embryonic development
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.
- MeSH
- antivirové látky * farmakologie chemie MeSH
- farmakoterapie COVID-19 MeSH
- HEK293 buňky MeSH
- inhibitory proteas farmakologie chemie MeSH
- koronavirové proteasy 3C * metabolismus antagonisté a inhibitory chemie genetika MeSH
- lidé MeSH
- mutace MeSH
- objevování léků * metody MeSH
- proteolýza MeSH
- SARS-CoV-2 * enzymologie účinky léků metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Tento článek shrnuje roli telomer a enzymu telomerázy v reprodukční fyziologii zvířat a možnosti jejich terapeutického ovlivnění. Telomery, nacházející se na koncích chromozomů, hrají klíčovou roli v ochraně genomové stability a buněčné proliferace. Jejich zkracování může negativně ovlivnit fertilitu, kvalitu gamet a embryonální vývoj. Telomeráza je enzym, který prodlužuje telomery a je aktivní především v kmenových a zárodečných buňkách. Poznatky z humánní medicíny naznačují, že podpora její aktivity může mít pozitivní vliv na repro dukční úspěšnost samců i samic. Protože biologie telomer je u lidí i zvířat velmi podobná, lze poznatky přenést do veterinární medicíny (Fick, 2012). Tento článek analyzuje dostupné studie a zkoumá potenciální využití telomerázové terapie ve veterinární praxi.
This article summarizes the role of telomeres and the enzyme telomerase in animal reproductive physiology and explores potential the rapeutic interventions. Telomeres, located at the ends of chromosomes, play a crucial role in maintaining genomic stability and cellular proliferation. Their shortening can negatively impact fertility, gamete quality, and embryonic development. Telomerase is an enzyme that extends telomeres and is primarily active in stem and germ cells. Findings from human medicine suggest that enhancing its activity may positively influence reproductive success in both males and females. Since telomere biology is highly similar between humans and animals, these insights can be translated into veterinary medicine (Fick, 2012). This article reviews current studies and examines the potential applications of telomerase therapy in veterinary practice.
Pancreas is a vital gland of gastrointestinal system with exocrine and endocrine secretory functions, interweaved into essential metabolic circuitries of the human body. Pancreatic ductal adenocarcinoma (PDAC) represents one of the most lethal malignancies, with a 5-year survival rate of 11%. This poor prognosis is primarily attributed to the absence of early symptoms, rapid metastatic dissemination, and the limited efficacy of current therapeutic interventions. Despite recent advancements in understanding the etiopathogenesis and treatment of PDAC, there remains a pressing need for improved individualized models, identification of novel molecular targets, and development of unbiased predictors of disease progression. Here we aim to explore the concept of precision medicine utilizing 3-dimensional, patient-specific cellular models of pancreatic tumors and discuss their potential applications in uncovering novel druggable molecular targets and predicting clinical parameters for individual patients.
- MeSH
- duktální karcinom slinivky břišní * patologie genetika metabolismus MeSH
- individualizovaná medicína * metody MeSH
- lidé MeSH
- nádory slinivky břišní * patologie genetika MeSH
- techniky 3D buněčné kultury metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: Proximal Femoral Focal Deficiency (PFFD) is the most proximal manifestation of a syndrome involving Congenitally Shortened lower Limbs (CSL), which also affects the fibula and midline metatarsals. This pattern of congenital human long bone deficiencies corresponds, in a time dependent manner, to the failed ingrowth pathways of new blood vessels of the growing embryonic limb. The distal femoral condyles are, in contrast, served by an alternative vascular supply from around the knee joint, and so remain resistant to the CSL deficiency. AIM: We hypothesize that embryonic vascular dysgenesis causes PFFD, as well as the cardinal features of the Femoral, Fibular and midline Metatarsal deficiencies (FFM) syndrome. RESULTS: Arteriography of CSL with PFFD reveals diminution or failed formation of the Femoral Artery (FA), which corresponds to downstream skeletal reductions. It may also reveal preservation of the primitive Axial Artery (AA) of the embryonic limb. The combination of missing and retained primitive vessels inform the time, place, and nature of the etiologic vascular events. This suggests that PFFD is the visible expression of a normally prefigured cartilaginous scaffold of the femur, which develops in conformity with the available pattern of blood vessels present. The teratogen thalidomide, known to affect the forming embryonic vasculature, also produces PFFD indistinguishable from the naturally occurring entity. CONCLUSION: The entire spectrum of PFFD, including phocomelia, fibular, and metatarsal dystrophisms, should thus be regarded as downstream skeletal results of embryonic arterial dysgeneses.
