Properties of Spontaneous rpsL Mutant of Streptomyces albus KO-1297.

The Streptomyces albus J1074 strain remains one of the most popular platforms for the discovery of new natural compounds due to the expression of biosynthetic gene clusters (BGCs) from the microorganisms of the Actinobacteria class. Different methods were tested to provide a maximal expression of heterologous BGCs in this strain. However, there is still no description of the properties of spontaneous J1074 mutants in the rpsL gene encoding a ribosomal protein S12. The interest in such mutations in actinobacteria is due to the fact that they provide a considerable increase in the antibiotic activity. In this work, we describe the isolation and characterization of the S. albus KO-1297 strain, which contains a spontaneous missense mutation in the rpsL gene leading to a Lys88Glu substitution in the protein S12. As compared with the initial strain, this mutant exhibits an increased resistance to streptomycin and higher antibiotic productivity. The KO-1297 strain and genetically engineered rpsL K88E mutant K88E are not identical in their ability to produce antibiotics. KO-1297 also exhibits a certain level of instability of rpsL mutation. The genomes of KO-1297 and its rpsL WT revertant contain the mutations that can cause phenotypic differences between these strains (as well as between them and SAM2 and K88E strains).

CHRONIC MYELOID LEUKEMIA COURSE IN PERSONS EXPOSED TO IONIZING RADIATION AS A RESULT OF THE CHORNOBYL ACCIDENT. / PEREBIG KhRONIChNOÏ MIIeLOÏDNOÏ LEIKEMIÏ U OSIB, IaKI ZAZNALY DIÏ IONIZUIuChOGO VYPROMINIuVANNIa VNASLIDOK ChORNOBYL'S'KOÏ AVARIÏ.

OBJECTIVE: Describe and characterize the peculiarities of the chronic myeloid leukemia (CML) course and responseto treatment in patients irradiated as a result of the Chornobyl nuclear power plant (ChNPP) accident based on theassessment of clinical-laboratory and clinical parameters. MATERIALS AND METHODS: The CML patients (n = 33) exposed to ionizing radiation as a result of the ChNPP accidentwere enrolled. The comparison group consisted of CML patients (n = 725) with no history of radiation exposure. Allpatients were in the chronic phase of the disease. Clinical, hematological and molecular genetic research methodswere applied. RESULTS: Patients exposed to ionizing radiation as a result of the ChNPP accident had no differences in CML manifestation, as well as in classical genetic markers at the onset of the disease compared with patients with no historyof radiation exposure. Reduction of tumor clone on imatinib therapy was significantly less effective in the patientsexposed to ionizing radiation than in cases of no history of radiation exposure. Cases of primary resistance were statistically significantly prevalent in the ChNPP accident consequences clean-up workers while in the residents ofradiologically contaminated areas a statistically significant increase in probability of loss of complete cytogeneticresponse (development of secondary resistance) to imatinib therapy was found. An association was found betweenthe radiation exposure and probability of loss of complete cytogenetic response to imatinib therapy in this group ofpatients. CONCLUSION: The radiation exposure in the history even many years before the onset of CML is an unfavorable exogenous factor responsible for the development of resistance to imatinib therapy.

[Penile angiomyxoma: a case report].

Angiomyxoma is a rare benign tumor prone to aggressive and invasive growth and relapse. Very few case reports of tumors with this morphology have been published in the world literature. In this paper, we report a rare case of penile angiomyxoma, which illustrates that any preoperative examination does not allow physicians to suspect this tumor type. This fact makes physicians be alert for cancer and use the active surgical tactics even if a tumor is supposed to be benign.

CASE OF DEVELOPMENT OF NON-HODGKIN'S MALIGNANT LYMPHOMA ON THE BACKGROUND OF CHRONIC MYELOID LEUKEMIA IN A PATIENT WHO SUFFERED OF THE CHORNOBYL ACCIDENT. / VYPADOK ROZVYTKU NEKhODZhKINS'KOÏ ZLOIaKISNOÏ LIMFOMY NA FONI KhRONIChNOÏ MIIeLOÏDNOÏ LEIKEMIÏ U PATsIIeNTKY, IaKA POSTRAZhDALA VNASLIDOK AVARIÏ NA ChAES.

