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The strategies of future medicine are aimed to modernize and integrate quality approaches including early molecular-genetic profiling, identification of new therapeutic targets and adapting design for clinical trials, personalized drug screening (PDS) to help predict and individualize patient treatment regimens. In the past decade, organoid models have emerged as an innovative in vitro platform with the potential to realize the concept of patient-centered medicine. Organoids are spatially restricted three-dimensional clusters of cells ex vivo that self-organize into complex functional structures through genetically programmed determination, which is crucial for reconstructing the architecture of the primary tissue and organs. Currently, there are several strategies to create three-dimensional (3D) tumor systems using (i) surgically resected patient tissue (PDTOs, patient-derived tumor organoids) or (ii) single tumor cells circulating in the patient's blood. Successful application of 3D tumor models obtained by co-culturing autologous tumor organoids (PDTOs) and peripheral blood lymphocytes have been demonstrated in a number of studies. Such models simulate a 3D tumor architecture in vivo and contain all cell types characteristic of this tissue, including immune system cells and stem cells. Components of the tumor microenvironment, such as fibroblasts and immune system cells, affect tumor growth and its drug resistance. In this review, we analyzed the evolution of tumor models from two-dimensional (2D) cell cultures and laboratory animals to 3D tissue-specific tumor organoids, their significance in identifying mechanisms of antitumor response and drug resistance, and use of these models in drug screening and development of precision methods in cancer treatment.

Cross-reacting material 197 (CRM197) is a non-toxic mutant of the diphtheria toxin and is widely used as a carrier protein in conjugate vaccines. This protein was first obtained from the supernatant of the mutant Corynebacterium diphtheriae strain. This pathogenic bacteria strain is characterized by a slow growth rate and a relatively low target protein yield, resulting in high production costs for CRM197. Many attempts have been made to establish high-yield protocols for the heterologous expression of recombinant CRM197 in different host organisms. In the present work, a novel CRM197-producing Escherichia coli strain was constructed. The target protein was expressed in the cytoplasm of SHuffle T7 E. coli cells without any additional tags and with a single potential mutation-an additional Met [-1]. The fine tuning of the mRNA structure (the disruption of the single hairpin in the start codon area) was sufficient to increase the CRM197 expression level several times, resulting in 150-270 mg/L (1.1-2.0 mg/g wet biomass) yields of pure CRM197 protein. Besides the high yield, the advantages of the obtained expression system include the absence of the necessity of CRM197 refolding or tag removal. Thus, an extensive analysis of the mRNA structure and the removal of the unwanted hairpins in the 5' area may significantly improve the target protein expression rate.

One of the latest methods in modern molecular biology is labeling genomic loci in living cells using fluorescently labeled Cas protein. The NIH Foundation has made the mapping of the 4D nucleome (the three-dimensional nucleome on a timescale) a priority in the studies aimed to improve our understanding of chromatin organization. Fluorescent methods based on CRISPR-Cas are a significant step forward in visualization of genomic loci in living cells. This approach can be used for studying epigenetics, cell cycle, cellular response to external stimuli, rearrangements during malignant cell transformation, such as chromosomal translocations or damage, as well as for genome editing. In this review, we focused on the application of CRISPR-Cas fluorescence technologies as components of multimodal imaging methods for in vivo mapping of chromosomal loci, in particular, attribution of fluorescence signal to morphological and anatomical structures in a living organism. The review discusses the approaches to the highly sensitive, high-precision labeling of CRISPR-Cas components, delivery of genetically engineered constructs into cells and tissues, and promising methods for molecular imaging.