- MeSH
- arteria femoralis * abnormality embryologie MeSH
- femur * abnormality krevní zásobení embryologie MeSH
- fibula abnormality krevní zásobení MeSH
- lidé MeSH
- metatarzální kosti abnormality MeSH
- vrozené deformity dolní končetiny * embryologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: The treatment of non-small cell lung cancer (NSCLC) patients is correlated with the efficacy of immune checkpoint blockade therapy (ICB) targeting programmed cell death ligand 1 (PD-L1) or its cognate receptor (PD-1) on cancer cells or infiltrating immune cells. Analysis of PD-L1/PD-1 expression in tumor tissue represents a crucial step before PD-L1/PD-1 blocker usage. METHODS: We used directed evolution of protein variants derived from a 13 kDa Myomedin loop-type combinatorial library with 12 randomized amino acid residues to select high-affinity binders of human PD-L1 (hPD-L1). After the ribosome display, individual clones were screened by ELISA. Detailed analysis of binding affinity and kinetics was performed using LigandTracer. The specificity of Myomedins was assessed using fluorescent microscopy on HEK293T-transfected cells and cultured cancer cells in vitro, formalin-fixed paraffin-embedded (FFPE) sections of human tonsils, and FFPE tumor samples of NSCLC patients. RESULTS: Seven identified PD-L1 binders, called MLE, showed positive staining for hPD-L1 on transfected HEK293T cells and cultured MCF-7 cells. MLE031, MLE105, MLE249, and MLE309 exhibited high affinity to both human and mouse PD-L1-transfected HEK293T cells measured with LigandTracer. The diagnostic potential of MLE variants was tested on human tonsillitis tissue and compared with diagnostic anti-PD-L1 antibody DAKO 28-8 and PD-L1 IHC 22C3 pharmDx antibody. MLE249 and MLE309 exhibited an excellent overlap with diagnostic DAKO 28-8 (Pearson ́s coefficient (r) = 0.836 and 0.731, respectively) on human tonsils on which MLE309 exhibited also excellent overlap with diagnostic 22C3 antibody (r = 0.876). Using three NSCLC tissues, MLE249 staining overlaps with 28-8 antibody (r = 0.455-0.883), and MLE309 exhibited overlap with 22C3 antibody (r = 0.534-0.619). Three MLE proteins fused with Fc fragments of rabbit IgG, MLE249-rFc, MLE309-rFc and MLE031-rFc, exhibited very good overlap with anti-PD-L1 antibody 28-8 on tonsil tissue (r = 0.691, 0.610, and 0.667, respectively). Finally, MLE249-rFc, MLE309-rFc and MLE031-rFc exhibited higher sensitivity in comparison to IHC 22C3 antibody using routine immunohistochemistry staining system Ventana, which is one of gold standards for PD-L1 diagnosis. CONCLUSIONS: We demonstrated the development of MLE Myomedins specifically recognizing hPD-L1 that may serve as a refinement tool for clinical PD-L1 detection.
