Dle současných doporučených kritérií pro diagnostiku a staging nemoci s Lewy tělísky je nezbytné hodnocení přítomnosti Lewyho tělísek a Lewyho neuritů v konkrétních oblastech mozku. Nejpoužívanější systémy pro staging určují míru postižení na podkladě přítomnosti Lewyho patologie v konkrétních anatomických oblastech mozku v kaudo-rostrálním směru. Výběr správného klonu protilátky a využití optimalizovaného protokolu včetně efektivního odmaskování epitopu je pro vizualizaci diagnostických depozit nezbytné. Cílem naší studie bylo zhodnocení využitelnosti některých komerčně dostupných a široce používaných primárních protilátek proti alfa-synukleinu v kontextu post mortem diagnostiky a stagingu nemoci s Lewyho tělísky. Zaměřili jsme se na imunohistochemický a imunofluorescenční průkaz Lewyho patologie pomocí protilátek proti všem základním částem polypeptidového řetězce alfa-synuklein, včetně konformačně specifického klonu a klonu pro detekci posttranslačně modifikovaného proteinu.
According to the current recommended criteria for the diagnosis and staging of the Lewy body disease, it is necessary to evaluate the presence of Lewy bodies and Lewy neurites in specific areas of the brain. The most widely used staging systems assess the degree of neurodegeneration based on the distribution of Lewy pathology across specific anatomical regions of the brain, progressing in a caudo-rostral trajectory. Choosing the right antibody clone and using an optimized protocol including effective antigen retrieval is essential for the visualization of diagnostic deposits. The aim of our study was to evaluate the utility of some commercially available and widely used primary antibodies against alpha-synuclein in the context of post mortem diagnosis and staging of Lewy body disease. We focused on immunohistochemical and immunofluorescence analysis of Lewy pathology using antibodies with epitopes in all parts of the alphasynuclein polypeptide chain, including a conformation-specific clone and a clone for the detection of post-translationally modified protein.
BACKGROUND AND OBJECTIVE: There is no standardized regimen for follow-up after radical cystectomy (RC) for bladder cancer (BC). To address this gap, we conducted a multicenter study involving urologist members from the European Association of Urology (EAU) bladder cancer guideline panels. Our objective was to identify consistent post-RC follow-up strategies and develop a practice-based framework based on expert opinion. METHODS: We surveyed 27 urologist members of the EAU guideline panels for non-muscle-invasive bladder cancer and muscle-invasive and metastatic bladder cancer using a pre-tested questionnaire with dichotomous responses. The survey inquired about follow-up strategies after RC and the use of risk-adapted strategies. Consistency was defined as >75% affirmative responses for follow-up practices commencing 3 mo after RC. Descriptive statistics were used for analysis. KEY FINDINGS AND LIMITATIONS: We received responses from 96% of the panel members, who provided data from 21 European hospitals. Risk-adapted follow-up is used in 53% of hospitals, with uniform criteria for high-risk (at least ≥pT3 or pN+) and low-risk ([y]pT0/a/1N0) cases. In the absence of agreement for risk-based follow up, a non-risk-adapted framework for follow-up was developed. Higher conformity was observed within the initial 3 yr, followed by a decline in subsequent follow-up. Follow-up was most frequent during the first year, including patient assessments, physical examinations, and laboratory tests. Computed tomography of the chest and abdomen/pelvis was the most common imaging modality, initially at least biannually, and then annually from years 2 to 5. There was a lack of consistency for continuing follow-up beyond 10 yr after RC. CONCLUSIONS AND CLINICAL IMPLICATIONS: This practice-based post-RC follow-up framework developed by EAU bladder cancer experts may serve as a valuable guide for urologists in the absence of prospective randomized studies. PATIENT SUMMARY: We asked urologists from the EAU bladder cancer guideline panels about their patient follow-up after surgical removal of the bladder for bladder cancer. We found that although urologists have varying approaches, there are also common follow-up practices across the panel. We created a practical follow-up framework that could be useful for urologists in their day-to-day practice.
