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1.
J Psychiatr Res ; 144: 102-109, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34610513

RESUMO

AIMS: Interventions to decrease readmissions in psychiatric patients are urgently needed. In Switzerland therapeutic leave (TL) composes a cornerstone of inpatient treatment. TL is a planned time-limited absence from the inpatient ward giving patients the opportunity to test their resilience in their usual environment. Evidence of its applicability as an intervention reducing readmissions is lacking. Therefore, our objective was to examine the association between TL and readmission risk. METHODS: Using the Kaplan-Meier curve we compared the time to readmission of 3'302 inpatients at the UPK Basel with and without TL. Cox regression was applied, integrating other covariates associated with readmission. RESULTS: The Kaplan-Meier curve indicated longer cumulative survival in patients with TL. The log-rank test implied statistical significance (χ2(1) = 18.8, p < .05). The Cox regression showed a reduced hazard for patients with TL (HR = 0.735, CI 95% = [0.639, 0.846], p < .001) and for involuntarily hospitalized patients (HR = 0.760, CI 95% = [0.618, 0.934], p < .01). A higher readmission risk was found for a history of psychiatric admissions (HR = 1.005, CI 95% = [1.004, 1.005], p < .001), higher severity of symptoms at admission (HR = 1.029, CI 95% = [1.018, 1.040], p < .001), comorbidity (HR = 1.178, CI 95% [1.024, 1.355], p = .022), and a diagnosis with schizophrenia-spectrum disorders (HR = 1.401, CI 95% [1.164, 1.687], p = .001). CONCLUSION: Linking TL with readmission risk, our results imply an easy way to improve quality of care, with possible implications for practice, policies and quality interventions. TL might be suitable to enhance recovery, reduce readmissions and health care costs. RCTs are needed for validation.

2.
Artigo em Inglês | MEDLINE | ID: mdl-34444055

RESUMO

Social distancing and the shortage of healthcare professionals during the COVID-19 pandemic, the impact of population aging on the healthcare system, as well as the rapid pace of digital innovation are catalyzing the development and implementation of new technologies and digital services in psychiatry. Is this transformation a blessing or a curse for psychiatry? To answer this question, we conducted a literature review covering a broad range of new technologies and eHealth services, including telepsychiatry; computer-, internet-, and app-based cognitive behavioral therapy; virtual reality; digital applied games; a digital medicine system; omics; neuroimaging; machine learning; precision psychiatry; clinical decision support; electronic health records; physician charting; digital language translators; and online mental health resources for patients. We found that eHealth services provide effective, scalable, and cost-efficient options for the treatment of people with limited or no access to mental health care. This review highlights innovative technologies spearheading the way to more effective and safer treatments. We identified artificially intelligent tools that relieve physicians from routine tasks, allowing them to focus on collaborative doctor-patient relationships. The transformation of traditional clinics into digital ones is outlined, and the challenges associated with the successful deployment of digitalization in psychiatry are highlighted.


Assuntos
Psiquiatria , Telemedicina , COVID-19 , Humanos , Pandemias , Psiquiatria/tendências , Telemedicina/tendências
3.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445776

RESUMO

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.


Assuntos
Anticorpos Monoclonais/química , Antineoplásicos Imunológicos/química , Cátions/química , Rituximab/química , Medicamentos Biossimilares/química , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Glicosilação , Espectrometria de Massas/métodos
4.
Environ Pollut ; 286: 117571, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34438494

RESUMO

Silver nanomaterials (AgNMs) are broadly used and among the most studied nanomaterials. The underlying molecular mechanisms (e.g. protein and metabolite response) that precede phenotypical effects have been assessed to a much lesser extent. In this paper, we assess differentially expressed proteins (DEPs) and metabolites (DEMs) by high-throughput (HTP) techniques (HPLC-MS/MS with tandem mass tags, reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) with mass spectrometric detection). In a time series (0, 7, 14 days), the standard soil model Enchytraeus crypticus was exposed to AgNM300K and AgNO3 at the reproduction EC20 and EC50. The impact on proteins/metabolites was clearly larger after 14 days. NM300K caused more upregulated DEPs/DEMs, more so at the EC20, whereas AgNO3 caused a dose response increase of DEPs/DEMs. Similar pathways were activated, although often via opposite regulation (up vs down) of DEPs, hence, dissimilar mechanisms underlie the apical observed impact. Affected pathways included e.g. energy and lipid metabolism and oxidative stress. Uniquely affected by AgNO3 was catalase, malate dehydrogenase and ATP-citrate synthase, and heat shock proteins (HSP70) and ferritin were affected by AgNM300K. The gene expression-based data in Adverse Outcome Pathway was confirmed and additional key events added, e.g. regulation of catalase and heat shock proteins were confirmed to be included. Finally, we observed (as we have seen before) that lower concentration of the NM caused higher biological impact. Data was deposited to ProteomeXchange, identifier PXD024444.