In this paper, a clinical case of combination of chronic myeloid leukemia and T-lymphoblastic lymphoma is present-ed, which is currently a rather rare finding for a clinician. The diagnosis of T-lymphoblastic lymphoma is establishedafter 2 years from the verification of chronic myeloid leukemia. The course of diseases and approaches to treatmentare described.The pathogenetic relationship between myeloid and lymphoid diseases remains unclear and is likely to be the resultof several factors - radiation, chemical and, consequently, genetic disorders.

Gene cloning system for sulfonamide-mineralizing Microbacterium sp. strain BR1.

The wide application of sulfonamide (SA) antibiotics in human and veterinary medicine contributes to the accumulation of these antibiotics in the environment and the corresponding onset of antibiotic resistance among bacteria. Microbacterium sp. BR1 is capable of mineralizing sulfamethoxazole and other SAs via a novel mechanism. The genetic basis of SA elimination by BR1 remains unknown. Development of an efficient plasmid transfer protocol for Microbacterium sp. BR1 is highly desirable, as it would open the door to genetic analysis and manipulation of its genome. Here we report that intergeneric Escherichia coli-Microbacterium spp. BR1 conjugation is an efficient way to introduce various plasmids into BR1. The generated transconjugants were stable in the presence of antibiotics and the plasmids showed no signs of rearrangements. Nevertheless, the plasmids were rapidly lost in the absence of selection. We also show that the cumate-inducible beta-glucuronidase reporter gene functions in BR1 and is strictly regulated. Our results set the working ground for further genetic manipulations of BR1, such as the overexpression of sulfonamide degradation genes or the selection of strong microbacterial promoters.

Characterization of Dielectric Nanocomposites with Electrostatic Force Microscopy.

Nanocomposites physical properties unexplainable by general mixture laws are usually supposed to be related to interphases, highly present at the nanoscale. The intrinsic dielectric constant of the interphase and its volume need to be considered in the prediction of the effective permittivity of nanodielectrics, for example. The electrostatic force microscope (EFM) constitutes a promising technique to probe interphases locally. This work reports theoretical finite-elements simulations and experimental measurements to interpret EFM signals in front of nanocomposites with the aim of detecting and characterizing interphases. According to simulations, we designed and synthesized appropriate samples to verify experimentally the ability of EFM to characterize a nanoshell covering nanoparticles, for different shell thicknesses. This type of samples constitutes a simplified electrostatic model of a nanodielectric. Experiments were conducted using either DC or AC-EFM polarization, with force gradient detection method. A comparison between our numerical model and experimental results was performed in order to validate our predictions for general EFM-interphase interactions.

[THE ROLE OF (p)ppGpp MOLECULES IN FORMATION OF "STRICT RESPONSE" IN BACTERIA AND BIOSYNTHESIS OF ANTIBIOTICS AND MORPHOLOGICAL DIFFERENTIATION IN ACTINOMYCETES].

Strict response is a pleiotropic physiological response of cells caused by lack of aminoacetylated tRNAs. Experimentally, this response occurs due to the lack of amino acids in the environment and the limitation of tRNA aminoacylation even in the presence of the corresponding amino acids in the cell. Many features of this response indicate its dependence on the accumulation of ppGpp molecules. There is a correlation between the growth rate of actinomycetes and biosynthesis of their secondary metabolites. Introduction of additional relA gene copies of ppGpp synthetase can affect the production of antibiotics in streptomycetes. The article presents the authors' own experimental data, dedicated to the influence of heterologous relA gene expression in Streptomyces nogalater cells.

[Using autonomous electrostimulation device Erektron in treating female overactive bladder].