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
- elektronová kryomikroskopie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- proteiny 14-3-3 * metabolismus chemie MeSH
- ubikvitinace MeSH
- ubikvitinligasy Nedd4 * metabolismus chemie genetika ultrastruktura MeSH
- vápník * metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Intracellular delivery is a bottleneck in the development of therapeutic peptides and proteins. Here, we demonstrate the efficient delivery of omoMYC, the first MYC inhibitor in clinical trials, using HBpep-SP, an engineered peptide forming liquid-liquid phase-separated coacervates. HBpep-SP coacervates facilitate efficient cellular uptake and intracellular delivery of the omoMYC peptide at concentrations lower than those required for spontaneous uptake. Strikingly, omoMYC coacervates result in reduced proliferation and apoptosis induction in the low c-MYC expressing cell lines HEK293 and SH-SY5Y cells, but not in HeLa and SK-N-BE(2) cells with high c-MYC/MYCN expression, respectively, suggesting that endogenous MYC/N levels may impact the effects of omoMYC. Importantly, our approach bypasses the need for cell penetration-enhancing chemical modifications, offering a novel strategy for the investigation of peptide drug mechanisms in therapeutic development.
- MeSH
- apoptóza účinky léků MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- lékové transportní systémy metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- peptidy * chemie MeSH
- proliferace buněk účinky léků MeSH
- protoonkogenní proteiny c-myc * antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Fibroblast growth factors (FGFs) control organ morphogenesis during development as well as tissue homeostasis and repair in the adult organism. Despite their importance, many mechanisms that regulate FGF function are still poorly understood. Interestingly, the thermodynamic stability of 22 mammalian FGFs varies widely, with some FGFs remaining stable at body temperature for more than 24 h, while others lose their activity within minutes. How thermodynamic stability contributes to the function of FGFs during development remains unknown. Here we show that FGF10, an important limb and lung morphogen, exists as an intrinsically unstable protein that is prone to unfolding and is rapidly inactivated at 37 °C. Using rationally driven directed mutagenesis, we have developed several highly stable (STAB) FGF10 variants with a melting temperature of over 19 °C more than that of wildtype FGF10. In cellular assays in vitro, the FGF10-STABs did not differ from wildtype FGF10 in terms of binding to FGF receptors, activation of downstream FGF receptor signaling in cells, and induction of gene expression. In mouse embryonal lung explants, FGF10-STABs, but not wildtype FGF10, suppressed branching, resulting in increased alveolarization and expansion of epithelial tissue. Similarly, FGF10-STAB1, but not FGF10 wildtype, inhibited the growth of mouse embryonic tibias and markedly altered limb morphogenesis when implanted into chicken limb buds, collectively demonstrating that thermal instability should be considered an important regulator of FGF function that prevents ectopic signaling. Furthermore, we show enhanced differentiation of human iPSC-derived lung organoids and improved regeneration in ex vivo lung injury models mediated by FGF10-STABs, suggesting an application in cell therapy.
- MeSH
- fibroblastový růstový faktor 10 * metabolismus genetika chemie MeSH
- lidé MeSH
- myši MeSH
- plíce metabolismus embryologie MeSH
- receptory fibroblastových růstových faktorů metabolismus MeSH
- signální transdukce * MeSH
- stabilita proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Aryl hydrocarbon receptor (AhR) is a transcription factor that is primarily known as an intracellular sensor of environmental pollution. After five decades, the list of synthetic and toxic chemicals that activate AhR signaling has been extended to include a number of endogenous compounds produced by various types of cells via their metabolic activity. AhR signaling is active from the very beginning of embryonal development throughout the life cycle and participates in numerous biological processes such as control of cell proliferation and differentiation, metabolism of aromatic compounds of endogenous and