RÉSUMÉ
The objective of the current investigation was to evaluate the induction of heat shock proteins (HSPs) in SP2/0 transgenic cells and the effect of these proteins on the production of monoclonal antibodies (mAbs). The SP2/0 cell line expressing the PSG-026 antibody, a biosimilar candidate of golimumab, the culture parameters, and the target protein expression were not justified for industrial production and were used for the experiments. Paracetamol and heat shock were used as chemical and physical inducers of HSPs, respectively. The results showed that paracetamol and heat shock increased the expression of HSP70 and HSP27 at the mRNA and protein levels. The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells. Paracetamol treatment at concentrations above 0.5 mM significantly reduced cell viability and mAb expression. However, treatment with 0.25 mM paracetamol results in delayed cell death and increased mAb production. Heat shock treatment at 45°C for 30 minutes after enhanced mAb expression was applied after pre-treatment with paracetamol. In bioreactor cultures, pretreatment of cells with paracetamol improved cell viability and shortened the lag phase, resulting in increased cell density. The production of mAbs in paracetamol-treated cultures was markedly greater than that in the control. Analysis of protein quality and charge variants revealed no significant differences between paracetamol-treated and control cultures, indicating that the induction of HSPs did not affect protein aggregation or charge variants. These findings suggest that inducing and manipulating HSP expression can be a valuable strategy for improving recombinant protein production in biopharmaceutical processes.
Sujet(s)
Acétaminophène , Anticorps monoclonaux , Survie cellulaire , Anticorps monoclonaux/pharmacologie , Animaux , Acétaminophène/pharmacologie , Survie cellulaire/effets des médicaments et des substances chimiques , Souris , Protéines du choc thermique HSP70/métabolisme , Protéines du choc thermique HSP70/génétique , Protéines du choc thermique/génétique , Protéines du choc thermique/métabolisme , Bioréacteurs , Réaction de choc thermique/effets des médicaments et des substances chimiques , Protéines du choc thermique HSP27/métabolisme , Protéines du choc thermique HSP27/génétique , Lignée cellulaireRÉSUMÉ
Vaccine potency is typically evaluated using an assay that acts as a surrogate for biological activity. Although in vivo vaccines better represent human immunological responses, in vitro assays are preferred due to lower variability, higher throughput, easier validation and ethical considerations. In in vitro determination of Human Papillomavirus (HPV), Virus-like particle (VLP) vaccine potency currently depends on monoclonal antibody assays. However, these reagents are hard to obtain and currently are not available commercially. In this work, a polyclonal antiserum-based immunoassay was developed to evaluate the relative potency of Alhydrogel formulated HPV 16 VLPs. The repeatability and specificity were evaluated, and found that the assay was sensitive to small amounts of non-VLP HPV 16 L1 proteins. Finally, the assay was tested in comparison to the mouse effective dose 50 (ED50) assay on a limited number of batches. The agreement between these results suggests this test as a suitable surrogate for the in vivo test.
Sujet(s)
Infections à papillomavirus , Vaccins contre les papillomavirus , Animaux , Souris , Humains , Papillomavirus humain de type 16 , Anticorps antiviraux , Dosage immunologique/méthodes , Protéines de capsideRÉSUMÉ
Protein L is a multidomain protein from Peptostreptococcus magnus with binding affinity to kappa light chain of human immunoglobulin (Ig) which is used for the purification of antibody fragments by affinity chromatography. The advances in protein engineering and computational biology approaches lead to the development of engineered affinity ligands with improved properties including binding affinity. In this study, molecular dynamics simulations (MDs) and Osprey software were used to design single B domains of the Protein L with higher affinity to antibody fragments. The modified B domains were then polymerized to ligand with six B domains by homology modeling methods. The results showed that single B domain mutants of MB1 (Thr865Trp) and MB2 (Thr847Met-Thr865Trp) had higher binding affinity to Fab compared to the wild single B domain. Also, MDs and molecular docking results showed that the polymerized Proteins L including the wild and mutated six B domains (6B0, 6B1, and 6B2) were stable during MDs and the two mutants of 6B1 and 6B2 showed higher binding affinity to Fab relative to the wild type.Communicated by Ramaswamy H. Sarma.