- MeSH
- Cystectomy * methods MeSH
- Humans MeSH
- Urinary Bladder Neoplasms * surgery pathology MeSH
- Aftercare standards methods MeSH
- Follow-Up Studies MeSH
- Surveys and Questionnaires MeSH
- Practice Guidelines as Topic MeSH
- Urology standards MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Geographicals
- Europe MeSH
In this study, we investigated the stability of the fully activated conformation of the orexin receptor 2 (OX2R) embedded in a pure POPC bilayer using MD simulations. Various thermodynamic ensembles (i.e., NPT, NVT, NVE, NPAT, μVT, and NPγT) were employed to explore the dynamical heterogeneity of the system in a comprehensive way. In addition, informational similarity metrics (e.g., Jensen-Shannon divergence) as well as Markov state modeling approaches were utilized to elucidate the receptor kinetics. Special attention was paid to assessing surface tension within the simulation box, particularly under NPγT conditions, where 21 nominal surface tension constants were evaluated. Our findings suggest that traditional thermodynamic ensembles such as NPT may not adequately control physical properties of the POPC membrane, impacting the plausibility of the OX2R model. In general, the performed study underscores the importance of employing the NPγT ensemble for computational investigations of membrane-embedded receptors, as it effectively maintains zero surface tension in the simulated system. These results offer valuable insights for future research aimed at understanding receptor dynamics and designing targeted therapeutics.
The vertebrate visual cycle hinges on enzymatically converting all-trans-retinol (at-ROL) into 11-cis-retinal (11c-RAL), the chromophore that binds to opsins in photoreceptors, forming light-responsive pigments. When struck by a photon, these pigments activate the phototransduction pathway and initiate the process of vision. The enzymatic isomerization of at-ROL, crucial for restoring the visual pigments and preparing them to receive new light stimuli, relies on various enzymes found in both the photoreceptors and retinal pigment epithelium cells. To function effectively, retinoids must shuttle between these two cell types. Retinol-binding protein 3 (RBP3), located in the interphotoreceptor matrix, probably plays a pivotal role in this transport mechanism. Comprised of four retinoid-binding modules, RBP3 also binds fatty acids, potentially aiding retinal function by facilitating the loading and unloading of different retinoids at specific cell types thereby directing the cycle. In this study, we present a 3.67 Å cryoEM structure of porcine RBP3, along with molecular docking analysis and corroborative in-solution small-angle X-ray scattering data for titration of RBP3 with relevant ligands, that also give insights on RBP3 conformational adaptability.
- MeSH
- X-Ray Diffraction MeSH
- Cryoelectron Microscopy methods MeSH
- Protein Conformation MeSH
- Scattering, Small Angle * MeSH
- Models, Molecular MeSH
- Eye Proteins MeSH
- Swine MeSH
- Retinol-Binding Proteins * chemistry metabolism MeSH
- Molecular Docking Simulation MeSH
- Protein Binding MeSH
- Vitamin A metabolism chemistry MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
MicroRNAs (miRNAs) are small non-coding RNAs (18-22 nucleotides) that regulate gene expression and are associated with various diseases, including Laryngeal Cancer (LCa), which has a high mortality rate due to late diagnosis. Traditional methods for miRNA detection present several drawbacks (time-consuming steps, high cost and high false positive rate). Early-stage diagnosis and selective detection of miRNAs remain challenging. This study proposes a 3D flexible biosensor that combines nanofibers (NFs), gold nanoparticles (AuNPs), and an inverse molecular sentinel (iMS) for enzyme-free, SERS-based detection of miRNA-223-3p, evaluated as a potential LCa biomarker. The electrospun flexible nanofibers decorated with AuNPs enhance Raman signal. Selective detection of miRNA-223-3p is achieved by immobilizing an iMS-DNA probe labeled with a Raman reporter (Cyanine 3) on the AuNPs. The iMS distinctive stem-and-loop structure undergoes a conformational change upon interaction with the miRNA-223-3p, producing an "on to off" SERS signal. The proposed sensor demonstrated a linear detection range from 10 to 250 fM, with a limit of detection (LOD) of 19.50 ± 0.05 fM. The sensor selectivity was confirmed by analyzing the SERS signal behaviour in the presence of both Non-complementary miRNA and miRNA with three mismatched base pairs. This easily fabricable sensor requires no amplification and offers key advantages, including sensitivity, flexibility, and cost-effectiveness.
