G protein-coupled receptor endocytosis generates spatiotemporal bias in ß-arrestin signaling.

The stabilization of different active conformations of G protein-coupled receptors is thought to underlie the varying efficacies of biased and balanced agonists. Here, profiling the activation of signal transducers by angiotensin II type 1 receptor (AT1R) agonists revealed that the extent and kinetics of ß-arrestin binding exhibited substantial ligand-dependent differences, which were lost when receptor internalization was inhibited. When AT1R endocytosis was prevented, even weak partial agonists of the ß-arrestin pathway acted as full or near-full agonists, suggesting that receptor conformation did not exclusively determine ß-arrestin recruitment. The ligand-dependent variance in ß-arrestin translocation was much larger at endosomes than at the plasma membrane, showing that ligand efficacy in the ß-arrestin pathway was spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shaped the effects of agonists on endosomal receptor-ß-arrestin binding and thus determined the extent of functional selectivity. Ligand dissociation rate and G protein activity had particularly strong, internalization-dependent effects on the receptor-ß-arrestin interaction. We also showed that endocytosis regulated the agonist efficacies of two other receptors with sustained ß-arrestin binding: the V2 vasopressin receptor and a mutant ß2-adrenergic receptor. In the absence of endocytosis, the agonist-dependent variance in ß-arrestin2 binding was markedly diminished. Our results suggest that endocytosis determines the spatiotemporal bias in GPCR signaling and can aid in the development of more efficacious, functionally selective compounds.

Cardiopulmonary rehabilitation programme improves physical health and quality of life in post-COVID syndrome.

BACKGROUND: Many patients with previous COVID-19 infection suffer from prolonged symptoms after their recovery: cough, dyspnea, chest pain, shortness of breath, fatigue, anxiety or depression, regardless of milder or severe coronavirus infection. Review of the literature demonstrates underrepresented complex cardiopulmonary rehabilitation of patients with post-COVID syndrome. The aim of our quasi-experimental study was to evaluate the effectiveness of complex cardiopulmonary rehabilitation and to assess the quality of life, functional parameters before and after a 14-day specific cardiopulmonary rehabilitation and two months later. METHODS: Sixty-eight patients participated in rehabilitation at Semmelweis University's Department of Pulmonology. Respiratory function: forced expiratory volume in 1 second (FEV1%pred), 6-minute walk test (6MWT), chest kinematics (CK), quality of life [EuroQol-5D (EQ-5D), Post-COVID-19 Functional Status (PCFS)] and Modified Medical Research Council (mMRC) dyspnea scale were measured at the beginning and end of the programme and two months after the rehabilitation. RESULTS: The 14-day rehabilitation programme resulted in significant improvement of 6MWT {492 [interquartile range (IQR), 435-547] vs. 523 (IQR, 477-580) m; P=0.031}, mMRC [1 (IQR, 0.25-1) vs. 0 (IQR, 0-1); P=0.003], EQ-VAS score [75 (IQR, 65-80) vs. 85 (IQR, 75-90); P=0.015], and PCFS [1 (IQR, 1-2) vs. 0.5 (IQR, 0-1); P=0.032]. Respiratory function and chest kinematics also improved, FEV1(%pred) [86 (IQR, 73-103) vs. 91 (IQR, 80-99); P=0.360], chest kinematics [3.5 (IQR, 2.75-4.25) vs. 4 (IQR, 1-5.25) cm; P=0.296], and breath-holding test (BHT) [33 (IQR, 23-44) vs. 41 (IQR, 28-58) s; P=0.041]. CONCLUSIONS: Complex cardiopulmonary rehabilitation improved workload, quality of life, respiratory function, complaints and clinical status of patients with post-COVID syndrome. Personalized complex pulmonary rehabilitation can be beneficial and recommended for patients suffer from post-COVID syndrome, who have good potential for recovery and are able to participate in the two weeks complex pulmonary rehabilitation.

Proteomic Changes of Osteoclast Differentiation in Rheumatoid and Psoriatic Arthritis Reveal Functional Differences.

Background: Osteoclasts play a crucial role in the maintenance, repair, and remodeling of bones of the adult vertebral skeleton due to their bone resorption capability. Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are associated with increased activity of osteoclasts. Objectives: Our study aimed to investigate the dynamic proteomic changes during osteoclast differentiation in healthy donors, in RA, and PsA. Methods: Blood samples of healthy donors, RA, and PsA patients were collected, and monocytes were isolated and differentiated into osteoclasts in vitro using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANK-L). Mass spectrometry-based proteomics was used to analyze proteins from cell lysates. The expression changes were analyzed with Gene Set Enrichment Analysis (GSEA). Results: The analysis of the proteomic changes revealed that during the differentiation of the human osteoclasts, expression of the proteins involved in metabolic activity, secretory function, and cell polarity is increased; by contrast, signaling pathways involved in the immune functions are downregulated. Interestingly, the differences between cells of healthy donors and RA/PsA patients are most pronounced after the final steps of differentiation to osteoclasts. In addition, both in RA and PsA the differentiation is characterized by decreased metabolic activity, associated with various immune pathway activities; furthermore by accelerated cytokine production in RA. Conclusions: Our results shed light on the characteristic proteomic changes during human osteoclast differentiation and expression differences in RA and PsA, which reveal important pathophysiological insights in both diseases.

