Feruloyl esterases (FAEs) are a crucial component of the hemicellulose-degrading enzyme family that facilitates the degradation of lignocellulose while releasing hydroxycinnamic acids such as ferulic acid with high added value. Currently, the low enzyme yield of FAEs is one of the primary factors limiting its application. Therefore, in this paper, we optimized the fermentation conditions for the expression of FAE BpFaeT132C-D143C with excellent thermal stability in Escherichia coli by experimental design. Firstly, we explored the effects of 11 factors such as medium type, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration, and inoculum size on BpFaeT132C-D143C activity separately by the single factor design. Then, the significance of the effects of seven factors, such as post-induction temperature, shaker rotational speed, and inoculum size on BpFaeT132C-D143C activity, was analyzed by Plackett-Burman design. We identified the main factors affecting the fermentation conditions of E. coli expressing BpFaeT132C-D143C as post-induction temperature, pre-induction period, and post-induction period. Finally, we used the steepest ascent path design and response surface method to optimize the levels of these three factors further. Under the optimal conditions, the activity of BpFaeT132C-D143C was 3.58 U/ml, which was a significant 6.6-fold increase compared to the pre-optimization (0.47 U/ml), demonstrating the effectiveness of this optimization process. Moreover, BpFaeT132C-D143C activity was 1.52 U/ml in a 3-l fermenter under the abovementioned optimal conditions. It was determined that the expression of BpFaeT132C-D143C in E. coli was predominantly intracellular in the cytoplasm. This study lays the foundation for further research on BpFaeT132C-D143C in degrading agricultural waste transformation applications.
- MeSH
- Escherichia coli * genetics metabolism enzymology MeSH
- Fermentation * MeSH
- Isopropyl Thiogalactoside metabolism MeSH
- Carboxylic Ester Hydrolases * genetics metabolism chemistry biosynthesis MeSH
- Culture Media chemistry MeSH
- Coumaric Acids metabolism MeSH
- Lignin MeSH
- Recombinant Proteins genetics metabolism biosynthesis chemistry MeSH
- Enzyme Stability MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
Glioblastomas are aggressive brain tumors for which effective therapy is still lacking, resulting in dismal survival rates. These tumors display significant phenotypic plasticity, harboring diverse cell populations ranging from tumor core cells to dispersed, highly invasive cells. Neuron navigator 3 (NAV3), a microtubule-associated protein affecting microtubule growth and dynamics, is downregulated in various cancers, including glioblastoma, and has thus been considered a tumor suppressor. In this study, we challenge this designation and unveil distinct expression patterns of NAV3 across different invasion phenotypes. Using glioblastoma cell lines and patient-derived glioma stem-like cell cultures, we disclose an upregulation of NAV3 in invading glioblastoma cells, contrasting with its lower expression in cells residing in tumor spheroid cores. Furthermore, we establish an association between low and high NAV3 expression and the amoeboid and mesenchymal invasive phenotype, respectively, and demonstrate that overexpression of NAV3 directly stimulates glioblastoma invasive behavior in both 2D and 3D environments. Consistently, we observed increased NAV3 expression in cells migrating along blood vessels in mouse xenografts. Overall, our results shed light on the role of NAV3 in glioblastoma invasion, providing insights into this lethal aspect of glioblastoma behavior.
- MeSH
- Phenotype * MeSH
- Glioblastoma * pathology genetics metabolism MeSH
- Neoplasm Invasiveness * genetics MeSH
- Humans MeSH
- Membrane Proteins MeSH
- Microtubules metabolism MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Brain Neoplasms * pathology genetics metabolism MeSH
- Cell Movement genetics physiology MeSH
- Nerve Tissue Proteins metabolism genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Using immunohistochemistry, we examined a large cohort of 135 ovarian tumors, made up of 96 low-grade serous carcinomas (LGSCs) and 39 serous borderline tumors (micropapillary variant, mSBT), with the aim of exploring their HER2 status (overexpression). We followed with comprehensive genomic analyses on this sample set from our previous study, which revealed HER2 mutation in 5% (4/75) of LGSC and 10% (3/29) of mSBT. No cases were evaluated as HER2-positive, but 6 LGSCs and 1 mSBT were scored as HER2 1+, and 2 LGSCs and 1 mSBT showed the so-called HER2 "ultra-low" phenotype. This could be of clinical value as a potential therapeutical target concerning emerging therapeutic treatments (antibody conjugates). However, the clinical significance of this expression still needs to be established.