exogenous origin, tissue regeneration and stratification, immune system development and polarization, control of stemness potential, and homeostasis maintenance. AhR signaling can be affected by various pharmaceuticals that may help modulate abnormal AhR signaling and drive pathological states. Given their role in immune system development and regulation, AhR antagonistic ligands are attractive candidates for immunotherapy of disease states such as advanced prostate cancer, where an aberrant immune microenvironment contributes to cancer progression and needs to be reeducated. Advanced stages of prostate cancer are therapeutically challenging and characterized by decreased overall survival (OS) due to the metastatic burden. Therefore, this review addresses the role of AhR signaling in the development and progression of prostate cancer and discusses the potential of AhR as a drug target for the treatment of advanced prostate cancer upon entering the phase of drug resistance and failure of first-line androgen deprivation therapy.Abbreviation: ADC: antibody-drug conjugate; ADT: androgen deprivation therapy; AhR: aryl hydrocarbon receptor; AR: androgen receptor; ARE: androgen response element; ARPI: androgen receptor pathway inhibitor; mCRPC: metastatic castration-resistant prostate cancer; DHT: 5a-dihydrotestosterone; FICZ: 6-formylindolo[3,2-b]carbazole; 3-MC: 3-methylcholanthrene; 6-MCDF: 6-methyl-1,3,8-trichlorodibenzofuran; MDSCs: myeloid-derived suppressor cells; PAHs: polycyclic aromatic hydrocarbons; PCa: prostate cancer; TAMs: tumor-associated macrophages; TF: transcription factor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TME: tumor microenvironment; TRAMP: transgenic adenocarcinoma of the mouse prostate; TROP2: tumor associated calcium signal transducer 2.
- MeSH
- cílená molekulární terapie MeSH
- lidé MeSH
- nádorové mikroprostředí účinky léků MeSH
- nádory prostaty * farmakoterapie metabolismus patologie MeSH
- protinádorové látky * terapeutické užití farmakologie MeSH
- receptory aromatických uhlovodíků * metabolismus antagonisté a inhibitory MeSH
- signální transdukce účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Syndrom střední aorty (MAS) je způsoben stenózou hrudní a/nebo břišní aorty, často doprovázenou současnou stenózou renálních nebo viscerálních tepen. Ačkoli je MAS vzácný (0,5-2 %), je závažnou příčinou hypertenze u dětí a dospívajících s vysokou morbiditou. Předpokládá se, že vzniká při selhání splynutí obou dorzálních aort během embryonálního vývoje, a vysoké procento případů je idiopatických. Klinicky probíhá se symptomatickou nebo asymptomatickou arteriální hypertenzí. Metodou volby je dnes angiografie, neinvazivní MR angiografie a CT angiografie mají podobnou diagnostickou přesnost. Ultrasonografie je primární screeningovou technikou. Léčba spočívá v kombinaci různých antihypertenziv. Chirurgická léčba může být kurativní. Naším cílem bylo přiblížit klinický obraz, diagnostiku a léčebný postup dvou nových případů s MAS.
Middle aortic syndrome (MAS) is caused by stenosis of the thoracic and/or abdominal aorta, often accompanied by concomitant stenosis of the renal or visceral arteries. Although MAS is rare (0.5-2%), it is a serious cause of hypertension in children and adolescents with high morbidity. It is thought to arise from failure of fusion of the two dorsal aortas during embryonic development, and a high percentage of cases are idiopathic. Clinically, it presents with symptomatic or asymptomatic arterial hypertension. The method of choice today is angiography, non-invasive MR angiography and CT angiography have similar diagnostic accuracy. Ultrasonography is the primary screening technique. Treatment consists of a combination of various antihypertensive drugs. Surgical treatment may be curative. Our aim was to present the clinical picture, diagnosis and treatment of two new cases with MAS.
- Klíčová slova
- syndrom střední aorty,
- MeSH
- angioplastika metody MeSH
- antihypertenziva farmakologie terapeutické užití MeSH
- aorta * diagnostické zobrazování patologie MeSH
- CT angiografie metody MeSH
- dítě MeSH
- hypertenze etiologie MeSH
- lidé MeSH
- mladiství MeSH
- obstrukce renální arterie etiologie MeSH
- radioisotopová scintigrafie metody MeSH
- ultrasonografie metody MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