High Fat High Sucrose Diet Modifies Uterine Contractility and Cervical Resistance in Pregnant Rats: The Roles of Sex Hormones, Adipokines and Cytokines.

BACKGROUND: In obesity, the adipose tissue becomes a very significant endocrine organ producing different factors called adipokines, such as leptin, adiponectin and kisspeptin; however, no data are available about their actions on uterine contraction in obese pregnant rats. Our aim was to study the impact of obesity on pregnant uterine contraction in a rat model. METHODS: Obesity was induced by the consumption of a high fat high sucrose diet (HFHSD) for 9 weeks, including pregnancy. Glucose tolerance, sex hormone, cytokine and adipokine levels were measured. Uterine contractions and cervical resistance, as well as their responses to adipokines, were tested along with the expressions of their uterine receptors. RESULTS: HFHSD increased body weight, and altered glucose tolerance and fat composition. The uterine leptin and kisspeptin pathway affect increased. The levels of proinflammatory cytokines were reduced, while the plasma level of progesterone was increased, resulting in weaker uterine contractions, and improving the uterine relaxing effects of adipokines. HFHSD reduced cervical resistance, but the core effect of adipokines is difficult to determine. CONCLUSIONS: Obesity in pregnant rats reduces uterine contractility and cytokine-induced inflammatory processes, and therefore obese pregnant rat methods are partially applicable for modelling human processes.

Impact of Medium-Sized Extracellular Vesicles on the Transduction Efficiency of Adeno-Associated Viruses in Neuronal and Primary Astrocyte Cell Cultures.

(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.

Influence of Post-Translational Modifications on Protein Identification in Database Searches.

Comprehensive analysis of post-translation modifications (PTMs) is an important mission of proteomics. However, the consideration of PTMs increases the search space and may therefore impair the efficiency of protein identification. Using thousands of proteomic searches, we investigated the practical aspects of considering multiple PTMs in Byonic searches for the maximization of protein and peptide hits. The inclusion of all PTMs, which occur with at least 2% frequency in the sample, has an advantageous effect on protein and peptide identification. A linear relationship was established between the number of considered PTMs and the number of reliably identified peptides and proteins. Even though they handle multiple modifications less efficiently, the results of MASCOT (using the Percolator function) and Andromeda (the search engine included in MaxQuant) became comparable to those of Byonic, in the case of a few PTMs.

PhaSePro: the database of proteins driving liquid-liquid phase separation.

Membraneless organelles (MOs) are dynamic liquid condensates that host a variety of specific cellular processes, such as ribosome biogenesis or RNA degradation. MOs form through liquid-liquid phase separation (LLPS), a process that relies on multivalent weak interactions of the constituent proteins and other macromolecules. Since the first discoveries of certain proteins being able to drive LLPS, it emerged as a general mechanism for the effective organization of cellular space that is exploited in all kingdoms of life. While numerous experimental studies report novel cases, the computational identification of LLPS drivers is lagging behind, and many open questions remain about the sequence determinants, composition, regulation and biological relevance of the resulting condensates. Our limited ability to overcome these issues is largely due to the lack of a dedicated LLPS database. Therefore, here we introduce PhaSePro (https://phasepro.elte.hu), an openly accessible, comprehensive, manually curated database of experimentally validated LLPS driver proteins/protein regions. It not only provides a wealth of information on such systems, but improves the standardization of data by introducing novel LLPS-specific controlled vocabularies. PhaSePro can be accessed through an appealing, user-friendly interface and thus has definite potential to become the central resource in this dynamically developing field.

DisProt: intrinsic protein disorder annotation in 2020.

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the 'dark' proteome.

[Assessment of the extent of myocardial necrosis following radiofrequency catheter ablation of different supraventricular arrhythmias]. / A myocardialis necrosis mértékének vizsgálata eltéro supraventricularis szívritmuszavarok rádiófrekvenciás katéterablatiós kezelését követoen.