RÉSUMÉ
Background: Post-translational modifications in bioprocessing and storage of recombinant mAbs are the main sources of charge variants. While the profile of these kinds of variants is considered an important attribute for the therapeutic mAbs, there is controversy about their direct role in safety and efficacy. In this study, the physicochemical and pharmacokinetic (PK) properties of the separated charge variants belonging to a trastuzumab potential biosimilar, were examined. Methods: The acidic peaks, basic peaks, and main variants of trastuzumab were separated and enriched by semi-preparative weak cation exchange. A panel of analytical techniques was utilized to characterize the physicochemical properties of these variants. The binding affinity to HER2 and FcγRs and the PK parameters were evaluated for each variant. Results: Based on the results, the charge variants of the proposed biosimilar had no significant influence on the examined efficacy and PK parameters. Conclusion: During the development and production of biosimilar monoclonal antibodies, evaluating the effect of their charge variants on efficacy and PK parameters is needed.
Sujet(s)
Produits pharmaceutiques biosimilaires , Trastuzumab/composition chimique , Produits pharmaceutiques biosimilaires/composition chimique , Produits pharmaceutiques biosimilaires/pharmacocinétique , Anticorps monoclonauxRÉSUMÉ
Background: Background: Downstream processing of therapeutic recombinant proteins expressed as the inclusion bodies (IBs) in E. coli is quite challenging. This study aimed to use the quality by design approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim. Methods: Methods: For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most critical quality attributes (CQAs) and effects of process parameters on these quality attributes. Results: Results: The solubilization of IBs was optimized by design of experiment on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in anion exchange chromatography that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on high molecular weight impurities. After polishing by gel filtration, the final product's biological activity showed no statistically significant differences among the groups received romiplostim and Nplate®, as the reference product. Conclusions: Conclusion: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters.
Sujet(s)
Escherichia coli , Corps d'inclusion , Protéines de fusion recombinantes , Escherichia coli/métabolisme , Protéines de fusion recombinantes/biosynthèseRÉSUMÉ
Protein L affinity chromatography is a useful method for the purification of antibody fragments containing kappa light chains. In affinity chromatography, increasing the binding affinity leads to increased product purity, recovery, and dynamic binding capacity (DBC). In this study, molecular docking and molecular dynamics simulation techniques were used to design the engineered Protein L with higher affinity to the kappa light chain. Each engineered ligand was produced as a recombinant protein and coupled to a solid matrix. The purity, recovery, and DBC of the engineered resins were evaluated and then compared to those of a commercially available resin. The results showed important parameters for engineering more efficient Protein L ligands for affinity chromatography.
Sujet(s)
Fragments d'immunoglobuline , Chromatographie d'affinité , Ligands , Simulation de docking moléculaire , Liaison aux protéines , Protéines recombinantes/génétique , Protéines recombinantes/métabolismeRÉSUMÉ
The most crucial role of granulocyte colony-stimulating factor (G-CSF) in the body is to increase the strength of immune system. In recent years, research on the use of nanoparticles in pharmaceuticals has been considered, most of which have been for drug-loading purposes. In this study, a novel G-CSF conjugated dendrimer was synthesized and characterized using different techniques. In vitro cytotoxicity was assessed on A549 and L929 cells, while abnormal toxicity was studied in mice. In vitro and in vivo biological activities were assessed in NFS60 cells and rats, respectively. In addition, in vivo distribution, plasma half-life, and histopathological effect were studied in rat. The characterization tests confirmed the successful conjugation. There was no difference between G-CSF cytotoxicity before and after conjugation, and no difference with the control group. No mice showed abnormal toxicity. Although in vitro biological activity revealed both conjugated and free G-CSF promote proliferation cells, biological activity decreased significantly after conjugation about one-third of the unconjugated form. Nonetheless, in vivo biological activity of conjugated G-CSF increased by more than 2.5-fold relative to the unconjugated form, totally. Fortunately, no histopathologic adverse effect was observed in vital rat tissues. Also, in vivo distribution of the conjugate was similar to the native protein with an enhanced terminal half-life. Our data revealed that G-CSF conjugated dendrimer could be considered as a candidate to improve the in vivo biological activity of G-CSF. Moreover, multivalent capability of the dendrimer may be used for other new potentials of G-CSF in future perspectives.
