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Here, we present a modular synthesis as well as physicochemical and biological evaluation of a new series of amphiphilic dendrons carrying triphenylphosphonium groups at their periphery. Within the series, the size and mutual balance of lipophilic and hydrophilic domains are systematically varied, changing the dendron shape from cylindrical to conical. In physiological solution, the dendrons exhibit very low critical micelle concentrations (2.6-4.9 μM) and form stable and uniform micelles 6-12 nm in diameter, depending on dendron shape; the results correlate well with molecular dynamics simulations. The compounds show relatively high cytotoxicity (IC 50 1.2-21.0 μM) associated with micelle formation and inversely related to the size of assembled particles. Depending on their shape, the dendrons show promising results in terms of dendriplex formation and antibacterial activity. In addition to simple amphiphilic dendrons, a fluorescently labeled analogue was also prepared and utilized as an additive visualizing the dendron's cellular uptake.

Background: The Slovak Republic, an Eastern European country, borders Ukraine, which has one of the highest rates of HIV/AIDS in Europe. This study aims to inform readers about the demographic characteristics of Ukrainian war refugees with HIV/AIDS seeking temporary protection status in Slovakia due to the Russian invasion of February 24, 2022. It focuses on their epidemiology, immunological profiles, co-infections, and treatment., Design: Multicenter, retrospective study between March 1, 2022, and March 31, 2023., Methods: Data were collected from all Ukrainian patients with refugee status living with HIV or newly diagnosed, who presented to any of the five HIV/AIDS centers in Slovakia. Information was obtained through medical interviews, physical examinations, laboratory tests and medical reports from the Ukrainian Ministry of Health., Results: From March 1, 2022, to March 31, 2023, 141 Ukrainian refugees were included, mostly women (56.7 %, n = 80), with a median age of 41 years. The median CD4 + count was 680 cells/mL; 69.2 % (n = 97) had a viral load below 40 copies/mL. On arrival, 87.8 % (n = 123) were on ART, 90.2 % (n = 111) on dolutegravir. Coinfections included hepatitis C (31 %, n = 41), hepatitis B (12.5 %, n = 17), and tuberculosis (11.3 %, n = 16). Three died from AIDS complications., Conclusion: The study provides a detailed overview of the epidemiological, demographic, immunological, co-infection, and treatment characteristics of Ukrainian PLWHA displaced by the war to the Slovak republic. With the conflict ongoing, it is necessary to be prepared for more refugees in the coming months., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Mumps data were analysed to assess the effect of vaccination on mumps complications and hospitalisation., Methods: The mumps cases reported to the Czech nationwide surveillance system from 2013 to 2022 were analysed using logistic regression with an odds ratio (aOR) adjusted for age, sex, year of onset and administrative region to measure the association between vaccination and complications or hospitalisation. Adjusted vaccine effectiveness (aVE) was calculated: aVE=(1-aOR)x100., Results: A total of 11,913 mumps cases were reported, of which 6,885 (58%) were male. The median age of the study participants was 16 (range: 0-89 years). No complications occurred in 91% of patients. Mumps orchitis occurred in 633 (9%) male cases. A total of 946 (8%) patients required hospitalisation. The highest proportion of complications and hospitalisations was in the age group 35-44 years. Two doses of vaccine reduced statistically significantly the risk of any complications and of hospitalisation compared with unvaccinated patients: aOR 0.48 (95% CI: 0.37, 0.62), aVE of 52% (95% CI: 38, 63); and aOR 0.43 (95% CI: 0.33, 0.56), aVE of 57% (95% CI: 44, 67), respectively. Two doses showed statistically significant aVE 50% (95% CI: 32, 64) against orchitis, and 59% (95% CI: 23, 79) against meningitis. Among the two-dose recipients, the proportion of complications increased gradually with the time from the second dose., Conclusions: Our findings demonstrated a protective effect of two-dose vaccination against mumps complications and hospitalisation for mumps. We recommend continuing routine childhood mumps vaccination and maintaining high MMR coverage in Czechia., (© 2024 Monika Liptáková et al., published by Sciendo.)