INTRODUCTION: Levels of cardiac necroenzymes, high-sensitive troponin (hsTnT) and creatine kinase muscle-brain (CKMB) increase as a result of a myocardial damage following catheter ablation. AIM: To analyze the mid-term alteration of hsTnT and CKMB levels following radiofrequency ablation (RFCA) for atrial fibrillation (AF), atrial flutter (AFlu), AV-nodal reentry tachycardia (AVNRT) and electrophysiological studies (EPS) without ablation. METHOD: Patients undergoing RFCA for various indications and EPS were consecutively enrolled in our prospective study. Concentrations of hsTnT and CKMB were measured from serial blood samples directly before and after the procedure, 4 and 20 hours later and at 3 months follow-up. RESULTS: Forty-seven patients (10 EPS, 12 AVNRT, 13 AFlu, 12 AF) with mean age of 55 ± 13 were included. hsTnT levels increased significantly in all groups after the procedures, while CKMB changed only in the AF group. hsTnT exceeded the reference value in all patients with ablation and in 80% of patients with EPS 4 hours post-ablation. Peak average hsTnT levels for EPS, AVNRT, AFlu were 24 ± 11, 260 ± 218 and 541 ± 233 ng/L, respectively. The highest hsTnT level was measured in the AF group (799 ± 433 ng/L). We found a positive correlation between hsTnT levels and ablation time after RFCA. CONCLUSIONS: The hsTnT levels significantly change after EPS and RFCA, in all patients who underwent ablation, and in 80% of those with EPS had hsTnT positivity in the early post-procedural phase. hsTnT levels depended significantly on the type of the subgroups and correlated with the ablation time. Awareness of those observations is essential to correctly interpret elevated hsTnT levels following RFCA. Orv Hetil. 2019; 160(14): 540-548.

Distinct In Vitro T-Helper 17 Differentiation Capacity of Peripheral Naive T Cells in Rheumatoid and Psoriatic Arthritis.

Background: The T-helper 17 (Th17) cells have a prominent role in inflammation as well as in bone and join destruction in both rheumatoid and psoriatic arthritis (RA and PsA). Here, we studied Th17 cell differentiation in RA and PsA. Methods: Blood samples from healthy donors, RA and PsA patients were collected. CD45RO- (naive) and CD45RO+ (memory) T cells were isolated from peripherial blood mononuclear cell by magnetic separation. Naive T cells were stimulated with anti-CD3, anti-CD28, and goat anti-mouse IgG antibodies and treated with transforming grow factor beta, interleukin (IL)-6, IL-1ß, and IL-23 cytokines and also with anti-IL-4 antibody. IL-17A and IL-22 production were measured by enzyme linked immunosorbent assay, RORC, and T-box 21 (TBX21) expression were analyzed by quantitative polymerase chain reaction and flow cytometry. C-C chemokine receptor 6 (CCR6), CCR4, and C-X-C motif chemokine receptor 3 expression were determined by flow cytometry. Cell viability was monitored by impedance-based cell analyzer (CASY-TT). Results: RORC, TBX21, CCR6, and CCR4 expression of memory T cells of healthy individuals (but not RA or PsA patients) were increased (p < 0.01; p < 0.001; p < 0.05; p < 0.05, respectively) compared to the naive cells. Cytokine-induced IL-17A production was different in both RA and PsA patients when compared to healthy donors (p = 0.0000026 and p = 0.0001047, respectively). By contrast, significant differences in IL-22 production were observed only between RA versus healthy or RA versus PsA patients (p = 0.000006; p = 0.0013454, respectively), but not between healthy donors versus PsA patients. Conclusion: The naive CD4 T-lymphocytes are predisposed to differentiate into Th17 cells and the in vitro Th17 cell differentiation is profoundly altered in both RA and PsA.

Extracellular vesicles regulate the human osteoclastogenesis: divergent roles in discrete inflammatory arthropathies.

OBJECTIVE: Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis. METHODS: Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14+ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7 days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted. RESULTS: EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n = 11) and RA-derived (n = 12) EXOs profoundly inhibited osteoclast differentiation (70%, p < 0.01; 65%, p < 0.01, respectively). In contrast, PsA-derived (n = 10) EXOs had a stimulatory effect (75%, p < 0.05). In cross-treatment experiments where EXOs and CD14+ cells were interchanged between the three groups, only healthy (n = 5) and RA (n = 5)-derived EXOs inhibited (p < 0.01, respectively) the generation of osteoclasts in all groups, whereas PsA (n = 7)-derived EXOs were unable to mediate this effect. CONCLUSIONS: Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.

Process analytical technology (PAT) approach to the formulation of thermosensitive protein-loaded pellets: Multi-point monitoring of temperature in a high-shear pelletization.

