Two-dimensional high-throughput on-cell screening of immunoglobulins against broad antigen repertoires.

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.

Dynamic interchange between two protonation states is characteristic of active sites of cholinesterases.

Cholinesterases are well-known and widely studied enzymes crucial to human health and involved in neurology, Alzheimer's, and lipid metabolism. The protonation pattern of active sites of cholinesterases influences all the chemical processes within, including reaction, covalent inhibition by nerve agents, and reactivation. Despite its significance, our comprehension of the fine structure of cholinesterases remains limited. In this study, we employed enhanced-sampling quantum-mechanical/molecular-mechanical calculations to show that cholinesterases predominantly operate as dynamic mixtures of two protonation states. The proton transfer between two non-catalytic glutamate residues follows the Grotthuss mechanism facilitated by a mediator water molecule. We show that this uncovered complexity of active sites presents a challenge for classical molecular dynamics simulations and calls for special treatment. The calculated proton transfer barrier of 1.65 kcal/mol initiates a discussion on the potential existence of two coupled low-barrier hydrogen bonds in the inhibited form of butyrylcholinesterase. These findings expand our understanding of structural features expressed by highly evolved enzymes and guide future advances in cholinesterase-related protein and drug design studies.

Air-Stable Arene Manganese Complexes as Catalysts for the Syngas-Assisted Direct Reductive Amination, Cyanation of Aldehyde, and CO2 Fixation by Epoxide with High Functional Groups Tolerance.

Manganese complexes [(arene)Mn(CO)3]+ were prepared in one step from arenes and Mn(CO)5Br. They were found to be efficient catalysts in the carbonyl cyanation with TMSCN, CO2 fixation by epoxides, and direct reductive amination in the presence of syngas. The amination reaction tolerated various reducible functional groups. The synergy of carbon monoxide and hydrogen in syngas provides high efficiency of the catalytic system. The developed protocols do not require an inert atmosphere, and the catalysts can be handled in air.

Plasticity of Response Properties of Mouse Visual Cortex Neurons Induced by Optogenetic Tetanization In Vivo.

Heterosynaptic plasticity, along with Hebbian homosynaptic plasticity, is an important mechanism ensuring the stable operation of learning neuronal networks. However, whether heterosynaptic plasticity occurs in the whole brain in vivo, and what role(s) in brain function in vivo it could play, remains unclear. Here, we used an optogenetics approach to apply a model of intracellular tetanization, which was established and employed to study heterosynaptic plasticity in brain slices, to study the plasticity of response properties of neurons in the mouse visual cortex in vivo. We show that optogenetically evoked high-frequency bursts of action potentials (optogenetic tetanization) in the principal neurons of the visual cortex induce long-term changes in the responses to visual stimuli. Optogenetic tetanization had distinct effects on responses to different stimuli, as follows: responses to optimal and orthogonal orientations decreased, responses to null direction did not change, and responses to oblique orientations increased. As a result, direction selectivity of the neurons decreased and orientation tuning became broader. Since optogenetic tetanization was a postsynaptic protocol, applied in the absence of sensory stimulation, and, thus, without association of presynaptic activity with bursts of action potentials, the observed changes were mediated by mechanisms of heterosynaptic plasticity. We conclude that heterosynaptic plasticity can be induced in vivo and propose that it may play important homeostatic roles in operation of neural networks by helping to prevent runaway dynamics of responses to visual stimuli and to keep the tuning of neuronal responses within the range optimized for the encoding of multiple features in population activity.

Gausemycin Antibiotic Family Acts via Ca2+-Dependent Membrane Targeting.

We report the molecular mechanism of action of gausemycins and the isolation of new members of the family, gausemycins C (1c), D (1d), E (1e), and F (1f), the minor components of the mixture. To elucidate the mechanism of action of gausemycins, we investigated the antimicrobial activity of the most active compounds, gausemycins A and B, in the presence of Ca2+, other metal ions, and phosphate. Gausemycins require a significantly higher Ca2+ concentration for maximum activity than daptomycin but lower than that required for malacidine and cadasides. Species-specific antimicrobial activity was found upon testing against a wide panel of Gram-positive bacteria. Membranoactivity of gausemycins was demonstrated upon their interactions with model lipid bilayers and micelles. The pore-forming ability was found to be dramatically dependent on the Ca2+ concentration and the membrane lipid composition. An NMR study of gausemycin B in zwitterionic and anionic micelles suggested the putative structure of the gausemycin/membrane complex and revealed the binding of Ca2+ by the macrocyclic domain of the antibiotic.

Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

Whole tumor analysis reveals early origin of the TERT promoter mutation and intercellular heterogeneity in TERT expression.

BACKGROUND: The TERT promoter mutation (TPM) is acquired in most IDH-wildtype glioblastomas (GBM) and IDH-mutant oligodendrogliomas (OD) enabling tumor cell immortality. Previous studies on TPM clonality show conflicting results. This study was performed to determine whether TPM is clonal on a tumor-wide scale. METHODS: We investigated TPM clonality in relation to presumed early events in 19 IDH-wildtype GBM and 10 IDH-mutant OD using 3-dimensional comprehensive tumor sampling. We performed Sanger sequencing on 264 tumor samples and deep amplicon sequencing on 187 tumor samples. We obtained tumor purity and copy number estimates from whole exome sequencing. TERT expression was assessed by RNA-seq and RNAscope. RESULTS: We detected TPM in 100% of tumor samples with quantifiable tumor purity (219 samples). Variant allele frequencies (VAF) of TPM correlate positively with chromosome 10 loss in GBM (R = 0.85), IDH1 mutation in OD (R = 0.87), and with tumor purity (R = 0.91 for GBM; R = 0.90 for OD). In comparison, oncogene amplification was tumor-wide for MDM4- and most EGFR-amplified cases but heterogeneous for MYCN and PDGFRA, and strikingly high in low-purity samples. TPM VAF was moderately correlated with TERT expression (R = 0.52 for GBM; R = 0.65 for OD). TERT expression was detected in a subset of cells, solely in TPM-positive samples, including samples equivocal for tumor. CONCLUSIONS: On a tumor-wide scale, TPM is among the earliest events in glioma evolution. Intercellular heterogeneity of TERT expression, however, suggests dynamic regulation during tumor growth. TERT expression may be a tumor cell-specific biomarker.

Toxic comments are associated with reduced activity of volunteer editors on Wikipedia.

Wikipedia is one of the most successful collaborative projects in history. It is the largest encyclopedia ever created, with millions of users worldwide relying on it as the first source of information as well as for fact-checking and in-depth research. As Wikipedia relies solely on the efforts of its volunteer editors, its success might be particularly affected by toxic speech. In this paper, we analyze all 57 million comments made on user talk pages of 8.5 million editors across the six most active language editions of Wikipedia to study the potential impact of toxicity on editors' behavior. We find that toxic comments are consistently associated with reduced activity of editors, equivalent to 0.5-2 active days per user in the short term. This translates to multiple human-years of lost productivity, considering the number of active contributors to Wikipedia. The effects of toxic comments are potentially even greater in the long term, as they are associated with a significantly increased risk of editors leaving the project altogether. Using an agent-based model, we demonstrate that toxicity attacks on Wikipedia have the potential to impede the progress of the entire project. Our results underscore the importance of mitigating toxic speech on collaborative platforms such as Wikipedia to ensure their continued success.

Bioengineering the Antimicrobial Activity of Yeast by Recombinant Thanatin Production.

The global spread of antibiotic resistance marks the end of the era of conventional antibiotics. Mankind desires new molecular tools to fight pathogenic bacteria. In this regard, the development of new antimicrobials based on antimicrobial peptides (AMPs) is again of particular interest. AMPs have various mechanisms of action on bacterial cells. Moreover, AMPs have been reported to be efficient in preclinical studies, demonstrating a low level of resistance formation. Thanatin is a small, beta-hairpin antimicrobial peptide with a bacterial-specific mode of action, predetermining its low cytotoxicity toward eukaryotic cells. This makes thanatin an exceptional candidate for new antibiotic development. Here, a microorganism was bioengineered to produce an antimicrobial agent, providing novel opportunities in antibiotic research through the directed creation of biocontrol agents. The constitutive heterologous production of recombinant thanatin (rThan) in the yeast Pichia pastoris endows the latter with antibacterial properties. Optimized expression and purification conditions enable a high production level, yielding up to 20 mg/L of rThan from the culture medium. rThan shows a wide spectrum of activity against pathogenic bacteria, similarly to its chemically synthesized analogue. The designed approach provides new avenues for AMP engineering and creating live biocontrol agents to fight antibiotic resistance.