The switch between planktonic and biofilm lifestyle correlates with intracellular concentration of the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). While bacteria possess cyclase and phosphodiesterase enzymes to catalyse formation or hydrolysis of c-di-GMP, both enzymatic domains often occur in a single protein. It is tacitly assumed that one of the two enzymatic activities is dominant, and that additional domains and protein interactions enable responses to environmental conditions and control activity. Here we report the structure of the phosphodiesterase domain of the membrane protein RbdA (regulator of biofilm dispersal) in a dimeric, activated state and show that phosphodiesterase activity is controlled by the linked cyclase. The phosphodiesterase region around helices α5/α6 forms the dimer interface, providing a rationale for activation, as this region was seen in contact with the cyclase domain in an auto-inhibited structure previously described. Kinetic analysis supports this model, as the activity of the phosphodiesterase alone is lower when linked to the cyclase. Analysis of a computed model of the RbdA periplasmatic domain reveals an all-helical architecture with a large binding pocket that could accommodate putative ligands. Unravelling the regulatory circuits in multi-domain phosphodiesterases like RbdA is important to develop strategies to manipulate or disperse bacterial biofilms., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background and Objectives: In a highly vaccinated population, an increasing number of previously vaccinated measles cases can be expected. The aim of this study was to assess the effect of vaccination on the clinical course and immune response in relation to the current measles case definition., Methods: The presence of fever, catarrhal symptoms, exanthema and complications, and specific IgM and IgG positivity were assessed in all 230 patients and compared in 193 patients with known vaccination status, divided into measles-containing vaccine (MCV) groups: MCV0 (85 patients), MCV1 (25 patients) and MCV2 (83 patients)., Results: Statistically significant differences between groups were found for catarrhal symptoms. Conjunctivitis and rhinitis were significantly less frequent in the MCV2 group (47% and 54%) compared to MCV0 (80% and 80%), p  < 0.001 and p  = 0.002 respectively. Typical exanthema was present in 74 (87%) MCV0 and 56 (67%) MCV2 patients, p  = 0.005. Complications were most common in the MCV0 group (29%). ECDC clinical case criteria were met in 81 (95%) MCV0, 18 (72%) MCV1 and 59 (71%) MCV2 patients, p  < 0.001. IgM were positive in 64 (83%) MCV0, 14 (74%) MCV1 and 36 (67%) MCV2 patients, differences were not statistically significant. There were highly significant differences in IgG between MCV0 and both vaccinated groups ( p  < 0.001)., Conclusions: A redefinition of the clinical case classification is essential to better capture modified measles and to raise awareness among healthcare workers of the differences in measles in vaccinated patients.

Objectives: The main objective of the questionnaire survey conducted by the National Institute of Public Health in Prague was to determine the level of knowledge and attitudes and to identify changes since the previous survey in 2015. A secondary objective was to gather more detailed information on how HIV/AIDS topics are taught in schools, as reported by school prevention coordinators., Methods: The questionnaire was distributed to 48 randomly selected educational institutions and the humanitarian organization MRIYA UA z.s. between October 2022 and January 2023. Data on 21 questions focusing on HIV/AIDS issues were collected anonymously from Czech and Ukrainian students. The survey included a representative sample of students from the 7th, 8th, and 9th grades in primary schools and multi-year grammar schools. Each institution received a questionnaire for the prevention coordinator, who answered 12 questions., Results: A total of 3,011 students completed the questionnaire. The average score for HIV/AIDS knowledge was 13.5 points out of a maximum of 22 points. Students' knowledge gradually increased with higher grades: students from multi-year grammar schools scored an average of 15.9 points, while primary school students scored an average of 13.2 points. Czech students scored an average of 13.6 points, whereas Ukrainian students scored an average of 12.4 points; the 1.2-point difference was statistically significant (p = 0.004). Compared to the 2015 survey, there was a slight overall decline in adolescents' knowledge levels. Students' attitudes towards people living with HIV/AIDS positively correlated with their knowledge about HIV/AIDS: the better their knowledge, the more favourable their attitudes toward people living with HIV/AIDS. Students' main sources of information about HIV/AIDS were school (37.1%) and the Internet (36.6%). According to school prevention coordinators, 95.7% of the surveyed schools address the topic of HIV/AIDS, most commonly in biology or health education classes, dedicating an average of 8.7 instructional hours to the subject. The most frequent teaching method is video lessons, used by 87.2% of the surveyed schools. Schools expressed a preference for improving the quality of education by utilizing presentations with professionally approved content, with 74.5% of schools supporting this option., Conclusions: The survey highlighted a gradual increase in HIV/AIDS knowledge with advancing school grades, a significant disparity in knowledge between Czech and Ukrainian students, and a slight overall decline in knowledge compared to 2015. The Internet and schools were the main information sources, though the role of schools declined significantly. It also underscored the need for enhanced educational programmes and continuous professional development for educators to improve health literacy and HIV/AIDS prevention among adolescents.