Assuntos
Nanopartículas Metálicas , Nanoestruturas , Poluentes do Solo , Íons , Metabolômica , Nanopartículas Metálicas/toxicidade , Nanoestruturas/toxicidade , Proteômica , Prata/toxicidade , Poluentes do Solo/análise , Espectrometria de Massas em Tandem , Transcriptoma
5.
Anal Chem ; 93(30): 10424-10434, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34288669

RESUMO

Modern analytical approaches employing high-resolution mass spectrometry (MS) facilitate the generation of a vast amount of structural data of highly complex glycoproteins. Nevertheless, systematic interpretation of this data at different structural levels remains an analytical challenge. The glycoprotein utilized as a model system in this study, human chorionic gonadotropin (hCG), exists as a heterodimer composed of two heavily glycosylated subunits. In order to unravel the multitude of glycoforms of recombinant hCG (drug product Ovitrelle), we combine established techniques, such as released glycan and glycopeptide analysis, with novel approaches employing high-performance liquid chromatography-mass spectrometry (HPLC-MS) to characterize protein subunits and native MS to analyze the noncovalent hCG complex. Starting from the deconvoluted mass spectrum of dimeric hCG comprising about 50 signals, it was possible to explore the chemical space of hCG glycoforms and elucidate the complexity that hides behind just 50 signals. Systematic, stepwise integration of data obtained at the levels of released glycans, glycopeptides, and subunits using a computational annotation tool allowed us to reveal 1031 underlying glycoforms. Additionally, critical quality attributes such as sialylation and core fucosylation were compared for two batches of Ovitrelle to assess the potential product variability.


Assuntos
Gonadotropina Coriônica , Expedições , Glicopeptídeos , Glicosilação , Humanos , Espectrometria de Massas , Polissacarídeos
6.
JMIR Ment Health ; 8(9): e28849, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34115606

RESUMO

BACKGROUND: During the COVID-19 pandemic in 2020, psychiatric hospitals all over the world had to adapt their services to the prevailing governmental regulations. As a consequence, home office use and telepsychiatry boomed. OBJECTIVE: The purpose of this study was to evaluate the potential of home office use, its adoption, and the association of home office use with employees' mental health in a large psychiatric university hospital in Switzerland. METHODS: We obtained and analyzed home office implementation and use data from the psychiatric university hospital's information technology services. We also conducted a cross-sectional web-based survey to assess the employees' attitudes toward the clinic's crisis management during the COVID-19 pandemic in early 2020. Part of this web-based survey consisted of questions about home office use between March and June 2020, attitudes toward home office implementation, and mental health. Three mental health measures assessed depressive symptoms (Patient Health Questionnaire [PHQ]-2), anxiety (General Anxiety Disorder [GAD]-2), and stress factors (stress module of the PHQ-D); a cut-off score ≥3 was used for the PHQ-2 and GAD-2. RESULTS: Of the 200 participating employees, 69 reported that they had worked from home at least partially (34.5%). Home office use differed significantly across professional groups (χ162=72.72, P≤.001, n=200). Employees experienced neither depressive symptoms (mean 0.76, SD 1.14) nor anxiety (mean 0.70, SD 1.03). The employees reported minor psychosocial stressors (mean 2.83, SD 2.92). The number of reported stress factors varied significantly across groups with different levels of home office use (χ42=9.72, P=.04). CONCLUSIONS: In general, home office implementation appears to be feasible for large psychiatric hospitals, however, it is not equally feasible for all professional groups. Professional groups that require personal contact with patients and technical or manual tasks must work onsite. Further evaluation of home office use in psychiatric hospitals up to the development of clinics that function merely online will follow in future research. The situation created by the COVID-19 pandemic served as a stepping stone to promote home office use and should be used to improve employees' work-life balance, to save employers costs and foster other benefits.

7.
J Biomol NMR ; 75(1): 71-82, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33475951

RESUMO

The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pKa values of isoAsp and C-terminal Asp in short peptides. The characteristic 1H-13C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4-5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.