INTRODUCTION: Overactive bladder (OAB) is one of the most common syndromes of lower urinary tract dysfunction. Besides standard therapy using anticholinergic medications, comprehensive management of overactive bladder includes physiotherapy. AIM: To test the clinical effectiveness and safety of autonomous electrostimulation device "Erektron" in treating OAB in women. MATERIALS AND METHODS: The study was conducted at the Urology and Gynecology Clinic of the Innovative Medical Technology Center between 25.04.2014 and 30.01.2015. It included 20 women with newly diagnosed OAB both with and without urinary urgency incontinence or urinary stress incontinence. The patients were divided into 2 groups. All patients were treated with the first line anticholinergic agent solifenacin 5 mg daily. In patients of group 1, anticholinergic therapy was administered concurrently with intravaginal electrostimulation using "Erektron" device. RESULTS: In both groups, the treatment resulted in positive results, but a more pronounced improvement was found in group 1 patients with mixed incontinence. CONCLUSION: Autonomous electrostimulation device MT-RV "Erektron" can be used in comprehensive management of patients with OAB, including those with stress urinary incontinence.

Transcriptional regulators of GntR family in Streptomyces coelicolor A3(2): analysis in silico and in vivo of YtrA subfamily.

Transcriptional factors of the GntR family regulate numerous physiological and morphological processes in response to the nutrient state of bacterial cells. The number of GntR transcriptional factors in genomes of soil-dwelling actinomycetes is one of the highest among bacteria, reflecting both the large size of their chromosomes and the complex ecological niche that they occupy. However, very little is known about the roles of GntRs in actinomycete biology. Here, we analyzed the genome of model actinomycete, Streptomyces coelicolor A3(2), in an attempt to gain new insights into the function of GntR family. All 56 GntR proteins of M145 strain were classified into FadR, HutC, MocR, YtrA, and DevA subfamilies according to their secondary structure. We then checked for the presence of GntR orthologs in six other sequenced Streptomyces and one Kitasatospora genomes, revealing that 12 GntRs were conserved in all analyzed strains. Genomic analysis of the less studied YtrA type regulators revealed 160 sequences present in 88 members of Coriobacteridae, Rubrobacteridae, and Actinobacteridae subclasses. These proteins form seven dense clusters on the consensus phylogenetic tree and their genes are usually co-located with the genes for transport proteins. Probable operator sites were identified for orthologous groups of Sco0823 and Sco3812 proteins. All S. coelicolor YtrA-like regulatory genes (SCO0823, SCO1728, SCO3812) were analyzed at transcriptional level, knocked out, and introduced on moderate copy number plasmid in M145 strain. Also, gene SCO0824, a part of putative SCO0823 operon, was studied. Results of these experiments are discussed here.

Assessment of response to imatinib therapy in patients with chronic myeloid leukemia with e13a2 and e14a2 transcripts of BCR/ABL1 gene. / OTsINKA VIDPOVIDI NA TERAPIIu IMATYNIBOM U PATsIIeNTIV Z KhRONIChNOIu MIIeLOIDNOIu LEYKEMIIeIu Z E13A2 TA E14A2 TRANSKRYPTAMY GENA BCR/ABL1.

OBJECTIVE: Assess the influence of e13a2 and e14a2 transcripts of BCR/ABL1 gene on the efficiency of imatinib ther apy in patients with chronic myeloid leukemia. MATERIALS AND METHODS: We examined 508 patients with the chronic phase of chronic myeloid leukemia without radi ation in anamnesis as well as 13 patients with the similar diagnosis and with confirmed presence of radiation expo sure due to the Chornobyl Nuclear Power Plant accident. RESULTS: No significant differences in hematologic parameters, rate of additional chromosomal aberrations and f vari ant translocations were observed between patients with е13а2 and е14а2 transcript. Cumulative probability of com plete cytogenetic response did not differ in patients with е13а2 and е14а2 transcript and was 76 and 80 % respec tively (р = 0,981). Median of achieving a complete cytogenetic response was 20 months in both patient groups. Significantly more patients with e14a2 transcript compared to patients with e13a2 achieved major molecular response by 12 month of therapy (61.5 % versus 23.0 %, p = 0.016). The higher incidence of deep molecular response by 24 month of therapy was revealed in this group (38.7 % versus 6.25 %, p = 0.018). The overall survival and pro gression free survival rates were not statistically different between two groups with different transcripts. However, the rate of event free survival was statistically lower for the patients with e13a2 transcript compared to the ones with e14a2 transcript (51 % versus 62.0 %, p = 0.039). The number of primary resistant patients was 40 % regardless of the transcript expressed. A significant prevalence in incidence either of lost complete cytogenetic response or fail ure of the major molecular response was shown in patients with e13a2 transcript compared to patients with e14a2 transcripts (43.5 % versus 24.8 %, p = 0.015). CONCLUSION: Imatinib therapy is more effective for CML patients with e14a2 transcript compared to patients with e13a2 transcript expression. The transcript e13a2can be viewed as a adverse prognostic factor for imatinib therapy of chronic myeloid leukemia.

[THE ROLE OF STREPTOMYCES NOGALATER Lv65 snoaM, snoaL and snoaE GENES IN NOGALAMYCIN BIOSYNTHESIS].

The results of phylogenetic analysis indicate high similarity of SnoaM, SnoaL SnoaE to the cyclases involved in the biosynthesis of various antibiotics. Genes snoaM, snoaL and snoaE disruption in S. nogalater chromosome was carried on and S. nogalater MI, LI and EI strains were generated. The gene replacement events in M1, L1 and E1 were verified by Southern hybridization. Recombinant strains were characterised by lack of nogalamycin biosynthesis. Originally, M1, L1 and E1 were complemented with plasmids expressing putative cyclase genes from S. nogalater leading to restoration of nogalamycine production. The complementation results clearly indicate that obtained strains are cyclase deficient mutants. Furthermore, complementation of M1, L1 and E1 with a cyclase genes from wild-type strain is consistent with the suggested function of these enzymes.

Testing the utility of site-specific recombinases for manipulations of genome of moenomycin producer Streptomyces ghanaensis ATCC14672.

Streptomyces ghanaensis ATCC14672 is the producer of phosphoglycolipid antibiotics moenomycins that for almost 40 years were used worldwide as an animal feed additive. As the use of moenomycins narrows down (due to bans in the EU and some other countries), it opens the opportunity to develop much-needed antibiotics against Gram-positive human pathogens, such as cocci. It is desirable to develop ATCC14672 strains accumulating only certain members of moenomycin family which would facilitate their purification, analysis and/or chemical modification. Here we tested site-specific recombinases (SSRs) as a tool to manipulate the genome of ATCC14672 and to achieve aforementioned goals. We show that of three SSRs tested--Cre, Dre, and Flp--the first two efficiently catalyzed recombination reactions, while Flp showed no activity in ATCC14672 cells. Cre recombinase can be reused at least three times to modify ATCC14672 genome without detrimental effects, such as large-scale inversions or deletions. Properties of the generated strains and SSRs are discussed.

The efficiency of tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia exposed to ionizing radiation due to the Chornobyl nuclear power plant accident. / Efektyvnist'' terapii' ingibitoramy tyrozynkinaz pacijentiv z hronichnoju mijeloi'dnoju lejkemijeju, jaki zaznaly vplyvu ionizujuchogo vyprominjuvannja vnaslidok avarii' na Chornobyl''s''kij AES.

Objective. To study the efficiency of tyrosine kinase inhibitors (TKI) therapy in patients with chronic myeloid leukemia (CML) exposed to ionizing radiation due to the Chornobyl NPP accident, based on the data of cytogenetic and molecular monitoring. Material and methods. 29 CML patients with confirmed radiation exposure due to Chornobyl NPP accident were examined. Of these, 20 patients were treated with imatinib; 103 patients with CML without radiation history treated with TKI were a comparison group. Cytogenetic and molecular genetic disturbances before and on the different stage of TKI therapy were analysed. Results. Additional chromosomal abnormalities as well as special pattern of BCR/ABL transcripts were not revealed in CML patients exposed to ionizing radiation. Complete cytogenetic response (CCR) was shown in 50 and 48.5 % of patients from study and comparison group, respectively. Major molecular response (MMR) was achieved in 20 % of patients with radiation exposure in anamnesis and in 27.6 % of patients from comparison group. The vast majority of CCR and MMR was reached in patients with the pretreatment term up to 6 months, when imatinib was used as a first line therapy. There were less cases of primary imatinib resistance in the same group of patients. In CML patients who had a history of radiation exposure, secondary resistance developed more frequently than in the comparison group and was 25 %. Conclusion. Laboratory monitoring based on the registration of CCR and MMR demonstrated high efficiency of TKI in the CML treatment of patients, exposed due to Chornobyl accident. Extension of pretreatment term leads to the loss of TKI therapy efficiency and increases the likelihood of primary resistance. CML patients exposed to ionizing radiation develop secondary resistence more often than CML patients without radiation exposure in anamnesis.