In recent years, an increasing amount of attention has been paid to medicinal products as possible risk factors in the development of eye diseases. The frequency of diagnosed drug-induced uveitis is growing yearly, which can be attributed to the appearance of new drugs - biological agents (immune checkpoint inhibitors, BRAF and MEK inhibitors, vascular endothelial growth factor inhibitors, tumor necrosis factor-α inhibitors), as well as systemic bisphosphonates and some antiviral drugs. The time interval between the beginning of the drug use and the appearance of uveitis symptoms varies from several days to months. Common symptoms include eye pain, photophobia, the appearance of floating opacities, and reduced vision associated with active inflammatory changes in the retina and optic nerve and outcomes of those inflammations. Timely diagnosis, cancellation of the drug that caused uveitis and appointment of adequate anti-inflammatory therapy in most cases effectively stops the symptoms of the disease, which determines the relevance of attention to the prevalence, pathogenesis, diagnosis and treatment of drug-induced uveitis.

Glaucoma is seen as a heterogeneous group of diseases characterized by optical neuropathy with associated visual field loss; one of the main risk factors for its development is increased intraocular pressure (IOP). In the case of drug-induced glaucoma (DIG), patients develop elevated IOP, optic neuropathy and visual field defects associated with the use of certain drugs. Corticosteroids are one of the most well-known classes of drugs that can cause an increase in IOP through the open-angle mechanism. Drug-induced glaucoma, which develops similarly to open-angle glaucoma, can also be caused by some non-steroidal anti-inflammatory agents, antibodies to the endothelial growth factor, etc. Classes of drugs that can cause angle-closure glaucoma include topical anticholinergic or sympathomimetic drops, tricyclic antidepressants, monoamine oxidase inhibitors, antihistamines, antiparkinsonian drugs, antipsychotic drugs, antispasmodics. Products containing sulfa group drugs can cause DIG due to a different closing angle mechanism involving a forward rotation of the ciliary body. It is important for medical practitioners to be aware of this unwanted drug reaction in order to prevent, detect and treat DIG. In the case of drug-induced increase in IOP, if the underlying disease allows discontinuation of drugs, this measure usually leads to normalization of IOP. In cases when the patient's IOP does not normalize after discontinuation of steroids or when they must continue to take corticosteroids, the administration of topical drugs for the treatment of glaucoma should be considered.

Drug-induced dysphonia is a non-life-threatening adverse drug reaction, however, this complication can significantly worsen the quality of life of patients, especially those in voice-speaking professions. The aim of the work was to search for information about the prevalence, etiology, pathogenesis, and features of treatment and prevention of drug-induced dysphonia. In the case of some drugs, the true prevalence may be higher than described in the literature, due to the fact that dysphonia is in most cases mild, reversible and, in comparison with other undesirable drug reactions, rarely attracts the attention of both the patient and practitioners.

One type of non-allergic non-infectious rhinitis is represented by a heterogeneous group of rhinitis medicamentosa, which can be divided into several pathogenetic types. The most common rebound nasal congestion associated with the use of topical decongestants. Excessive use of intranasal decongestants leads to a decrease in the number of alpha-adrenoreceptors on the surface of cell membranes and uncoupling their connection with the G-protein and the development of tachyphylaxis. To prevent the development of rebound nasal congestion caused by topical decongestants, it is important to limit the frequency of their use. In most cases, the duration of the use of vasoconstrictor drugs should be limited to 5-7 days, according to Patient information leaflets for the drugs. However, in patients who have had a history of episodes of rebound nasal congestion, which develops including the previously indicated periods, the duration of decongestant therapy should be limited to 3 days. Rhinitis associated with local inflammation is caused by the intake of acetylsalicylic acid (ASA) or other non-steroidal anti-inflammatory drugs. Currently, the so-called "aspirin triad" is well known - a combination of bronchial asthma, rhinosinusitis (often polyposis) and intolerance to ASA. Neurogenic rhinitis develops due to the use of drugs that violate vascular tone, for example, antihypertensive drugs or type 5 phosphodiesterase inhibitors. Drug-induced rhinitis has a significant impact on the patient's quality of life: nasal congestion, rhinorrhea, secondary night apnea, insomnia as a result of nasal breathing disturbances, headaches, irritability, weakness after sleepless nights disturb patients to a large extent. Timely diagnosis and withdrawal of a provocative drug, the use of topical corticosteroids in case of severe rhinitis are the basis of the treatment of rhinitis medicamentosa. In severe cases, there is a need, including surgical treatment, such as, for example, submucosal laser destruction of the lower nasal concha.

Drug-induced optic neuropathy is a group of disorders in which medications cause degeneration of the optic nerve. The true prevalence of drug-induced neuropathy has not been studied, although the percentage of patients who develop optic nerve damage is known for individual medications. The common pathophysiological mechanisms are believed to be mitochondrial damage and imbalance of intracellular and extracellular free radical homeostasis. Typical symptoms of drug-induced neuropathy are reduced visual acuity in the central area, which is often bilateral, visual field disturbances, dyschromatopsia, and edema of the optic nerve head. Early detection of drug-induced optic neuropathy can potentially prevent or minimize serious complications. For patients who develop drug-induced optic neuropathy, treatment is based on timely diagnosis and cancellation of the provoking drug. In most patients, vision usually recovers a few weeks or months after discontinuation of previous therapy, but there have been cases of irreversible vision loss. In addition to withdrawal of the drug that caused optic nerve lesion, treatment of drug-induced neuropathy may include use of drugs and treatment methods prescribed by neurologists for peripheral neuropathy, however, such treatment is seldom based on evidence.

The ability of drugs to have an ototoxic effect has been studied for a long time, however, the true prevalence of this undesirable phenomenon is unknown, which is due to the use of various audiological protocols, a wide range of reactions to drugs in different ethnic groups, and most importantly, the lack of caution with regard to otological symptoms due to their reversibility or lack of immediate threat to life. Drug-induced ototoxicity is a functional disorder of the inner ear (cochlea and/or vestibular apparatus) or eighth pair of cranial nerves. Pharmacotherapy, associated with the development of ototoxic drug reactions, may remain undervalued for a long time, often until irreversible hearing impairment is formed. The most frequently prescribed drugs that cause ototoxic phenomena include anticancer drugs, antibacterial drugs of the aminoglycoside group, loop diuretics, calcium channel blockers, non-steroidal anti-inflammatory drugs, antimalarial drugs, salicylates, etc. Monitoring the degree of hearing impairment before and during therapy is important in preventing the development of drug-induced ototoxicity and makes it possible to consider alternative treatment regimens in a timely manner. It is in this connection that the role of participation in the appointment of rational pharmacotherapy to patients with a potential risk of developing otological phenomena of a clinical pharmacologist and audiologist undoubtedly increases.

Type II restriction⁻modification (RM) systems are the most widespread bacterial antiviral defence mechanisms. DNA methyltransferase SsoII (M.SsoII) from a Type II RM system SsoII regulates transcription in its own RM system in addition to the methylation function. DNA with a so-called regulatory site inhibits the M.SsoII methylation activity. Using circular permutation assay, we show that M.SsoII monomer induces DNA bending of 31° at the methylation site and 46° at the regulatory site. In the M.SsoII dimer bound to the regulatory site, both protein subunits make equal contributions to the DNA bending, and both angles are in the same plane. Fluorescence of TAMRA, 2-aminopurine, and Trp was used to monitor conformational dynamics of DNA and M.SsoII under pre-steady-state conditions by stopped-flow technique. Kinetic data indicate that M.SsoII prefers the regulatory site to the methylation site at the step of initial protein⁻DNA complex formation. Nevertheless, in the presence of S -adenosyl-l-methionine, the induced fit is accelerated in the M.SsoII complex with the methylation site, ensuring efficient formation of the catalytically competent complex. The presence of S -adenosyl-l-methionine and large amount of the methylation sites promote efficient DNA methylation by M.SsoII despite the inhibitory effect of the regulatory site.