Sujet(s)
Dendrimères , Facteur de stimulation des colonies de granulocytes , Animaux , Lignée cellulaire , Dendrimères/administration et posologie , Dendrimères/composition chimique , Dendrimères/pharmacocinétique , Facteur de stimulation des colonies de granulocytes/administration et posologie , Facteur de stimulation des colonies de granulocytes/composition chimique , Facteur de stimulation des colonies de granulocytes/pharmacocinétique , Coeur/anatomie et histologie , Coeur/effets des médicaments et des substances chimiques , Humains , Rein/anatomie et histologie , Rein/effets des médicaments et des substances chimiques , Foie/anatomie et histologie , Foie/effets des médicaments et des substances chimiques , Mâle , Souris , Rats , Distribution tissulaireRÉSUMÉ
Viruses are obligatory intracellular parasites that use cell proteins to take the control of the cell functions in order to accomplish their life cycle. Studying the viral-host interactions would increase our knowledge of the viral biology and mechanisms of pathogenesis. Studies on pathogenesis mechanisms of lyssaviruses, which are the causative agents of rabies, have revealed some important host protein partners for viral proteins, especially for most studied species, i.e. Rabies virus. In this review article, the key physical lyssavirus-host protein interactions, their contributions to rabies infection, and their exploitation are discussed to improve the knowledge about rabies pathogenesis.
Sujet(s)
Interactions hôte-microbes/physiologie , Lyssavirus/métabolisme , Virus de la rage/métabolisme , Rage (maladie)/métabolisme , Animaux , Humains , Phagocytose/physiologie , Liaison aux protéines/physiologie , Rage (maladie)/transmissionRÉSUMÉ
OBJECTIVE: Chinese hamster ovary (CHO) cells are the leading cell factories for producing recombinant proteins in the biopharmaceutical industry. In this regard, constraint-based metabolic models are useful platforms to perform computational analysis of cell metabolism. These models need to be regularly updated in order to include the latest biochemical data of the cells, and to increase their predictive power. Here, we provide an update to iCHO1766, the metabolic model of CHO cells. RESULTS: We expanded the existing model of Chinese hamster metabolism with the help of four gap-filling approaches, leading to the addition of 773 new reactions and 335 new genes. We incorporated these into an updated genome-scale metabolic network model of CHO cells, named iCHO2101. In this updated model, the number of reactions and pathways capable of carrying flux is substantially increased. CONCLUSIONS: The present CHO model is an important step towards more complete metabolic models of CHO cells.
Sujet(s)
Cellules CHO/métabolisme , Génome/génétique , Voies et réseaux métaboliques/génétique , Modèles biologiques , Biologie des systèmes/méthodes , Animaux , Cricetinae , Cricetulus , Protéines recombinantes/génétique , Protéines recombinantes/métabolismeRÉSUMÉ
Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.
Sujet(s)
Cytosquelette d'actine/métabolisme , Interactions hôte-microbes , Virus de la rage/composition chimique , Virus de la rage/métabolisme , Rage (maladie)/métabolisme , Rage (maladie)/virologie , Protéines de la matrice virale/métabolisme , Cytosquelette d'actine/composition chimique , Animaux , Technique de Western/méthodes , Lignée cellulaire , Électrophorèse bidimensionnelle sur gel/méthodes , Glyceraldehyde 3-phosphate dehydrogenases/composition chimique , Glyceraldehyde 3-phosphate dehydrogenases/métabolisme , Mâle , Souris , Simulation de docking moléculaire , Liaison aux protéines , Rats , Rat Wistar , Protéines recombinantes/métabolisme , Tubuline/composition chimique , Tubuline/métabolisme , Protéines de la matrice virale/composition chimiqueRÉSUMÉ
OBJECTIVE: Heart dysfunctions are the major complications of trastuzumab in patients with Human Epidermal growth factor Receptor-2 (HER2)-positive breast cancers. METHODS: In this study, the cytotoxicity of trastuzumab on H9c2 cardiomyoblasts was demonstrated, and the proteome changes of cells were investigated by a tandem mass tagging quantitative approach. The Differentially Abundant Proteins (DAPs) were identified and functionally enriched. RESULTS: We determined that carvedilol, a non-selective beta-blocker, could effectively inhibit trastuzumab toxicity when administrated in a proper dose and at the same time. The proteomics analysis of carvedilol co-treated cardiomyoblasts showed complete or partial reversion in expressional levels of trastuzumab-induced DAPs. CONCLUSION: Downregulation of proteins involved in the translation biological process is one of the most important changes induced by trastuzumab and reversed by carvedilol. These findings provide novel insights to develop new strategies for the cardiotoxicity of trastuzumab.