- MeSH
- Biosensing Techniques * methods MeSH
- Early Detection of Cancer methods MeSH
- Metal Nanoparticles * chemistry MeSH
- Humans MeSH
- Limit of Detection MeSH
- MicroRNAs * analysis genetics MeSH
- Laryngeal Neoplasms * diagnosis genetics MeSH
- Nanofibers * chemistry MeSH
- Spectrum Analysis, Raman * methods MeSH
- Gold * chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Acute lymphoblastic leukemia expressing the gamma delta T-cell receptor (γδ T-ALL) is a poorly understood disease. We studied 200 children with γδ T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. γδ T-ALL diagnosed in children under 3 years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High-throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by poly(ADP-ribose) polymerase inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric γδ T-ALL. Significance: Patients with acute lymphoblastic leukemia expressing the gamma delta T-cell receptor under 3 years old or measurable residual disease ≥1% at end of induction showed dismal outcomes and should be classified as having high-risk disease. The STAG2/LMO2 subtype was enriched in this very young age group. STAG2 inactivation may perturb chromatin conformation and cell differentiation and confer vulnerability to poly(ADP-ribose) polymerase inhibition.
- MeSH
- Adaptor Proteins, Signal Transducing * genetics metabolism MeSH
- Child MeSH
- Gene Rearrangement MeSH
- Infant MeSH
- Humans MeSH
- Precursor T-Cell Lymphoblastic Leukemia-Lymphoma genetics pathology MeSH
- Child, Preschool MeSH
- Cell Cycle Proteins genetics metabolism MeSH
- LIM Domain Proteins * genetics MeSH
- Proto-Oncogene Proteins MeSH
- Check Tag
- Child MeSH
- Infant MeSH
- Humans MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Alcohol use is one of the leading public health concerns in the Czech Republic. Drinking motives play a vital role in both initiation and subsequent alcohol use. A revised version of the self-report Drinking Motives Questionnaire (DMQ-R) has been proposed to assess these motives. The present study aims to validate the DMQ-R in the Czech general population. METHODS: A total sample of 1,784 Czech participants completed a national survey. For the analysis, only a sub-sample of the past 12 months alcohol users was used: N = 1,123; 52.8% male; mean (SD) age = 40.2 (13.3). Drinking motives were assessed by the adopted Czech version of the DMQ-R. Both confirmatory (CFA) and exploratory factor analysis (EFA) were conducted to examine the factorial structure of the instrument. The age of the participant was additionally considered in the analysis (15-24 years as opposed to 25-64 years). RESULTS: The CFA supported the four-factor model in the 25-64 age group. The analysis supported the construct validity of the Social, Conformity, and Coping factors. The Enhancement factor retained only two items and was found to refer more to a domain of 'Pleasant Feeling'. For the 15-24 age group, the hypothesised four-factor structure was not corroborated. CONCLUSIONS: The Czech version of the DMQ-R was found to be a reliable measurement tool of the Social, Conformity, and Coping motives. Future research should investigate the dimensionality of the instrument items presumed to correspond to the Enhancement motives. This should be conducted particularly among adolescents and young adults aged 15-24 years, where administering the DMQ-R with a large enough sample is also needed.
