Creation of Recombinant Biocontrol Agents by Genetic Programming of Yeast.

Bacterial infections caused by antibiotic-resistant pathogens pose an extremely serious and elusive problem in healthcare. The discovery and targeted creation of new antibiotics are today among the most important public health issues. Antibiotics based on antimicrobial peptides (AMPs) are of particular interest due to their genetically encoded nature. A distinct advantage of most AMPs is their direct mechanism of action that is mediated by their membranolytic properties. The low rate of emergence of antibiotic resistance associated with the killing mechanism of action of AMPs attracts heightened attention to this field. Recombinant technologies enable the creation of genetically programmable AMP producers for large-scale generation of recombinant AMPs (rAMPs) or the creation of rAMP-producing biocontrol agents. The methylotrophic yeast Pichia pastoris was genetically modified for the secreted production of rAMP. Constitutive expression of the sequence encoding the mature AMP protegrin-1 provided the yeast strain that effectively inhibits the growth of target gram-positive and gram-negative bacteria. An antimicrobial effect was also observed in the microculture when a yeast rAMP producer and a reporter bacterium were co-encapsulated in droplets of microfluidic double emulsion. The heterologous production of rAMPs opens up new avenues for creating effective biocontrol agents and screening antimicrobial activity using ultrahigh-throughput technologies.

B Cell Profiling in Patients with Pemphigus Vulgaris.

Pemphigus vulgaris is a severe, socially significant autoimmune disease associated with autoantibodies to the desmoglein 3 antigen. The disease affects all age groups, beginning at 18 years of age; the mortality rate of pemphigus can reach as high as 50%, depending on a patient's age and a number of other factors. There is no highly selective or personalized therapy for pemphigus vulgaris at the moment. One of the well-known therapeutic approaches to the disease is to use rituximab, an anti-CD20 antibody that can help achieve B cell depletion in peripheral blood. To solve the problem of nonspecific elimination of B cells in patients with pemphigus vulgaris, it is reasonable to use specific immunoligands, their choice being based on an assessment of the level of autoantibodies specific to each of the fragments of desmoglein. In this work, the proportion of autoreactive B cells in patients diagnosed with pemphigus vulgaris is found to be 0.09-0.16%; a positive correlation was revealed between the antibody level and the number of autoreactive B cells to various fragments of desmoglein.

An ELISA Platform for the Quantitative Analysis of SARS-CoV-2 RBD-neutralizing Antibodies As an Alternative to Monitoring of the Virus-Neutralizing Activity.

Monitoring of the level of the virus-neutralizing activity of serum immunoglobulins ensures that one can reliably assess the effectiveness of any protection against the SARS-CoV-2 infection. For SARS-CoV-2, the RBD-ACE2 neutralizing activity of sera is almost equivalent to the virus-neutralizing activity of their antibodies and can be used to assess the level of SARS-CoV-2 neutralizing antibodies. We are proposing an ELISA platform for performing a quantitative analysis of SARS-CoV-2 RBD-neutralizing antibodies, as an alternative to the monitoring of the virus-neutralizing activity using pseudovirus or "live" virus assays. The advantage of the developed platform is that it can be adapted to newly emerging virus variants in a very short time (1-2 weeks) and, thereby, provide quantitative data on the activity of SARS-CoV-2 RBD-neutralizing antibodies. The developed platform can be used to (1) study herd immunity to SARS-CoV-2, (2) monitor the effectiveness of the vaccination drive (revaccination) in a population, and (3) select potential donors of immune plasma. The protective properties of the humoral immune response in hospitalized patients and outpatients, as well as after prophylaxis with the two most popular SARS-CoV-2 vaccines in Russia, were studied in detail using this platform. The highest RBD-neutralizing activity was observed in the group of hospitalized patients. The protective effect in the group of individuals vaccinated with Gam-COVID-Vac vaccine was 25% higher than that in outpatients and almost four times higher than that in individuals vaccinated with the CoviVac vaccine.

A new mouse unilateral model of diffuse alveolar damage of the lung.