Sujet(s)
Antagonistes bêta-adrénergiques/pharmacologie , Antinéoplasiques immunologiques/toxicité , Carvédilol/pharmacologie , Myoblastes cardiaques/effets des médicaments et des substances chimiques , Protéome/métabolisme , Trastuzumab/toxicité , Antagonistes bêta-adrénergiques/usage thérapeutique , Antinéoplasiques immunologiques/usage thérapeutique , Tumeurs du sein/traitement médicamenteux , Tumeurs du sein/métabolisme , Cardiomyopathies/prévention et contrôle , Carvédilol/usage thérapeutique , Lignée cellulaire , Survie cellulaire/effets des médicaments et des substances chimiques , Simulation numérique , Régulation négative , Femelle , Humains , Myoblastes cardiaques/métabolisme , Myoblastes cardiaques/anatomopathologie , Protéomique , Récepteur ErbB-2/métabolisme , Trastuzumab/usage thérapeutiqueRÉSUMÉ
Chinese hamster ovary (CHO) cells are the main workhorse in the biopharmaceutical industry for the production of recombinant proteins, such as monoclonal antibodies. To date, a variety of metabolic engineering approaches have been used to improve the productivity of CHO cells. While genetic manipulations are potentially laborious in mammalian cells, rational design of CHO cell culture medium or efficient fed-batch strategies are more popular approaches for bioprocess optimization. In this study, a genome-scale metabolic network model of CHO cells was used to design feeding strategies for CHO cells to improve monoclonal antibody (mAb) production. A number of metabolites, including threonine and arachidonate, were suggested by the model to be added into cell culture medium. The designed composition has been experimentally validated, and then optimized, using design of experiment methods. About a two-fold increase in the total mAb expression has been observed using this strategy. Our approach can be used in similar bioprocess optimization problems, to suggest new ways of increasing production in different cell factories.
Sujet(s)
Anticorps monoclonaux/biosynthèse , Bioréacteurs , Techniques de culture cellulaire , Animaux , Anticorps monoclonaux/génétique , Cellules CHO , Cricetulus , Milieux de culture , Protéines recombinantes/biosynthèse , Protéines recombinantes/génétiqueRÉSUMÉ
Cardiac dysfunction is a major side effect of trastuzumab therapy for patients with HER2-positive breast cancer. Beta blockers, such as carvedilol, have been used for protection of trastuzumab cardiotoxicity but there is no definitive conclusive clinical report on their efficacy. In the present study, the preservability effects of carvedilol on trastuzumab-induced left ventricular (LV) dysfunction and the reversibility of trastuzumab-induced cardiotoxicity were evaluated in Wistar rats by echocardiography method. We showed that trastuzumab treatment of rats could induce the LV dysfunction through increasing the LV internal systolic diameter (LVIDs), increasing the end-systolic volume (ESV), decreasing the ejection fraction (EF), and decreasing the fractional shortening (FS). These parameters were not reversed after 14 days of stopping trastuzumab administration. Interestingly, carvedilol improved LVIDs, ESV, EF, and FS. Collectively, the results of this study have verified clinical observations which simultaneously administration of carvedilol may be considered as a possible therapeutic strategy to prevent trastuzumab-mediated LV dysfunction.