Assuntos
Ácido Aspártico/química , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Sequência de Aminoácidos , Asparagina/química , Isomerismo , Peptídeos/química , Relação Estrutura-Atividade
8.
Front Psychiatry ; 11: 580885, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33192724

RESUMO

Open doors in psychiatry have been a subject of controversy in recent years. While some studies postulate the clinical necessity of closed doors, others challenge the theoretical advantages of this setting, mention numerous drawbacks of closed wards, and focus on the advantages of open-door settings. With regard to patients diagnosed with substance use disorders (SUD), other standards may apply. Very little research has been done on this topic. Some studies adopted a consumer perspective (i.e. asking involved parties about their experience of the door status). To the authors' knowledge, no study has so far addressed the ideal setting for the treatment of SUD. With our data from the opening of a specialized SUD ward, we take one step to closing this knowledge gap. Applying a qualitative design, we asked patients and health care professionals (HCP) to report changes following the opening of the ward. The results are mainly in line with the literature on the general psychiatric population. The newly introduced open-door setting was mostly perceived as positive, but some disadvantages were mentioned (e.g. less protection of patients, less control over who enters/leaves the ward, the theoretically increased risk of patients absconding). Moreover, HCP (but not patients) mentioned potentially increased substance use on the ward as an additional disadvantage that could arise. Opening a previously closed ward was generally perceived as a positive and progressive decision. These findings support the trend towards an overall open-door policy in psychiatry.

9.
Sci Rep ; 10(1): 19052, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33149258

RESUMO

Flagellins are the protein components of bacterial flagella and assemble in up to 20,000 copies to form extracellular flagellar filaments. An unusual family of flagellins was recently discovered that contains a unique metalloprotease domain within its surface-exposed hypervariable region. To date, these proteolytic flagellins (also termed flagellinolysins) have only been characterized in the Gram-positive organism Clostridium haemolyticum, where flagellinolysin was shown to be proteolytically active and capable of cleaving extracellular protein substrates. The biological function of flagellinolysin and its activity in other organisms, however, remain unclear. Here, using molecular biochemistry and proteomics, we have performed an initial characterization of a novel flagellinolysin identified from Hylemonella gracilis, a Gram-negative organism originally isolated from pond water. We demonstrate that H. gracilis flagellinolysin (HgrFlaMP) is an active calcium-dependent zinc metallopeptidase and characterize its cleavage specificity profile using both trypsin and GluC-derived peptide libraries and protein substrates. Based on high-throughput degradomic assays, HgrFlaMP cleaved 784 unique peptides and displayed a cleavage site specificity similar to flagellinolysin from C. haemolyticum. Additionally, by using a set of six protein substrates, we identified 206 protein-embedded cleavage sites, further refining the substrate preference of HgrFlaMP, which is dominated by large hydrophobic amino acids in P1', and small hydrophobic or medium-sized polar residues on the amino-terminal side of the scissile bond. Intriguingly, recombinant HgrFlaMP was also capable of cleaving full-length flagellins from another species, suggesting its potential involvement in interbacterial interactions. Our study reports the first experimentally characterized proteolytic flagellin in a Gram-negative organism, and provides new insights into flagellum-mediated enzymatic activity.


Assuntos
Comamonadaceae/metabolismo , Flagelina/metabolismo , Água Doce/microbiologia , Microbiologia da Água , Aminoácidos , Comamonadaceae/classificação , Comamonadaceae/genética , Flagelina/genética , Genoma Bacteriano , Fases de Leitura Aberta , Filogenia , Proteólise , Proteoma , Proteômica/métodos , Especificidade por Substrato
10.
PLoS One ; 15(11): e0241560, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33151990

RESUMO

The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8-86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals.


Assuntos
Anticorpos/imunologia , Aptâmeros de Nucleotídeos/metabolismo , Temperatura Alta , Rituximab/farmacologia , Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Simulação por Computador , Humanos , Conformação de Ácido Nucleico , Técnica de Seleção de Aptâmeros
11.
Sci Rep ; 10(1): 18080, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093535

RESUMO

In recent years, advanced HPLC-MS strategies based on intact protein ("top-down") or protein subunit ("middle-up/middle-down") analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.


Assuntos
Anticorpos/imunologia , Antígenos de Plantas/imunologia , Cromatografia Líquida de Alta Pressão/métodos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/classificação , Imunoglobulina G/imunologia , Espectrometria de Massas/métodos , Alérgenos/imunologia , Animais , Feminino , Glicosilação , Fragmentos Fc das Imunoglobulinas/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Vacinação
12.
Front Immunol ; 11: 1824, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013833

RESUMO

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from -5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.