Background: Atherosclerosis is a major cause of ischemic stroke, and early detection of advanced atherosclerosis in the carotid artery is important for reducing morbidity and mortality. What is even more important is not only detection of atherosclerosis but early determination whether the patients are at high risk of an event with adverse effects as the size of the plaque does not necessarily reflect its potential to trigger such events., Aim: We studied whether plasma lipidomics profile can be used as a diagnostic tool for stratification of stable or unstable plaques without the need of removing the carotid plaque., Methods: This study used liquid chromatography high-resolution tandem mass spectrometry lipidomics to characterize lipid profiles in patients' plasma and found that patients with significant and complicated (vulnerable) atherosclerotic plaque had distinct lipid profiles compared to those with insignificant plaques., Results: The lipid classes that were most predictive of vulnerable plaque were lysophosphoethanolamines, fatty acyl esters of hydroxy fatty acids, free fatty acids, plasmalogens, and triacylglycerols. Most of these compounds were found decreased in plasma of patients with unstable plaques which enabled sufficient performance of a statistical model used for patient stratification., Conclusions: Plasma lipidomes measured by liquid chromatography-mass spectrometry show differences in patients with stable and unstable carotid plaques, therefore these compounds could potentially be used as biomarkers for unstable plaque in future clinical diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Thrombosis is linked to neutrophil release of neutrophil extracellular traps (NETs). NETs are proposed as a mechanism of resistance to thrombolysis. This study intends to analyze the composition of thrombi retrieved after mechanical thrombectomy, estimate the age and organization of thrombi, and evaluate associations with the use of thrombolysis, antiplatelets, and heparin., Methods: This retrospective observational study involved 72 samples (44 from cerebral and 28 coronary arteries), which were stained with hematoxylin and eosin, anti-NE (neutrophil elastase) antibody, and anti-histone H2B (histone H2B) antibody, representing different components in NET formation, all detectable during the later stages of NETosis, for histochemical and digital quantification of NET content. The histological and morphological evaluations of the specimens were correlated, through univariate and mediation analyses, with clinical information and therapy administered before intervention., Results: The results demonstrated that the composition of cerebral and coronary thrombi differs, and there were significantly more lytic cerebral thrombi than coronary thrombi (66% versus 14%; P =0.005). There was a considerably higher expression of NETs in the cerebral thrombi as testified by the higher expression of H2B ( P =0.031). Thrombolysis was remarkably associated with higher NE positivity (average marginal effect, 6.461 [95% CI, 0.7901-12.13]; P =0.02555), regardless of the origin of thrombi. There was no notable association between the administration of antiaggregant therapy/heparin and H2B/NE amount when adjusted for the thrombus location. Importantly, the age of the thrombus was the only independent predictor of NET content without any mediation of the thrombolytic treatment ( P =0.014)., Conclusions: The age of the thrombus is the driving force for NET content, which correlates with impaired clinical outcomes. The therapy that is currently administered does not modify NET content. This study supports the need to investigate new pharmacological approaches added to thrombolysis to prevent NET formation or enhance their disruption, such as recombinant human DNase I (deoxyribonuclease I)., Competing Interests: Disclosures None.

Liposomal carrier systems have emerged as a promising technology for pulmonary drug delivery. This study focuses on two selected liposomal systems, namely, dipalmitoylphosphatidylcholine stabilized by phosphatidic acid and cholesterol (DPPC-PA-Chol) and dipalmitoylphosphatidylcholine stabilized by polyethylene glycol and cholesterol (DPPC-PEG-Chol). First, the research investigates the stability of these liposomal systems during the atomization process using different kinds of nebulizers (air-jet, vibrating mesh, and ultrasonic). The study further explores the aerodynamic particle size distribution of the aerosol generated by the nebulizers. The nebulizer that demonstrated optimal stability and particle size was selected for more detailed investigation, including Andersen cascade impactor measurements, an assessment of the influence of flow rate and breathing profiles on aerosol particle size, and an in vitro deposition study on a realistic replica of the upper airways. The most suitable combination of a nebulizer and liposomal system was DPPC-PA-Chol nebulized by a Pari LC Sprint Star in terms of stability and particle size. The influence of the inspiration flow rate on the particle size was not very strong but was not negligible either (decrease of D v 50 by 1.34 μm with the flow rate increase from 8 to 60 L/min). A similar effect was observed for realistic transient inhalation. According to the in vitro deposition measurement, approximately 90% and 70% of the aerosol penetrated downstream of the trachea using the stationary flow rate and the realistic breathing profile, respectively. These data provide an image of the potential applicability of liposomal carrier systems for nebulizer therapy. Regional lung drug deposition is patient-specific; therefore, deposition results might vary for different airway geometries. However, deposition measurement with realistic boundary conditions (airway geometry, breathing profile) brings a more realistic image of the drug delivery by the selected technology. Our results show how much data from cascade impactor testing or estimates from the fine fraction concept differ from those of a more realistic case.

