OBJECTIVE: Transgenic mice with fluorescent protein (FP) reporters take full advantage of new in vivo imaging technologies. Therefore, we generated a TRPC5- and a TRPA1-reporter mouse based on FP C-terminal fusion, providing us with better alternatives for studying the physiology, interaction and coeffectors of these two TRP channels at the cellular and tissue level. METHODS: We generated transgenic constructs of the murine TRPC5- and TRPA1-gene with a 3*GGGGS linker and C-terminal fusion to mCherry and mTagBFP, respectively. We microinjected zygotes to generate reporter mice. Reporter mice were examined for visible fluorescence in trigeminal ganglia with two-photon microscopy, immunohistochemistry and calcium imaging. RESULTS: Both TRPC5-mCherry and TRPA1-mTagBFP knock-in mouse models were successful at the DNA and RNA level. However, at the protein level, TRPC5 resulted in no mCherry fluorescence. In contrast, sensory neurons derived from the TRPA1-reporter mice exhibited visible mTag-BFP fluorescence, although TRPA1 had apparently lost its ion channel function. CONCLUSIONS: Creating transgenic mice with a TRP channel tagged at the C-terminus with a FP requires detailed investigation of the structural and functional consequences in a given cellular context and fine-tuning the design of specific constructs for a given TRP channel subtype. Different degrees of functional impairment of TRPA1 and TRPC5 constructs suggest a specific importance of the distal C-terminus for the regulation of these two channels in trigeminal neurons.
- MeSH
- Red Fluorescent Protein MeSH
- Trigeminal Ganglion metabolism MeSH
- Gene Knock-In Techniques * MeSH
- TRPC Cation Channels * genetics metabolism MeSH
- TRPA1 Cation Channel * genetics metabolism MeSH
- Luminescent Proteins * genetics metabolism MeSH
- Mice, Transgenic * MeSH
- Mice MeSH
- Recombinant Fusion Proteins metabolism genetics MeSH
- Calcium metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
INTRODUCTION: Crystalloid and colloid solutions commonly used in intensive and perioperative care can affect haemocoagulation status. This in vitro study assessed the impact of Plasma-Lyte, albunorm 5%, and Gelaspan 4% solutions on primary and secondary haemostasis using rotational thromboelastometry and platelet function analyser. METHODS: In this prospective study, we examined blood samples from 20 healthy volunteers using rotational thromboelastometry and platelet function analyser. Simultaneously, we analysed the blood samples subjected to 10% dilution using Plasma-Lyte, albunorm 5%, and Gelaspan 4% solutions. RESULTS: Compared to controls, Plasma-Lyte shortened EXTEM-CT (p = 0.005) and reduced FIBTEM-MCF (p = 0.017). albunorm 5% prolonged EXTEM-CFT (p = 0.001), decreased EXTEM-alpha (p < 0.001) and MCF in EXTEM, INTEM, and FIBTEM tests (p < 0.001, p = 0.038, p = 0.001, respectively), along with MCE in the PLTEM test (p < 0.001). Gelaspan 4% also prolonged EXTEM-CFT (p < 0.001), decreased EXTEM-alpha (p < 0.001) and MCF in EXTEM, INTEM, and FIBTEM tests (p < 0.001, p < 0.001, p = 0.009, respectively), along with MCE in the PLTEM test (p < 0.001). Gelaspan 4% also reduced EXTEM-CT (p = 0.021). All solution prolonged CT in PFA Col/ADP (p = 0.003 for Plasma-Lyte, p < 0.001 for albunorm and Gelaspan) and albunorm 5% also prolonged CT in Col/Epi (p = 0.003). CONCLUSION: Plasma-Lyte had the least effect on secondary haemostasis, whereas albunorm 5% had the least effect among colloids. Gelaspan 4% adversely affected the propagation phase of coagulation, maximal strength and elasticity of the coagulum, and the level of functional fibrinogen. All solutions adversely affected platelet function in primary haemostasis, with Plasma-Lyte showing the least effect.