Assuntos
Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Apresentação do Antígeno/imunologia , Simulação por Computador , Ativação Linfocitária/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Animais , Células Dendríticas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação Puntual , Dobramento de Proteína , Estabilidade Proteica , Linfócitos T/imunologia
13.
J Biol Chem ; 2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037072

RESUMO

Identification of antibody binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome that is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort is frequently involved in this cross-reactivity, but no antibody binding epitopes have been determined so far.  To reveal human IgE binding regions of Art v 3, we produced three murine high-affinity monoclonal antibodies (mAbs), which showed 70-90% coverage of the allergenic epitopes from mugwort pollen allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4, as well as a novel Art v 3-specific epitope at the C-terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP-allergy and facilitates design of therapeutics.

14.
Pathog Dis ; 78(7)2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866262

RESUMO

Persistent infections with the bacterial group-I carcinogen Helicobacter pylori (H. pylori) have been associated with a broad range of gastric disorders, including gastritis, ulceration, gastric cancer or mucosa-associated lymphoid tissue (MALT) lymphoma. Pathogenesis of H. pylori requires a balance between immune tolerance and defense. Although H. pylori induces inflammatory responses, the immune system cannot eliminate the pathogen. The detailed molecular mechanisms of how H. pylori interferes with cells of the immune system, in particular infiltrated B cells, are not well investigated. Previously, it was shown that the bacterial effector and oncoprotein cytotoxin-associated gene A (CagA) is delivered into B cells followed by its tyrosine-phosphorylation. To investigate the functional consequences in B cells colonized by CagA-positive H. pylori, we analyzed the global transcriptome of H. pylori-infected Mec-1 cells by RNA sequencing. We found 889 differentially expressed genes (DEGs) and validated JUN, FOSL2, HSPA1B, SRC, CXCR3, TLR-4, TNF-α, CXCL8, CCL2, CCL4, MHC class I and MHC class II molecules by qPCR, western blot, flow cytometry and ELISA assays. The H. pylori-specific mRNA expression signature reveals a downregulation of inflammation- and migration-associated genes, whereas central signal transduction regulators of cell survival and death are upregulated.

15.
Eng Life Sci ; 20(8): 368-378, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32774209

RESUMO

Large-scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2-Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large-scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N-glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large-scale bioreactors.

16.
Anal Bioanal Chem ; 412(24): 6583-6593, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32691086

RESUMO

Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development. Graphical abstract.


Assuntos
Bevacizumab/química , Contaminação de Medicamentos , Etanercepte/química , Rituximab/química , Produtos Biológicos/química , Cromatografia Líquida de Alta Pressão/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Recombinantes de Fusão/química , Espectrometria de Massas em Tandem/métodos
17.
Anal Chem ; 92(14): 9666-9673, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32530275

RESUMO

Therapeutic proteins are an indispensable class of drugs and often therapeutics of last resort. They are sensitive to oxidation, which is of critical concern, because it can affect drug safety and efficacy. Protein oxidation, with methionine and tryptophan as the most susceptible moieties, is mainly monitored by HPLC-MS techniques. However, since several oxidation products display the same mass difference, their identification by MS is often ambiguous. Therefore, an alternative analytical method able to unambiguously identify and, ideally, also quantify oxidation species in proteins is highly desired. Here, we present an NMR-based approach to monitor oxidation in full-length proteins under denaturing conditions, as demonstrated on two biotherapeutic monoclonal antibodies (mAbs). We show that methionine sulfoxide, methionine sulfone, N-formylkynurenine, kynurenine, oxindolylalanine, hydroxypyrroloindole, and 5-hydroxytryptophan result in characteristic chemical shift correlations suited for their identification and quantification. We identified the five most abundant oxidation products in forced degradation studies of two full-length therapeutic mAbs and can also unambiguously distinguish oxindolylalanine from 5-hydroxytryptophan, which are undistinguishable by MS due to the same mass shift. Quantification of the abundant methionine sulfoxide by NMR and MS gave highly comparable values. These results underline the suitability of NMR spectroscopy for the identification and quantification of critical quality attributes of biotherapeutics.


