Saturation Mutagenesis and Molecular Modeling: The Impact of Methionine 182 Substitutions on the Stability of ß-Lactamase TEM-1.

Serine ß-lactamase TEM-1 is the first ß-lactamase discovered and is still common in Gram-negative pathogens resistant to ß-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a "global suppressor", has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of ß-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the ß-lactamase to be reactivated.

Multiplex Microarrays in 96-Well Plates Photoactivated with 4-Azidotetrafluorobenzaldehyde for the Identification and Quantification of ß-Lactamase Genes and Their RNA Transcripts.

Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin-peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to ß-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.

Drug Repurposing of the Unithiol: Inhibition of Metallo-ß-Lactamases for the Treatment of Carbapenem-Resistant Gram-Negative Bacterial Infections.

The increasing antibiotic resistance is a clinical problem worldwide. Numerous Gram-negative bacteria have already become resistant to the most widely used class of antibacterial drugs, ß-lactams. One of the main mechanisms is inactivation of ß-lactam antibiotics by bacterial ß-lactamases. Appearance and spread of these enzymes represent a continuous challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. Drug repurposing is a prospective approach for finding new targets for drugs already approved for use. We describe here the inhibitory potency of known detoxifying antidote 2,3-dimercaptopropane-1-sulfonate (unithiol) against metallo-ß-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-ß-lactamase NDM-1 with the KI of 16.7 µM. It is an order of magnitude lower than the KI for l-captopril, the inhibitor of angiotensin-converting enzyme approved as a drug for the treatment of hypertension. Phenotypic methods demonstrate that the unithiol inhibits natural metallo-ß-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant K. pneumoniae and P. aeruginosa bacterial strains. The 3D full atom structures of unithiol complexes with NDM-1 and VIM-2 are obtained using QM/MM modeling. The thiol group is located between zinc cations of the active site occupying the same place as the catalytic hydroxide anion in the enzyme-substrate complex. The sulfate group forms both a coordination bond with a zinc cation and hydrogen bonds with the positively charged residue, lysine or arginine, responsible for proper orientation of antibiotics upon binding to the active site prior to hydrolysis. Thus, we demonstrate both experimentally and theoretically that the unithiol is a prospective competitive inhibitor of metallo-ß-lactamases and it can be utilized in complex therapy together with the known ß-lactam antibiotics.

Synthesis, SAR and molecular docking study of novel non-ß-lactam inhibitors of TEM type ß-lactamase.

The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type ß-lactamase enzyme. Two compounds 4g and 5c reveal the inhibition potency in micromolar range and show their action by non-covalent binding in the vicinity of the TEM-171 active site. The structure activity relationship around carbon chain length and different substituents in ortho- and para-positions of acylated phenoxyanilide as well as molecular modelling study has been performed.

Structural diversity of tick-borne encephalitis virus particles in the inactivated vaccine based on strain Sofjin.

The main approach to preventing tick-borne encephalitis (TBE) is vaccination. Formaldehyde-inactivated TBE vaccines have a proven record of safety and efficiency but have never been characterized structurally with atomic resolution. We report a cryoelectron microscopy (cryo-EM) structure of the formaldehyde-inactivated TBE virus (TBEV) of Sofjin-Chumakov strain representing the Far-Eastern subtype. A 3.8 Å resolution reconstruction reveals the structural integrity of the envelope E proteins, specifically the E protein ectodomains. The comparative study shows a high structural similarity to the previously published structures of the TBEV European subtype strains Hypr and Kuutsalo-14. A fraction of inactivated virions exhibits asymmetric features including the deformations of the membrane profile. We propose that the heterogeneity is caused by inactivation and perform a local variability analysis on the small parts of the envelope protein shell to reveal membrane curvature features possibly induced by the inactivation. The results of this study will have implications for the design of novel vaccines against diseases caused by flaviviruses.

Preparation and characterization of inactivated tick-borne encephalitis virus samples for single-particle imaging at the European XFEL.

