Bacterial β sliding clamp (β-clamp) is an emerging drug target currently lacking small-molecule inhibitors with good in vivo activity. Thus, there is a need for fast and simple screening methods for identifying inhibitor candidates. Here we demonstrate the use of nuclear magnetic resonance spectroscopy (NMR) for evaluating compound binding to the E. coli β-clamp. To identify suitable molecular probes, a series of tetrahydrocarbazoles were synthesized, some of which contain fluorine. Key challenges in the synthesis were formation of regioisomers during the Fischer indole reaction and reducing racemization at the stereogenic center. The tetrahydrocarbazoles were assayed against the E. coli β-clamp by saturation-transfer difference (STD) NMR, waterLOGSY and T1ρ. Analysis by isothermal titration calorimetry gave KD-values of 1.7-14 μM for three fluorinated probe candidates, and NMR chemical shift perturbation experiments confirmed these molecules to directly interact with the β-clamp binding pocket. Binding of the fluorinated molecules to β-clamp was easily observed with 19F-observed T2-based binding experiments, and proof of concept for a fluorine-based binding assay for E. coli β-clamp binders is provided.

• MeSH
• Escherichia coli * drug effects   MeSH
• Halogenation MeSH
• Carbazoles * chemistry   chemical synthesis   pharmacology   metabolism   MeSH
• Magnetic Resonance Spectroscopy * MeSH
• Molecular Probes chemistry   chemical synthesis   metabolism   MeSH
• Molecular Structure MeSH
• Escherichia coli Proteins metabolism   antagonists & inhibitors   chemistry   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH

INTRODUCTION: The aging process is intricately linked to alterations in cellular and tissue structures, with the respiratory system being particularly susceptible to age-related changes. Therefore, this study aimed to profile the activity of proteases using activity-based probes in lung tissues of old and young rats, focusing on the expression levels of different, in particular cathepsins G and X and matrix Metalloproteinases (MMPs). Additionally, the impact on extracellular matrix (ECM) components, particularly fibronectin, in relation to age-related histological and ultrastructural changes in lung tissues was investigated. MATERIALS AND METHODS: Lung tissues from old and young rats were subjected to activity-based probe profiling to assess the activity of different proteases. Expression levels of cathepsins G and X were quantified, and zymography was performed to evaluate matrix metalloproteinases activity. Furthermore, ECM components, specifically fibronectin, were examined for signs of degradation in the old lung tissues compared to the young ones. Moreover, histological, immunohistochemical and ultrastructural assessments of old and young lung tissue were also conducted. RESULTS: Our results showed that the expression levels of cathepsins G and X were notably higher in old rat lung tissues in contrast to those in young rat lung tissues. Zymography analysis revealed elevated MMP activity in the old lung tissues compared to the young ones. Particularly, significant degradation of fibronectin, an essential ECM component, was observed in the old lung tissues. Numerous histological and ultrastructural alterations were observed in old lung tissues compared to young lung tissues. DISCUSSION AND CONCLUSION: The findings indicate an age-related upregulation of cathepsins G and X along with heightened MMP activity in old rat lung tissues, potentially contributing to the degradation of fibronectin within the ECM. These alterations highlight potential mechanisms underlying age-associated changes in lung tissue integrity and provide insights into protease-mediated ECM remodeling in the context of aging lungs.

• MeSH
• Extracellular Matrix metabolism   ultrastructure   MeSH
• Fibronectins * metabolism   MeSH
• Cathepsin G metabolism   MeSH
• Rats MeSH
• Lysosomes ultrastructure   metabolism   MeSH
• Matrix Metalloproteinases metabolism   MeSH
• Lung * ultrastructure   metabolism   MeSH
• Peptide Hydrolases metabolism   MeSH
• Aging * metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Photon-upconversion nanoparticles (UCNP) have already been established as labels for affinity assays in analog and digital formats. Here, advanced, or smart, systems based on UCNPs coated with active shells, fluorescent dyes, and metal and semiconductor nanoparticles participating in energy transfer reactions are reviewed. In addition, switching elements can be embedded in such assemblies and provide temporal and spatial control of action, which is important for intracellular imaging and monitoring activities. Demonstration and critical comments on representative approaches demonstrating the progress in the use of such UCNPs in bioanalytical assays, imaging, and monitoring of target molecules in cells are reported, including particular examples in the field of cancer theranostics.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Photons * MeSH
• Humans MeSH
• Neoplasms diagnostic imaging   diagnosis   MeSH
• Nanoparticles * chemistry   MeSH
• Optical Imaging MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