Sujet(s)
Antagonistes bêta-adrénergiques/administration et posologie , Tumeurs du sein/traitement médicamenteux , Carvédilol/administration et posologie , Trastuzumab/effets indésirables , Dysfonction ventriculaire gauche/prévention et contrôle , Administration par voie orale , Animaux , Cardiotoxicité/diagnostic , Cardiotoxicité/étiologie , Cardiotoxicité/prévention et contrôle , Modèles animaux de maladie humaine , Échocardiographie , Femelle , Humains , Injections péritoneales , Adulte d'âge moyen , Rats , Débit systolique/effets des médicaments et des substances chimiques , Trastuzumab/administration et posologie , Dysfonction ventriculaire gauche/induit chimiquement , Dysfonction ventriculaire gauche/diagnosticRÉSUMÉ
The demand for reliable comparability studies of biosimilars grows with their increased market share. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. In the current study, Enbrel® (etanercept, the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. "Bottom-up" mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted in complete identification of the primary structure. It was confirmed that total ion chromatograms are valuable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality. Analysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary structure and in-vitro functionality.
RÉSUMÉ
BACKGROUND: Etanercept is prescribed for the rapid and effective treatment of chronic immune-mediated inflammatory disorders. Due to the expiration of etanercept patents and worldwide demand for comparable and more affordable substitutes, several biosimilars of etanercept have been approved in different countries and new ones are in the process of approval. OBJECTIVES: In the present study, Altebrel™ as an etanercept proposed biosimilar was investigated in a side by side comparison using various orthogonal analytical methods. MATERIALS AND METHODS: Three batches of the Altebrel™ and Enbrel® samples were used for the study. Several physicochemical properties of samples were compared according to international guidelines, incliding; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion high performance liquid chromatography (SE-HPLC), hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and its biological activity was evaluated using surface plasmon resonance affinity analysis and tumor necrosis factor alpha (TNFα) neutralization biological assay. Amino acid analysis was applied to check the primary sequence and far-UV circular dichroism (CD) spectroscopy investigated the secondary structure. RESULTS: The obtained results indicated a high degree of similarity between Altebrel™ and Enbrel®. Results of SDS-PAGE, CE-SDS, HIC-HPLC and SE-HPLC implied a comparable pattern of size variants for all samples. Based on the data achieved via in vitro bioactivity assays and SPR analysis, the functional property of Altebrel™ was proved comparable to that of the reference product. Moreover, amino acid analysis indicated similar primary structure and circular dichroism study implied a similar secondary structure for Altebrel™ and Enbrel®. CONCLUSION: Overall, our data provide analytical evidence for structural and in vitro functional similarity between Altebrel™ and Enbrel®.
RÉSUMÉ
Background: Chinese hamster ovary (CHO) cell line is considered as the most common cell line in the biopharmaceutical industry because of its capability in performing efficient post-translational modifications and producing the recombinant proteins, which are similar to natural human proteins. The optimization of the upstream process via different feed strategies has a great impact on the target molecule expression and yield. Methods: To determine and understand the molecular events beneath the feed effects on the CHO cell, a label-free quantitative proteomic analysis was applied. The proteome changes followed by the addition of a designed amino acid feed to the monoclonal antibody producing CHO cell line culture medium were investigated. Results: The glutathione synthesis, the negative regulation of the programmed cell death, proteasomal catabolic process, and the endosomal transport pathway were up-regulated in the group fed with a designed amino acid feed compared to the control group. Conclusion: Our findings could be helpful to identify new targets for metabolic engineering.