X-ray imaging of virus particles at the European XFEL could eventually allow their complete structures to be solved, potentially approaching the resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, about 1 ml of purified virus suspension containing at least 1012 particles per millilitre is required. Such large amounts of concentrated suspension have never before been obtained for enveloped viruses. Tick-borne encephalitis virus (TBEV) represents an attractive model system for the development of enveloped virus purification and concentration protocols, given the availability of large amounts of inactivated virus material provided by vaccine-manufacturing facilities. Here, the development of a TBEV vaccine purification and concentration scheme is presented combined with a quality-control protocol that allows substantial amounts of highly concentrated non-aggregated suspension to be obtained. Preliminary single-particle imaging experiments were performed for this sample at the European XFEL, showing distinct diffraction patterns.

Development of Nonwoven Fibrous Materials Based on Poly-3-Hydroxybutyrate with a High Content of α-Tricalcium Phosphate.

α-tricalcium (α-TCP) phosphate is widely used as an osteoinductive biocompatible material, serving as an alternative to synthetic porous bone materials. The objective of this study is to obtain a highly filled fibrous nonwoven material composed of poly-3-hydroxybutyrate (PHB) and α-TCP and to investigate the morphology, structure, and properties of the composite obtained by the electrospinning method (ES). The addition of α-TCP had a significant effect on the supramolecular structure of the material, allowing it to control the crystallinity of the material, which was accompanied by changes in mechanical properties, FTIR spectra, and XRD curves. The obtained results open the way to the creation of new osteoconductive materials with a controlled release of the source of calcium into the living organism.

Size Distribution of Inactivated Tick-Borne Encephalitis Virus Particles Revealed by a Comprehensive Physicochemical Approach.

Tick-borne encephalitis virus (TBEV) is an enveloped RNA virus, a member of the genus Flavivirus (family Flaviviridae). Here, we provide a detailed analysis of the size and structure of the inactivated TBEV vaccine strain Sofjin-Chumakov. Four analytical methods were used to analyze individual TBEV particles-negative staining TEM, cryo-EM, atomic force microscopy (AFM), and nanoparticle tracking analysis (NTA). All methods confirmed that the particles were monodisperse and that their mean size was ~50 nm. Cryo-EM data allowed us to obtain a 3D electron density model of the virus with clearly distinguishable E protein molecules. STEM-EELS analysis detected phosphorus in the particles, which was interpreted as an indicator of RNA presence. Altogether, the described analytical procedures can be valuable for the characterization of inactivated vaccine virus samples.

Crystal structures of the molecular class A ß-lactamase TEM-171 and its complexes with tazobactam.

The resistance of bacteria to ß-lactam antibiotics is primarily caused by the production of ß-lactamases. Here, novel crystal structures of the native ß-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical ß-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex resembled that in published ß-lactamase-tazobactam structures, in the second complex, which was obtained after longer soaking of the native crystals in the inhibitor solution, a new and previously unreported tazobactam conformation was observed. It is proposed that the two complexes correspond to different stages along the deacylation path of the acyl-enzyme intermediate. The results provide a novel structural basis for the rational design of new ß-lactamase inhibitors.

Structural characterization of ß-propiolactone inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles.

The severe COVID-19 pandemic drives the research toward the SARS-CoV-2 virion structure and the possible therapies against it. Here, we characterized the ß-propiolactone inactivated SARS-CoV-2 virions using transmission electron microscopy (TEM) and atomic force microscopy (AFM). We compared the SARS-CoV-2 samples purified by two consecutive chromatographic procedures (size exclusion chromatography [SEC], followed by ion-exchange chromatography [IEC]) with samples purified by ultracentrifugation. The samples prepared using SEC and IEC retained more spikes on the surface than the ones prepared using ultracentrifugation, as confirmed by TEM and AFM. TEM showed that the spike (S) proteins were in the pre-fusion conformation. Notably, the S proteins could be recognized by specific monoclonal antibodies. Analytical TEM showed that the inactivated virions retained nucleic acid. Altogether, we demonstrated that the inactivated SARS-CoV-2 virions retain the structural features of native viruses and provide a prospective vaccine candidate.

