G protein-coupled receptors (GPCRs) play a crucial role in cell function by transducing signals from the extracellular environment to the inside of the cell. They mediate the effects of various stimuli, including hormones, neurotransmitters, ions, photons, food tastants and odorants, and are renowned drug targets. Advancements in structural biology techniques, including X-ray crystallography and cryo-electron microscopy (cryo-EM), have driven the elucidation of an increasing number of GPCR structures. These structures reveal novel features that shed light on receptor activation, dimerization and oligomerization, dichotomy between orthosteric and allosteric modulation, and the intricate interactions underlying signal transduction, providing insights into diverse ligand-binding modes and signalling pathways. However, a substantial portion of the GPCR repertoire and their activation states remain structurally unexplored. Future efforts should prioritize capturing the full structural diversity of GPCRs across multiple dimensions. To do so, the integration of structural biology with biophysical and computational techniques will be essential. We describe in this review the progress of nuclear magnetic resonance (NMR) to examine GPCR plasticity and conformational dynamics, of atomic force microscopy (AFM) to explore the spatial-temporal dynamics and kinetic aspects of GPCRs, and the recent breakthroughs in artificial intelligence for protein structure prediction to characterize the structures of the entire GPCRome. In summary, the journey through GPCR structural biology provided in this review illustrates how far we have come in decoding these essential proteins architecture and function. Looking ahead, integrating cutting-edge biophysics and computational tools offers a path to navigating the GPCR structural landscape, ultimately advancing GPCR-based applications. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.

• MeSH
• konformace proteinů MeSH
• lidé MeSH
• receptory spřažené s G-proteiny * chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Current antibiotics and chemotherapeutics are becoming ineffective because pathogenic bacteria and tumor cells have developed multiple drug resistance. Therefore, it is necessary to find new substances that can be used in treatment, either alone or as sensitizing molecules in combination with existing drugs. Peptaibols are bioactive, membrane-active peptides of non-ribosomal origin, mainly produced by filamentous fungi such as Trichoderma spp. This study focused on producing peptaibol-rich extracts from Trichoderma atroviride O1, cultivated on malt extract agar (MA) under circadian and constant darkness conditions for 13 days. Peptaibol production was detected by MALDI-TOF mass spectrometry after six days of cultivation. The extracts demonstrated antibacterial activity against Staphylococcus aureus strains, particularly the methicillin-resistant variant, but not against the Gram-negative Pseudomonas aeruginosa. Quorum sensing interference revealed that a peptaibol-rich extract suppressed Vibrio campbellii BAA-1119's AI-2 signaling system to a degree comparable with gentamycin. Beyond antibacterial properties, the extracts exhibited notable antiproliferative activity against human ovarian cancer cells and their adriamycin-resistant subline in both 2D and 3D models. Specifically, MA-derived extracts reduced ovarian cancer cell viability by 70% at 50 μg/mL, especially under light/dark regime of cultivation. Compared to previously published results for PDA-based extracts, MA cultivation shifted the biological effects of peptaibol-containing extracts toward anticancer potential. These findings support the idea that modifying fungal cultivation parameters, the bioactivity of secondary metabolite mixtures can be tailored for specific therapeutic applications.

