Small protein blockers of human IL-6 receptor alpha inhibit proliferation and migration of cancer cells.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

A First Look at the Relationship Between Large Herbivore-Induced Landscape Modifications and Ixodes ricinus Tick Abundance in Rewilding Sites.

Background: While the influence of landscape and microclimatic conditions on tick populations is well-documented, there remains a gap in more specific data regarding their relationship to rewilding efforts with large herbivore activity. Objective: This pilot study, spanning from 2019 to 2021, explores the effects of naturalistic grazing by large semi-wild ungulates on tick abundance in the Milovice Reserve, Czechia. Methods: Tick collection was observed using flagging techniques at two distinct sites of rewilding area: one grazed, actively utilized by animals involved in the rewilding project, and one ungrazed, left fallow in neighboring areas utilized only by wild animals. Transects, each measuring 150 m in length and 5 m in width (750 m2), were established at these two sampling locations from March to September between 2019 and 2021. To minimize potential bias resulting from tick movement, a 300 m buffer zone separated the two sites. Data analysis employed a generalized estimating equations (GEE) model with negative binomial regression. The study assessed potential variations in tick abundance between selected transects, considering factors such as plant cover seasonality, temperature, and humidity. Results: During the collection periods, we gathered 586 live ticks, with 20% found in grazed areas and 80% in ungrazed areas. Notably, tick abundance was significantly higher in ungrazed areas. Peaks in tick abundance occurred in both grazed and ungrazed areas during spring, particularly in April. However, tick numbers declined more rapidly in grazed areas. Microclimatic variables like temperature and humidity did not significantly impact tick abundance compared to landscape management and seasonal factors. Conclusion: Rewilding efforts, particularly natural grazing by large ungulates, influence tick abundance and distribution. This study provides empirical data on tick ecology in rewilded areas, highlighting the importance of landscape management and environmental factors in tick management and conservation. Trophic rewilding plays a crucial role in shaping ecosystems and tick population dynamics in transformed landscapes.

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging.

BACKGROUND: Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. METHODS: We designed a 13 kDa ß-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. RESULTS: Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. CONCLUSIONS: Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.

Genetic background of adaptation of Crimean-Congo haemorrhagic fever virus to the different tick hosts.

Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a negative-sense, single-stranded RNA virus with a segmented genome and the causative agent of a severe Crimean-Congo haemorrhagic fever (CCHF) disease. The virus is transmitted mainly by tick species in Hyalomma genus but other ticks such as representatives of genera Dermacentor and Rhipicephalus may also be involved in virus life cycle. To improve our understanding of CCHFV adaptation to its tick species, we compared nucleotide composition and codon usage patterns among the all CCHFV strains i) which sequences and other metadata as locality of collection and date of isolation are available in GenBank and ii) which were isolated from in-field collected tick species. These criteria fulfilled 70 sequences (24 coding for S, 23 for M, and 23 for L segment) of virus isolates originating from different representatives of Hyalomma and Rhipicephalus genera. Phylogenetic analyses confirmed that Hyalomma- and Rhipicephalus-originating CCHFV isolates belong to phylogenetically distinct CCHFV clades. Analyses of nucleotide composition among the Hyalomma- and Rhipicephalus-originating CCHFV isolates also showed significant differences, mainly in nucleotides located at the 3rd codon positions indicating changes in codon usage among these lineages. Analyses of codon adaptation index (CAI), effective number of codons (ENC), and other codon usage statistics revealed significant differences between Hyalomma- and Rhipicephalus-isolated CCHFV strains. Despite both sets of strains displayed a higher adaptation to use codons that are preferred by Hyalomma ticks than Rhipicephalus ticks, there were distinct codon usage preferences observed between the two tick species. These findings suggest that over the course of its long co-evolution with tick vectors, CCHFV has optimized its codon usage to efficiently utilize translational resources of Hyalomma species.

Outcomes of the EMDataResource Cryo-EM Ligand Modeling Challenge.

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein/nucleic-acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: E. coli beta-galactosidase with inhibitor, SARS-CoV-2 RNA-dependent RNA polymerase with covalently bound nucleotide analog, and SARS-CoV-2 ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. We found that (1) the quality of submitted ligand models and surrounding atoms varied, as judged by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics, and contact scores, and (2) a composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

Structural, Biochemical, and Computational Characterization of Sulfamides as Bimetallic Peptidase Inhibitors.

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

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.

Disease-associated nonsense and frame-shift variants resulting in the truncation of the GluN2A or GluN2B C-terminal domain decrease NMDAR surface expression and reduce potentiating effects of neurosteroids.

N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

Are kuravirus capsid diameters quantized? The first all-atom genome tracing method for double-stranded DNA viruses.

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.

Mapping the potential distribution of the principal vector of Crimean-Congo haemorrhagic fever virus Hyalomma marginatum in the Old World.

