Downstream process
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Despite the efficacy and potential therapeutic benefits that poly(lactic-co-glycolic acid) (PLGA) nanomedicine formulations can offer, challenges related to large-scale processing hamper their clinical and commercial development. Major hurdles for the launch of a polymeric nanocarrier product on the market are batch-to-batch variations and lack of product consistency in scale-up manufacturing. Therefore, a scalable and robust manufacturing technique that allows for the transfer of nanomedicine production from the benchtop to an industrial scale is highly desirable. Downstream processes for purification, concentration, and storage of the nanomedicine formulations are equally indispensable. Here, we develop an inline sonication process for the production of polymeric PLGA nanomedicines at the industrial scale. The process and formulation parameters are optimized to obtain PLGA nanoparticles with a mean diameter of 150 ± 50 nm and a small polydispersity index (PDI < 0.2). Downstream processes based on tangential flow filtration (TFF) technology and lyophilization for the washing, concentration, and storage of formulations are also established and discussed. Using the developed manufacturing and downstream processing technologies, production of two PLGA nanoformulations encasing ritonavir and celecoxib was achieved at 84 g/h rate. As a measure of actual drug content, encapsulation efficiencies of 49.5 ± 3.2% and 80.3 ± 0.9% were achieved for ritonavir and celecoxib, respectively. When operated in-series, inline sonication and TFF can be adapted for fully continuous, industrial-scale processing of PLGA-based nanomedicines.
- Publikační typ
- časopisecké články MeSH
Trichothecene mycotoxin deoxynivalenol (DON) negatively regulates immune response by damaging host immune system and harming the organism's health. We hypothesized that DON can initiate an active immunosuppressive mechanism similar to "immune evasion" to alter the cellular microenvironment and evade immune surveillance. We tested this hypothesis using the RAW264.7 macrophage model. DON rapidly increased the expression of immune checkpoints PD-1 and PD-L1, inflammatory cytokine TGF-β, and key immune evasion factors STAT3, VEGF, and TLR-4, and caused cellular hypoxia. Importantly, hypoxia-inducible factor-1α (HIF-1α) acts as a key regulator of DON-induced immunosuppression. HIF-1α accumulated in the cytoplasm and was gradually transferred to the nucleus following DON treatment. Moreover, DON activated HIF-1α through STAT3 signaling to upregulate downstream signaling, including PD-1/PD-L1. Under DON treatment, immunosuppressive miR-210-3p, lncRNA PVT1, lncRNA H19, and lncRNA HOTAIR were upregulated by the STAT3/HIF-1α axis. Moreover, DON damaged mitochondrial function, causing mitophagy, and suppressed immune defenses. Collectively, DON triggered RAW264.7 intracellular hypoxia and rapidly activated HIF-1α via STAT3 signaling, activating immune evasion signals, miRNAs, and lncRNAs, thereby initiating the key link of immune evasion. This study offers further clues for accurate prevention and treatment of immune diseases caused by mycotoxins.
In chronic myeloid leukemia, the identification of individual BCR-ABL1 fusions is required for the development of personalized medicine approach for minimal residual disease monitoring at the DNA level. Next generation sequencing (NGS) of amplicons larger than 1000 bp simplified and accelerated a process of characterization of patient-specific BCR-ABL1 genomic fusions. NGS of large regions upstream and downstream the individual breakpoints in BCR and ABL1 genes, respectively, also provided information about the sequence variants such are single nucleotide polymorphisms.
The integrated electrodialysis (ED) process supports valorisation of a lactose-rich side stream from the dairy industry, creating an important source of milk sugar used in various branches of the industry. This work focuses on the optimization of the downstream processes before the crystallization of lactose. The process line includes a pre-treatment and desalination by ED of the industrial waste solution of the lactose mother liquor (LML). The LML was diluted to 25% total solids to overcome hydraulic issues with the ED desalination process. Two different levels of electrical conductivity reduction (70% and 90%) of the LML solutions were applied to decrease the mineral components and organic acids of the LML samples. The ED performance parameters such as ash transfer rate (J), the specific capacity (CF) of the ED and specific electric energy consumption (E) were determined and the influence of the LML solution on the monopolar ion-exchange membranes has been investigated. A higher degree of desalination is associated with higher electric energy consumption (by 50%) and lower specific capacity (by 40%). A noticeable decrease (by 12.8%) in the resistance of the anion exchange membranes was measured after the trials whereas the resistance of the cation exchange membranes remained practically unchanged. Any deposition of the alkaline earth metals on the membrane surface was not observed.
- Publikační typ
- časopisecké články MeSH
The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications.
- MeSH
- buněčná diferenciace genetika MeSH
- buněčné linie MeSH
- fibroblasty cytologie metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie metabolismus MeSH
- metylace DNA * MeSH
- přeprogramování buněk genetika MeSH
- shluková analýza MeSH
- stanovení celkové genové exprese MeSH
- vývojová regulace genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The ability to flexibly switch between tasks is an important faculty in daily life. One process that has been suggested to be an important aspect of flexible task switching is the inhibition of a recently performed task. This is called backward inhibition. Several studies suggest that task switching performance can be enhanced by rewards. However, it is less clear in how far backward inhibition mechanisms are also affected by rewards, especially when it comes to the neuronal mechanisms underlying reward-related modulations of backward inhibition. We therefore investigated this using a system neurophysiological approach combining EEG recordings with source localization techniques. We demonstrate that rewards reduce the strength of backward inhibition processes. The neurophysiological data shows that these reward-related effects emerge from response and/or conflict monitoring processes within medial frontal cortical structures. Upstream processes of perceptual gating and attentional selection, as well as downstream processes of context updating and stimulus-response mapping are not modulated by reward, even though they also play a role in backward inhibition effects.
- MeSH
- dospělí MeSH
- elektroencefalografie metody MeSH
- exekutivní funkce fyziologie MeSH
- inhibice (psychologie) * MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mozek fyziologie MeSH
- odměna * MeSH
- psychomotorický výkon fyziologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.
- MeSH
- Caenorhabditis elegans enzymologie metabolismus účinky záření MeSH
- diglyceridy chemie metabolismus účinky záření MeSH
- fotochemické procesy účinky záření MeSH
- myši MeSH
- optické jevy MeSH
- proteinkinasa C chemie metabolismus účinky záření MeSH
- signální transdukce účinky záření MeSH
- ultrafialové záření * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
... interactions, 91 Specificity and cross-reactivity of antibodies, 92 What the T-cell sees, 95 -- Processing ... ... of intracellular antigen for presentation by Class I MHC, 95 Processing of antigen for Class II MHC ... ... two signals, 173 -- Protein tyrosine phosphorylation is an early event in T-cell signaling, 173 Downstream ...
11th ed. xvi, 474 s. : il., tabs. ; 28 cm
- MeSH
- imunita MeSH
- imunitní systém MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- alergologie a imunologie
Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.
- MeSH
- aktivace lymfocytů MeSH
- buněčná membrána metabolismus MeSH
- Drosophila melanogaster MeSH
- gating iontového kanálu MeSH
- HEK293 buňky MeSH
- konformace proteinů MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- mutace MeSH
- proteasy chemie MeSH
- protein ORAI1 chemie MeSH
- serinové endopeptidasy metabolismus MeSH
- signální transdukce MeSH
- stochastické procesy MeSH
- vápník metabolismus MeSH
- vápníková signalizace fyziologie MeSH
- vápníkové kanály chemie MeSH
- vazba proteinů MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