- MeSH
- Adult MeSH
- Immunohistochemistry MeSH
- Middle Aged MeSH
- Humans MeSH
- Mutation MeSH
- Biomarkers, Tumor genetics metabolism MeSH
- Ovarian Neoplasms * pathology genetics metabolism MeSH
- Receptor, ErbB-2 * genetics metabolism MeSH
- Aged MeSH
- Cystadenocarcinoma, Serous * pathology genetics metabolism MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
N-Methyl-d-aspartate receptors (NMDARs) play a crucial role in excitatory neurotransmission, with numerous pathogenic variants identified in the GluN subunits, including their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs, but this was tested in a limited number of studies. Using microscopy and electrophysiology in the human embryonic kidney 293 (HEK293) cells, we found that surface expression of GluN1/GluN2A receptors containing a set of alanine substitutions within the LBDs correlated with the measured EC50 values for glycine (GluN1 subunit mutations) while not correlating with the measured EC50 values for l-glutamate (GluN2A subunit mutations). The mutant cycle of GluN1-S688 residue, including the pathogenic GluN1-S688Y and GluN1-S688P variants, showed a correlation between relative surface expression of the GluN1/GluN2A receptors and the measured EC50 values for glycine, as well as with the calculated ΔGbinding values for glycine obtained from molecular dynamics simulations. In contrast, the mutant cycle of GluN2A-S511 residue did not show any correlation between the relative surface expression of the GluN1/GluN2A receptors and the measured EC50 values for l-glutamate or calculated ΔGbinding values for l-glutamate. Coexpression of both mutated GluN1 and GluN2A subunits led to additive or synergistic alterations in the surface number of GluN1/GluN2A receptors. The synchronized ER release by ARIAD technology confirmed the altered early trafficking of GluN1/GluN2A receptors containing the mutated LBDs. The microscopical analysis from embryonal rat hippocampal neurons (both sexes) corroborated our conclusions from the HEK293 cells.
- MeSH
- Glycine metabolism MeSH
- HEK293 Cells MeSH
- Hippocampus cytology metabolism MeSH
- Rats MeSH
- Glutamic Acid metabolism MeSH
- Humans MeSH
- Ligands MeSH
- Mutation genetics MeSH
- Protein Domains MeSH
- Nerve Tissue Proteins MeSH
- Receptors, N-Methyl-D-Aspartate * metabolism genetics chemistry MeSH
- Protein Transport physiology genetics MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Pacienti se syndromem suchého oka tvoří významnou část každodenní oftalmologické péče. Mezi rizikové faktory rozvoje syndromu suchého oka patří i diabetes mellitus. Významnou roli zde hrají změny homeostázy slzného filmu, chronický zánět povrchu oka a doprovázející patologie povrchových nervových vláken rohovky. Cílem této práce bylo popsat stav moderních biomarkerů poškození povrchu oka u pacientů s diabetem 1. typu a posoudit jejich využitelnost pro časný záchyt rozvoje syndromu suchého oka. Materiál a metodika: Do studie bylo zahrnuto celkem 19 pacientů s diabetem 1. typu a 15 pacientů v kontrolní skupině. Pacienti podstoupili detailní vyšetření povrchu oka, odběr biomateriálu k laboratorní analýze hladin matrixové metaloproteinázy 9 (MMP-9), exprese HLA-DR epiteliemi a snímání rohovky pomocí rohovkového konfokálního mikroskopu. Výsledky: Ve skupině pacientů s diabetem 1. typu byla statisticky významně nižší celková délka nervových vláken (p = 0,0482). Osmolarita, citlivost rohovky, vitální barvení povrchu oka, tear break-up time (TBUT) ani MMP-9 v slzném filmu se mezi skupinami statisticky významně nelišily (p = 0,8272, p = 0,6029, p = 0,3507, p = 0,7561 a p = 0,0826 respektive). HLA-DR exprese byla testována na skupině 10 pacientů s diabetem 1. typu a 8 pacientech kontrolní skupiny a u obou skupin byla mírná nebo žádná (p > 0,9999). Závěr: V souladu s dříve publikovanou literaturou jsme v naší studii potvrdili snížení celkové délky nervových vláken subbazálního plexu rohovky u pacientů s diabetem 1. typu oproti subjektům v kontrolní skupině. Neprokázali jsme však změny povrchu oka pomocí standardních ani nových markerů (hladina MMP-9, exprese HLA-DR antigenu na povrchu oka) u pacientů s DM1 oproti kontrolní skupině.
