Repeat-based holocentromeres influence genome architecture and karyotype evolution.

The centromere represents a single region in most eukaryotic chromosomes. However, several plant and animal lineages assemble holocentromeres along the entire chromosome length. Here, we compare genome organization and evolution as a function of centromere type by assembling chromosome-scale holocentric genomes with repeat-based holocentromeres from three beak-sedge (Rhynchospora pubera, R. breviuscula, and R. tenuis) and their closest monocentric relative, Juncus effusus. We demonstrate that transition to holocentricity affected 3D genome architecture by redefining genomic compartments, while distributing centromere function to thousands of repeat-based centromere units genome-wide. We uncover a complex genome organization in R. pubera that hides its unexpected octoploidy and describe a marked reduction in chromosome number for R. tenuis, which has only two chromosomes. We show that chromosome fusions, facilitated by repeat-based holocentromeres, promoted karyotype evolution and diploidization. Our study thus sheds light on several important aspects of genome architecture and evolution influenced by centromere organization.

The giant diploid faba genome unlocks variation in a global protein crop.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

Assembly of the 81.6 Mb centromere of pea chromosome 6 elucidates the structure and evolution of metapolycentric chromosomes.

Centromeres in the legume genera Pisum and Lathyrus exhibit unique morphological characteristics, including extended primary constrictions and multiple separate domains of centromeric chromatin. These so-called metapolycentromeres resemble an intermediate form between monocentric and holocentric types, and therefore provide a great opportunity for studying the transitions between different types of centromere organizations. However, because of the exceedingly large and highly repetitive nature of metapolycentromeres, highly contiguous assemblies needed for these studies are lacking. Here, we report on the assembly and analysis of a 177.6 Mb region of pea (Pisum sativum) chromosome 6, including the 81.6 Mb centromere region (CEN6) and adjacent chromosome arms. Genes, DNA methylation profiles, and most of the repeats were uniformly distributed within the centromere, and their densities in CEN6 and chromosome arms were similar. The exception was an accumulation of satellite DNA in CEN6, where it formed multiple arrays up to 2 Mb in length. Centromeric chromatin, characterized by the presence of the CENH3 protein, was predominantly associated with arrays of three different satellite repeats; however, five other satellites present in CEN6 lacked CENH3. The presence of CENH3 chromatin was found to determine the spatial distribution of the respective satellites during the cell cycle. Finally, oligo-FISH painting experiments, performed using probes specifically designed to label the genomic regions corresponding to CEN6 in Pisum, Lathyrus, and Vicia species, revealed that metapolycentromeres evolved via the expansion of centromeric chromatin into neighboring chromosomal regions and the accumulation of novel satellite repeats. However, in some of these species, centromere evolution also involved chromosomal translocations and centromere repositioning.

Disruption of the standard kinetochore in holocentric Cuscuta species.

The segregation of chromosomes depends on the centromere. Most species are monocentric, with the centromere restricted to a single region per chromosome. In some organisms, the monocentric organization changed to holocentric, in which the centromere activity is distributed over the entire chromosome length. However, the causes and consequences of this transition are poorly understood. Here, we show that the transition in the genus Cuscuta was associated with dramatic changes in the kinetochore, a protein complex that mediates the attachment of chromosomes to microtubules. We found that in holocentric Cuscuta species, the KNL2 genes were lost; the CENP-C, KNL1, and ZWINT1 genes were truncated; the centromeric localization of CENH3, CENP-C, KNL1, MIS12, and NDC80 proteins was disrupted; and the spindle assembly checkpoint (SAC) degenerated. Our results demonstrate that holocentric Cuscuta species lost the ability to form a standard kinetochore and do not employ SAC to control the attachment of microtubules to chromosomes.

Multivalency of nucleosome recognition by LEDGF.

Eukaryotic transcription is dependent on specific histone modifications. Their recognition by chromatin readers triggers complex processes relying on the coordinated association of transcription regulatory factors. Although various modification states of a particular histone residue often lead to differential outcomes, it is not entirely clear how they are discriminated. Moreover, the contribution of intrinsically disordered regions outside of the specialized reader domains to nucleosome binding remains unexplored. Here, we report the structures of a PWWP domain from transcriptional coactivator LEDGF in complex with the H3K36 di- and trimethylated nucleosome, indicating that both methylation marks are recognized by PWWP in a highly conserved manner. We identify a unique secondary interaction site for the PWWP domain at the interface between the acidic patch and nucleosomal DNA that might contribute to an H3K36-methylation independent role of LEDGF. We reveal DNA interacting motifs in the intrinsically disordered region of LEDGF that discriminate between the intra- or extranucleosomal DNA but remain dynamic in the context of dinucleosomes. The interplay between the LEDGF H3K36-methylation reader and protein binding module mediated by multivalent interactions of the intrinsically disordered linker with chromatin might help direct the elongation machinery to the vicinity of RNA polymerase II, thereby facilitating productive elongation.