Inflammatory bowel disease, encompassing Crohn's disease and ulcerative colitis, is a persistent immune-mediated inflammatory gastrointestinal disease. This study investigates the role of growth differentiation factor 15 in severe IBD cases, aiming to identify a reliable parameter to assess disease severity and monitor activity. We analyzed plasma samples from 100 patients undergoing biologic therapy for severe IBD and 50 control subjects. Our analysis included evaluations of GDF-15 levels, inflammatory markers, and clinical features. We employed statistical methods such as the Mann-Whitney U test, ANOVA, and Spearman's correlation for an in-depth analysis. Our results demonstrated consistently higher GDF-15 levels in patients with both Crohn's disease and ulcerative colitis compared to the control group, irrespective of the biologic treatment received. The correlation analysis indicated significant relationships between GDF-15 levels, patient age, fibrinogen, and IL-6 levels. This study positions GDF-15 as a promising biomarker for severe IBD, with notable correlations with age and inflammatory markers. These findings underscore GDF-15's potential in enhancing disease monitoring and management strategies in an IBD context and encourage further research to clarify GDF-15's role in the IBD pathophysiology.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins., Competing Interests: Competing interests statement:The authors declare no competing interest.

The numerical simulation of inhaled aerosols in medical research starts to play a crucial role in understanding local deposition within the respiratory tract, a feat often unattainable experimentally. Research on children is particularly challenging due to the limited availability of in vivo data and the inherent morphological intricacies. CFD solvers based on Finite Volume Methods (FVM) have been widely employed to solve the flow field in such studies. Recently, Lattice Boltzmann Methods (LBM), a mesoscopic approach, have gained prominence, especially for their scalability on High-Performance Computers. This study endeavours to compare the effectiveness of LBM and FVM in simulating particulate flows within a child's respiratory tract, supporting research related to particle deposition and medication delivery using LBM. Considering a 5-year-old child's airway model at a steady inspiratory flow, the results are compared with in vitro experiments. Notably, both LBM and FVM exhibit favourable agreement with experimental data for the mean velocity field and the turbulence intensity. For particle deposition, both numerical methods yield comparable results, aligning well with in vitro experiments across a particle size range of 0.1-20 µm. Discrepancies are identified in the upper airways and trachea, indicating a lower deposition fraction than in the experiment. Nonetheless, both LBM and FVM offer invaluable insights into particle behaviour for different sizes, which are not easily achievable experimentally. In terms of practical implications, the findings of this study hold significance for respiratory medicine and drug delivery systems - potential health impacts, targeted drug delivery strategies or optimisation of respiratory therapies., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography., (open access.)

The revolution in cryo-electron microscopy has resulted in unprecedented power to resolve large macromolecular complexes including viruses. Many methods exist to explain density corresponding to proteins and thus entire protein capsids have been solved at the all-atom level. However methods for nucleic acids lag behind, and no all-atom viral double-stranded DNA genomes have been published at all. We here present a method which exploits the spiral winding patterns of DNA in icosahedral capsids. The method quickly generates shells of DNA wound in user-specified, idealized spherical or cylindrical spirals. For transition regions, the method allows guided semiflexible fitting. For the kuravirus SU10, our method explains most of the density in a semiautomated fashion. The results suggest rules for DNA turns in the end caps under which two discrete parameters determine the capsid inner diameter. We suggest that other kuraviruses viruses may follow the same winding scheme, producing a discrete rather than continuous spectrum of capsid inner diameters. Our software may be used to explain the published density maps of other double-stranded DNA viruses and uncover their genome packaging principles., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Knowledge of precipitation composition is important, among other things, to reveal changes in atmospheric chemistry. Here we present the long-term time trends in ratios of major ions in precipitation, namely nitrate to sulphate (NO 3 - /SO 4 2- ), ammonium to sulphate (NH 4 + /SO 4 2- ) and ammonium to nitrate (NH 4 + /NO 3 - ). For this we explore the long-term time series recorded by the Czech Hydrometeorological Institute at eight monitoring sites situated in urban, rural and mountain regions of the Czech Republic between 1980 and 2020. To that end, we use innovative Bayesian inference with the Integrated Nested Laplace Approximation (INLA) computational method appropriate for investigating complicated large-scale data. Our results indicated: (i) increasing NO 3 - /SO 4 2- ratio in precipitation over time and distinct seasonal behaviour with higher values in winter and lower values in summer, (ii) increasing NH 4 + /SO 4 2- ratio in precipitation and distinct seasonal behaviour with higher values in summer and lower values in winter and (iii) relatively stable NH 4 + /NO 3 - ratio in precipitation with a mild recent increase and distinct seasonal behaviour with higher values in summer and lower values in winter. This behaviour pattern holds true for all the sites analysed, irrespective of their geographical position, altitude or environment. Though explored in detail rarely, the ion ratios are important to study as they reflect changes in atmospheric chemistry, mirroring changes in emissions and meteorology and suggesting changing impacts on ecosystems and the environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tularemia is a zoonosis caused by Francisella tularensis, a gram-negative aerobic bacterium belonging to the class of Gammaproteobacteria and the family Francisellaceae. Despite its undeniable importance for human health, there is little data on the current distribution of F. tularensis in various hematophagous arthropods. The aim of this study was to perform a mass molecular screening of different possible hematophagous vectors: ticks (4348 ticks of the species Ixodes ricinus, Dermacentor reticulatus, and Haemaphysalis concinna), mosquitoes (4100 specimens of Aedes vexans), and blackflies (6900 specimens of the Simulium spp.) for the presence of F. tularensis in the Břeclav district in 2022. Only two specimens were positive for the specific DNA of Francisella tularensis subsp. holarctica. Both samples originated from D. reticulatus, one collected from infested roe deer and the other included in a pooled sample (n = 10). Both positive samples were sequenced, and the presence of F. tularensis subsp. holarctica was confirmed. In addition, the absence of F. tularensis in mosquitoes and black flies was documented.