The quantification of cellular metabolic activity via MTT assay has become a widespread practice in eukaryotic cell studies and is progressively extending to bacterial cell investigations. This study pioneers the application of MTT assay to evaluate the metabolic activity of biofilm-forming cells within bacterial biofilms on nanofibrous materials. The biofilm formation of Staphylococcus aureus and Escherichia coli on nanomaterials electrospun from polycaprolactone (PCL), polylactic acid (PLA), and polyamide (PA) was examined. Various parameters of the MTT assay were systematically investigated, including (i) the dissolution time of the formed formazan, (ii) the addition of glucose, and (iii) the optimal wavelength for spectrophotometric determination. Based on interim findings, a refined protocol suitable for application to nanofibrous materials was devised. We recommend 2 h of the dissolution, the application of glucose, and spectrophotometric measurement at 595 nm to obtain reliable data. Comparative analysis with the reference CFU counting protocol revealed similar trends for both tested bacteria and all tested nanomaterials. The proposed MTT protocol emerges as a suitable method for assessing the metabolic activity of bacterial biofilms on PCL, PLA, and PA nanofibrous materials.

• MeSH
• Biofilms * growth & development   MeSH
• Escherichia coli * physiology   MeSH
• Glucose metabolism   MeSH
• Nanofibers * chemistry   MeSH
• Nylons chemistry   MeSH
• Polyesters * chemistry   MeSH
• Spectrophotometry methods   MeSH
• Staphylococcus aureus * physiology   MeSH
• Tetrazolium Salts * metabolism   chemistry   MeSH
• Thiazoles metabolism   MeSH
• Publication type
• Journal Article MeSH

Úvod: Práce na pracovištích nukleární medicíny představuje pracovní činnosti, při kterých dochází k expozici ionizujícím zářením. Expozice může být ovlivněna nejen druhem aplikovaného radiofarmaka a jeho aktivitou, ale i způsobem aplikace. Metoda: Práce sleduje a hodnotí výsledky osobní dozimetrie lékařů na pracovišti Oddělení nukleární medicíny Masarykova onkologického ústavu v Brně ovlivněné instrumentací pro aplikaci radiofarmak. Výsledky: Využití instrumentace k aplikaci radiofarmak umožňuje redukci profesního ozáření aplikujících lékařů o více než 90 % v prstové a celotělové dozimetrii. Závěr: Na základě našich zkušeností s instrumentací v nukleární medicíně lze tento princip radiační ochrany (RO) označit za velmi účinný nástroj optimalizace RO také při aplikaci radiofarmak. Takto optimalizovanou RO dochází k posílení bezpečnostních prvků ve vztahu k aplikujícím pracovníkům, čímž se vytváří prostor k možnému navyšování provozu a zvyšování dostupnosti péče v oblasti nukleární medicíny ve vztahu k pacientům.

Introduction: Exposition to radiation is necessarily connected with working at departments of nuclear medicine. The exposure can be influenced by the type of a radiopharmaceutical, its activity and the way of application. Method: This paper analyses the personal dosimetry of physicians at the Department of Nuclear Medicine, Masaryk Memorial Cancer Institute in Brno and evaluates the influence of instrumental application of radiopharmaceuticals on radiation exposure. Results: Use of instrumentation for application of radiopharmaceuticals results in reduction of occupational exposure of physicians by more than 90 % in ring and whole-body dosimetry. Conclusion: Based on our experience with instrumentation at department of nuclear medicine, this principle of radiation protection (RP) has proved to be a very effective tool of RP optimization in application of radiopharmaceuticals. Such optimization of RP reinforces safety elements in relation to the applicating staff, therefore enabling the increase in number of examined patients and increase in availability of nuclear medicine care in relation of patients.