New Regions With Molecular Alterations in a Rare Case of Insulinomatosis: Case Report With Literature Review.

Insulinomatosis is characterized by monohormonality of multiple macro-tumors and micro-tumors that arise synchronously and metachronously in all regions of the pancreas, and often recurring hypoglycemia. One of the main characteristics of insulinomatosis is the presence of insulin-expressing monohormonal endocrine cell clusters that are exclusively composed of proliferating insulin-positive cells, are less than 1 mm in size, and show solid islet-like structure. It is presumed that insulinomatosis affects the entire population of ß-cells. With regards to molecular genetics, this phenomenon is not related to mutation in MEN1 gene and is more similar to sporadic benign insulinomas, however, at the moment molecular genetics of this disease remains poorly investigated. NGS sequencing was performed with a panel of 409 cancer-related genes. Results of sequencing were analyzed by bioinformatic algorithms for detecting point mutations and copy number variations. DNA copy number variations were detected that harbor a large number of genes in insulinoma and fewer genes in micro-tumors. qPCR was used to confirm copy number variations at ATRX, FOXL2, IRS2 and CEBPA genes. Copy number alterations involving FOXL2, IRS2, CEBPA and ATRX genes were observed in insulinoma as well as in micro-tumors samples, suggesting that alterations of these genes may promote malignization in the ß-cells population.

Challenges and Scientific Prospects of the Newest Generation of mRNA-Based Vaccines against SARS-CoV-2.

In the context of the current COVID-19 pandemic, traditional, complex and lengthy methods of vaccine development and production would not have been able to ensure proper management of this global public health crisis. Hence, a number of technologies have been developed for obtaining a vaccine quickly and ensuring a large scale production, such as mRNA-based vaccine platforms. The use of mRNA is not a new concept in vaccine development but has leveraged on previous knowledge and technology. The great number of human resources and capital investements for mRNA vaccine development, along with the experience gained from previous studies on infectious diseases, allowed COVID-19 mRNA vaccines to be developed, conditionally approved and commercialy available in less than one year, thanks to decades of basic research. This review critically presents and discusses the COVID-19 mRNA vaccine-induced immunity, and it summarizes the most common anaphylactic and autoimmune adverse effects that have been identified until now after massive vaccination campaigns.

Pre-Steady-State Kinetics of the SARS-CoV-2 Main Protease as a Powerful Tool for Antiviral Drug Discovery.

The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, Mpro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific Mpro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of Mpro-targeting molecules is urgently needed. Here, we propose a pre-steady-state kinetic analysis of the interaction of Mpro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type Mpro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of Mpro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre-steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A Mpro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A Mpro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 Mpro.

Increase of Productivity and Neutralization of Pathological Processes in Plants of Grain and Fruit Crops with the Help of Aqueous Solutions Activated by Plasma of High-Frequency Glow Discharge.

In this work, we, for the first time, manufactured a plasma-chemical reactor operating at a frequency of 0.11 MHz. The reactor allows for the activation of large volumes of liquids in a short time. The physicochemical properties of activated liquids (concentration of hydrogen peroxide, nitrate anions, redox potential, electrical conductivity, pH, concentration of dissolved gases) are characterized in detail. Antifungal activity of aqueous solutions activated by a glow discharge has been investigated. It was shown that aqueous solutions activated by a glow discharge significantly reduce the degree of presence of phytopathogens and their effect on the germination of such seeds. Seeds of cereals (sorghum and barley) and fruit (strawberries) crops were studied. The greatest positive effect was found in the treatment of sorghum seeds. Moreover, laboratory tests have shown a significant increase in sorghum drought tolerance. The effectiveness of the use of glow-discharge-activated aqueous solutions was shown during a field experiment, which was set up in the saline semi-desert of the Northern Caspian region. Thus, the technology developed by us makes it possible to carry out the activation of aqueous solutions on an industrial scale. Water activated by a glow discharge exhibits antifungicidal activity and significantly accelerates the development of the grain and fruit crops we studied. In the case of sorghum culture, glow-discharge-activated water significantly increases drought resistance.