Crimean-Congo haemorrhagic fever (CCHF) is the most widely distributed tick-borne viral disease in humans and is caused by the Crimean-Congo haemorrhagic fever virus (CCHFV). The virus has a broader distribution, expanding from western China and South Asia to the Middle East, southeast Europe, and Africa. The historical known distribution of the CCHFV vector Hyalomma marginatum in Europe includes most of the Mediterranean and the Balkan countries, Ukraine, and southern Russia. Further expansion of its potential distribution may have occurred in and out of the Mediterranean region. This study updated the distributional map of the principal vector of CCHFV, H. marginatum, in the Old World using an ecological niche modeling approach based on occurrence records from the Global Biodiversity Information Facility (GBIF) and a set of covariates. The model predicted higher suitability of H. marginatum occurrences in diverse regions of Africa and Asia. Furthermore, the model estimated the environmental suitability of H. marginatum across Europe. On a continental scale, the model anticipated a widespread potential distribution encompassing the southern, western, central, and eastern parts of Europe, reaching as far north as the southern regions of Scandinavian countries. The distribution of H. marginatum also covered countries across Central Europe where the species is not autochthonous. All models were statistically robust and performed better than random expectations (p < 0.001). Based on the model results, climatic conditions could hamper the successful overwintering of H. marginatum and their survival as adults in many regions of the Old World. Regular updates of the models are still required to continually assess the areas at risk using up-to-date occurrence and climatic data in present-day and future conditions.

Bulk and single-nucleus RNA sequencing highlight immune pathways induced in individuals during an Ixodes scapularis tick bite.

Ticks are hematophagous arthropods that use a complex mixture of salivary proteins to evade host defenses while taking a blood meal. Little is known about the immunological and physiological consequences of tick feeding on humans. Here, we performed the first bulk and single-nucleus RNA sequencing (snRNA-seq) of skin and blood of four persons presenting with naturally acquired, attached Ixodes scapularis ticks. Pathways and individual genes associated with innate and adaptive immunity were identified based on bulk RNA sequencing, including interleukin-17 signaling and platelet activation pathways at the site of tick attachment or in peripheral blood. snRNA-seq further revealed that the Hippo signaling, cell adhesion, and axon guidance pathways were involved in the response to an I. scapularis bite in humans. Features of the host response in these individuals also overlapped with that of laboratory guinea pigs exposed to I. scapularis and which acquired resistance to ticks. These findings offer novel insights for the development of new biomarkers for I. scapularis exposure and anti-tick vaccines for human use.

When will RNA get its AlphaFold moment?

The protein structure prediction problem has been solved for many types of proteins by AlphaFold. Recently, there has been considerable excitement to build off the success of AlphaFold and predict the 3D structures of RNAs. RNA prediction methods use a variety of techniques, from physics-based to machine learning approaches. We believe that there are challenges preventing the successful development of deep learning-based methods like AlphaFold for RNA in the short term. Broadly speaking, the challenges are the limited number of structures and alignments making data-hungry deep learning methods unlikely to succeed. Additionally, there are several issues with the existing structure and sequence data, as they are often of insufficient quality, highly biased and missing key information. Here, we discuss these challenges in detail and suggest some steps to remedy the situation. We believe that it is possible to create an accurate RNA structure prediction method, but it will require solving several data quality and volume issues, usage of data beyond simple sequence alignments, or the development of new less data-hungry machine learning methods.

First Detection of SARS-CoV-2 in White Rhinoceros during a Small-Scale Coronavirus Surveillance in the Bandia Reserve, Senegal.

The SARS-CoV-2 pandemic has heightened interest in the monitoring and surveillance of coronaviruses in wildlife. Testing for the virus in animals can provide valuable insights into viral reservoirs, transmission, and pathogenesis. In this study, we present the results of the molecular surveillance project focused on coronaviruses in Senegalese wildlife. During the project, we screened fecal samples of the wild animals living in the Bandia Reserve (ten non-human primates, one giraffe, and two white rhinoceros) and the free-living urban population of African four-toed hedgehogs in Ngaparou. The results showed the absence of coronaviruses in hedgehogs, non-human primates, and a giraffe. A single positive sample was obtained from a white rhinoceros. The sequencing results of amplified RdRp gene confirmed that the detected virus was SARS-CoV-2. This study represents the first documented instance of molecular detection of SARS-CoV-2 in white rhinoceros and, therefore, extends our knowledge of possible SARS-CoV-2 hosts.

Assessment and strategy development for SARS-CoV-2 screening in wildlife: A review.

Coronaviruses (members of the Coronaviridae family) are prominent in veterinary medicine, with several known infectious agents commonly reported. In contrast, human medicine has disregarded coronaviruses for an extended period. Within the past two decades, coronaviruses have caused three major outbreaks. One such outbreak was the coronavirus disease 2019 (COVID-19) caused by the coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Over the 3-year COVID-19 outbreak, several instances of zooanthroponosis have been documented, which pose risks for virus modifications and possible re-emergence of the virus into the human population, causing a new epidemic and possible threats for vaccination or treatment failure. Therefore, widespread screening of animals is an essential technique for mitigating future risks and repercussions. However, mass detection of SARS-CoV-2 in wild animals might be challenging. In silico prediction modeling, experimental studies conducted on various animal species, and natural infection episodes recorded in various species might provide information on the potential threats to wildlife. They may be useful for diagnostic and mass screening purposes. In this review, the possible methods of wildlife screening, based on experimental data and environmental elements that might play a crucial role in its effective implementation, are reviewed.