Quantifying the Impact of the Peptide Identification Framework on the Results of Fast Photochemical Oxidation of Protein Analysis.

Fast Photochemical Oxidation of Proteins (FPOP) is a promising technique for studying protein structure and dynamics. The quality of insight provided by FPOP depends on the reliability of the determination of the modification site. This study investigates the performance of two search engines, Mascot and PEAKS, for the data processing of FPOP analyses. Comparison of Mascot and PEAKS of the hemoglobin--haptoglobin Bruker timsTOF data set (PXD021621) revealed greater consistency in the Mascot identification of modified peptides, with around 26% of the IDs being mutual for all three replicates, compared to approximately 22% for PEAKS. The intersection between Mascot and PEAKS results revealed a limited number (31%) of shared modified peptides. Principal Component Analysis (PCA) using the peptide-spectrum match (PSM) score, site probability, and peptide intensity was applied to evaluate the results, and the analyses revealed distinct clusters of modified peptides. Mascot showed the ability to assess confident site determination, even with lower PSM scores. However, high PSM scores from PEAKS did not guarantee a reliable determination of the modification site. Fragmentation coverage of the modification position played a crucial role in Mascot assignments, while the AScore localizations from PEAKS often become ambiguous because the software employs MS/MS merging.

Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis.

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

Data-Independent Acquisition Represents a Promising Alternative for Fast Photochemical Oxidation of Proteins (FPOP) Samples Analysis.

Fast Photochemical Oxidation of Proteins (FPOP) is a protein footprinting method utilizing hydroxyl radicals to provide valuable information on the solvent-accessible surface area. The extensive number of oxidative modifications that are created by FPOP is both advantageous, leading to great spatial resolution, and challenging, increasing the complexity of data processing. The precise localization of the modification together with the appropriate reproducibility is crucial to obtain relevant structural information. In this paper, we propose a novel approach combining validated spectral libraries together with utilizing DIA data. First, the DDA data searched by FragPipe are subsequently validated using Skyline software to form a spectral library. This library is then matched against the DIA data to filter out nonrepresentative IDs. In comparison with FPOP data processing using only a search engine followed by generally applied filtration steps, the manually validated spectral library offers higher confidence in identifications and increased spatial resolution. Furthermore, the reproducibility of quantification was compared for DIA, DDA, and MS-only acquisition modes on timsTOF SCP. Comparison of coefficients of variation (CV) showed that the DIA and MS acquisition modes exhibit significantly better reproducibility in quantification (CV medians 0.1233 and 0.1494, respectively) compared to the DDA mode (CV median 0.2104).

Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting.

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

Does modulation of tau hyperphosphorylation represent a reasonable therapeutic strategy for Alzheimer's disease? From preclinical studies to the clinical trials.

Protein kinases (PKs) have emerged as one of the most intensively investigated drug targets in current pharmacological research, with indications ranging from oncology to neurodegeneration. Tau protein hyperphosphorylation was the first pathological post-translational modification of tau protein described in Alzheimer's disease (AD), highlighting the role of PKs in neurodegeneration. The therapeutic potential of protein kinase inhibitors (PKIs)) and protein phosphatase 2 A (PP2A) activators in AD has recently been explored in several preclinical and clinical studies with variable outcomes. Where a number of preclinical studies demonstrate a visible reduction in the levels of phospho-tau in transgenic tauopathy models, no reduction in neurofibrillary lesions is observed. Amongst the few PKIs and PP2A activators that progressed to clinical trials, most failed on the efficacy front, with only a few still unconfirmed and potential positive trends. This suggests that robust preclinical and clinical data is needed to unequivocally evaluate their efficacy. To this end, we take a systematic look at the results of preclinical and clinical studies of PKIs and PP2A activators, and the evidence they provide regarding the utility of this approach to evaluate the potential of targeting tau hyperphosphorylation as a disease modifying therapy.

Revealing the nuclearity of iron citrate complexes at biologically relevant conditions.