• MeSH
• agar * chemie   MeSH
• antibakteriální látky * farmakologie   metabolismus   MeSH
• Hypocreales MeSH
• kultivační média chemie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• nádorové buněčné linie MeSH
• peptaiboly * farmakologie   metabolismus   biosyntéza   chemie   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky * farmakologie   metabolismus   MeSH
• Pseudomonas aeruginosa účinky léků   MeSH
• Staphylococcus aureus účinky léků   MeSH
• Trichoderma * metabolismus   růst a vývoj   chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: The immunosuppressive roles of galectin-3 (Gal-3) in carcinogenesis make this lectin an attractive target for pharmacological inhibition in immunotherapy. Although current clinical immunotherapies appear promising in the treatment of solid tumors, their efficacy is significantly weakened by the hostile immunosuppressive tumor microenvironment (TME). Gal-3, a prominent TME modulator, efficiently subverts the elimination of cancer, either directly by inducing apoptosis of immune cells or indirectly by binding essential effector molecules, such as interferon-gamma (IFNγ). METHODS: N-(2-Hydroxypropyl)methacrylamide (HPMA)-based glycopolymers bearing poly-N-acetyllactosamine-derived tetrasaccharide ligands of Gal-3 were designed, synthesized, and characterized using high-performance liquid chromatography, dynamic light scattering, UV-Vis spectrophotometry, gel permeation chromatography, nuclear magnetic resonance, high-resolution mass spectrometry and CCK-8 assay for evaluation of glycopolymer non-toxicity. Pro-immunogenic effects of purified glycopolymers were tested by apoptotic assay using flow cytometry, competitive ELISA, and in vitro cell-free INFγ-based assay. RESULTS: All tested glycopolymers completely inhibited Gal-3-induced apoptosis of monocytes/macrophages, of which the M1 subtype is responsible for eliminating cancer cells during immunotherapy. Moreover, the glycopolymers suppressed Gal-3-induced capture of glycosylated IFNγ by competitive inhibition to Gal-3 carbohydrate recognition domain (CRD), which enables further inherent biological activities of this effector, such as differentiation of monocytes into M1 macrophages and repolarization of M2-macrophages to the M1 state. CONCLUSION: The prepared glycopolymers are promising inhibitors of Gal-3 and may serve as important supportive anti-cancer nanosystems enabling the infiltration of proinflammatory macrophages and the reprogramming of unwanted M2 macrophages into the M1 subtype.

• MeSH
• akrylamidy chemie   farmakologie   MeSH
• apoptóza účinky léků   MeSH
• galektin 3 * antagonisté a inhibitory   MeSH
• galektiny MeSH
• interferon gama * metabolismus   MeSH
• krevní proteiny MeSH
• lidé MeSH
• makrofágy účinky léků   MeSH
• monocyty * účinky léků   MeSH
• nádorové mikroprostředí účinky léků   MeSH
• polymery * chemie   farmakologie   MeSH
• protinádorové látky * farmakologie   chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BackgroundThe ischemia-reperfusion injury (IRI) is unavoidable in vascular surgery. Damage to the microcirculation and endothelial glycocalyx might set up a shock with loss of circulatory coherence and organ failure. Sulodexide may help to protect endothelial glycocalyx and alleviate the ischemia-reperfusion injury.MethodsTwenty female piglets underwent surgery with a 30-min-long suprarenal aortic clamp, followed by two hours of reperfusion. Ten piglets received sulodexide before the clamp, and 10 received normal saline. Blood and urine samples were taken at baseline and in 20-min intervals until the 120th minute to analyze the serum syndecan-1, E-selectin, and thrombomodulin. Albumin and glycosaminoglycans were examined in the urine. The kidney biopsies before and after the protocol were examined by light microscopy with hematoxylin-eosin staining. The sublingual microcirculation was recorded by side-stream dark field imaging at the time as blood and urine.ResultsBased on the 2-way ANOVA testing, there was no statistically significant difference in the parameters of sublingual microcirculation. Serum markers of endothelial cell activation and damage (E-selectin and thrombomodulin) did not show any statistically significant difference either. Syndecan-1, a marker of glycocalyx damage, showed statistically significantly higher values based on the 2-way ANOVA testing (p < 0.0001) with the highest difference in the 80th minute: 7.8 (3.9-44) ng/mL in the control group and 1.8 (0.67-2.8) ng/mL in the sulodexide group. In the urine, the albuminuria was higher in the control group, although not statistically significant. Glycosaminoglycans were statistically significantly higher in the sulodexide group based on the mixed-effect analysis due to the intervention itself. Histological analysis of the renal biopsies showed necrosis in both groups after reperfusion.ConclusionAdministering sulodexide significantly reduced the level of endothelial markers of IRI. The study results support further research into using preemptive administration of sulodexide to modulate IRI in clinical medicine.

