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

Celotělovápostmortem CTangiografie(PMCTA) představujevcelosvětovémměřítkuexperimentálnímetoduzávisloupředevšímnadostupnostiinterdisciplinární spolupráce, personálním a technickém vybavení a finančních možnostech pracoviště. Autoři prezentují technické poznámky k etablovaní PMCTA na pracovišti, které od roku 2015 rutinně provádělo nativní CT vyšetření. Aplikaci této diagnosticky i vědecko-výzkumně výtěžné metody do soudnělékařské praxe umožnila mezioborová spolupráce společně s institucionální podporou rozvoje nových diagnostických metod.

Whole-body post mortem CT angiography (PMCTA) is an innovative and experimental imaging technique that relies heavily on interdisciplinary collaboration, access to skilled personnel, advanced technical equipment and the financial possibilities of the workplace. Native CT examinations (PMCT) prior to autopsy are already a standard procedure in certain forensic departments in the Czech Republic (e.g., murders, suicides, deaths of children, traffic accidents etc.). Nonetheless, the progression of forensic sciences all over the world shows the necessity to integrate other advanced imaging modalities in routine forensic practice. Incorporating PMCTA into standard forensic workflows enhances the precision of forensic diagnostics, supplements traditional autopsy findings, and elevates the objectivity of forensic outputs. This paper presents technical notes on the development of PMCTA in forensic practice in a department that since 2015 until now has routinely performed native CT examinations. Institutional support was crucial in enabling the adoption of the imaging technique, which has so far been applied to more than thirty cases. The department is currently conducting a comparative study focused on the application of three different types of perfusion media – polyethylene glycol (PEG), saline, paraffin oil – and assessing the diagnostic efficacy of PMCTA relative to conventional autopsy. Based on our experience, PMCTA is suitable for all corpses except those with advanced post-mortem decomposition or extensive open injuries. The highest diagnostic yield is achieved in cases involving suspected gastrointestinal bleeding or vascular pathologies and lesions especially of large vessels (e.g., dissection/rupture of the aorta). The protocol for whole-body PMCTA can be adapted to meet the specific needs and conditions of individual forensic departments, providing a flexible yet robust framework for enhancing forensic medical investigations.

• MeSH
• CT angiografie metody   MeSH
• diagnostické zobrazování metody   MeSH
• lidé MeSH
• pitva * metody   MeSH
• počítačová rentgenová tomografie * metody   MeSH
• soudní lékařství metody   trendy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Ubiquinone (UQ), the only known electron carrier in the mammalian electron transport chain (ETC), preferentially delivers electrons to the terminal electron acceptor oxygen (O2). In hypoxia, ubiquinol (UQH2) diverts these electrons onto fumarate instead. Here, we identify rhodoquinone (RQ), an electron carrier detected in mitochondria purified from certain mouse and human tissues that preferentially delivers electrons to fumarate through the reversal of succinate dehydrogenase, independent of environmental O2 levels. The RQ/fumarate ETC is strictly present in vivo and is undetectable in cultured mammalian cells. Using genetic and pharmacologic tools that reprogram the ETC from the UQ/O2 to the RQ/fumarate pathway, we establish that these distinct ETCs support unique programs of mitochondrial function and that RQ confers protection upon hypoxia exposure in vitro and in vivo. Thus, in discovering the presence of RQ in mammals, we unveil a tractable therapeutic strategy that exploits flexibility in the ETC to ameliorate hypoxia-related conditions.

• MeSH
• elektrony MeSH
• fumaráty metabolismus   MeSH
• hypoxie metabolismus   MeSH
• kyslík metabolismus   MeSH
• lidé MeSH
• mitochondrie * metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• sukcinátdehydrogenasa metabolismus   MeSH
• transport elektronů MeSH
• ubichinon * metabolismus   analogy a deriváty   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Úvod: Technologie trojrozměrného exoskopu ORBEYE™ Olympus je unikátním řešením v oblasti vizualizace s jasnějším obrazem ve srovnání se standardním obrazem, který umožňuje přepnutí i do režimu úzkopásmového zobrazování. ORBEYE™ poskytuje přesné zobrazení anatomických detailů a struktur tkání. Metoda a materiál: Od roku 2021 je na našem pracovišti k dispozici přístroj ORBEYETM. V období od 11/2021 do 11/2023 jsme s jeho využitím operovali 97 pacientů. Výsledky: Technologii jsme používali v otorinolaryngologii pro tyto operační výkony: navigovaná mikrochirurgie hrtanu, biopsie z orofaryngu/hypofaryngu v kombinaci s využitím módu úzkopásmového světla (n = 75) a navigované biopsie z kořene jazyka a hypofaryngu. Dále pro ušní operace v rozsahu myringoplastika, atikoantrotomie, meatoplastika (n = 7). Dále pro primární a revizní navigované operace příštítných tělísek, navigované operace štítné žlázy – v rozsahu hemithyreoidektomie, totální thyreoidektomie (n = 15). Závěr: Exoskop ORBEYE™ poskytuje nový pohled na zobrazení tkání hrtanu, orofaryngu, hypofaryngu, středního ucha, štítné žlázy a příštítných tělísek při chirurgických zákrocích. Jeho výhodami jsou přirozená poloha těla chirurga při operaci, operování bez okulárních čoček, nelimitovaný operační prostor. Systém umožňuje účast celého chirurgického týmu na operačních výkonech a předávání zkušeností dalším kolegům.