In the literature there are some publications about the effect of impeller and chopper speeds on product parameters. However, there is no information about the effect of temperature. Therefore our main aim was the investigation of elevated temperature and temperature distribution during pelletization in a high shear granulator according to process analytical technology. During our experimental work, pellets containing pepsin were formulated with a high-shear granulator. A specially designed chamber (Opulus Ltd.) was used for pelletization. This chamber contained four PyroButton-TH® sensors built in the wall and three PyroDiff® sensors 1, 2 and 3cm from the wall. The sensors were located in three different heights. The impeller and chopper speeds were set on the basis of 32factorial design. The temperature was measured continuously in 7 different points during pelletization and the results were compared with the temperature values measured by the thermal sensor of the high-shear granulator. The optimization parameters were enzyme activity, average size, breaking hardness, surface free energy and aspect ratio. One of the novelties was the application of the specially designed chamber (Opulus Ltd.) for monitoring the temperature continuously in 7 different points during high-shear granulation. The other novelty of this study was the evaluation of the effect of temperature on the properties of pellets containing protein during high-shear pelletization.

Respiratory gating algorithm helps to reconstruct more accurate electroanatomical maps during atrial fibrillation ablation performed under spontaneous respiration.

PURPOSE: Electroanatomical mapping is a useful tool during the ablation of atrial fibrillation. Respiratory movement might influence the mapping accuracy and merging. This study aims to investigate the effect of respiratory gating on the accuracy of magnetic-field-based electroanatomical mapping under spontaneous respiration. METHODS: Fifty-one consecutive patients (35 male, aged 30-78 years) who underwent left atrial radiofrequency catheter ablation due to atrial fibrillation were included. Electroanatomical mapping was performed with CARTO 3 System under conscious sedation. Respiratory gating was achieved with the AccuResp algorithm (Biosense Webster). Average surface match and maximum distance of the pre-acquired and electroanatomical maps, as well as left atrial volume, were recorded with and without respiratory gating after merging. RESULTS: The average surface match of the electroanatomical map with the left atrial reconstruction was significantly better with respiratory gating than without using the algorithm (3.81 ± 1.09 vs 4.11 ± 1.61 mm, p = 0.0119). It was not dependent of the rhythm during mapping or the image modality used for left atrial reconstruction. The maximal distance between the two maps did not depend on the use of the algorithm (19.81 ± 6.24 mm for gated and 20.87 ± 7.99 mm for non-gated, p = 0.3161). Left atrial volume of the map was significantly lower when using the respiratory compensation module (106.3 ± 31.6 vs 127.0 ± 36.4 ml, p < 0.0001) and showed a significant correlation with the pre-recorded 3D reconstruction volumes (r = 0.66, p < 0.0001). CONCLUSIONS: The use of the novel respiratory gating algorithm might improve the accuracy of electroanatomical mapping during left atrial ablation under conscious sedation. The possible impact on the effectiveness of the ablation needs to be further evaluated.

[A rare case of fatal outcome due to extensive facial trauma caused by dogbite]. / Kiterjedt arcsérüléssel járó, halálos kimenetelu kutyatámadás ritka esete.

Statistically 10% of head and neek trauma is caused by animal bite, more often inflicted by dogs. More than 50% of victims are infants and small children. Generally, the result of dogbite is superficial trauma but there are cases of deep soft tissue defects with amputation and multiple bone fractures. In case described in this article, an elderly woman suffered a dog attack, wich resulted in extensive face and head injuries. In spite of receiving proper treatment, the patient passed away. It is highly probable that underlying Syncumar therapy caused complications which contributed to the fatal outcome. Because of it's unusual nature we found this case to be of interest for publication.

Chemoprevention and inhibition of P-glycoprotein in cancer cells by Chinese medicinal herbs.

Many of the herbal extracts used in the Chinese clinical medical routine inhibit the growth of tumor cells. In the present work, extracts of 12 selected herbs were prepared with methanol, chloroform, ethyl acetate and water, and the effects of these on the multidrug resistance (MDR) and P-glycoprotein of mouse lymphoma cells transfected with the human mdr1 gene and on a human lung alveolar epithelial cell line were investigated. The extracts were tested for antiproliferative effects, and the reversal of MDR in mouse lymphoma cells. The possible chemopreventive effect of the chloroform extracts was studied on the expression of cytomegalovirus (CMV) immediate-early (IE) antigen in human lung cancer cells (A549). The antimicrobial effects of the extracts were tested on some representative micro-organisms. Certain of the chloroform extracts of the plant materials were the most effective compounds on the reversal of MDR. Two of the chloroform extracts enhanced the antiproliferative effect of doxorubicin on MDR mouse lymphoma cells. The selected extracts did not show any antibacterial effect with the agar diffusion method. Certain chloroform extracts decreased the intermediate IE antigen expression of CMV in A459 cells.