Cross-regulation of antibody responses against the SARS-CoV-2 Spike protein and commensal microbiota via molecular mimicry.

The commensal microflora provides a repertoire of antigens that illicit mucosal antibodies. In some cases, these antibodies can cross-react with host proteins, inducing autoimmunity, or with other microbial antigens. We demonstrate that the oral microbiota can induce salivary anti-SARS-CoV-2 Spike IgG antibodies via molecular mimicry. Anti-Spike IgG antibodies in the saliva correlated with enhanced abundance of Streptococcus salivarius 1 month after anti-SARS-CoV-2 vaccination. Several human commensal bacteria, including S. salivarius, were recognized by SARS-CoV-2-neutralizing monoclonal antibodies and induced cross-reactive anti-Spike antibodies in mice, facilitating SARS-CoV-2 clearance. A specific S. salivarius protein, RSSL-01370, contains regions with homology to the Spike receptor-binding domain, and immunization of mice with RSSL-01370 elicited anti-Spike IgG antibodies in the serum. Additionally, oral S. salivarius supplementation enhanced salivary anti-Spike antibodies in vaccinated individuals. Altogether, these data show that distinct species of the human microbiota can express molecular mimics of SARS-CoV-2 Spike protein, potentially enhancing protective immunity.

Comparative Study of the Relationship between Microstructure and Mechanical Properties of Aluminum Alloy 5056 Fabricated by Additive Manufacturing and Rolling Techniques.

In recent years, additive manufacturing of products made from 5000 series alloys has grown in popularity for marine and automotive applications. At the same time, little research has been aimed at determining the permissible load ranges and areas of application, especially in comparison with materials obtained by traditional methods. In this work, we compared the mechanical properties of aluminum alloy 5056 produced by wire-arc additive technology and rolling. Structural analysis of the material was carried out using EBSD and EDX. Tensile tests under quasi-static loading and impact toughness tests under impact loading were also carried out. SEM was used to examine the fracture surface of the materials during these tests. The mechanical properties of the materials under quasi-static loading conditions exhibit a striking similarity. Specifically, the yield stress σ0.2 was measured at 128 MPa for the industrially manufactured AA5056_IM and 111 MPa for the AA5056_AM. In contrast, impact toughness tests showed that AA5056_AM KCVfull was 190 kJ/m2, half that of AA5056_IM KCVfull, which was 395 kJ/m2.

Doxorubicin-Loaded Polyelectrolyte Multilayer Capsules Modified with Antitumor DR5-Specific TRAIL Variant for Targeted Drug Delivery to Tumor Cells.

Recently, biodegradable polyelectrolyte multilayer capsules (PMC) have been proposed for anticancer drug delivery. In many cases, microencapsulation allows to concentrate the substance locally and prolong its flow to the cells. To reduce systemic toxicity when delivering highly toxic drugs, such as doxorubicin (DOX), the development of a combined delivery system is of paramount importance. Many efforts have been made to exploit the DR5-dependent apoptosis induction for cancer treatment. However, despite having a high antitumor efficacy of the targeted tumor-specific DR5-B ligand, a DR5-specific TRAIL variant, its fast elimination from a body limits its potential use in a clinic. A combination of an antitumor effect of the DR5-B protein with DOX loaded in the capsules could allow to design a novel targeted drug delivery system. The aim of the study was to fabricate PMC loaded with a subtoxic concentration of DOX and functionalized with the DR5-B ligand and to evaluate a combined antitumor effect of this targeted drug delivery system in vitro. In this study, the effects of PMC surface modification with the DR5-B ligand on cell uptake both in 2D (monolayer culture) and 3D (tumor spheroids) were studied by confocal microscopy, flow cytometry and fluorimetry. Cytotoxicity of the capsules was evaluated using an MTT test. The capsules loaded with DOX and modified with DR5-B demonstrated synergistically enhanced cytotoxicity in both in vitro models. Thus, the use of the DR5-B-modified capsules loaded with DOX at a subtoxic concentration could provide both targeted drug delivery and a synergistic antitumor effect.

Increasing the Thermal Stability and High-Temperature Strength of Vanadium Alloys by Strengthening with Nanosized Non-Metallic Particles.