Assuntos
Adalimumab/química , Espectroscopia de Ressonância Magnética/métodos , Rituximab/química , Aminoácidos/química , Antirreumáticos/química , Peróxido de Hidrogênio , Fatores Imunológicos/química , Oxidantes , Oxirredução
18.
Cell Commun Signal ; 18(1): 99, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576205

RESUMO

BACKGROUND: Aberrant hedgehog (HH) signaling is implicated in the development of various cancer entities such as medulloblastoma. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Increased phosphorylation of essential effectors such as Smoothened (SMO) and GLI proteins by kinases including Protein Kinase A, Casein Kinase 1, and Glycogen Synthase Kinase 3 ß controls effector activity, stability and processing. However, a deeper and more comprehensive understanding of phosphorylation in the signal transduction remains unclear, particularly during early response processes involved in SMO activation and preceding GLI target gene regulation. METHODS: We applied temporal quantitative phosphoproteomics to reveal phosphorylation dynamics underlying the short-term chemical activation and inhibition of early hedgehog signaling in HH responsive human medulloblastoma cells. Medulloblastoma cells were treated for 5.0 and 15 min with Smoothened Agonist (SAG) to induce and with vismodegib to inhibit the HH pathway. RESULTS: Our phosphoproteomic profiling resulted in the quantification of 7700 and 10,000 phosphosites after 5.0 and 15 min treatment, respectively. The data suggest a central role of phosphorylation in the regulation of ciliary assembly, trafficking, and signal transduction already after 5.0 min treatment. ERK/MAPK signaling, besides Protein Kinase A signaling and mTOR signaling, were differentially regulated after short-term treatment. Activation of Polo-like Kinase 1 and inhibition of Casein Kinase 2A1 were characteristic for vismodegib treatment, while SAG treatment induced Aurora Kinase A activity. Distinctive phosphorylation of central players of HH signaling such as SMO, SUFU, GLI2 and GLI3 was observed only after 15 min treatment. CONCLUSIONS: This study provides evidence that phosphorylation triggered in response to SMO modulation dictates the localization of hedgehog pathway components within the primary cilium and affects the regulation of the SMO-SUFU-GLI axis. The data are relevant for the development of targeted therapies of HH-associated cancers including sonic HH-type medulloblastoma. A deeper understanding of the mechanisms of action of SMO inhibitors such as vismodegib may lead to the development of compounds causing fewer adverse effects and lower frequencies of drug resistance. Video Abstract.

19.
Cells ; 9(6)2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32503348

RESUMO

Cancer stem cells (CSCs), a small subset of the tumor bulk with highly malignant properties, are deemed responsible for tumor initiation, growth, metastasis, and relapse. In order to reveal molecular markers and determinants of their tumor-initiating properties, we enriched rare stem-like pancreatic tumor-initiating cells (TICs) by harnessing their clonogenic growth capacity in three-dimensional multicellular spheroid cultures. We compared pancreatic TICs isolated from three-dimensional tumor spheroid cultures with nontumor-initiating cells (non-TICs) enriched in planar cultures. Employing differential proteomics (PTX), we identified more than 400 proteins with significantly different expression in pancreatic TICs and the non-TIC population. By combining the unbiased PTX with mRNA expression analysis and literature-based predictions of pro-malignant functions, we nominated the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14) as well as galactin-3-binding protein LGALS3BP (MAC-2-BP) as putative determinants of pancreatic TICs. In silico pathway analysis followed by candidate-based RNA interference mediated loss-of-function analysis revealed a critical role of S100A8, S100A9, and LGALS3BP as molecular determinants of TIC proliferation, migration, and in vivo tumor growth. Our study highlights the power of combining unbiased proteomics with focused gene expression and functional analyses for the identification of novel key regulators of TICs, an approach that warrants further application to identify proteins and pathways amenable to drug targeting.


Assuntos
Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Nus , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Análise de Componente Principal , Proteoma/metabolismo , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Angew Chem Int Ed Engl ; 59(37): 16225-16232, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32496655

RESUMO

N-glycosylation may affect the safety and efficacy of biopharmaceuticals and is thus monitored during manufacturing. Mass spectrometry of the intact protein is increasingly used to reveal co-existing glycosylation variants. However, quantification of N-glycoforms via this approach may be biased by single hexose residues as introduced by glycation or O-glycosylation. Herein, we describe a simple strategy to reveal actual N-glycoform abundances of therapeutic antibodies, involving experimental determination of glycation levels followed by computational elimination of the "hexosylation bias". We show that actual N-glycoform abundances may significantly deviate from initially determined values. Indeed, glycation may even obscure considerable differences in N-glycosylation patterns of drug product batches. Our observations may thus have implications for biopharmaceutical quality control. Moreover, we solve an instance of the problem of isobaricity, which is fundamental to mass spectrometry.


Assuntos
Produtos Biológicos/metabolismo , Hexoses/metabolismo , Algoritmos , Animais , Bevacizumab/metabolismo , Células CHO , Cricetulus , Denosumab/metabolismo , Glicosilação
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