- Publication type
- Journal Article MeSH
Východiska: Krvácení do horní části gastrointestinálního traktu je relativně častou, ale potenciálně fatální náhlou příhodou břišní. Na diagnostice a léčbě tohoto onemocnění se podílí řada odborností, ale nemocní jsou obvykle primárně hospitalizováni na lůžkách chirurgických oddělení. Ošetřující lékař, tj. chirurg je tedy zodpovědný za léčbu pacienta a indikaci konzilií spolupracujících specialistů. Operace může být také ultimum refugium postupem při selhání méně invazivních léčebných postupů. Cíl: Cílem práce je přehled současných postupů a metod užívaných v léčbě pacientů s krvácením do horní části trávicího traktu na základě literární rešerše i vlastních zkušeností s léčbou těchto nemocných. Závěr: Krvácení do horní části gastrointestinalniho traktu je relativně častou náhlou příhodou. Jde o krvácení, jehož zdroj je proximálně od Treitzova ligamenta. Diagnostika a léčba vyžaduje multioborovou spolupráci více oborů. Klíčovou roli v diagnostice a terapii dnes představuje endoskopie. Správné načasování jednotlivých kroků je zásadní pro přežití pacienta. Článek v přehledné formě shrnuje současné doporučené postupy od iniciální resuscitace, tekutinové léčby, podání krevních náhrad přes úpravu koagulačních parametrů u pacientů s antikoagulační a antitrombotickou léčbou, možnosti endoskopické diagnostiky a terapie a také postupy při recidivě krvácení, vč. angiointervenční a chirurgické léčby s hlavním zaměřením na nevariceální krvácení.
Background: Upper gastrointestinal bleeding is a relatively common but potentially fatal medical emergency. Many medical disciplines are involved in the diagnosis and treatment of this condition. The patients are usually admitted primarily to surgical wards and the attending surgeon is responsible for management of the patients. Surgery may also be an ultimatum refugium when less invasive treatments fail. Objective: The aim of this study is to review the current practice in the management of patients with upper gastrointestinal bleeding based on a literature review and our own experience in the management of these patients. Conclusions: Upper gastrointestinal bleeding is a relatively common emergency. It is a hemorrhage whose the source is proximal to the ligament of Treitz. The diagnosis and treatment require a multidisciplinary approach. Today, endoscopy plays a key role in the diagnosis and treatment. The correct timing of each step is essential for patient survival. This article provides a clear summary of the current recommended procedures from initial resuscitation, fluid therapy, administration of blood substitutes, adjustment of coagulation parameters in patients on anticoagulant and antithrombotic therapy, endoscopic diagnostic and therapeutic options, and procedures for recurrent bleeding, including angiointervention and surgical treatment, with a main focus on nonvariceal bleeding.
- MeSH
- Anticoagulants adverse effects therapeutic use MeSH
- Endoscopy, Digestive System MeSH
- Gastrointestinal Hemorrhage * diagnosis etiology therapy MeSH
- Upper Gastrointestinal Tract * surgery pathology drug effects MeSH
- Humans MeSH
- Practice Guidelines as Topic MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
Cíl: Referovat výsledky léčby pacientů s centrální serózní chorioretinopatií léčených pomocí laseru Navilas. Materiál a metody: Retrospektivně byly vyhodnoceny výsledky ošetření 39 očí 37 pacientů s akutní formou centrální serózní chorioretinopatie, u kterých nedošlo při konzervativní terapii k vstřebání serózní ablace smyslového epitelu sítnice. U těchto pacientů jsme provedli ošetření tvrdou stopou v místě průsaku (hotspot) pomocí navigovaného laseru Navilas. Výsledky: 3 pacienti se na kontrolu nedostavili, ze zbylých 36 došlo u 32 očí k úplnému vstřebání tekutiny. U 2 pacientů bylo nutné opětovné ošetření, u jednoho pacienta se rozvinula choroideální neovaskularizace a u jednoho nedošlo ani při opětovném ošetření ke vstřebání tekutiny. Závěr: Laserové ošetření hotspotu u pacientů s centrální serózní chorioretinopatií je stále velmi efektivní léčebná metoda. Pomocí navigačního laseru Navilas lze ošetřit i léze juxtafoveolární s vysokou mírou bezpečnosti.
Aim: To report the results of treatment of patients with central serous chorioretinopathy treated with a Navilas laser. Material and Methods: We retrospectively evaluated the results of the treatment of 39 eyes of 37 patients with acute form of central serous chorioretinopathy, who did not respond to conventional treatment. In these patients we performed focal laser treatment at the point of leakage (hotspot) using a Navilas guided laser. Results: 3 patients did not report for the check-up, of the remaining 36 eyes, complete liquid absorption was achieved in 32. Retreatment was necessary in 2 patients, choroidal neovascularization developed in one patient, and in one patient fluid absorption was not achieved even after retreatment. Conclusion: Focal laser treatment of hotspots in patients with central serous chorioretinopathy is still a very effective treatment method. Juxtafoveolar lesions can also be treated with a high degree of safety using a Navilas navigation laser.