OBJECTIVE AND DESIGN: The existing biological models of diffuse alveolar damage (DAD) in mice have many shortcomings. To offset these shortcomings, we have proposed a simple, nonsurgical, and reproducible method of unilateral total damage of the left lung in ICR mice. This model is based on the intrabronchial administration of a mixture of bacterial lipopolysaccharide (LPS) from the cell wall of S. enterica and α-galactosylceramide (inducing substances) to the left lung. METHODS: Using computer tomography of the lungs with endobronchial administration of contrast material, we have been able to perform an operative intravital verification of the targeted delivery of the inducer. The model presented is characterized by more serious and homogeneous damage of the affected lung compared to the existing models of focal pneumonia; at the same time, our model is characterized by longer animal survival since the right lung remains intact. RESULTS: The model is also characterized by diffuse alveolar damage of the left lung, animal survival of 100%, abrupt increases in plasma levels of TNFa, INFg, and IL-6, and significant myocardial overload in the right heart. It can be used to assess the efficacy of innovative drugs for the treatment of DAD and ARDS as the clinical manifestations that are developed in patients infected with SARS-CoV-2. Morphological patterns of lungs in the noninfectious ("sterile") model of DAD induced by LPS simultaneously with α-galactosylceramide (presented here) and in the infectious model of DAD induced by SARS-CoV-2 have been compared. CONCLUSION: The DAD model we have proposed can be widely used for studying the efficacy of candidate molecules for the treatment of infectious respiratory diseases, such as viral pneumonias of different etiology, including SARS-CoV-2.

Analysis of the Specificity of Auto-Reactive Antibodies to Individual Fragments of the Extracellular Domain of Desmoglein 3 in Patients with Pemphigus Vulgaris.

A method for the analysis of the epitope specificity of auto-reactive antibodies to desmoglein 3 (Dsg3) using competitive ELISA has been developed. It is based on a two-stage solid-phase ELISA with initial "depletion" of auto-reactive antibodies against the studied epitope and subsequent quantitative assessment of antibodies against full-length extracellular domain Dsg3. The proposed approach for assessing the specificity of the autoimmune response in patients with pemphigus vulgaris can provide in the future the possibility to personalize the therapy using plasmapheresis by preliminary selection of the antigenic composition of the extracorporeal immunosorbent.

Recombinant Fragment of the Extracellular Domain of Human Desmoglein 3 Fused with the Fc-Fragment of Human IgG1 Selectively Adsorbs Autoreactive Antibodies from the Sera of Pemphigus Patients.

Using the recombinant second fragment of the extracellular domain (EC2) of human desmoglein type 3 (Dsg3) as an affinity ligand, an immunosorbent was obtained that selectively binds autoreactive antibodies to this domain from the immune sera of patients with pemphigus. The EC2 protein was obtained in the form of a fusion protein with the Fc-fragment of human IgG1. The production was carried out in CHO cells using the method of transient expression.

COVID-19 in Russia: Clinical and Immunological Features of the First-Wave Patients.

The coronavirus disease outbreak in 2019 (COVID-19) has now achieved the level of a global pandemic and affected more than 100 million people on all five continents and caused over 2 million deaths. Russia is, needless to say, among the countries affected by SARS-CoV-2, and its health authorities have mobilized significant efforts and resources to fight the disease. The paper presents the result of a functional analysis of 155 patients in the Moscow Region who were examined at the Central Clinical Hospital of the Russian Academy of Sciences during the first wave of the pandemic (February-July, 2020). The inclusion criteria were a positive PCR test and typical, computed tomographic findings of viral pneumonia in the form of ground-glass opacities. A clinical correlation analysis was performed in four groups of patients: (1) those who were not on mechanical ventilation, (2) those who were on mechanical ventilation, and (3) those who subsequently recovered or (4) died. The correlation analysis also considered confounding comorbidities (diabetes, metabolic syndrome, hypertension, etc.). The immunological status of the patients was examined (levels of immunoglobulins of the M, A, G classes and their subclasses, as well as the total immunoglobulin level) using an original SARS-CoV-2 antibody ELISA kit. The ELISA kit was developed using linear S-protein RBD-SD1 and NTD fragments, as well as the N-protein, as antigens. These antigens were produced in the prokaryotic E. coli system. Recombinant RBD produced in the eukaryotic CHO system (RBD CHO) was used as an antigen representing conformational RBD epitopes. The immunoglobulin A level was found to be the earliest serological criterion for the development of a SARS-CoV-2 infection and it yielded the best sensitivity and diagnostic significance of ELISA compared to that of class M immunoglobulin. We demonstrated that the seroconversion rate of "early" N-protein-specific IgM and IgA antibodies is comparable to that of antibodies specific to RBD conformational epitopes. At the same time, seroconversion of SARS-CoV-2 N-protein-specific class G immunoglobulins was significantly faster compared to that of other specific antibodies. Our findings suggest that the strong immunogenicity of the RBD fragment is for the most part associated with its conformational epitopes, while the linear RBD and NTD epitopes have the least immunogenicity. An analysis of the occurrence rate of SARS-CoV-2-specific immunoglobulins of different classes revealed that RBD- and N-specific antibodies should be evaluated in parallel to improve the sensitivity of ELISA. An analysis of the immunoglobulin subclass distribution in sera of seropositive patients revealed uniform induction of N-protein-specific IgG subclasses G1-G4 and IgA subclasses A1-A2 in groups of patients with varying severity of COVID-19. In the case of the S-protein, G1, G3, and A1 were the main subclasses of antibodies involved in the immune response.

Peculiarities of the Presentation of the Encephalitogenic MBP Peptide by HLA-DR Complexes Providing Protection and Predisposition to Multiple Sclerosis.

Predisposition to multiple sclerosis (MS), a chronic autoimmune disease of the central nervous system, is due to various factors. The genetic component is considered one of the most important factors. HLA class II genes contribute the most to the development of MS. The HLA-DRB1*15 allele group is considered one of the main genetic risk factors predisposing to MS. The group of HLA-DRB1*01 alleles was shown to have a protective effect against this disease in the Russian population. In this work, we compared the binding of the encephalitogenic fragment of the myelin basic protein (MBP) to two HLA-DR complexes that provide protection against and predisposition to MS: HLA-DR1 (HLA-DRB1*0101) and HLA-DR15 (HLA-DRB1*1501), respectively. We found that the myelin peptide MBP88-100 binds to HLA-DR1 at a rate almost an order of magnitude lower than the viral peptide of hemagglutinin (HA). The same was true for the binding of MBP85-97 to HLA-DR15 in comparison with viral pp65. The structure of the C-terminal part of the peptide plays a key role in the binding to HLA-DR1 for equally high-affinity N-terminal regions of the peptides. The IC50 of the myelin peptide MBP88-100 competing with viral HA for binding to HLA-DR1 is almost an order of magnitude higher than that of HA. As for HA, the same was also true for the binding of MBP85-97 to HLA-DR15 in comparison with viral pp65. Thus, autoantigenic MBP cannot compete with the viral peptide for binding to protective HLA-DR1. However, it is more competitive than viral peptide for HLA-DR15.

X-Ray Diffraction and Multifrequency EPR Study of Radiation-Induced Room Temperature Stable Radicals in Octacalcium Phosphate.

Octacalcium phosphate (OCP) {Ca8H2(PO4)6×5H2O] has attracted increasing attention over the last decade as a transient intermediate to the biogenic apatite for bone engineering and in studies involving the processes of pathological calcification. In this work, OCP powders obtained by hydrolysis of dicalcium phosphate dehydrate were subjected to X- and γ-ray irradiation and studied by means of stationary and pulsed electron paramagnetic resonance at 9, 36 and 94 GHz microwave frequencies. Several types of paramagnetic centers were observed in the investigated samples. Their spectroscopic parameters (components of the g and hyperfine tensors) were determined. Based on the extracted parameters, the induced centers were ascribed to H0, CO33-, CO2- and nitrogen-centered (presumably NO32-) radicals. The spectroscopic parameters of the nitrogen-centered stable radical in OCP powders were found to be markedly different from those in hydroxyapatite. According to X-ray diffraction data, γ-ray irradiation allowed the phase composition of calcium phosphates to change; all minor phases with the exception of OCP and hydroxyapatite disappeared, while the OCP crystal lattice parameters changed after irradiation. The obtained results could be used for the tracing of mineralization processes from their initiation to completion of the final product, identification of the OCP phase, and to follow the influence of radiation processes on phase composition of calcium phosphates.