• MeSH
• Safety MeSH
• Radiation Dosage MeSH
• Infusion Pumps * classification   MeSH
• Radiation, Ionizing MeSH
• Humans MeSH
• Occupational Diseases prevention & control   MeSH
• Nuclear Medicine methods   instrumentation   statistics & numerical data   MeSH
• Radiation Protection methods   instrumentation   MeSH
• Radiopharmaceuticals * administration & dosage   MeSH
• Radiometry methods   instrumentation   adverse effects   statistics & numerical data   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Advancements in genomics are transforming the clinical management of chronic myeloid leukemia (CML) toward precision medicine. The impact of somatic mutations on treatment outcomes is still under debate. We studied the association of somatic mutations in epigenetic modifier genes and activated signaling/myeloid transcription factors (AS/MTFs) with disease progression and treatment failure in patients with CML after tyrosine kinase inhibitor (TKI) therapy. A total of 394 CML samples were sequenced, including 254 samples collected at initial diagnosis and 140 samples taken during follow-up. Single-molecule molecular inversion probe (smMIP)-based next-generation sequencing (NGS) was conducted targeting recurrently mutated loci in 40 genes, with a limit of detection of 0.2%. Seventy mutations were detected in 57 diagnostic samples (22.4%), whereas 64 mutations were detected in 39 of the follow-up samples (27.9%). Carrying any mutation at initial diagnosis was associated with worse outcomes after TKI therapy, particularly in AS/MTF genes. Patients having these mutations at initial diagnosis and treated with imatinib showed higher risks of treatment failure (hazard ratio, 2.53; 95% confidence interval, 1.13-5.66; P = .0239). The adverse prognostic impact of the mutations was not clear for patients treated with second-generation TKIs. The multivariate analysis affirmed that mutations in AS/MTF genes independently serve as adverse prognostic factors for molecular response, failure-free survival, and progression risk. Additionally, there was an observable nonsignificant trend indicating a heightened risk of progression to advanced disease and worse overall survival. In conclusion, mutations in the AS/MTF genes using smMIP-based NGS can help identify patients with a potential risk of both treatment failure and progression and may help upfront TKI selection.

• MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive * genetics   drug therapy   mortality   diagnosis   MeSH
• Adult MeSH
• Protein Kinase Inhibitors therapeutic use   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Mutation * MeSH
• Prognosis MeSH
• Disease Progression MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Signal Transduction MeSH
• Transcription Factors genetics   MeSH
• Treatment Outcome MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

• MeSH
• Cathepsin B * antagonists & inhibitors   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Schistosoma mansoni * enzymology   drug effects   MeSH
• Schistosomicides pharmacology   chemistry   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Úvod: Depresivní porucha, jako jedno z nejčastějších duševních onemocnění, nepříznivě ovlivňuje životy a zdravotní stav mnoha lidí po celém světě. Vyskytuje se v jakémkoliv věku, nejčastěji však mezi 20. a 40. rokem života. Celosvětově trpí každoročně depresivními poruchami přibližně 100 milionů lidí, přičemž u řady jedinců je terapie doprovázena částečnou léčebnou odpovědí nebo přímo rezistencí k léčbě, což trvale zvyšuje potřebu hledání efektivních strategií prevence a léčby, ale také průběžné monitorování a vyhodnocování aktuálně využívaných přístupů v běžné klinické praxi. Materiály a metody: Šířeji koncipovaná studie s pracovním názvem UMBRELLA byla neintervenční, observační, multicentrická studie, jejíž první etapa je zaměřená na tzv. "real world evidence" důvodů pro změnu psychofarmakologické léčby a jejího detailního charakteru u pacientů trpících depresivní epizodou periodické depresivní poruchy. Do závěrečného vyhodnocení byla zařazena data 267 pacientů léčených v psychiatrických ambulancích v České republice, které na sondě mapující reálnou klinickou praxi participovaly. Výsledky: Analyzována byla data od 74 mužů (27,7 %) a 193 žen (72,3 %) s průměrným věkem 49 let. Změny léčby byly prováděny lékařem na základě vyhodnocení přítomných reziduálních příznaků onemocnění, přičemž nejčastějšími problémy byly perzistující depresivní nálada, únava a anhedonie. Nejčastěji používané léčivé přípravky zahrnovaly antidepresiva (např. escitalopram, venlafaxin, sertralin), atypická antipsychotika (např. quetiapin) a klasická antipsychotika (např. chlorprothixen). Úprava farmakologické léčby byla nejčastěji provedena manipulací s antidepresivy, změnou účinné látky či úpravami jejich denních dávek. Diskuse a závěr: Předkládaná naturalistická observační studie zmapovala důvody pro změnu farmakologického přístupu a vlastní charakter nově zvolené léčebné strategie. Zjištěná heterogenita způsobů manipulace s farmakologickou léčbou potvrzuje, že léčba depresivních epizod v klinické praxi probíhá individualizovaným postupem u každého jednotlivého pacienta. Zde narážíme na snahu po uplatňování doporučených postupů v léčbě tohoto rozšířeného duševního onemocnění, které velmi efektivně popisují léčbu iniciálních epizod a v případě opakovaných epizod by také měly obsahovat základní principy terapeutického přístupu k řešení této problematiky. Zjištění, že i v rámci opakovaných epizod periodické depresivní poruchy lze efektivně zasáhnout zvýšením dávky antidepresiva nebo jeho změnou, podporuje snahu po edukaci specialistů i praktických lékařů v těchto bazálních postupech léčby. Celkově studie přináší informace reflektující běžnou klinickou praxi a podtrhuje význam iniciace strategie změny farmakologického přístupu u každého pacienta, v kterékoliv etapě léčby tohoto duševního onemocnění.

