous, compared to just heating the substrate surface.
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Substâncias Explosivas/análise , Lasers Semicondutores , Praguicidas/análise , Preparações Farmacêuticas/análise , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
The nutrient-responsive ß-O-linked N-acetylglucosamine (O-GlcNAc) modification of critical effector proteins modulates signaling and transcriptional pathways contributing to cellular development and survival. An elevation in global protein O-GlcNAc modification occurs during the early stages of osteoblast differentiation and correlates with enhanced transcriptional activity of RUNX2, a key regulator of osteogenesis. To identify other substrates of O-GlcNAc transferase in differentiating MC3T3E1 osteoblasts, O-GlcNAc-modified peptides were enriched by wheat germ agglutinin lectin weak affinity chromatography and identified by tandem mass spectrometry using electron transfer dissociation. This peptide fragmentation approach leaves the labile O-linkage intact permitting direct identification of O-GlcNAc-modified peptides. O-GlcNAc modification was observed on enzymes involved in post-translational regulation, including MAST4 and WNK1 kinases, a ubiquitin-associated protein (UBAP2l), and the histone acetyltransferase CREB-binding protein. CREB-binding protein, a transcriptional co-activator that associates with CREB and RUNX2, is O-GlcNAcylated at Ser-147 and Ser-2360, the latter of which is a known site of phosphorylation. Additionally, O-GlcNAcylation of components of the TGFß-activated kinase 1 (TAK1) signaling complex, TAB1 and TAB2, occurred in close proximity to known sites of Ser/Thr phosphorylation and a putative nuclear localization sequence within TAB2. These findings demonstrate the presence of O-GlcNAc modification on proteins critical to bone formation, remodeling, and fracture healing and will enable evaluation of this modification on protein function and regulation.
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Acetilgalactosamina/metabolismo , Glicoproteínas/metabolismo , Osteoblastos/metabolismo , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem/métodos , Acetilgalactosamina/química , Sequência de Aminoácidos , Animais , Configuração de Carboidratos , Células Cultivadas , Cromatografia de Afinidade , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Osteogênese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteoma/química , Proteoma/isolamento & purificação , Proteoma/metabolismoRESUMO
The mechanism which leads to a dielectric barrier electrospray (DB-ES) was investigated by measurements of the electrical current. It comprises several components, namely, the displacement current, currents which are initiated by electromagnetic transmission, an ion current inside the capillary, which is responsible for the built-up of the potential at the tip, and the electrospray current as a result of the displacement current and the ion transfer current. An augmented interpretation of the current signal of the DB-ES is presented, and features are described in more detail. The behavior of the ions inside the capillary and the charge transfer are described from the chemical and the electrical point of view.
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A novel electrospray interface is presented which induces an electric field by dielectric polarization through a non-conductive barrier. Therefore, a square-wave high-voltage signal is applied. This technique allows mass spectrometric measurements in the positive as well as in the negative mass spectrometry mode without changing the polarity of the potential applied, and it decreases the risk of undesired discharges, induced by high electric currents. The applicability of this technique is demonstrated by mass spectrometric determination of reserpine.
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Cellulose acetate, developed about 100 years ago as a versatile, semisynthetic plastic material, is used in a variety of applications and is perhaps best known as the basis of photographic film stock. Objects made wholly or partly from cellulose acetate are an important part of modern and contemporary cultural heritage, particularly in museum collections. Given the potential instability of the material, however, it is imperative to understand the aging mechanisms and deterioration pathways of cellulose ester plastics to mitigate decomposition and formulate guidelines for storage, exhibition, and conservation. One important aspect of this process is the ability to fully characterize the plastic, because variations in composition affect its aging properties and ultimate stability. In this Account, we assess the potential of a range of analytical techniques for plastics made from cellulose acetate, cellulose propionate, and cellulose butyrate. Comprehensive characterization of cellulose ester plastics is best achieved by applying several complementary analytical techniques. Fourier-transform IR (FTIR) and Raman spectroscopy provide rapid means for basic characterization of plastic objects, which can be useful for quick, noninvasive screening of museum collections with portable instruments. Pyrolysis GC/MS is capable of differentiating the main types of cellulose ester polymers but also permits a richly detailed compositional analysis of additives. Thermal analysis techniques provide a wealth of compositional information and thermal behavior. Thermogravimetry (TG) allows for quantitative analysis of thermally stable volatile additives, and weight-difference curves offer a novel means for assessing oxidative stability. The mechanical response to temperature, such as the glass transition, can be measured with dynamic mechanical analysis (DMA), but results from other thermal analysis techniques such as TG, differential scanning calorimetry (DSC), and dynamic load thermomechanical analysis (DLTMA) are often required to more accurately interpret the results. The analytical results from this study form the basis for in-depth studies of works of art fabricated from cellulose acetate. These objects, which are particularly at risk when stored in tightly sealed containers (as is often the case with photographic film), warrant particular attention for conservation given their susceptibility toward sudden onset of deterioration.
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Immobilized metal affinity chromatography (IMAC) based on Fe (3+) or Ga (3+) chelation is the most widely employed technique for the enrichment of phosphopeptides from biological samples prior to mass spectrometric analysis. An IMAC resin geared mainly toward phosphoprotein enrichment, Pro-Q Diamond, has been assessed for its utility in phosphopeptide isolation. Using both single phosphoprotein tryptic digests of beta-casein and ovalbumin and synthetic mixtures composed of tryptic digests of phosphorylated and nonphosphorylated protein standards, the selectivity and sensitivity of Pro-Q Diamond resin in an immobilized metal affinity-reversed phase microcolumn format were compared to an alternate titanium dioxide approach. The biphasic microcolumn method was found to be superior to metal oxide-based phosphopeptide capture in biological samples of increasing complexity. The lower limit of mass spectrometric detection for the immobilized metal affinity-reversed phase microcolumn approach was determined to be 10 pmol of beta-casein monophosphorylated peptide in 20 microL of a solution of human serum protein digest (from 200 microg total serum protein after digestion and desalting).
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Cromatografia de Afinidade/métodos , Metais/química , Fosfopeptídeos/química , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Humanos , Dados de Sequência Molecular , Padrões de Referência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A non-conductive piezo ceramic plate has been used to induce an electric field to generate an electrospray as ionization method for mass spectrometric determination. This technique decreases the risk of undesired discharges, induced by high electric currents. The applicability of the technique is demonstrated and compared with a commercial electrospray for mass spectrometric determination of reserpine and myoglobin.
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Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Cerâmica/química , Mioglobina/análise , Reserpina/análiseRESUMO
For the first time, we report a miniaturized approach for isotachophoresis employing the technique of free-flow electrophoresis. Using a micromachined separation chamber with a volume of 200 nL, a sample mixture of fluorescein, eosin G, and acetylsalicylic acid was separated, stacked, and concentrated in less than a minute. Additionally, an isotachophoretic separation of a reaction mixture of myoglobin and fluoresceinisothiocyanate as a fluorescence label has shown the potential of this method for on-line sample preparation.
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The electrostatic induction of an applied voltage causes electrophoretic separation under free-flow conditions and no electrolysis or electric current flowing between the metal electrodes was observed.