Engineering Artificial Biodiversity of Lantibiotics to Expand Chemical Space of DNA-Encoded Antibiotics.

The discovery of antibiotics was one of the fundamental stages in the development of humanity, leading to a dramatic increase in the life expectancy of millions of people all over the world. The uncontrolled use of antibiotics resulted in the selection of resistant strains of bacteria, limiting the effectiveness of antimicrobial therapy nowadays. Antimicrobial peptides (AMPs) were considered promising candidates for next-generation antibiotics for a long time. However, the practical application of AMPs is restricted by their low therapeutic indices, impaired pharmacokinetics, and pharmacodynamics, which is predetermined by their peptide structure. Nevertheless, the DNA-encoded nature of AMPs enables creating broad repertoires of artificial biodiversity of antibiotics, making them versatile templates for the directed evolution of antibiotic activity. Lantibiotics are a unique class of AMPs with an expanded chemical space. A variety of post-translational modifications, mechanisms of action on bacterial membranes, and DNA-encoded nature make them a convenient molecular template for creating highly representative libraries of antimicrobial compounds. Isolation of new drug candidates from this synthetic biodiversity is extremely attractive but requires high-throughput screening of antibiotic activity. The combination of synthetic biology and ultrahigh-throughput microfluidics allows implementing the concept of directed evolution of lantibiotics for accelerated creation of new promising drug candidates.

Involvement of the N Domain Residues E34, K35, and R38 in the Functionally Active Structure of Escherichia coli Lon Protease.

ATP-dependent Lon protease of Escherichia coli (EcLon), which belongs to the superfamily of AAA+ proteins, is a key component of the cellular proteome quality control system. It is responsible for the cleavage of mutant, damaged, and short-lived regulatory proteins that are potentially dangerous for the cell. EcLon functions as a homooligomer whose subunits contain a central characteristic AAA+ module, a C-terminal protease domain, and an N-terminal non-catalytic region composed of the actual N-terminal domain and the inserted α-helical domain. An analysis of the N domain crystal structure suggested a potential involvement of residues E34, K35, and R38 in the formation of stable and active EcLon. We prepared and studied a triple mutant LonEKR in which these residues were replaced with alanine. The introduced substitutions were shown to affect the conformational stability and nucleotide-induced intercenter allosteric interactions, as well as the formation of the proper protein binding site.

Production of Recombinant Human Transferrin in Eukaryotic Pichia pastoris Expression System.

The development and manufacturing of serum-free culture media allowing reducing the costs of preparations and standardizing the biotechnological process are important trends in biotechnology. Substitution of protein compounds in the serum-free media with recombinant analogues reduces the risk of contamination with various infectious agents. Human transferrin is a protein component of serum-free media responsible for the transport of Fe3+ ions into cells. We generated a producing strain P. pastoris secreting human transferrin to the culture medium. The use of constitutive GAP promoter and maintenance of medium pH at 6.5 allows attaining maximum level of transferrin expression (20 mg/liter).

"Shielding" of Cytokine Induction by the Periodontal Microbiome in Patients with Periodontitis Associated with Type 2 Diabetes Mellitus.

Periodontal diseases, especially those with polymicrobial etiology, are often associated with type 2 diabetes mellitus, proceeding more severely and affecting the course of diabetes mellitus. Recently, this feature has been associated with the ability of periodontopathogen microflora to cause not only a local infectious process in the oral cavity, but also to interact with the human immune system and induce various systemic effects. We investigated changes in the salivary cytokine profile of patients with chronic periodontitis, associated and not associated with type 2 diabetes mellitus. We observed a statistically significant decrease of MCP-1/CCL2, GM-CSF, IL-5, IL-6, and IFN-γ in the saliva of patients with chronic periodontitis associated with type 2 diabetes mellitus in comparison with patients with chronic periodontitis only. All of these cytokines are associated with macrophage activation. These data are an important contribution to the elucidation of the mechanism of periodontopathogens involvement in the manifestation of the systemic effects of type 2 diabetes.