The abnormalities in blood coagulation in patients with diabetes can lead to a prothrombotic state and requirement for the administration of direct anticoagulants. However, no comparative studies have been conducted on the effects of different direct anticoagulants. A head-to-head investigation of the impact of anticoagulants in 50 patients of type 1 diabetes mellitus (DMT1) was performed, and the data were compared to 50 generally healthy individuals. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured in plasma treated with vehicle, heparin, or four direct anticoagulants at 1 μM. In addition to common biochemical parameters, novel inflammatory markers (neopterin, kynurenine/tryptophan ratio) and major vitamin K forms were measured. Heparin and dabigatran treatments resulted in prolonged coagulation in DMT1 patients compared to healthy individuals in both tests (both p < 0.001). The same phenomenon was observed for rivaroxaban and apixaban-treated samples in PT (p < 0.001). Interestingly, healthy volunteers had higher total vitamin K levels than DMT1. Further analysis suggested that observed coagulation differences were not caused by differences in glycemia but were rather associated with an unexpected, better lipid profile of our DMT1 group. There were also correlations between prolongation of coagulation brought about by the most active anticoagulants and inflammatory markers, and hence inflammatory state probably also contributed to the differences, as well as the mentioned differences in vitamin K levels. Conclusively, this paper suggests the suitability for controlling the effects of direct anticoagulants in DMT1 patients.
- MeSH
- Anticoagulants * pharmacology therapeutic use MeSH
- Dabigatran pharmacology MeSH
- Diabetes Mellitus, Type 1 * blood drug therapy MeSH
- Adult MeSH
- Blood Coagulation drug effects MeSH
- Heparin pharmacology MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Partial Thromboplastin Time MeSH
- Prothrombin Time MeSH
- Rivaroxaban pharmacology MeSH
- Case-Control Studies MeSH
- Vitamin K * blood MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Vápník a fosfor jsou důležitými minerály, na jejichž metabolismu a homeostáze se v organismu podílí celá řada hormonů a orgánů, zejména skelet, příštítná tělíska, gastrointestinální trakt a ledviny. Poruchy fosfokalciového metabolismu mohou být spojeny s nefyziologickými koncentracemi těchto minerálů v séru, patologickými ději v kostní tkáni a poruchami hlavních regulačních systémů, hlavně příštítných tělísek, ledvin a gastrointestinálního traktu.
Calcium and phosphorus are important minerals whose metabolism and homeostasis in the body involve many hormones and organs, especially the skeleton, parathyroid glands, digestive tract and kidneys. Disturbances in phospho-calcium metabolism may be associated with non-physiological serum concentrations of these minerals, pathological processes in bone tissue, and disturbances in major regulatory systems, particularly the parathyroid glands, kidneys, and gastrointestinal tract.
- MeSH
- Phosphorus * blood MeSH
- Hyperphosphatemia diagnosis complications MeSH
- Hypercalcemia diagnosis drug therapy complications MeSH
- Hypophosphatemia diagnosis complications MeSH
- Hypocalcemia diagnosis drug therapy complications MeSH
- Calcitriol blood metabolism MeSH
- Humans MeSH
- Metabolic Diseases diagnosis drug therapy complications metabolism MeSH
- Vitamin D Deficiency diagnosis drug therapy blood MeSH
- Calcium * blood MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review 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
Unfractionated heparin has long been considered the standard anticoagulation in ECMO, despite some pitfalls such as heparin resistance, heparin induced thrombocytopenia (HIT), etc Recently, some centres started to increasingly use argatroban for this purpose, typically using activated partial thromboplastin time (aPTT) for its monitoring. Direct monitoring of the efficacy of argatroban using Anti-IIa is not yet an established method, although it might be more appropriate as it targets the same pathway.An observational study was performed in adult veno-venous ECMO patients hospitalized with SARS-CoV-2 infection anticoagulated with argatroban to an aPTT target of 40-60 s and Anti-IIa target of 0.4-0.6 μg/mL. Bleeding and thrombotic complications were monitored.Forty-four VV ECMO patients were included, with an overall hospital mortality of approx. 50%. No life-threatening thrombotic events were recorded. The risk of bleeding complications significantly increased with aPTT above 52.7 s and with Anti-IIa values over 0.78 μg/mL. Using the above cut-offs for both the aPTT and Anti-IIa and their combination, the negative predictive value for bleeding was approximately 90%.It seems that the generally recommended limits for Anti-IIa of 1.5 μg/mL may be high. However, further data are needed to confirm lower limits.Trial Registration:retrospectively registered in ClinicalTrials.gov, NCT06038682.
- MeSH
- Anticoagulants * therapeutic use adverse effects administration & dosage MeSH
- Arginine analogs & derivatives MeSH
- COVID-19 * blood therapy mortality complications MeSH
- Adult MeSH
- COVID-19 Drug Treatment * MeSH
- Hemorrhage chemically induced MeSH
- Pipecolic Acids * therapeutic use administration & dosage adverse effects MeSH
- Middle Aged MeSH
- Humans MeSH
- Extracorporeal Membrane Oxygenation * methods MeSH
- Drug Monitoring methods MeSH
- Partial Thromboplastin Time MeSH
- SARS-CoV-2 MeSH
- Aged MeSH
- Sulfonamides MeSH
- Thrombosis prevention & control etiology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Observational Study MeSH
Mitochondria are key to cellular energetics, metabolism, and signaling. Their dysfunction is linked to devastating diseases, including mitochondrial disorders, diabetes, neurodegenerative diseases, cardiac disorders, and cancer. Here, we present a knockout mouse model lacking the complex IV assembly factor SMIM20/MITRAC7. SMIM20-/- mice display cardiac pathology with reduced heart weight and cardiac output. Heart mitochondria present with reduced levels of complex IV associated with increased complex I activity, have altered fatty acid oxidation, and display elevated levels of ROS production. Interestingly, mutant mouse ventricular myocytes show unphysiological Ca2+ handling, which can be attributed to the increase in mitochondrial ROS production. Our study presents an example of a tissue-specific phenotype in the context of OXPHOS dysfunction. Moreover, our data suggest a link between complex IV dysfunction and Ca2+ handling at the endoplasmic reticulum through ROS signaling.
- MeSH
- Endoplasmic Reticulum metabolism MeSH
- Myocytes, Cardiac metabolism MeSH
- Membrane Proteins * metabolism genetics MeSH
- Mitochondrial Proteins * metabolism genetics MeSH
- Myocardium * metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Oxidative Phosphorylation MeSH
- Zebrafish Proteins MeSH
- Reactive Oxygen Species metabolism MeSH
- Electron Transport Complex IV * metabolism MeSH
- Mitochondria, Heart metabolism MeSH
- Calcium metabolism MeSH
- Calcium Signaling * MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Trvalá antikoagulační terapie u pacientů v chronickém hemodialyzačním programu přináší řadu klinických a farmakologických výzev. Fibrilace síní, často komplikovaná nerovnováhou mezi pro- a antikoagulačními mechanismy, vyžaduje individuální přístup s ohledem na vysoké riziko krvácivých komplikací a trombembolismu. V posledních letech přibývá důkazů o použití přímých perorálních antikoagulancií (DOAC) u této populace na úkor warfarinu, jehož podávání nebylo prokázáno jako efektivní, a je navíc spojeno se zvýšeným rizikem krvácení a negativním vlivem na kalcium-fosfátový metabolismus. Tato práce se zaměřuje na zhodnocení současných možností antikoagulace s důrazem na nefrologické a koagulační aspekty, včetně přehodnocení zastaralých režimů a potenciální aplikace moderních přístupů.
Long-term anticoagulation therapy in patients on chronic hemodialysis presents a few clinical and pharmacological challenges. Atrial fibrillation, often complicated by an imbalance between pro- and anticoagulant factors, requires an individualized approach considering the high risk of bleeding complications and thromboembolism. In recent years, we have an increasing evidence for the use of direct oral anticoagulants (DOAC) in this population at the expense of warfarin The administration of warfarin has not been proven to be effective and is also associated with an increased risk of bleeding and a negative effect on calcium-phosphate metabolism. This work focuses on the evaluation of current anticoagulation options with an emphasis on nephrological and coagulation aspects, including a re-evaluation of outdated regimens and the potential application of modern approaches
- Keywords
- apixaban,
- MeSH
- Renal Insufficiency, Chronic * complications therapy MeSH
- Renal Dialysis MeSH
- Atrial Fibrillation * drug therapy MeSH
- Heparin, Low-Molecular-Weight administration & dosage therapeutic use MeSH
- Humans MeSH
- Pyrazoles therapeutic use MeSH
- Risk Factors MeSH
- Thromboembolism drug therapy prevention & control MeSH
- Warfarin therapeutic use MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