Using the methods of scanning and transmission electron microscopy, the features of the structural-phase state of a vanadium alloy of the V-Cr-Ta-Zr system after a combined treatment, which consisted in cyclic alternation of thermomechanical and chemical-heat treatments, were studied. The values of yield strength and ductility of the V-Cr-Ta-Zr alloy were determined, depending on the stabilization and test temperatures. It was established that, after the combined treatment, the structural-phase state of the V-Cr-Ta-Zr alloy was composite, in which the joint implementation of dispersion and substructural strengthening ensured the formation of a gradient grain structure with a polygonal state, the elements of which were fixed by nanosized ZrO2 particles characterized by a high thermal stability. Such modification of the microstructure was accompanied by an increase in the high-temperature strength and a shift in the upper limit of the temperature stability interval towards high temperatures, of up to 900 °C. It was assumed that the polygonal state inside the grains contributed to the implementation of cooperative mechanisms of the dislocation-disclination type, which ensured the accommodation of the material in the "high-strength state" under loading.

Mixed Electronic-Ionic Conductivity and Stability of Spark Plasma Sintered Graphene-Augmented Alumina Nanofibres Doped Yttria Stabilized Zirconia GAlN/YSZ Composites.

Graphene-doped ceramic composites with mixed electronic-ionic conductivity are currently attracting attention for their application in electrochemical devices, in particular membranes for solid electrolyte fuel cells with no necessity to use the current collector. In this work, composites of the Y2O3-ZrO2 matrix with graphene-augmented γ-Al2O3 nanofibres (GAlN) were spark plasma sintered. The conductivity and electrical stability in cyclic experiments were tested using electrical impedance spectroscopy. Composites with 0.5 and 1 wt.% GAlN show high ionic conductivity of 10-2-10-3 S/cm at 773 K. Around 3 wt.% GAlN percolation threshold was achieved and a gradual increase of electronic conductivity from ~10-2 to 4 × 10-2 S/cm with an activation energy of 0.2 eV was observed from 298 to 773 K while ionic conductivity was maintained at elevated temperatures. The investigation of the evolution of conductivity was performed at 298-973 K. Besides, the composites with 1-3 wt.% of GAlN addition show a remarkable hardness of 14.9-15.8 GPa due to ZrC formation on the surfaces of the materials.

Paired optogenetic and visual stimulation can change the orientation selectivity of visual cortex neurons.

Synaptic plasticity is currently considered the main mechanism underlying the plastic modification of neural networks. The vast majority of studies of synaptic plasticity are carried out on reduced preparations, but the situation in vivo is fundamentally different from that in vitro. In this work, we used the Hebbian paradigm, which is known to induce long-term changes in synaptic strength in vitro, to manipulate the properties of a single pyramidal neuron in the mouse visual cortex. We have shown that optogenetic stimulation of a ChR2-expressing pyramidal neuron in the primary visual cortex of Thy-ChR2 mice paired with the presentation of a visual stimulus of non-optimal orientation induces long-term changes in the properties of the receptive field, manifested in alteration of the orientation selectivity of the cell. Non-paired stimulation did not lead to changes in the properties of the receptive field of the neuron during the experiment. Thus, we have demonstrated the role of associative plasticity in the dynamic organization of the receptive fields of neurons in the visual cortex.

Bidirectional regulation by "star forces": Ionotropic astrocyte's optical stimulation suppresses synaptic plasticity, metabotropic one strikes back.