Introduction: Depressive disorder, as one of the most common mental illnesses, adversely affects the lives and health of many people around the world. It occurs at any age, but most commonly between the ages of 20 and 40. Worldwide, approximately 100 million people suffer from depressive disorders each year, with many individuals experiencing partial treatment response or treatment resistance, which continually increases the need to find effective prevention and treatment strategies, as well as to monitor and evaluate currently used approaches in routine clinical practice. Materials and methods: The more broadly conceived study, working title UMBRELLA, was a non-interventional, observational, multicentre study, the first phase of which focused on the so-called "real world evidence? of reasons for changing psychopharmacological treatment and its detailed nature in patients suffering from a depressive episode of periodic depressive disorder. The final evaluation included data from 267 patients treated in psychiatric outpatient clinics in the Czech Republic that participated in the probe into their own clinical practice. Results: Data from 74 men (27.7%) and 193 women (72.3%) with a mean age of 49 years were analysed. Changes in treatment were made by the physician based on an assessment of the residual symptoms present, with persistent depressed mood, fatigue and anhedonia being the most common problems. The most commonly used medications included antidepressants (e.g., escitalopram, venlafaxine, sertraline), atypical antipsychotics (e.g., quetiapine), and classic antipsychotics (e.g., chlorprothixene). Adjustments to pharmacological treatment were most commonly made by manipulating antidepressants, changing the active ingredient, or modifying their daily doses. Discussion and conclusion: The present naturalistic observational study mapped the reasons for the change in pharmacological approach and the actual nature of the newly chosen treatment strategy. The observed heterogeneity of pharmacological treatment manipulation patterns confirms that the treatment of depressive episodes in clinical practice follows an individualized approach for each individual patient. Here, we encounter a desire for the application of recommended practices in the treatment of this widespread mental illness, which very effectively describe the treatment of initial episodes and, in the case of recurrent episodes, should provide the basic principles of a therapeutic approach to the management of this problem. The finding that, even in the context of recurrent episodes of periodic depressive disorder, effective intervention can be achieved by increasing the dose of antidepressant medication or changing it, supports the drive to educate specialists and practitioners in these basic treatment approaches. Overall, the study provides information that reflects common clinical practice and underscores the importance of initiating a strategy to change the pharmacological approach in any patient, at any stage of the treatment of this mental illness.

• MeSH
• Ambulatory Care MeSH
• Antidepressive Agents classification   therapeutic use   MeSH
• Antipsychotic Agents classification   therapeutic use   MeSH
• Depressive Disorder, Treatment-Resistant drug therapy   physiopathology   prevention & control   MeSH
• Drug Therapy, Combination MeSH
• Evidence-Based Practice MeSH
• Middle Aged MeSH
• Humans MeSH
• Medication Review MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Publication type
• Multicenter Study MeSH
• Observational Study MeSH

Advancements in small-molecule research have created the need for sensitive techniques to accurately study biological processes in living systems. Fluorescent-labeled probes have become indispensable tools, particularly those that use boron-dipyrromethene (BODIPY) dyes. Terpenes and terpenoids are organic compounds found in nature that offer diverse biological activities, and BODIPY-based probes play a crucial role in studying these compounds. Monoterpene-BODIPY conjugates have exhibited potential for staining bacterial and fungal cells. Sesquiterpene-BODIPY derivatives have been used to study sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), indicating their potential for drug development. Owing to their unique properties, diterpenes have been investigated using BODIPY conjugates to evaluate their mechanisms of action. Triterpene-BODIPY conjugates have been synthesized for biological studies, with different spacers affecting their cytotoxicity. Fluorescent probes, inspired by terpenoid-containing vitamins, have also been developed. Derivatives of tocopherol, coenzyme Q10, and vitamin K1 can provide insights into their oxidation-reduction abilities. All these probes have diverse applications, including the study of cell membranes to investigate immune responses and antioxidant properties. Further research in this field can help better understand and use terpenes and terpenoids in various biological contexts.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Humans MeSH
• Molecular Structure MeSH
• Boron Compounds * chemistry   pharmacology   MeSH
• Terpenes * chemistry   pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

• MeSH
• Action Potentials physiology   MeSH
• Cell Membrane MeSH
• Myocytes, Cardiac * metabolism   MeSH
• Membrane Potentials MeSH
• Optogenetics * MeSH
• Sarcolemma metabolism   MeSH
• Publication type
• Journal Article MeSH