Citric acid plays an ubiquitous role in the complexation of essential metals like iron and thus it has a key function making them biologically available. For this, iron(III) citrate complexes are considered among the most significant coordinated forms of ferric iron that take place in biochemical processes of all living organisms. Although these systems hold great biological relevance, their coordination chemistry has not been fully elucidated yet. The current study aimed to investigate the speciation of iron(III) citrate using Mössbauer and electron paramagnetic resonance spectroscopies. Our aim was to gain insights into the structure and nuclearity of the complexes depending on the pH and iron to citrate ratio. By applying the frozen solution technique, the results obtained directly reflect the iron speciation present in the aqueous solution. At 1:1 iron:citrate molar ratio, polynuclear species prevailed forming most probably a trinuclear structure. In the case of citrate excess, the coexistence of several monoiron species with different coordination environments was confirmed. The stability of the polynuclear complexes was checked in the presence of organic solvents.

Predicting nurses' safety compliance behaviour in a developing economy, using the theory of planned behaviour: A configurational approach.

AIM: The study's main objective was to use a fuzzy set qualitative comparative analysis to identify the configuration of recipes that predict nurses' safety compliance behaviour. DESIGN: A cross-sectional design. METHODS: A survey was used where questionnaires were collected from 285 nurses across four primary healthcare hospitals within the Ashanti Region, Ghana. The data collection happened between June 1 to August 2, 2022. A fuzzy set qualitative comparative analysis was used to identify the recipes of psychological factors that determine nurses' safety compliance behaviour. RESULTS: Results from the study suggest that the necessary configurations that explained nurses' safety compliance behaviour came from the presence of subjective norm, attitude, perceived behavioural control, perceived organizational support and negation of intention. The result highlights the need for safety protocols to be conscious of the interplay between nurses' assessment of self, social clues and perception of management care and support since such psychological factors must be considered concurrently to achieve the optimal safety compliance behaviour among nurses. CONCLUSION: A health and safety protocol that fails to recognize the importance of psychological antecedents on subordinates' safety compliance behaviour could limit the safety policy's usefulness in bringing the appropriate behavioural change in nurses. IMPACT: To date, no study has combined the antecedents of theory planned behaviour with perceived organizational support and cue to action to assess how they collectively predict nurses' safety compliance behaviour. Findings from the study suggest that nurses in primary health facilities inform their safety compliance behaviour by assessing self-capabilities, social signals from superiors and colleagues and perception of management support. Hospital administrators and nursing managers in sub-Saharan Africa may rely on these psychological forces to persuade nurses to develop positive safety compliance behaviour at the health facility. PATIENT OR PUBLIC CONTRIBUTION: No Patient or Public Contribution.

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

In Vitro Evolution Reveals Noncationic Protein-RNA Interaction Mediated by Metal Ions.

RNA-peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA-protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal uL11 C-terminal domain was selected from an approximately 1010 library of partially randomized sequences, all composed of ten prebiotically plausible canonical amino acids. The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with molecular dynamics simulations uncovers qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant uL11 domain uses ion bridging (K+/Mg2+) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein-RNA interactions evolved, where the lack of aromatic/basic residues may have been compensated by acidic residues plus metal ions.

Top-Down Proteoform Analysis by 2D MS with Quadrupolar Detection.

Two-dimensional mass spectrometry (2D MS) is a multiplexed tandem mass spectrometry method that does not rely on ion isolation to correlate the precursor and fragment ions. On a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), 2D MS instead uses the modulation of precursor ion radii inside the ICR cell before fragmentation and yields 2D mass spectra that show the fragmentation patterns of all the analytes. In this study, we perform 2D MS for the first time with quadrupolar detection in a dynamically harmonized ICR cell. We discuss the advantages of quadrupolar detection in 2D MS and how we adapted existing data processing techniques for accurate frequency-to-mass conversion. We apply 2D MS with quadrupolar detection to the top-down analysis of covalently labeled ubiquitin with ECD fragmentation, and we develop a workflow for label-free relative quantification of biomolecule isoforms in 2D MS.

The rapid detection of procalcitonin in septic serum using immunoaffinity MALDI chips.

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

Early-onset pulmonary and cutaneous vasculitis driven by constitutively active SRC-family kinase HCK.

BACKGROUND: Inborn errors of immunity are genetic disorders characterized by various degrees of immune dysregulation that can manifest as immune deficiency, autoimmunity, or autoinflammation. The routine use of next-generation sequencing in the clinic has facilitated the identification of an ever-increasing number of inborn errors of immunity, revealing the roles of immunologically important genes in human pathologies. However, despite this progress, treatment is still extremely challenging. OBJECTIVE: We sought to report a new monogenic autoinflammatory disorder caused by a de novo activating mutation, p.Tyr515∗, in hematopoietic cell kinase (HCK). The disease is characterized by cutaneous vasculitis and chronic pulmonary inflammation that progresses to fibrosis. METHODS: Whole-exome sequencing, Sanger sequencing, mass spectrometry, and western blotting were performed to identify and characterize the pathogenic HCK mutation. Dysregulation of mutant HCK was confirmed ex vivo in primary cells and in vitro in transduced cell lines. RESULTS: Mutant HCK lacking the C-terminal inhibitory tyrosine Tyr522 exhibited increased kinase activity and enhanced myeloid cell priming, migration and effector functions, such as production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α, and production of reactive oxygen species. These aberrant functions were reflected by inflammatory leukocyte infiltration of the lungs and skin. Moreover, an overview of the clinical course of the disease, including therapies, provides evidence for the therapeutic efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in inflammatory lung disease. CONCLUSIONS: We propose HCK-driven pulmonary and cutaneous vasculitis as a novel autoinflammatory disorder of inborn errors of immunity.

Safety and Performance of Narhinel 0.9% Sodium Chloride Monodose and Otrisal 0.74% Sodium Chloride Monodose Nasal Saline Solutions and Nasal Aspirators in Real-World Settings: Postmarket Clinical Follow-up Study Results.

Background: Blocked or stuffy nose is a common and bothersome symptom of colds, particularly for young children who are unable to clear their noses on their own. Nasal saline solutions and nasal aspirators are designed to gently cleanse and remove blocking nasal secretions. Objective: To assess the safety and performance of 2 monodose isotonic saline solutions (Narhinel 0.9% and Otrisal 0.74% sodium chloride; GSK Consumer Healthcare SARL, a Haleon company, Nyon, Switzerland) and 2 nasal aspirators with disposable hard- and soft-nozzle refills used as a standalone or combination treatment. Methods: We conducted 2 observational, online questionnaire-based, postmarket clinical follow-up studies in Europeans who had used any of the devices ≥1 time in the past 6 months. Coprimary objectives were to confirm the safety and performance of the saline solutions (Narhinel and Otrisal, Study 1) and nasal aspirators (with hard- and soft-nozzle refills, Study 2). Safety was assessed via the proportion of patients reporting adverse events and/or device malfunctions while using the devices within the previous 6 months, and performance was assessed by satisfaction rated on a 5-point scale, with "satisfied" and "very satisfied" being the highest performance ratings. Results: A total of 1136 (Study 1) and 1237 (Study 2) questionnaires were initiated by volunteer participants. Less than 2% of participants reported adverse events for any evaluated product in the previous 6 months. Most participants were "satisfied" or "very satisfied" with the devices for their intended use, with 78% to 91% of participants in the Narhinel arm, 73%-94% in the Otrisal arm, 71% to 95% in the soft-nozzle arm, and 71% to 80% in the hard-nozzle arm giving these ratings. Conclusions: These data support the safety and performance of 2 monodose saline solutions (Narhinel and Otrisal) for nasal cleansing, nasal moisturization, and/or loosening nasal secretions, and of nasal aspirators (with hard- and soft-nozzle refills) for clearing a blocked nose and removing nasal secretions.

[A case of botulism in the Czech Republic and current possibilities for detecting the neurotoxin produced by Clostridium botulinum].

In the Czech Republic, botulism is a rare life-threatening disease. A total of 155 cases have been reported since 1960; according to the ISIN (formerly EPIDAT) database, there have been only three isolated cases since 2013, with the exception of a single occurrence of familial botulism in 2013. In our work, we present the occurrence of botulism after ingestion of pâté of untraceable origin by a couple who were hospitalized for botulotoxin food poisoning in July 2022. Their neurological symptoms were dominated by dysarthria. After administration of antibotulinum serum, their condition improved significantly. Patient samples were analyzed using affinity carriers and MALDI mass spectrometry, a modern highly sensitive technique for detecting the presence of botulinum neurotoxins. Unlike traditional detection by a difficult and costly biological experiment on mice, the above analysis does not require the killing of laboratory animals.