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.

• Publication type
• Journal Article MeSH

• Publication type
• Meeting Abstract MeSH

Cell communication systems based on polypeptide ligands use transmembrane receptors to transmit signals across the plasma membrane. In their biogenesis, receptors depend on the endoplasmic reticulum (ER)-Golgi system for folding, maturation, transport and localization to the cell surface. ER stress, caused by protein overproduction and misfolding, is a well-known pathology in neurodegeneration, cancer and numerous other diseases. How ER stress affects cell communication via transmembrane receptors is largely unknown. In disease models of multiple myeloma, chronic lymphocytic leukemia and osteogenesis imperfecta, we show that ER stress leads to loss of the mature transmembrane receptors FGFR3, ROR1, FGFR1, LRP6, FZD5 and PTH1R at the cell surface, resulting in impaired downstream signaling. This is caused by downregulation of receptor production and increased intracellular retention of immature receptor forms. Reduction of ER stress by treatment of cells with the chemical chaperone tauroursodeoxycholic acid or by expression of the chaperone protein BiP resulted in restoration of receptor maturation and signaling. We show a previously unappreciated pathological effect of ER stress; impaired cellular communication due to altered receptor processing. Our findings have implications for disease mechanisms related to ER stress and are particularly important when receptor-based pharmacological approaches are used for treatment.

• MeSH
• Endoplasmic Reticulum Chaperone BiP MeSH
• Taurochenodeoxycholic Acid pharmacology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Receptors, Cell Surface * metabolism   MeSH
• Signal Transduction * drug effects   MeSH
• Endoplasmic Reticulum Stress * drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Error detection in motor behavior is a fundamental cognitive function heavily relying on local cortical information processing. Neural activity in the high-gamma frequency band (HGB) closely reflects such local cortical processing, but little is known about its role in error processing, particularly in the healthy human brain. Here we characterize the error-related response of the human brain based on data obtained with noninvasive EEG optimized for HGB mapping in 31 healthy subjects (15 females, 16 males), and additional intracranial EEG data from 9 epilepsy patients (4 females, 5 males). Our findings reveal a multiscale picture of the global and local dynamics of error-related HGB activity in the human brain. On the global level as reflected in the noninvasive EEG, the error-related response started with an early component dominated by anterior brain regions, followed by a shift to parietal regions, and a subsequent phase characterized by sustained parietal HGB activity. This phase lasted for more than 1 s after the error onset. On the local level reflected in the intracranial EEG, a cascade of both transient and sustained error-related responses involved an even more extended network, spanning beyond frontal and parietal regions to the insula and the hippocampus. HGB mapping appeared especially well suited to investigate late, sustained components of the error response, possibly linked to downstream functional stages such as error-related learning and behavioral adaptation. Our findings establish the basic spatio-temporal properties of HGB activity as a neural correlate of error processing, complementing traditional error-related potential studies.

• MeSH
• Adult MeSH
• Electroencephalography MeSH
• Electrocorticography MeSH
• Gamma Rhythm physiology   MeSH
• Humans MeSH
• Brain Mapping methods   MeSH
• Young Adult MeSH
• Brain physiology   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Fusion Proteins, bcr-abl genetics   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics   MeSH
• Polymorphism, Single Nucleotide genetics   MeSH
• Humans MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH

Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.

• MeSH
• Oligonucleotides, Antisense pharmacology   MeSH
• Cell Nucleus drug effects   metabolism   MeSH
• Endothelial Cells metabolism   MeSH
• Trinucleotide Repeat Expansion genetics   MeSH
• Fuchs' Endothelial Dystrophy genetics   pathology   MeSH
• Genetic Predisposition to Disease * MeSH
• Cohort Studies MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Organ Specificity MeSH
• RNA Processing, Post-Transcriptional MeSH
• RNA Precursors genetics   MeSH
• Risk Factors MeSH
• Endothelium, Corneal pathology   MeSH
• Aged MeSH
• RNA Splicing Factors metabolism   MeSH
• Transcription Factor 4 genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Nitric oxide (NO) is considered as a signalling molecule involved in a variety of important physiological and pathological processes in plant and animal systems. The major pathway of NO reactions in vivo represents S-nitrosation of thiols to form S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is the key enzyme in the degradation pathway of S-nitrosoglutathione (GSNO), a low-molecular weight adduct of NO and glutathione. GSNOR indirectly regulates the level of protein S-nitrosothiol in the cells. This study was focused on the dynamic regulation of the activity of plant GSNORs through reversible S-nitrosation and/or oxidative modifications of target cysteine residues. Pre-incubation with NO/NO- donors or hydrogen peroxide resulted in a decreased reductase and dehydrogenase activity of all studied plant GSNORs. Incubation with thiol reducing agent completely reversed inhibitory effects of nitrosative modifications and partially also oxidative inhibition. In biotin-labelled samples, S-nitrosation of plant GSNORs was confirmed after immunodetection and using mass spectrometry S-nitrosation of conserved Cys271 was identified in tomato GSNOR. Negative regulation of constitutive GSNOR activity in vivo by nitrosative or oxidative modifications might present an important mechanism to control GSNO levels, a critical mediator of the downstream signalling effects of NO, as well as for formaldehyde detoxification in dehydrogenase reaction mode.

• MeSH
• Aldehyde Oxidoreductases antagonists & inhibitors   chemistry   metabolism   MeSH
• Cysteine chemistry   metabolism   MeSH
• Nitric Oxide Donors pharmacology   MeSH
• Nitrosation MeSH
• Nitric Oxide metabolism   MeSH
• Oxidation-Reduction MeSH
• Hydrogen Peroxide pharmacology   MeSH
• Protein Processing, Post-Translational MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Plant Proteins antagonists & inhibitors   chemistry   metabolism   MeSH
• S-Nitrosoglutathione metabolism   MeSH
• S-Nitrosothiols metabolism   MeSH
• Signal Transduction MeSH
• Solanum lycopersicum genetics   growth & development   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

• MeSH
• Enzyme Activation MeSH
• Biological Assay methods   MeSH
• Chloramphenicol O-Acetyltransferase genetics   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Gene Expression * MeSH
• Protein Processing, Post-Translational MeSH
• Proteolysis MeSH
• Genes, Reporter * MeSH
• Ubiquitination * MeSH
• Ubiquitin-Protein Ligases metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Skriptum určené k výuce na VŠCHT Praha.

• MeSH
• Biopharmaceutics MeSH
• Biological Therapy MeSH
• Biotechnology MeSH
• Technology, Pharmaceutical MeSH

• Conspectus
• Farmacie. Farmakologie
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• farmacie a farmakologie
• biomedicínské inženýrství
• farmacie a farmakologie
• NML Publication type
• učebnice vysokých škol

Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.

• MeSH
• eIF-2 Kinase genetics   metabolism   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 * genetics   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Protein Isoforms metabolism   MeSH
• Endoplasmic Reticulum Stress * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH