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 β-lactam antibiotics. The detection of
genes in 65 clinical isolates of
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
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.
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.
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, β-lactams. ...One of the main mechanisms is inactivation of β-lactam antibiotics by bacterial β-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-β-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-β-lactamase NDM-1 with the K
of 16.7 µM. It is an order of magnitude lower than the K
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-β-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant
and
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-β-lactamases and it can be utilized in complex therapy together with the known β-lactam antibiotics.
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.
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 β-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.
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.
A purification and concentration scheme for the tick‐borne encephalitis virus (TBEV) vaccine was developed along with a quality‐control protocol that enabled substantial amounts of highly concentrated non‐aggregated suspension to be produced. This approach allowed single‐particle imaging experiments to be conducted for this sample at the European XFEL, which resulted in distinct patterns of diffraction. In order to illustrate the advantages of the proposed protocols, the cryo‐EM structure of TBEV has been determined.
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, M
, 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 M
inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of M
-targeting molecules is urgently needed. Here, we propose a pre-steady-state kinetic analysis of the interaction of M
with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type M
and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of M
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 M
mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A M
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 M
.
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.
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The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type β-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.
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