Objectives: HIV positivity diagnosis is often accompanied by stigmatization and discrimination, even in developed societies. The aim of the study was to obtain current information on the quality of life of people living with HIV in the Czech Republic, to assess the level and perception of stigmatization and discrimination, and to identify existing problems of people living with HIV concerning health services, family and the public., Methods: Data for the pilot study were collected by convenience sampling between 2021 and 2022. Respondents were recruited from residents of Czech nationality and foreigners living with HIV in the Czech Republic who participated in an ECDC online questionnaire study or attended a convalescent stay for people living with HIV. Likert scales were used to capture key opinions, attitudes and beliefs of respondents. Data were processed using descriptive analysis., Results: The study involved 42 people living with HIV. A total of 77% of the respondents reported that they had learned to live with their HIV-positive status, but 21% admitted to having low self-esteem due to their status. A total of 81% of the respondents had disclosed their HIV-positive status to someone outside of the healthcare setting, however, 40% of the respondents found it difficult to do so. Assault or threats from a sexual partner were reported by 17% of the respondents. A total of 27% of the respondents admitted having been denied or suspended health care, 44% reported inappropriate comments from healthcare workers, and 32% of the respondents avoided visiting the healthcare facility. Alarmingly, 41% of the respondents had been refused dental care at some point in the past because of their HIV-positive status., Conclusions: Although the study presents only limited findings due to the small number of respondents, it is clear that stigmatization and discrimination of people living with HIV exists in the Czech society. It brings the risk of loss of motivation and involvement of people living with HIV in working together with healthcare providers on their treatment. There is a clear need for a larger study to identify the causes of stigmatization and to find ways to prevent it.

The representativeness of ambient air quality of an in situ measurement is key in the use and correct interpretation of the measured concentration values. Though the horizontal representativeness aspect is generally not neglected in air pollution studies, a detailed, high-resolution vertical distribution of ambient air pollutant concentrations is rarely addressed. The aim of this study is twofold: (i) to explore the vertical distribution of ground-level ozone (O 3 ) concentrations measured at four heights above the ground-namely at 2, 8, 50, and 230 m-and (ii) to examine in detail the vertical O 3 concentration gradient in air columns between 2 and 8, 8 and 50, and 50 and 230 m above the ground. We use the daily mean O 3 concentrations measured continuously at the Košetice station, representing the rural Central European background ambient air quality observed during 2015-2021. We use the semiparametric GAM (generalised additive model) approach (with complexity or roughness-penalised splines implementation) to analyse the data with sufficient flexibility. Our models for both O 3 concentrations and O 3 gradients use (additive) decomposition into annual trend and seasonality (plus an overall intercept). The seasonal and year-to-year patterns of the modelled O 3 concentrations look very similar at first glance. Nevertheless, a more detailed look through O 3 gradients shows that they differ substantially with respect to their seasonal and long-term dynamics. The vertical O 3 concentration gradient in 2-230 m is not uniform but changes substantially with increasing height and shows by far the highest dynamics near the ground between 2 and 8 m, differing in both the seasonal and annual aspects for all the air columns inspected. We speculate that non-linear changes of both seasonal and annual components of vertical O 3 gradients are due to atmospheric-terrestrial interactions and to meteorological factors, which we will explore in a future study., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The resistance of the emerging human pathogen Stenotrophomonas maltophilia to tetracycline antibiotics mainly depends on multidrug efflux pumps and ribosomal protection enzymes. However, the genomes of several strains of this Gram-negative bacterium code for a FAD-dependent monooxygenase (SmTetX) homologous to tetracycline destructases. This protein was recombinantly produced and its structure and function were investigated. Activity assays using SmTetX showed its ability to modify oxytetracycline with a catalytic rate comparable to those of other destructases. SmTetX shares its fold with the tetracycline destructase TetX from Bacteroides thetaiotaomicron; however, its active site possesses an aromatic region that is unique in this enzyme family. A docking study confirmed tetracycline and its analogues to be the preferred binders amongst various classes of antibiotics., (open access.)

Animal testing has been prohibited for the safety assessment of cosmetic ingredients or finished products. Thus, alternative non-animal methods, followed by confirmatory clinical studies on human volunteers, should be used as the sole legally acceptable approach within the EU. The safety assessment of cosmetic products requires the involvement of multiple scientific disciplines, including analytical chemistry and biomedicine, as well as in chemico , in vitro and in silico toxicology. Recent data suggest that fragrance components may exert multiple adverse biological effects, e.g. cytotoxicity, skin sensitisation, (photo)genotoxicity, mutagenicity, reprotoxicity and endocrine disruption. Therefore, a pilot study was conducted with selected samples of fragrance-based products, such as deodorant, eau de toilette and eau de parfum, with the aim of integrating results from a number of alternative non-animal methods suitable for the detection of the following toxicological endpoints: cytotoxicity (with 3T3 Balb/c fibroblasts); skin sensitisation potential ( in chemico method, DPRA); skin sensitisation potential (LuSens in vitro method, based on human keratinocytes); genotoxicity potential ( in vitro Comet assay with 3T3 Balb/c cells); and endocrine disruption ( in vitro YES/YAS assay). The presence of twenty-four specific known allergens in the products was determined by using GC-MS/MS. The strategies for estimation of the NOAEL of a mixture of allergens, which were proposed by the Scientific Committee on Consumer Products in their 'Opinion on Tea tree oil' document and by the Norwegian Food Safety Authority in their 'Risk Profile of Tea tree oil' report, were used as models for the NOAEL estimation of the mixtures of allergens that were identified in the individual samples tested in this study.

Binder H33 is a small protein binder engineered by ribosome display to bind human interleukin 10. Crystals of binder H33 display severe diffraction anisotropy. A set of data files with correction for diffraction anisotropy based on different local signal-to-noise ratios was prepared. Paired refinement was used to find the optimal anisotropic high-resolution diffraction limit of the data: 3.13-2.47 Å. The structure of binder H33 belongs to the 2% of crystal structures with the highest solvent content in the Protein Data Bank., (© Petr Kolenko et al. 2023.)

Objectives: The aim of this work was to analyze the relationship between new cases of clinical tick-borne encephalitis (TBE) and various meteorological and seasonal predictors., Material and Methods: The modelling is based on national data from the Czech Republic for the period 2001-2016 in daily resolution, namely on average temperatures, average relative air humidity and the number of TBE cases classified according to the date of the first symptoms. Four variants of a negative binomial model from the generalized additive model class are used. The basic model relates the occurrence of TBE to the lagged ambient daily average temperature and daily average relative air humidity and their interaction with the lag reflecting the incubation period and other factors. The lag value was estimated via the optimization procedure based on Akaike information criterion. The model also includes the effect of the season and the effect of the day of the week. To increase the biological plausibility, the basic model has been expanded to account for possible time-varying effects of meteorological variables and to incorporate multiple lags., Results: The most statistically significant effect is the within-year seasonality and then the interaction of the temperature and relative air humidity. The relationship of both meteorological factors and their interactions vary throughout the activities season of the hostquesting Ixodes ricinus. This also changes the conditions of occurrence of the new clinical cases of TBE. The time-varying effect of meteorological factors on the incidence of TBE shows non-trivial changes within a year. In the period before the middle of the calendar year (around the week 22) the effect decreases, then it is followed by an increase until the week 35., Conclusion: Flexible models were developed with quantitatively characterized effects of temperature, air humidity and their interaction, with the delay of the effect estimated through the optimization process. Performance of the model with multiple lags was checked using independent data to verify the possibility of using the results to improve the prediction of the risk of clinical cases of TBE uprise.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world., (open access.)

Objectives: Pregnant women are among the priority groups to receive influenza vaccines in the Czech Republic since 2011, data on vaccination coverage are not yet available. The aim of the study was to determine the influenza vaccination coverage (IVC) and provide source data for further activities., Methods: A prospective observational study was performed in a large maternity hospital in Prague. The self-completed questionnaire was distributed to 5,475 pregnant women between September 1, 2020 and August 31, 2021. Questions included maternal sociodemographic characteristics, influenza vaccination status and sources of maternal vaccination recommendations during pregnancy., Results: A total of 4,617 completed questionnaires have been analysed. The median age of study participants (N = 4,592) was 33 years (range: 18-51 years). The majority (69.7%) of women had completed their university education, most women were childless (58.5%) or had one child (32.5%) before the start of the study. Less than 2% of women reported being vaccinated against influenza during their pregnancy (1.5%; 95% CI, 1.1-1.9%). Only 21% of women knew that it's possible to get vaccinated against influenza during pregnancy. Participants considered influenza vaccination in pregnancy as important (3.3%), useful (41.1%) and useless (44.4%). Out of 959 pregnant women who had information about influenza vaccination during pregnancy, only 6.9% were vaccinated, while among those who did not have this information, 0.1% were vaccinated during pregnancy (p < 0.001). The most frequent source of information was Internet, then media and a general practitioner., Conclusions: The IVC during pregnancy in our study was extremely low. In order to improve IVC among pregnant women, it is necessary to increase awareness of recommendations and vaccination options among the public and professionals and incorporating vaccination recommendation in routine antenatal practice., (© 2023. The Author(s).)

Objectives: For the proper diagnosis of toxoplasmosis it is essential to determine the stage of the infection, for which the most preferred method is IgG avidity test. The avidity index (AI) should initially be low (AI≤0.3) in the acute phase and increase during the infection. However, persistent low avidity can occur in patients with latent toxoplasmosis, which can complicate the interpretation of the results. The aim of the study is to explain the causes of this phenomenon., Methodology: A retrospective study was carried out with 717 serum samples collected from 442 patients from the categories of pregnant and non-pregnant women, men, and newborns + infants (age < 0.5 year). The trends of AI kinetics were evaluated in repeatedly examined patients. The frequency of cases with low avidity was compared in individual categories of patients and in groups of people with acute and non-acute toxoplasmosis., Results: The proportion of patients with initially low avidity was 42.1% in the acute toxoplasmosis group while it was 13.0% in the non-acute groups. In uninfected newborns with anti-Toxoplasma antibodies transmitted from the mother, a decrease in IgG avidity levels over time was observed, resulting in 29.2% of samples showing low (improper) avidity. While the dynamics of IgG avidity and the frequency of cases of improperly low avidity were similar in men and pregnant and non-pregnant women, the category of newborns and infants differed substantially for these indicators., Conclusions: Due to acceptable specificity and negative predictive value, high avidity can rule out acute toxoplasmosis, but moderate sensitivity complicates the possibility of its confirmation. The results of the avidity test must be interpreted in the context of the results of other methods., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kodym et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: The completeness and timeliness of the pertussis questionnaire-based enhanced surveillance system (ESS) among infants and reported pertussis data within the electronic nationwide notification system (NNS) in the years 2015, 2017 and 2019 were evaluated in a pilot study., Methods: The completeness of the variables for demographic characteristics, date of symptom onset, hospitalisation and vaccination status were assessed in both systems. Timeliness of reporting in the NNS was analysed as the interval between symptom onset and a) the date of first specimen collection (diagnostic delay), and b) the date of the Regional Public Health Authority receiving notification (notification delay)., Results: A total of 121 confirmed pertussis cases were reported to the NNS in the study years, while in the ESS a total of 104 confirmed cases were reported in infants. In both systems most cases were in the age group of one completed month of life (20% versus 23%) and males (55% versus 55%). The majority of cases were hospitalised (81% versus 85%) and unvaccinated (77% versus 78%). Within the NNS, the first dose of vaccine was reported in 13 cases, the second dose in 11, and third dose in three cases. Within the NNS, 100% completeness of following variables was found: symptom onset, week and region of reporting, age, gender and place of isolation. Median diagnostic delay was nine days. Median notification delay was 18 days., Conclusions: Data completeness was high in the NNS, except for lack of vaccination data in those eligible by age. Efforts to improve the completeness of laboratory-related variables and timeliness are essential. Based on the study results, the project of improving the ESS for infants will continue with regular evaluation., (© 2023 Monika LIPTÁKOVÁ et al., published by Sciendo.)

Background: Predicting stroke risk in patients with carotid artery stenosis (CS) remains challenging. Circulating biomarkers seem to provide improvements with respect to risk stratification. Methods: Study patients who underwent carotid endarterectomy were categorized into four groups according to symptomatology and compared as follows: symptomatic with asymptomatic patients; and asymptomatic patients including amaurosis fugax (AF) (asymptomatic + AF group) with patients with a transient ischemic attack (TIA) or brain stroke (BS) (hemispheric brain stroke group). Carotid specimens were histologically analyzed and classified based on the American Heart Classification (AHA) standard. As a marker of OS, the plasma levels of malondialdehyde (MDA) were measured. Comparisons of MDA plasma levels between groups were analyzed. Results : In total, 35 patients were included in the study. There were 22 (63%) patients in the asymptomatic group and 13 (37%) in the symptomatic group. Atheromatous plaque ( p = 0.03) and old hemorrhage ( p = 0.05), fibrous plaque ( p = 0.04), myxoid changes ( p = 0.02), plaques without hemorrhage ( p = 0.04), significant neovascularization ( p = 0.04) and AHA classification ( p = 0.006) had significant correlations with clinical presentation. There were 26 (74%) patients in the asymptomatic group and 9 (26%) in the hemispheric brain stroke group. Atheromatous plaque ( p = 0.02), old hemorrhage ( p = 0.05) and plaques without neovascularization ( p = 0.02), fibrous plaque ( p = 0.03), plaques without hemorrhage ( p = 0.02) and AHA classification ( p = 0.01) had significant correlations with clinical presentation. There was no significant difference between symptomatic and asymptomatic groups with respect to MDA plasma levels ( p = 0.232). A significant difference was observed when MDA plasma levels were compared to asymptomatic + AF and the hemispheric stroke group ( p = 0.002). Conclusions : MDA plasma level correlates with the risk of hemispheric stroke (TIA or BS) and is a reliable marker of plaque vulnerability in carotid artery stenosis.