Introduction: The 4K 3-dimensional ORBEYE™ exoscope/video microscope technology (Olympus) is a revolutionary solution in visualization with clearer precise images compared to the standard one, allowing the switch to a Narrow Band Imaging (NBI) mode. It is mainly used in neurosurgery. Materials and methods: Between 11/2021 and 11/2023, we operated on 97 patients using ORBEYE™, which has been available in our institution since 2021. Results: We used ORBEYE™ in otorhinolaryngology on head and neck surgery for this spectrum of surgical procedures: Group I: navigated laryngeal microsurgery and navigated oropharyngeal and hypopharyngeal biopsies. Seventy-five patients were operated on with a diagnosis of premalignant and malignant lesions of the larynx, oropharynx, and hypopharynx. Group II: navigated parathyroid surgery and thyroid surgery. A total of 15 patients were operated on parathyroid and thyroid lesions. Group III: ear surgery. A total of 7 patients were operated on for diagnosis of tympanic membrane perforation, chronic otitis media, and exostoses of the external auditory canal. Conclusion: ORBEYE™ provided a unique resolution and highly detailed view of the laryngeal, oropharyngeal, hypopharyngeal, middle ear, thyroid, and parathyroid tissues in high-resolution 4K and 3-dimensional modes of surgical procedures. For us, the advantages of the ORBEYE™ system were a comfortable upright position of the surgeon‘s body, the ability to perform surgery without eyepiece lenses, flexible positioning, and a large surgical space. This method allowed the participation of the entire surgical team in the surgical procedures and the transfer of experience to other colleagues. Thus, we expect a rapid development of ORBEYE™ technology in many surgical directions.

• Klíčová slova
• ORBEYE exoskop,
• MeSH
• digitální technologie metody   MeSH
• farynx chirurgie   patologie   MeSH
• lidé MeSH
• mikrochirurgie klasifikace   metody   přístrojové vybavení   MeSH
• otorinolaryngologické chirurgické výkony * klasifikace   metody   přístrojové vybavení   MeSH
• paratyreoidea chirurgie   patologie   MeSH
• štítná žláza chirurgie   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• klinická studie MeSH
• práce podpořená grantem MeSH

BACKGROUND: High-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive subtype of epithelial ovarian carcinoma. It is primarily diagnosed at stage III or IV when the 5-year survival rate ranges between 20% and 40%. Here, we aimed to validate the hypothesis, based on HGSOC cell lines, that proposed the existence of two distinct groups of HGSOC cells with high and low oxidative phosphorylation (OXPHOS) metabolism, respectively, which are associated with their responses to glucose and glutamine withdrawal. METHODS: We isolated and cultivated primary cancer cell cultures from HGSOC and nontransformed ovarian fibroblasts from the surrounding ovarium of 45 HGSOC patients. We tested the metabolic flexibility of the primary cells, particularly in response to glucose and glutamine depletion, analyzed and modulated endoplasmic reticulum stress, and searched for indices of the existence of previously reported groups of HGSOC cells with high and low OXPHOS metabolism. RESULTS: The primary HGSOC cells did not form two groups with high and low OXPHOS that responded differently to glucose and glutamine availabilities in the cell culture medium. Instead, they exhibited a continuum of OXPHOS phenotypes. In most tumor cell isolates, the responses to glucose or glutamine withdrawal were mild and surprisingly correlated with those of nontransformed ovarian fibroblasts from the same patients. The growth of tumor-derived cells in the absence of glucose was positively correlated with the lipid trafficking regulator FABP4 and was negatively correlated with the expression levels of HK2 and HK1. The correlations between the expression of electron transport chain (ETC) proteins and the oxygen consumption rates or extracellular acidification rates were weak. ER stress markers were strongly expressed in all the analyzed tumors. ER stress was further potentiated by tunicamycin but not by the recently proposed ER stress inducers based on copper(II)-phenanthroline complexes. ER stress modulation increased autophagy in tumor cell isolates but not in nontransformed ovarian fibroblasts. CONCLUSIONS: Analysis of the metabolism of primary HGSOC cells rejects the previously proposed hypothesis that there are distinct groups of HGSOC cells with high and low OXPHOS metabolism that respond differently to glutamine or glucose withdrawal and are characterized by ETC protein levels.