- MeSH
- Adaptation, Psychological MeSH
- Adult MeSH
- Factor Analysis, Statistical MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Motivation * MeSH
- Alcohol Drinking * psychology epidemiology MeSH
- Surveys and Questionnaires standards MeSH
- Psychometrics MeSH
- Reproducibility of Results MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
- Geographicals
- Czech Republic MeSH
This study investigates the factors modulating the reactivity of 5'-deoxyadenosyl (5'dAdo ̇) radical, a potent hydrogen atom abstractor that forms in the active sites of radical SAM enzymes and that otherwise undergoes a rapid self-decay in aqueous solution. Here, we compare hydrogen atom abstraction (HAA) reactions between native substrates of radical SAM enzymes and 5'dAdo ̇ in aqueous solution and in two enzymatic microenvironments. With that we reveal that HAA efficiency of 5'dAdo ̇ is due to (i) the in situ formation of 5'dAdo ̇ in a pre-ordered complex with a substrate, which attenuates the unfavorable effect of substrate:5'dAdo ̇ complex formation, and (ii) the prevention of the conformational changes associated with self-decay by a tight active-site cavity. The enzymatic cavity, however, does not have a strong effect on the HAA activity of 5'dAdo ̇. Thus, we performed an analysis of in-water HAA performed by 5'dAdo ̇ based on a three-component thermodynamic model incorporating the diagonal effect of the free energy of reaction, and the off-diagonal effect of asynchronicity and frustration. To this aim, we took advantage of the straightforward relationship between the off-diagonal thermodynamic effects and the electronic-structure descriptor - the redistribution of charge between the reactants during the reaction. It allows to access HAA-competent redox and acidobasic properties of 5'dAdo ̇ that are otherwise unavailable due to its instability upon one-electron reduction and protonation. The results show that all reactions feature a favourable thermodynamic driving force and tunneling, the latter of which lowers systematically barriers by ∼2 kcal mol-1. In addition, most of the reactions experience a favourable off-diagonal thermodynamic contribution. In HAA reactions, 5'dAdo ̇ acts as a weak oxidant as well as a base, also 5'dAdo ̇-promoted HAA reactions proceed with a quite low degree of asynchronicity of proton and electron transfer. Finally, the study elucidates the crucial and dual role of asynchronicity. It directly lowers the barrier as a part of the off-diagonal thermodynamic contribution, but also indirectly increases the non-thermodynamic part of the barrier by presumably controlling the adiabatic coupling between proton and electron transfer. The latter signals that the reaction proceeds as a hydrogen atom transfer rather than a proton-coupled electron transfer.
We currently lack antivirals for most human viruses. In a quest for new molecules, focusing on viral RNA, instead of viral proteins, can represent a promising strategy. In this study, new inhibitors were identified starting from a published crystal structure of the tertiary SARS-CoV-2 RNA involved in the -1 programmed ribosomal frameshift. The pseudoknot structure was refined, and a virtual screening was performed using the repository of binders to the nucleic acid library, taking into consideration RNA flexibility. Hit compounds were validated against the wild-type virus and with a dual-luciferase assay measuring the frameshift efficiency. Several active molecules were identified. Our study reveals new inhibitors of SARS-CoV-2 but also highlights the feasibility of targeting RNA starting from virtual screening, a strategy that could be broadly applied to drug development.
- MeSH
- Antiviral Agents * pharmacology chemistry MeSH
- Nucleic Acid Conformation MeSH
- Humans MeSH
- Models, Molecular MeSH
- Drug Evaluation, Preclinical * methods MeSH
- RNA, Viral * metabolism antagonists & inhibitors genetics MeSH
- SARS-CoV-2 * drug effects MeSH
- Molecular Docking Simulation MeSH
- User-Computer Interface MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Computational exploration of chemical space is crucial in modern cheminformatics research for accelerating the discovery of new biologically active compounds. In this study, we present a detailed analysis of the chemical library of potential glucocorticoid receptor (GR) ligands generated by the molecular generator, Molpher. To generate the targeted GR library and construct the classification models, structures from the ChEMBL database as well as from the internal IMG library, which was experimentally screened for biological activity in the primary luciferase reporter cell assay, were utilized. The composition of the targeted GR ligand library was compared with a reference library that randomly samples chemical space. A random forest model was used to determine the biological activity of ligands, incorporating its applicability domain using conformal prediction. It was demonstrated that the GR library is significantly enriched with GR ligands compared to the random library. Furthermore, a prospective analysis demonstrated that Molpher successfully designed compounds, which were subsequently experimentally confirmed to be active on the GR. A collection of 34 potential new GR ligands was also identified. Moreover, an important contribution of this study is the establishment of a comprehensive workflow for evaluating computationally generated ligands, particularly those with potential activity against targets that are challenging to dock.
- MeSH
- Small Molecule Libraries * pharmacology chemistry MeSH
- Humans MeSH
- Ligands MeSH
- Receptors, Glucocorticoid * metabolism chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