Patients with dry eye syndrome form a significant proportion of those treated in everyday ophthalmology practice. Diabetes mellitus is a major risk factor for the development of dry eye syndrome. Changes in tear film homeostasis, chronic inflammation and subsequent corneal nerve fiber pathology play a key role in its progression. The aim of this study was to describe the status of modern biomarkers of ocular surface damage in patients with type 1 diabetes and asses their utility in early diagnosis of dry eye syndrome. Material and methods: In total the pilot study included 19 patients with type 1 diabetes (T1D) and 15 patients in the control group. All patients underwent a detailed ocular surface examination, sample collection for matrix metalloproteinase-9 (MMP-9) laboratory analysis and epithelial HLA-DR expression evaluation, and in-vivo corneal confocal microscopy. Results: T1D patients showed statistically significantly reduced corneal nerve fiber length (p = 0.0482). The differences between the groups in terms of osmolarity, corneal sensitivity, Oxford score, tear break-up time and MMP-9 level were not statistically significant (p = 0.8272, p = 0.6029, p = 0.3507, p = 0.7561 and p = 0.0826 respectively). HLA-DR expression was examined in 10 T1D patients and 8 patients in the control group. Both groups showed minimal or no expression (p > 0.9999). Conclusion: The previously published literature supports our finding of corneal nerve fiber length reduction in T1D patients compared to controls. However, we did not find any significant changes in standard or modern ocular surface markers (MMP-9 levels, HLA-DR expression) measured in patients with dry eye syndrome.
The study focuses on the effects of fluvastatin on immunomarkers of the M1 and M2 macrophages and its direct role in macrophage (M0) polarization. Moreover, it investigates the dependency of immunomodulatory properties of fluvastatin on the mevalonate pathway. Macrophages (M0, M1, M2), differentiated from human blood monocytes, were treated with fluvastatin. Mevalonate and geranylgeranyl pyrophosphate intermediates were introduced to assess the mevalonate pathway dependence. The immunomarkers were evaluated with qPCR, ELISA, Griess assay, and flow cytometry. Fluvastatin significantly reduces the pro-inflammatory gene expression (NFκB, IL-1β, IL-6, iNOS) in M1 while enhancing the anti-inflammatory markers (Arg-1, TGFβ) in M2 macrophages. The production of the TNFα, IL-1β, and IL-6 cytokines is reduced in M1, and IL-10 production increased in M2 macrophages. Fluvastatin decreases the iNOS activity in M1 macrophages. The intermediates reverse the fluvastatin's effects on anti-inflammatory gene expression by M2 macrophages, cytokine production (by M1 and M2 macrophages), and iNOS activity (by M1 macrophages). Their impact on surface marker expression was somewhat limited. These findings demonstrate that fluvastatin exerts anti-inflammatory effects on polarized macrophages without affecting polarization per se and also highlight the dependency on the mevalonate pathway. This study deepens the understanding of statins' immunomodulatory mechanisms, suggesting potential applications in treating inflammatory diseases.
- MeSH
- Anti-Inflammatory Agents * pharmacology MeSH
- Cytokines metabolism MeSH
- Fluvastatin * pharmacology MeSH
- Mevalonic Acid * metabolism MeSH
- Humans MeSH
- Macrophages * drug effects metabolism immunology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Nedd4-2 E3 ligase regulates Na+ homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca2+-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca2+ alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.
- MeSH
- Cryoelectron Microscopy MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Models, Molecular MeSH
- Protein Domains MeSH
- 14-3-3 Proteins * metabolism chemistry MeSH
- Ubiquitination MeSH
- Nedd4 Ubiquitin Protein Ligases * metabolism chemistry genetics ultrastructure MeSH
- Calcium * metabolism MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Lipoprotein (a) [Lp(a)] has been recognized as an independent, inherited, causal risk factor for atherosclerotic cardiovascular disease (ASCVD) and aortic valve stenosis, thus representing a major target of residual CV risk. Currently, no drug has been officially approved for lowering Lp(a) levels, and in clinical practice, Lp(a) is mainly used to (re)define CV risk, particularly in individuals at borderline CV risk and people with a family history of premature coronary heart disease, according to various guidelines. Specific Lp(a)-targeted antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) agents have been developed to produce substantial Lp(a) reductions via the inhibition of apo(a) synthesis in the liver. These drugs are conjugated to N-acetylgalactosamine (GalNAc) to ensure their binding to asialoglycoproteins, which are specifically expressed on the surface of the hepatocytes. Such drugs include pelacarsen (an injectable ASO) and olpasiran, zerlasiran, and lepodisiran (injectable siRNA agents). Muvalaplin represents another therapeutic option to lower Lp(a) levels, since it is an oral selective small molecule inhibitor of Lp(a) formation, thus potentially exerting certain advantages in terms of its clinical use. The present narrative review summarizes the available clinical data on the efficacy and safety of these investigational Lp(a)-lowering therapies, as reported in phase 1 and 2 trials. The effects of these drugs on other [aside from Lp(a)] lipid parameters are also discussed. The phase 3 CV trial outcomes are ongoing for some of these agents (i.e., pelacarsen, olpasiran, and lepodisiran) and are briefly mentioned. Overall, there is an urgent need for evidence-based guidelines on Lp(a) reduction in daily clinical practice, following the results of the phase 3 CV trials, as well as for establishing the ideal Lp(a) quantification method (i.e., using an apo(a) isoform-independent assay with appropriate calibrators, reporting the Lp(a) level in molar units).