Sujet(s)
Acides aminés/métabolisme , Anticorps monoclonaux/biosynthèse , Survie cellulaire/physiologie , Protéomique/méthodes , Acides aminés/pharmacologie , Animaux , Cellules CHO , Survie cellulaire/effets des médicaments et des substances chimiques , Cricetinae , Cricetulus , SourisRÉSUMÉ
Since physiological and pathological processes occur at nano-environments, nanotechnology has considered as an efficient tool for designing of next generation specific biomolecules with enhanced pharmacodynamic and pharmacodynamic properties. In the current investigation, by control of the size and hydrodynamic volume at the nanoscale, for the first time, physicochemical and pharmacokinetic properties of an anti-VEGFA nanobody was remarkably improved by attachment of a Proline-Alanine-Serine (PAS) rich sequence. The results elucidated unexpected impressive effects of PAS sequence on physicochemical properties especially on size, hydrodynamics radius, and even solubility of nanobody. CD analysis revealed an increment in random coil structure of the PASylated protein in comparison to native one without any change in charge state or binding kinetic parameters of nanobody assessed by isoelectric focusing and surface plasmon resonance measurements, respectively. In vitro biological activities of nanobody were not affected by coupling of the PAS sequence. In contrast, the terminal half-life was significantly increased by a factor of 14 for the nanobody-PAS after single dose IV injection to the mice. Our study demonstrated that the control of size in the design of small therapeutic proteins has a promising effect on the stability and solubility, in addition to their physiochemical and pharmacokinetic properties. The designed new anti-VEGFA nanobody could promise a better therapeutic agent with a long administration intervals and lower dose, which in turn leads to a better patient compliance. Size adjustment of an anti-VEGF nanobody at the nanoscale by the attachment of a natural PAS polymer remarkably improves physicochemical properties, as well as a pharmacokinetic profile without any change in biological activity of the miniaturized antibody.
Sujet(s)
Phénomènes chimiques , Anticorps à domaine unique/composition chimique , Anticorps à domaine unique/métabolisme , Facteur de croissance endothéliale vasculaire de type A/immunologie , Animaux , Prolifération cellulaire/effets des médicaments et des substances chimiques , Femelle , Cellules HEK293 , Humains , Cinétique , Souris , Modèles moléculaires , Masse moléculaire , Agrégats de protéines , Structure secondaire des protéines , Anticorps à domaine unique/immunologie , Anticorps à domaine unique/pharmacologie , Thermodynamique , Distribution tissulaireRÉSUMÉ
Autoantibody profiling with a systems medicine approach can help identify critical dysregulated signaling pathways (SPs) in cancers. In this way, immunoglobulins G (IgG) purified from the serum samples of 92 healthy controls, 10 pre-treated (PR) non-Hodgkin lymphoma (NHL) patients, and 20 NHL patients who underwent chemotherapy (PS) were screened with a phage-displayed random peptide library. Protein-protein interaction networks of the PR and PS groups were analyzed and visualized by Gephi. The results indicated AXIN2, SENP2, TOP2A, FZD6, NLK, HDAC2, HDAC1, and EHMT2, in addition to CAMK2A, PLCG1, PLCG2, GRM5, GRIN2B, GRIN2D, CACNA2D3, and SPTAN1 as hubs in 11 and 7 modules of PR and PS networks, respectively. PR- and PS-specific hubs were evaluated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. The PR-specific hubs were involved in Wnt SP, signaling by Notch1 in cancer, telomere maintenance, and transcriptional misregulation. In contrast, glutamate receptor SP, Fc receptor-related pathways, growth factors-related SPs, and Wnt SP were statistically significant enriched pathways, based on the pathway analysis of PS hubs. The results revealed that the most PR-specific proteins were associated with events involved in tumor development, while chemotherapy in the PS group was associated with side effects of drugs and/or cancer recurrence. As the findings demonstrated, PR- and PS-specific proteins in this study can be promising therapeutic targets in future studies.