Tetracycline-modifying enzyme SmTetX from Stenotrophomonas maltophilia.

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.

Overview of game meat utilisation challenges and opportunities: A European perspective.

Re-wilding and similar initiatives have resulted in an increase in wildlife suitable for human consumption in Europe. However, game meat production and consumption present several challenges, including infectious diseases which pose risks to livestock, processers, and consumers. This review provides insights into the infectious diseases and toxic contaminants associated with game meat. The effect of killing method on the meat quality is also discussed and means of improving the meat quality of game meat is elucidated. The use of different food safety systems that could be applied to provide safe meat is reported. The importance of collaborative multi-sector approaches is emphasized, to generate and distribute knowledge and implement One Health strategies that ensure the safe, traceable, sustainable, and professional development of commercial game meat supply chains.

Conformation-based refinement of 18-mer DNA structures.

Nine new crystal structures of CG-rich DNA 18-mers with the sequence 5'-GGTGGGGGC-XZ-GCCCCACC-3', which are related to the bacterial repetitive extragenic palindromes, are reported. 18-mer oligonucleotides with the central XZ dinucleotide systematically mutated to all 16 sequences show complex behavior in solution, but all ten so far successfully crystallized 18-mers crystallized as A-form duplexes. The refinement protocol benefited from the recurrent use of geometries of the dinucleotide conformer (NtC) classes as refinement restraints in regions of poor electron density. The restraints are automatically generated at the dnatco.datmos.org web service and are available for download. This NtC-driven protocol significantly helped to stabilize the structure refinement. The NtC-driven refinement protocol can be adapted to other low-resolution data such as cryo-EM maps. To test the quality of the final structural models, a novel validation method based on comparison of the electron density and conformational similarity to the NtC classes was employed.

Postindustrial Landscapes Are Neglected Localities That May Play an Important Role in the Urban Ecology of Ticks and Tick-Borne Diseases-A Pilot Study.

BACKGROUND: Numerous recent studies have shown that ticks and tick-borne pathogens pose a considerable threat in urban areas, such as parks, playgrounds, zoos, cemeteries, etc. Abandoned postindustrial localities, and other types of vague terrain, are other examples of urban wilderness areas that have been absolutely neglected in respect to ticks and tick-borne pathogens thus far, even though they provide ideal biotopes for ticks. METHODS: The abundance of ticks and prevalence of Borrelia burgdorferi sensu lato spirochetes were compared between a city park and an adjacent abandoned construction waste disposal site in Prague, Czechia from June to October 2021. RESULTS: The results showed that ticks and borrelia spirochetes are present at the city park as well as at the abandoned construction waste disposal site, although in lower numbers. DISCUSSION: According to the best of our knowledge, this is the first report describing the presence of ticks and tick-borne pathogens in an urban postindustrial landscape. More detailed studies are needed to uncover the role of these localities in the ecology of ticks and ecoepidemiology of tick-borne diseases in urban areas.

Effects of Pregnanolone Glutamate and Its Metabolites on GABAA and NMDA Receptors and Zebrafish Behavior.

Multiple molecular targets have been identified to mediate membrane-delimited and nongenomic effects of natural and synthetic steroids, but the influence of steroid metabolism on neuroactive steroid signaling is not well understood. To begin to address this question, we set out to identify major metabolites of a neuroprotective synthetic steroid 20-oxo-5ß-pregnan-3α-yl l-glutamyl 1-ester (pregnanolone glutamate, PAG) and characterize their effects on GABAA and NMDA receptors (GABARs, NMDARs) and their influence on zebrafish behavior. Gas chromatography-mass spectrometry was used to assess concentrations of PAG and its metabolites in the hippocampal tissue of juvenile rats following intraperitoneal PAG injection. PAG is metabolized in the peripheral organs and nervous tissue to 20-oxo-17α-hydroxy-5ß-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5ß-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5ß-pregnan-20-one (17-hydroxypregnanolone, 17-OH-PA). Patch-clamp electrophysiology experiments in cultured hippocampal neurons demonstrate that PA and 17-OH-PA are potent positive modulators of GABARs, while PAG and 17-OH-PA have a moderate inhibitory effect at NMDARs. PAG, 17-OH-PA, and PA diminished the locomotor activity of zebrafish larvae in a dose-dependent manner. Our results show that PAG and its metabolites are potent modulators of neurotransmitter receptors with behavioral consequences and indicate that neurosteroid-based ligands may have therapeutic potential.

The Epidemiology of Infectious Diseases Meets AI: A Match Made in Heaven.

Infectious diseases remain a major threat to public health [...].