• MeSH
• E-selektin krev   MeSH
• glykokalyx MeSH
• glykosaminoglykany * farmakologie   terapeutické užití   MeSH
• ledviny patologie   krevní zásobení   MeSH
• mikrocirkulace účinky léků   MeSH
• modely nemocí na zvířatech MeSH
• prasata MeSH
• reperfuzní poškození * prevence a kontrola   MeSH
• syndekan-1 krev   MeSH
• trombomodulin krev   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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
• difrakce rentgenového záření MeSH
• elektronová kryomikroskopie metody   MeSH
• konformace proteinů MeSH
• maloúhlový rozptyl * MeSH
• molekulární modely MeSH
• oční proteiny MeSH
• prasata MeSH
• proteiny vázající retinol * chemie   metabolismus   MeSH
• simulace molekulového dockingu MeSH
• vazba proteinů MeSH
• vitamin A metabolismus   chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The GPCR signalling cascade is a key pathway responsible for the signal transduction of a multitude of physical and chemical stimuli, including light, odorants, neurotransmitters and hormones. Understanding the structural and functional properties of the GPCR cascade requires direct observation of signalling processes in high spatial and temporal resolution, with minimal perturbation to endogenous systems. Optical microscopy and spectroscopy techniques are uniquely suited to this purpose because they excel at multiple spatial and temporal scales and can be used in living objects. Here, we review recent developments in microscopy and spectroscopy technologies which enable new insights into GPCR signalling. We focus on advanced techniques with high spatial and temporal resolution, single-molecule methods, labelling strategies and approaches suitable for endogenous systems and large living objects. This review aims to assist researchers in choosing appropriate microscopy and spectroscopy approaches for a variety of applications in the study of cellular signalling. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.

• MeSH
• lidé MeSH
• mikroskopie * metody   MeSH
• receptory spřažené s G-proteiny * chemie   metabolismus   MeSH
• signální transdukce MeSH
• spektrální analýza * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Background: This study aimed to evaluate the effects of a six-week visual training protocol, based on the Science Vision Training Academy (SVTA) method, on reaction times and executive functions in high-ranking fencers. Methods: Twenty-seven fencers, aged 17.34 ± 3.63 years, were randomly assigned to an experimental Visual Training Group (VTG = 16) and a Control Group (CG = 11). The VTG, in addition to regular fencing training, underwent SVTA training two times per week using six different visual modules, while the CG followed only their traditional fencing training. Simple and complex reaction times and movement times were assessed before and after the intervention using the Fit-Light System. Results: Both groups showed a significant improvement in all four reaction time tests: simple reaction time with and without a weapon and complex reaction time ability (motor inhibition ability) with and without a weapon (p < 0.001). No significant differences were observed between the groups. A significant Time* Group interaction was found in the short reaction time and movement time (p < 0.001). This trend suggests that, although genetically determined and difficult to significantly improve through training, short reaction time can be stimulated through SVTA protocols. Conclusions: Training in realistic conditions is always preferable to non-ecological protocols; however, the SVTA method may be beneficial to enhance simple reaction time in elite fencers.

• Publikační typ
• časopisecké články MeSH

Brain imaging studies in complex regional pain syndrome (CRPS) have found mixed evidence for functional and structural changes in CRPS. In this cross-sectional study, we evaluated two patient cohorts from different centers and examined functional connectivity (rsFC) in 51 CRPS patients and 50 matched controls. rsFC was compared in predefined ROI pairs, but also in non-hypothesis driven analyses. Resting state (rs)fMRI changes in default mode network (DMN) and the degree rank order disruption index (kD) were additionally evaluated. Finally, imaging parameters were correlated with clinical severity and somatosensory function. Among predefined pairs, we found only weakly to moderately lower functional connectivity between the right nucleus accumbens and bilateral ventromedial prefrontal cortex in the infra-slow oscillations (ISO) band. The unconstrained ROI-to-ROI analysis revealed lower rsFC between the periaqueductal gray matter (PAG) and left anterior insula, and higher rsFC between the right sensorimotor thalamus and nucleus accumbens. In the correlation analysis, pain was positively associated with insulo-prefrontal rsFC, whereas sensorimotor thalamo-cortical rsFC was positively associated with tactile spatial resolution of the affected side. In contrast to previous reports, we found no group differences for kD or rsFC in the DMN, but detected overall lower data quality in patients. In summary, while some of the previous results were not replicated despite the larger sample size, novel findings from two independent cohorts point to potential down-regulated antinociceptive modulation by the PAG and increased connectivity within the reward system as pathophysiological mechanisms in CRPS. However, in light of the detected systematic differences in data quality between patients and healthy subjects, validity of rsFC abnormalities in CRPS should be carefully scrutinized in future replication studies.