• Publikační typ
• časopisecké články MeSH

There is a lack of experimental electron ionization high-resolution mass spectra available to assist compound identification. The in silico generation of mass spectra by quantum chemistry can aid annotation workflows, in particular to support the identification of compounds that lack experimental reference spectra, such as environmental chemicals. We present an open-source, semiautomated workflow for the in silico prediction of electron ionization high-resolution mass spectra at 70 eV based on the QCxMS software. The workflow was applied to predict the spectra of 367 environmental chemicals, and the accuracy was evaluated by comparison to experimental reference spectra acquired. The molecular flexibility, number of rotatable bonds, and number of electronegative atoms of a compound were negatively correlated with prediction accuracy. Few analytes are predicted to sufficient accuracy for the direct application of predicted spectra in spectral matching workflows (overall average score 428). The m/z values of the top 5 most abundant ions of predicted spectra rarely match ions in experimental spectra, evidencing the disconnect between simulated fragmentation pathways and empirical reaction mechanisms.

• Publikační typ
• časopisecké články MeSH

PURPOSE OF THE STUDY: Surgical options for paediatric femoral fractures include fl exible intramedullary nailing (FIN), plating, and external fi xators. Length unstable fractures are usually spiral, long oblique, or comminuted and are often associated with > 2 cm of shortening. The purpose of this study was to see whether FIN is effective for managing unstable femur fractures in children. MATERIAL AND METHODS: An electronic literature search was performed up to 25 February 2022 in Cochrane Library, PubMed, and Embase databases using a combination of MeSH search terms and keywords related to the population (e.g., "child" AND "diaphyses" AND "femur"), and intervention (e.g., "nail" OR "ESIN"). The data extracted included the study details, Demographic data, surgical details, postoperative immobilization, complications, and outcome. RESULTS: Eight studies with a total sample size of 369 patients were reviewed. The mean operative time, blood loss, and length of stay in the hospital were 67.62±12.32 minutes, 33.82±16.82 ml, and 4.9±1.27 days, respectively. The results were excellent in 61.92% of the patients, satisfactory in 32.61%, and poor in 5.43%. 4.54% of patients had major complications requiring reoperation and 32.46% of patients had minor complications. the most common complication was nail prominence seen in 26.30% of patients. Locked Ender's nail was associated with the least reoperation, malunion, and LLD rate compared to other types of FIN. CONCLUSIONS: FIN along with a single walking spica cast is a good choice in all forms of paediatric femoral fracture patterns allowing proper alignment and rotation. Locked Ender's nail is safe and effective for managing unstable paediatric femur fracture. KEY WORDS: pediatric femur fracture, length unstable, fl exible intramedullary nailing, submuscular plating, Flynn criterion.

• MeSH
• dítě MeSH
• dolní končetina MeSH
• femur MeSH
• fraktury femuru * chirurgie   MeSH
• interní fixátory MeSH
• intramedulární fixace fraktury * škodlivé účinky   MeSH
• lidé MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• systematický přehled MeSH

• Klíčová slova
• lidský kapitál, lidský faktor, green skills,
• MeSH
• analýza dat MeSH
• asertivita MeSH
• digitální technologie MeSH
• hygiena práce * zákonodárství a právo   MeSH
• lidé MeSH
• práce na dálku MeSH
• pracovní stres MeSH
• technologie zelené chemie MeSH
• technologie metody   organizace a řízení   MeSH
• úrazy a nehody zákonodárství a právo   MeSH
• Check Tag
• lidé MeSH

The pre-tetramerization loop (PTL) of the human tumor suppressor protein p53 is an intrinsically disordered region (IDR) necessary for the tetramerization process, and its flexibility contributes to the essential conformational changes needed. Although the IDR can be accurately simulated in the traditional manner of molecular dynamics (MD) with the end-to-end distance (EEdist) unhindered, we sought to explore the effects of restraining the EEdist to the values predicted by electron microscopy (EM) and other distances. Simulating the PTL trajectory with a restrained EEdist , we found an increased agreement of nuclear magnetic resonance (NMR) chemical shifts with experiments. Additionally, we observed a plethora of secondary structures and contacts that only appear when the trajectory is restrained. Our findings expand the understanding of the tetramerization of p53 and provide insight into how mutations could make the protein impotent. In particular, our findings demonstrate the importance of restraining the EEdist in studying IDRs and how their conformations change under different conditions. Our results provide a better understanding of the PTL and the conformational dynamics of IDRs in general, which are useful for further studies regarding mutations and their effects on the activity of p53.

• MeSH
• konformace proteinů MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• nádorový supresorový protein p53 chemie   MeSH
• sekundární struktura proteinů MeSH
• simulace molekulární dynamiky * MeSH
• vnitřně neuspořádané proteiny * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson's correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion.

• Publikační typ
• časopisecké články MeSH