- Publication type
- Journal Article MeSH
- Review MeSH
Léčba pacientů s karcinomem žaludku by měla být plánována prakticky ve všech případech v prostředí multidisciplinárního týmu. V případě resekabilního onemocnění používáme ve většině případů kombinaci systémové léčby a chirurgie, chemoterapie je podávána jako perioperační, méně často jako adjuvantní. Indikace chemoradioterapie v adjuvantní indikaci je vyhrazena jako možnost jen pro případy, kdy operace nebyla dostatečně radikální. Základem léčby pokročilého a metastatického onemocnění je paliativní chemoterapie FOLFOX nebo FLOT. Podle výsledků prediktorů je volena cílená léčba a/nebo imunoterapie. V případě pozitivity HER2 je do kombinace s chemoterapií indikován trastuzumab, v případě exprese claudinu 18.2 lze do kombinace s chemoterapií nově zvážit zolbetuximab. Chemoimunoterapie s nivolumabem nebo pembrolizumabem je vhodná pro pacienty s expresí PD-L1, pacienti s nádory s MSI-high jsou kandidáty pro imunoterapii.
Treatment of patients with gastric cancer should be planned in practically all cases in a multidisciplinary team. In the case of resectable disease, we use in most cases a combination of systemic treatment and surgery. Chemotherapy is administered perioperatively, less often as adjuvant treatment. The indication of chemoradiotherapy in adjuvant indication is reserved as an option only for cases where the surgery was not radical enough. The basis of treatment of advanced and metastatic disease is palliative chemotherapy FOLFOX or FLOT. According to the results of predictors, targeted therapy and/or immunotherapy is indicated. In the case of HER2 positivity, trastuzumab is indicated in combination with chemotherapy; in the case of claudin 18.2 expression, zolbetuximab can be newly considered in combination with chemotherapy. Chemoimmunotherapy with nivolumab or pembrolizumab is suitable for patients with PD-L1 expression, patients with MSI-high tumours are candidates for immunotherapy.
- MeSH
- Molecular Targeted Therapy MeSH
- Humans MeSH
- Microsatellite Instability MeSH
- Stomach Neoplasms * pathology therapy MeSH
- Antineoplastic Agents, Immunological pharmacology therapeutic use MeSH
- Antineoplastic Agents pharmacology therapeutic use MeSH
- Antineoplastic Combined Chemotherapy Protocols administration & dosage therapeutic use MeSH
- Antineoplastic Protocols MeSH
- Receptor, ErbB-2 MeSH
- Check Tag
- Humans MeSH
Interferon‐induced transmembrane proteins (IFITMs) are frequently overexpressed in cancer cells, including cervical carcinoma cells, and play a role in the progression of various cancer types. However, their mechanisms of action remain incompletely understood. In the present study, by employing a combination of surface membrane protein isolation and quantitative mass spectrometry, it was comprehensively described how the IFITM1 protein influences the composition of the cervical cancer cell surfaceome. Additionally, the effects of interferon‐γ on protein expression and cell surface exposure were evaluated in the presence and absence of IFITM1. The IFITM1‐regulated membrane and membrane‐associated proteins identified are involved mainly in processes such as endocytosis and lysosomal transport, cell‐cell and cell‐extracellular matrix adhesion, antigen presentation and the immune response. To complement the proteomic data, gene expression was analyzed using reverse transcription‐quantitative PCR to distinguish whether the observed changes in protein levels were attributable to transcriptional regulation or differential protein dynamics. Furthermore, the proteomic and gene expression data are supported by functional studies demonstrating the impact of the IFITM1 and IFITM3 proteins on the adhesive, migratory and invasive capabilities of cervical cancer cells, as well as their interactions with immune cells.
- MeSH
- Cell Adhesion MeSH
- Antigens, Differentiation * metabolism genetics MeSH
- Phenotype MeSH
- Interferon-gamma pharmacology metabolism MeSH
- Humans MeSH
- Membrane Proteins * metabolism genetics MeSH
- Cell Line, Tumor MeSH
- Uterine Cervical Neoplasms * pathology genetics metabolism immunology MeSH
- Cell Movement MeSH
- RNA-Binding Proteins * metabolism genetics MeSH
- Proteome * MeSH
- Proteomics methods MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