Sujet(s)
Antinéoplasiques/pharmacologie , Découverte de médicament , Lymphome malin non hodgkinien/métabolisme , Biologie des systèmes , Adolescent , Adulte , Sujet âgé , Sujet âgé de 80 ans ou plus , Antinéoplasiques/usage thérapeutique , Marqueurs biologiques , Études cas-témoins , Biologie informatique/méthodes , Résistance aux médicaments antinéoplasiques , Gene Ontology , Réseaux de régulation génique , Humains , Lymphome malin non hodgkinien/diagnostic , Lymphome malin non hodgkinien/traitement médicamenteux , Adulte d'âge moyen , Peptides/antagonistes et inhibiteurs , Peptides/métabolisme , Liaison aux protéines , Cartes d'interactions protéiques , Récidive , Transduction du signal/effets des médicaments et des substances chimiques , Biologie des systèmes/méthodes , Résultat thérapeutique , Jeune adulteRÉSUMÉ
BACKGROUND: The emergence of multidrug resistant Acinetobacter baumannii within hospitals poses a significant threat to patients. The inherent rate of mutation of these strains has not been described nor has the mechanism by which drug resistance arises. METHODS: Here, we determined the spontaneous mutation rates in 93 clinical strains of A. baumannii using fluctuation analysis. To rule out the clonal relatedness of hypermutator strains, pulsed-field gel electrophoresis (PFGE) was conducted. Using a combination of two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry, the differentially expressed proteins of a hypermutator and a reference strain were identified. RESULTS: The spontaneous mutation rate of multi-drug resistant A. baumannii strains varied broadly from 0 to 2.1×10-6 mutation per cell division. The mutation rate in three multidrug resistant A. baumannii (MDR-AB) strains was found to be 1.63×10-6 (95% confidence interval (CI): 1×10-6-2×10-6), 2.1×10-6 (95% CI: 2×10-6 - 3×10-6), and 1.78×10-8 (95% CI: 9.29×10-9 2.95×10-8), consistent with a hypermutator phenotype. This rate is approximately 1000-fold higher than the average mutation rate in other MDR-ABs. PFGE of the three hypermutator strains indicate that they belong to distinct clones. Proteomic analysis of one hypermutator strain revealed 31 differentially expressed proteins including three with sizes of 51.2, 20.9, and 11.9kDa, which corresponded to a serine protease, a polyisoprenoid-binding protein, and the peptidoglycan binding protein, LysM. The serine protease was expressed only in the hypermutator strain, whereas the polyisoprenoid-binding protein and the peptidoglycan binding protein LysM were down-regulated 1.6 and 3-fold, respectively, in the hypermutators strain. CONCLUSION: Hypermutator A. baumannii strains occur with a low, but appreciable frequency among clinical multi-drug resistant isolates. The presence of hypermutator clinical isolates raises concerns that they may contribute to the failure of antibiotic treatment in infected patients and confound the interpretation of in vitro antibiotic susceptibility testing. The differentially expressed proteins involved in biofilm suppression and oxidative stress response, may represent adaptations derived from the hypermutator phenotype, a hypothesis that needs further testing.
Sujet(s)
Acinetobacter baumannii/génétique , Protéines bactériennes/génétique , Multirésistance bactérienne aux médicaments/génétique , Régulation de l'expression des gènes bactériens , Taux de mutation , Acinetobacter baumannii/effets des médicaments et des substances chimiques , Antibactériens/pharmacologie , Électrophorèse en champ pulsé , Électrophorèse bidimensionnelle sur gel , Tests de sensibilité microbienne , Protéome/génétique , Spectrométrie de masse MALDIRÉSUMÉ
Because of high mortality caused by cardiovascular diseases, various fibrinolytic agents with diverse pharmacokinetic and pharmacodynamic properties have been developed. A novel mutated chimeric tissue plasminogen activator (mt-PA) was developed by the removal of first three domains of t-PA, insertion of GHRP sequence and mutation towards resistance to plasminogen activator inhibitor-1 (PAI-1). Mt-PA protein was expressed in Expi293F cells. The expression level of mt-PA was found to be 5000 IU/mL. Following purification, the pharmacokinetic properties of mt-PA were evaluated in three doses in rats. Data related to mt-PA were best fitted to two compartment model. With the increase in dose, the Area Under the plasma concentration-time Curve (AUC0â∞) increased. The elimination half-life (t1/2) of mt-PA was in the range of 19.1-26.1 min in three doses while that of Alteplase was 8.3 min. The plasma clearance (CLp) of mt-PA ranged from 3.8 to 5.9 mL/min in three doses, which was several times lower than that of Alteplase (142.6 mL/min). The mean residence time (MRT) of mt-PA ranged from 23.3-31.8 min in three doses, which was 4-5 times greater than that of Alteplase (6 min). Mt-PA showed extended half-life and mean residence time and is a good candidate for further clinical studies.