Background: Comparative data regarding the effect of percutaneous and thoracoscopic ablation of atrial fibrillation (AF) on cognitive function are very limited. The aim of the study was to determine and compare the effect of both types of ablations on patient cognitive functions in the mid-term., Methods: Patients with AF indicated for ablation procedure were included. Forty-six patients underwent thoracoscopic, off-pump ablation using the COBRA Fusion radiofrequency system, followed by a catheter ablation three months afterward (Hybrid group). A comparative cohort of 53 AF patients underwent pulmonary vein isolation only (PVI group). Neuropsychological examinations were done before and nine months after the surgical or catheter ablation procedure. Neuropsychological testing comprised 13 subtests of seven domains, and the results were expressed as post-operative cognitive dysfunction (POCD) nine months after the procedure., Results: Patients in both groups were similar with respect to the baseline clinical characteristics; only non-paroxysmal AF was more common in the hybrid group (98% vs. 34%). Major POCD was present in eight (17.4%) of hybrid patients versus three (5.7%) of PVI patients (p = 0.11), combined (major/minor) worsened cognitive decline was present in 10 (21.7%) hybrid patients versus three (5.6%) PVI patients (p = 0.034). On the other hand, combined (major/minor) improvement was present in 15 (32.6%) hybrid patients versus nine (16.9%) patients in the PVI group (p = 0.099)., Conclusion: Hybrid ablation, a combination of thoracoscopic and percutaneous ablation, is associated with a higher risk of cognitive decline compared to sole percutaneous ablation., (© 2022 Wiley Periodicals LLC.)

Introduction: Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified., Methods: In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes., Results: In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro ., Discussion: Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Daniel Lišková, Kosztyu, Kuchař, Černý, Bharadwaj, Petroková, Vroblová, Křupka, Malý, Zosinčuková, Šulc, Rašková Kafková, Raška and Malý.)

S1 nuclease from Aspergillus oryzae is a single-strand-specific nuclease from the S1/P1 family that is utilized in biochemistry and biotechnology. S1 nuclease is active on both RNA and DNA but with differing catalytic efficiencies. This study clarifies its catalytic properties using a thorough comparison of differences in the binding of RNA and DNA in the active site of S1 nuclease based on X-ray structures, including two newly solved complexes of S1 nuclease with the products of RNA cleavage at atomic resolution. Conclusions derived from this comparison are valid for the whole S1/P1 nuclease family. For proper model building and refinement, multiple lattice-translocation defects present in the measured diffraction data needed to be solved. Two different approaches were tested and compared. Correction of the measured intensities proved to be superior to the use of the dislocation model of asymmetric units with partial occupancy of individual chains. As the crystals suffered from multiple lattice translocations, equations for their correction were derived de novo. The presented approach to the correction of multiple lattice-translocation defects may help to solve similar problems in the field of protein X-ray crystallography.

One of the main limitations of laser powder bed fusion technology is the residual stress (RS) introduced into the material by the local heating of the laser beam. RS restricts the processability of some materials and causes shape distortions in the process. Powder bed preheating is a commonly used technique for RS mitigation. Therefore, the objective of this study was to investigate the effect of powder bed preheating in the range of room temperature to 400 °C on RS, macrostructure, microstructure, mechanical properties, and properties of the unfused powder of the nickel-based superalloy Inconel 939. The effect of base plate preheating on RS was determined by an indirect method using deformation of the bridge-shaped specimens. Inconel 939 behaved differently than titanium and aluminum alloys when preheated at high temperatures. Preheating at high temperatures resulted in higher RS, higher 0.2% proof stress and ultimate strength, lower elongation at brake, and higher material hardness. The increased RSs and the change in mechanical properties are attributed to changes in the microstructure. Preheating resulted in a larger melt pool, increased the width of columnar grains, and led to evolution of the carbide phase. The most significant microstructure change was in the increase of the size and occurrence of the carbide phase when higher preheating was applied. Furthermore, it was detected that the evolution of the carbide phase strongly corresponds to the build time when high-temperature preheating is applied. Rapid oxidation of the unfused powder was not detected by EDX or XRD analyses.