Long-term humoral immunogenicity, safety and protective efficacy of inactivated vaccine against COVID-19 (CoviVac) in preclinical studies.

The unprecedented in recent history global COVID-19 pandemic urged the implementation of all existing vaccine platforms to ensure the availability of the vaccines against COVID-19 to every country in the world. Despite the multitude of high-quality papers describing clinical trials of different vaccine products, basic detailed data on general toxicity, reproductive toxicity, immunogenicity, protective efficacy and durability of immune response in animal models are scarce. Here, we developed a ß-propiolactone-inactivated whole virion vaccine CoviVac and assessed its safety, protective efficacy, immunogenicity and stability of the immune response in rodents and non-human primates. The vaccine showed no signs of acute/chronic, reproductive, embryo- and fetotoxicity, or teratogenic effects, as well as no allergenic properties in studied animal species. The vaccine induced stable and robust humoral immune response both in form of specific anti-SARS-CoV-2 IgG and NAbs in mice, Syrian hamsters, and common marmosets. The NAb levels did not decrease significantly over the course of one year. The course of two immunizations protected Syrian hamsters from severe pneumonia upon intranasal challenge with the live virus. Robustness of the vaccine manufacturing process was demonstrated as well. These data encouraged further evaluation of CoviVac in clinical trials.

The impact of long-distance mutations on the Ω-loop conformation in TEM type ß-lactamases.

ß-lactamases are hydrolytic enzymes primarily responsible for occurrence and abundance of bacteria resistant to ß-lactam antibiotics. TEM type ß-lactamases are formed by the parent enzyme TEM-1 and more than two hundred of its mutants. Positions for the known amino acid substitutions cover ∼30% of TEM type enzyme's sequence. These substitutions are divided into the key mutations that lead to changes in catalytic properties of ß-lactamases, and the secondary ones, which role is poorly understood. In this study, Residue Interaction Networks were constructed from molecular dynamic trajectories of ß-lactamase TEM-1 and its variants with two key substitutions, G238S and E240K, and their combinations with secondary ones (M182T and Q39K). Particular attention was paid to a detailed analysis of the interactions that affect conformation and mobility of the Ω-loop, representing a part of the ß-lactamase active site. It was shown that key mutations weakened the stability of contact inside the Ω-loop thus increasing its mobility. Combination of three amino acid substitutions, including the 182 residue, leads to the release of R65 promoting its new contacts with N175 and D176. As a result, Ω-loop is fixed on the protein globule. The second distal mutation Q39K prevents changes in spatial position of R65, which lead to the weakening of the effect of M182T substitution and the recovery of the Ω-loop mobility. Thus, the distal secondary mutations are directed for recovering the mobility of enzyme disturbed by the key mutations responsible for expansion of substrate specificity. AbbreviationsESBLextended spectrum beta-lactamasesIRinhibitor resistant beta-lactamasesMDmolecular dynamicsRINresidue interaction networksRMSDroot mean square deviationRMSFroot mean square fluctuations.Communicated by Ramaswamy H. Sarma.

Diagnostic Value of Combinatorial Markers in Colorectal Carcinoma.