Background: Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity., Methods: The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used., Results and Conclusions: The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed., General Significance: The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage., (Copyright © 2016 Elsevier B.V. All rights reserved.)

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) consists of a methyltransferase domain (residues 72-379) and an N-terminal region (residues 1-71) which regulates transcription in SsoII restriction-modification system. Small-angle X-ray scattering (SAXS) is employed here to study the low resolution structure of M.SsoII and its complex with DNA containing the methylation site. The shapes reconstructed ab initio from the SAXS data reveal two distinct protein domains of unequal size. The larger domain matches the crystallographic structure of a homologous DNA methyltransferase HhaI (M.HhaI), and the cleft in this domain is occupied by DNA in the model of the complex reconstructed from the SAXS data. This larger domain can thus be identified as the methyltransferase domain whereas the other domain represents the N-terminal region. Homology modeling of the M.SsoII structure is performed by using the model of M.HhaI for the methyltransferase domain and representing the N-terminal region either as a flexible chain of dummy residues or as a rigid structure of a homologous protein (phage 434 repressor) connected to the methyltransferase domain by a short flexible linker. Both models are compatible with the SAXS data and demonstrate high mobility of the N-terminal region. The linker flexibility might play an important role in the function of M.SsoII as a transcription factor.

Transcription regulation in bacterial restriction-modification (R-M) systems is an important process, which provides coordinated expression levels of tandem enzymes, DNA methyltransferase (MTase) and restriction endonuclease (RE) protecting cells against penetration of alien DNA. The present study focuses on (cytosine-5)-DNA methyltransferase Ecl18kI (M.Ecl18kI), which is almost identical to DNA methyltransferase SsoII (M.SsoII) in terms of its structure and properties. Each of these enzymes inhibits expression of the intrinsic gene and activates expression of the corresponding RE gene via binding to the regulatory site in the promoter region of these genes. In the present work, complex formation of M.Ecl18kI and RNA polymerase from Escherichia сoli with the promoter regions of the MTase and RE genes is studied. The mechanism of regulation of gene expression in the Ecl18kI R-M system is thoroughly investigated. M.Ecl18kI and RNA polymerase are shown to compete for binding to the promoter region. However, no direct contacts between M.Ecl18kI and RNA polymerase are detected. The properties of M.Ecl18kI and M.SsoII mutants are studied. Amino acid substitutions in the N-terminal region of M.Ecl18kI, which performs the regulatory function, are shown to influence not only M.Ecl18kI capability to interact with the regulatory site and to act as a transcription factor, but also its ability to bind and methylate the substrate DNA. The loss of methylation activity does not prevent MTase from performing its regulatory function and even increases its affinity to the regulatory site. However, the presence of the domain responsible for methylation in the M.Ecl18kI molecule is necessary for M.Ecl18kI to perform its regulatory function.

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) functions as a methyltransferase and also as a transcription factor. Chemical and photochemical crosslinking was used for exploring the structure of M.SsoII-DNA complexes and M.SsoII in the absence of DNA. Photocrosslinking with 4-(N-maleimido)benzophenone demonstrated that in the M.SsoII complex with DNA containing the regulatory site, the M.SsoII region responsible for methylation was bound to DNA flanking the regulatory site, which contained no methylation sequence. This required high flexibility of the linker connecting the M.SsoII N-terminal domain and the M.SsoII region responsible for methylation. The flexibility was demonstrated by crosslinking with bis-maleimidoethane and 1,11-bis-maleimidotetraethyleneglycol.

The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5'-CCNGG-3' (N=A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5'-AGGACAAATTGTCCT-3' in the promoter region of SsoII restriction-modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, K(D), of the M.SsoII-DNA complexes decreased in the order 60oct>60reg>60met, suggesting a higher stability of M.SsoII-60met complex in comparison with the others. The association rate constant, k(a), was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell., (This journal is © The Royal Society of Chemistry 2011)