The role of astrocytes in modulating synaptic plasticity is an important question that until recently was not addressed due to limitations of previously existing technology. In the present study, we took an advantage of optogenetics to specifically activate astrocytes in hippocampal slices in order to study effects on synaptic function. Using the AAV-based delivery strategy, we expressed the ionotropic channelrhodopsin-2 (ChR2) or the metabotropic Gq-coupled Opto-a1AR opsins specifically in hippocampal astrocytes to compare different modalities of astrocyte activation. In electrophysiological experiments, we observed a depression of basal field excitatory postsynaptic potentials (fEPSPs) in the CA1 hippocampal layer following light stimulation of astrocytic ChR2. The ChR2-mediated depression increased under simultaneous light and electrical theta-burst stimulation (TBS). Application of the type 2 purinergic receptor antagonist suramin prevented depression of basal synaptic transmission, and switched the ChR2-dependent depression into potentiation. The GABAB receptor antagonist, phaclofen, did not prevent the depression of basal fEPSPs, but switched the ChR2-dependent depression into potentiation comparable to the values for TBS in control slices. In contrast, light stimulation of Opto-a1AR expressed in astrocytes led to an increase in basal fEPSPs, as well as a potentiation of synaptic responses to TBS significantly. A specific blocker of the Gq protein downstream target, the phospholipase C, U73122, completely prevented the effects of Opto-a1AR stimulation on basal fEPSPs or Opto + TBS responses. To understand molecular basis for the observed effects, we performed an analysis of gene expression in these slices using quantitative PCR approach. We observed a significant upregulation of "immediate-early" gene expression in hippocampal slices after light activation of Opto-a1AR-expressing astrocytes alone (cRel, Arc, Fos, JunB, and Egr1) or paired with TBS (cRel, Fos, and Egr1). Activation of ChR2-expressing hippocampal astrocytes was insufficient to affect expression of these genes in our experimental conditions. Thus, we concluded that optostimulation of astrocytes with ChR2 and Opto-a1AR optogenetic tools enables bidirectional modulation of synaptic plasticity and gene expression in hippocampus.

Animal Microbiomes as a Source of Novel Antibiotic-Producing Strains.

Natural compounds continue to serve as the most fruitful source of new antimicrobials. Analysis of bacterial genomes have revealed that the biosynthetic potential of antibiotic producers by far exceeds the number of already discovered structures. However, due to the repeated discovery of known substances, it has become necessary to change both approaches to the search for antibiotics and the sources of producer strains. The pressure of natural selection and the diversity of interactions in symbiotic communities make animal microbiomes promising sources of novel substances. Here, microorganisms associated with various animals were examined in terms of their antimicrobial agents. The application of alternative cultivation techniques, ultrahigh-throughput screening, and genomic analysis facilitated the investigation of compounds produced by unique representatives of the animal microbiota. We believe that new strategies of antipathogen defense will be discovered by precisely studying cell-cell and host-microbe interactions in microbiomes in the wild.

Conformational Dynamics of the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

Variants of SARS-CoV-2 keep emerging and causing new waves of COVID-19 around the world. Effective new approaches in drug development are based on the binding of agents, such as neutralizing monoclonal antibodies to a receptor-binding domain (RBD) of SARS-CoV-2 spike protein. However, mutations in RBD may lower the affinity of previously developed antibodies. Therefore, rapid analysis of new variants and selection of a binding partner with high affinity is of great therapeutic importance. Here, we explore a computational approach based on molecular dynamics simulations and conformational clusterization techniques for the wild-type and omicron variants of RBD. Biochemical experiments support the hypothesis of the presence of several conformational states within the RBD assembly. The development of such an approach will facilitate the selection of neutralization drugs with higher affinity based on the primary structure of the target antigen.

Unique Structural Fold of LonBA Protease from Bacillus subtilis, a Member of a Newly Identified Subfamily of Lon Proteases.

ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as Bacillus subtilis LonB, combined with the modeled structure of its ATPase domain. Several structural features present in both domains differ from their counterparts in either LonA or LonB subfamilies. We thus postulate that this enzyme is the founding member of a newly identified LonBA subfamily, so far found only in the gene sequences of firmicutes.

Highly Similar Sequences of Mature IgA1 Proteases from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae.

The mature serine-type IgA1 protease from Neisseria meningitidis serogroup B strain H44/76 (IgA1pr1_28-1004) is considered here as the basis for creating a candidate vaccine against meningococcal meningitis. In this work, we examine the primary structure similarity of IgA1 proteases from various strains of a number of Gram-negative bacteria (N. meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae) in order to find a structural groundwork for creating a broad-spectrum vaccine based on fragments of this enzyme. BLAST has shown high similarity between the primary structure of IgA1pr1_28-1004 and hypothetical sequences of mature IgA1 proteases from N. meningitidis (in 1060 out of 1061 examined strains), N. gonorrhoeae (in all 602 examined strains) and H. influenzae (in no less than 137 out of 521 examined strains). For these enzymes, common regions of sequence correspond to IgA1pr1_28-1004 fragments 28-84, 146-193, 253-539, 567-628, 639-795 and 811-1004, with identity of at least 85%. We believe that these fragments can be used in the development of a vaccine to prevent diseases caused by pathogenic strains of N. meningitidis and N. gonorrhoeae as well as a significant number of strains of H. influenzae.