Objectives: Blood-based tests have been shown to be an effective strategy for colorectal cancer (CRC) detection in screening programs. This study was aimed to test the performance of 20 blood markers including tumor antigens, inflammatory markers, and apolipoproteins as well as their combinations. Methods: In total 203 healthy volunteers and 102 patients with CRC were enrolled into the study. Differences between healthy and cancer subjects were evaluated using Wilcoxon rank-sum test. Several multivariate classification algorithms were employed using information about different combinations of biomarkers altered in CRC patients as well as age and gender of the subjects; random sub-sampling cross-validation was done to overcome overfitting problem. Diagnostic performance of single biomarkers and multivariate classification models was evaluated by receiver operating characteristic (ROC) analysis. Results: Of 20 biomarkers, 16 were significantly different between the groups (p-value ≤ 0.001); ApoA1, ApoA2 and ApoA4 levels were decreased, whereas levels of tumor antigens (e.g. carcinoembriogenic antigen) and inflammatory markers (e.g., C-reactive protein) were increased in CRC patients vs. healthy subjects. Combinatorial markers including information about all 16 significant analytes, age and gender of patients, demonstrated better performance over single biomarkers with average accuracy on test datasets ≥95% and area under ROC curve (AUROC) ≥98%. Conclusions: Combinatorial approach was shown to be a valid strategy to improve performance of blood-based CRC diagnostics. Further evaluation of the proposed models in screening programs will be performed to gain a better understanding of their diagnostic value.

The Role of the Ω-Loop in Regulation of the Catalytic Activity of TEM-Type ß-Lactamases.

Bacterial resistance to ß-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial enzymes that are termed ß-lactamases (ßLs). The evolution of serine-class A ß-lactamases from penicillin-binding proteins (PBPs) is related to the formation of the Ω-loop at the entrance to the enzyme's active site. In this loop, the Glu166 residue plays a key role in the two-step catalytic cycle of hydrolysis. This residue in TEM-type ß-lactamases, together with Asn170, is involved in the formation of a hydrogen bonding network with a water molecule, leading to the deacylation of the acyl-enzyme complex and the hydrolysis of the ß-lactam ring of the antibiotic. The activity exhibited by the Ω-loop is attributed to the positioning of its N-terminal residues near the catalytically important residues of the active site. The structure of the Ω-loop of TEM-type ß-lactamases is characterized by low mutability, a stable topology, and structural flexibility. All of the revealed features of the Ω-loop, as well as the mechanisms related to its involvement in catalysis, make it a potential target for novel allosteric inhibitors of ß-lactamases.

The role of microwave ablation in management of functioning pancreatic neuroendocrine tumors.

BACKGROUND: Surgical resection is considered to be the only potentially curative option for patients with pancreatic neuroendocrine tumors (pNETs). High risk rates of perioperative complications make minimally invasive ablative techniques a novel perspective and alternative treatment option for pancreatic neuroendocrine tumors. This study aims to present the first experience of using a microwave ablation in management of pNETs. METHODS: Sechenov University has an experience of treating more than 400 patients with hormone-producing tumors of the pancreas, 7 of which were treated by microwave ablation (MWA). RESULTS: In all patients that underwent MWA, a regression of hormonal symptomatic was achieved. Two patients required readmission a month later for draining of pseudocyst and abscess. CONCLUSIONS: The exact role of microwave ablation in the treatment of non-metastatic pancreatic neuroendocrine tumors has not been defined yet. There is a lack of large prospective randomized studies and the reason for this is that local tumor destruction is indicated in selected cases only, thus making it difficult to analyze a large group of patients and assess long-term results of the treatment. However, microwave ablation allows to take a better control of symptoms in patients with hormone overproduction and in those with high risk of postoperative complications.

Mutual influence of secondary and key drug-resistance mutations on catalytic properties and thermal stability of TEM-type ß-lactamases.

Highly mutable ß-lactamases are responsible for the ability of Gram-negative bacteria to resist ß-lactam antibiotics. Using site-directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM-type ß-lactamases, bearing the secondary substitution Q39K and key mutations related to the extended-spectrum (E104K, R164S) and inhibitor-resistant (M69V) ß-lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of ß-lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of ß-lactamase TEM-1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of ß-lactamase TEM-1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N-terminal α-helix, towards the residues R244 and G236, located in the vicinity of the enzyme's catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of ß-lactamases can be used as a new potential target for inhibiting the enzyme.