• MeSH
• default mode network diagnostické zobrazování   patofyziologie   MeSH
• dospělí MeSH
• komplexní regionální syndromy bolesti * patofyziologie   diagnostické zobrazování   MeSH
• konektom metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• magnetická rezonanční tomografie * MeSH
• mozek patofyziologie   diagnostické zobrazování   MeSH
• nervová síť patofyziologie   diagnostické zobrazování   MeSH
• průřezové studie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH

BACKGROUND AND AIMS: The pathophysiology of haemophilic arthropathy (HA) is complex and largely undefined. Proteomic analyses provide insights into the intricate mechanisms of the HA.Our study aimed to identify differentially expressed proteins in relation to the severity of HA, explore their pathophysiological roles, and evaluate their potential as HA biomarkers. METHODS: Our cross-sectional observational study encompassed 30 HA patients and 15 healthy subjects. Plasma samples were pooled into three groups of 15 samples from those with severe haemophilic arthropathy (sHA), mild haemophilic arthropathy (mHA) and healthy controls. Proteomic analysis was performed using liquid chromatography-mass spectrometry. The severity of HA was assessed using the World Federation of Haemophilia Physical Examination Score and ultrasonography following the Haemophilia Early Arthropathy Detection with Ultrasound (HEAD-US) guidelines. RESULTS: A total of 788 proteins were identified, with 97% of the uniquely identified proteins being expressed in all analysed groups. We identified several up and downregulated proteins across the groups that were mainly related to inflammatory and immunity-modulating processes, as well as joint degeneration. We highlighted ten proteins relevant for the development of HA: cathepsin G, endoplasmic reticulum aminopeptidase 2, S100-A9, insulin-like growth factor I, apolipoprotein (a), osteopontin, pregnancy zone protein, cartilage oligomeric matrix protein, CD44, and cadherin-related family member 2. CONCLUSION: Our analysis identified several proteins that shed further light on the distinctive pathogenesis of HA and could serve for biomarker research. However, these results need to be validated on a larger patient group.

• Publikační typ
• časopisecké články MeSH

BACKGROUND: The consequences at the molecular level and the mechanisms involved in a possible cardioprotective effect of antihypertensive treatment are not yet fully understood. Here, the efficacy of pyridostigmine (PYR) and trandolapril (TRA) as antihypertensive and antihypertrophic agents was investigated and compared in hypertensive SHR and normotensive WKY rats. In parallel, we investigated the effects of these drugs on myocardial β-adrenergic and cholinergic signaling pathways and protein expression profiles. METHODS: Age-matched male SHR and WKY rats were chronically (8 weeks) treated with PYR or TRA in drinking water. Blood pressure (BP) and heart rate (HR) were monitored telemetrically prior to tissue sampling for biochemical analysis. Baroreceptor reflex sensitivity (BRS) and methylatropine HR response as a measure of vagal tone were evaluated in separate groups of animals. RESULTS: PYR slightly lowered BP and HR in SHR rats during the dark phase of the day, while TRA effectively reduced BP during the light and dark phases without affecting HR. PYR enhanced BRS and improved vagal tone. There were no significant alterations in myocardial β-adrenergic and cholinergic signaling, with the exception of decreased forskolin-stimulated adenylyl cyclase (AC) activity in SHR rats, which was restored by TRA. Proteomic analysis revealed numerous differences induced by both treatments. Notable were changes in TGFβ-related signaling pathways as well as proteins involved in modifying hemodynamic parameters and cardiac hypertrophy. CONCLUSIONS: PYR is able to slightly decrease BP and HR in SHR rats but effectively increase BRS through vagal potentiation. The specific differences in protein expression profiles in rat myocardium induced by treatment with PYR and TRA reflect different mechanisms of action of these two agents at the molecular level.

• Publikační typ
• časopisecké články MeSH