High-Throughput Screening of Biodiversity for Antibiotic Discovery.

The increasing number of infections caused by antibiotic-resistant strains of pathogens challenges modern technologies of drug discovery. Combinatorial chemistry approaches are based on chemical libraries. They enable the creation of high-affinity low-molecular-weight ligands of the therapeutically significant molecular targets of human cells, thus opening an avenue toward a directed design of highly effective therapeutic agents. Nevertheless, these approaches face insurmountable difficulties in antibiotic discovery. Natural compounds that have evolved for such important characteristics as broad specificity and efficiency are a good alternative to chemical libraries. However, unrestricted use of natural antibiotics and their analogues leads to avalanche-like spread of resistance among bacteria. The search for new natural antibiotics, in its turn, is extremely complicated nowadays by the problem of antibiotic rediscovery. This calls for the application of alternative high-throughput platforms for antibiotic activity screening, cultivation of "unculturable" microorganisms, exploration of novel antibiotic biosynthetic gene clusters, as well as their activation and heterologous expression. Microfluidic technologies for the screening of antibiotic activity at the level of single cells are, therefore, of great interest, since they enable the use of a single platform to combine the technology of ultrahigh-throughput screening, next-generation sequencing, and genome mining, thus opening up unique opportunities for antibiotic discovery.

Cloning and characterization of serpin from red king crab Paralithodes camtschaticus.

Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin-like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K-specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant kass=(2.3 ±â€¯0.7) × 106 M-1s-1 and an SI of 4.7 ±â€¯0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K-specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes.

Generation of Highly Specific Proteolytic Biocatalysts by Screening Technologies.

We propose a yeast display-based system for screening of proteolytic enzyme libraries that utilizes substrate protein adsorbed on the yeast cell surface and containing a desired cleavage sequence. Specific cleavage of the substrate protein releases its biotin-binding center. The cells carrying the target proteinase can be selected by cytofluorometry due to interaction with biotinylated fluorescent protein. Using human enterokinase light chain as the model proteinase we showed that the proposed screening system highly effectively selects the proteolytic enzymes with preset specificity.

Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics.

BACKGROUND: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. RESULTS: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. CONCLUSIONS: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.

The Preferable Binding Pose of Canonical Butyrylcholinesterase Substrates Is Unproductive for Echothiophate.

In this paper, we, for the first time, describe the interaction between the butyrylcholinesterase enzyme and echothiophate, a popular model compound and an analogue of the chemical warfare agents VX and VR, at the atomistic level. Competition between the two echothiophate conformations in the active site was found using molecular modeling techniques. The first one is close to the mode of binding of the substrates of choline series (butyrylcholine and butyrylthiocholine) and is inhibitory, since it is unable to react with the enzyme. The second one is characterized by a significantly worse estimated binding affinity and is reactive. Thus, echothiophate combines the features of two types of inhibitors: competitive and suicidal. This observation will help clarify the kinetic reaction scheme in order to accurately assess the kinetic constants, which is especially important when designing new butyrylcholinesterase variants capable of full-cycle hydrolysis of organophosphorus compounds.

Mouse Model for Assessing the Subchronic Toxicity of Organophosphate Pesticides.

The development of antidotes to organophosphate poisons is an important aspect of modern pharmacology. Recombinant acetylcholinesterase and butyrylcholinesterase are effective DNA-encoded acceptors of organophosphate poisons and, in particular, pesticides. Here, we present the results of a study on the effectiveness of recombinant butyrylcholinesterase (BChE) in modeling organophosphate poisoning caused by oral administration of paraoxon at a dose of 2 mg / kg. The study showed a high activity of BChE as a protective agent for subchronic anticholinesterase poisoning in an in vivo model. The administration of BChE in a dose of 20 mg / kg allows one to avoid mortality, and also contributed to rapid recovery after model poisoning.

[Peculiarities of the Mechanism of Interactions of Catalytic Antibodies with Organophosphorus Substrates].

Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min^(-1)М^(-1) for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.