[Gene networks that regulate secondary metabolism in actinomycetes: pleiotropic regulators].

Current advances in the research and practical applications of pleiotropic regulatory genes for antibiotic production in actinomycetes are reviewed. The basic regulatory mechanisms found in these bacteria are outlined. Examples described in the review show the importance of the manipulation of regulatory systems that affect the synthesis of antibiotics for the metabolic engineering of the actinomycetes. Also, the study of these genes is the basis for the development of genetic engineering approaches towards the induction of "cryptic" part of the actinomycetes secondary metabolome, which capacity for production of biologically active compounds is much bigger than the diversity of antibiotics underpinned by traditional microbiological screening. Besides the practical problems, the study of regulatory genes for antibiotic biosynthesis will provide insights into the process of evolution of complex regulatory systems that coordinate the expression of gene operons, clusters and regulons, involved in the control of secondary metabolism and morphogenesis of actinomycetes.

Generation and study of the strains of streptomycetes - heterologous hosts for production of moenomycin.

Moenomycins (Mm) are family of phosphoglycolipid natural products that is considered a blueprint to develop new class of antibiotics. The natural Mm producer, Streptomyces ghanaensis (ATCC14672), produces very low amounts of moenomycin, fueling the investigations on genetic approaches to improve its titers. Heterologous expression of moenomycin biosynthesis gene cluster (moe) would be one of the ways to reach this goal. Here we report the generation of a number of novel heterologous streptomycete hosts producing nosokomycin A2 (one of the members of Mm family), and determine their potential for the antibiotic production. The rpoB point mutation in the model strain of Streptomyces genetics, S. coelicolor (strain M1152) significantly improved nosokomycin A2 production compared to parental strains (M145 and M512), while double rpoBrpsL mutation in the same species (strain M1154) decreased it. Our results point to the previously unanticipated epistatic interactions between mutations that individually are known to be highly beneficial for antibiotic production. We also showed here for the first time that facultative chemolitotrophic streptomycete S. thermospinosisporus and chloramphenicol producer S. venezuelae can be used as the hosts for moe genes.

Genes for biosynthesis of butenolide-like signalling molecules in Streptomyces ghanaensis, their role in moenomycin production.

Moenomycins (Mm) - phosphoglycolipid compounds produced by Streptomyces ghanaensis ATCC14672 - are considered a promising model for development of novel class of antibiotics. In this regard it is important to generate Mm overproducing strains which would be a basis for economically justified production of this antibiotic. In this work a set of genes for synthesis and reception of low-molecular weight signaling molecules (LSM) in ATCC14672 were described and their significance for Mm production was studied. The ATCC14672 genome carries structural and regulatory genes for production of LSMs of avenolide and γ-butyrolactone families. Additional copies of LSM biosynthetic genes ssfg_07848 and ssfg_07725 did not alter the Mm production level. ATCC14672 LSMs are not capable of restoring the sporulation of butyrolactone-nonproducing mutant of S. griseus. Likewise, while the heterologous host S. lividans 1326 produced Mm, its mutant M707 (deficient in the butyrolactone synthase gene scbA) did not. Thus, while the natural level of LSMs production by ATCC14672 does not limit Mm synthesis, the former is essential for the synthesis of moenomycins.

[Generation and study of the strains of streptomycetes--heterologous hosts for the production of moenomycin].

In this paper, we report the generation of a number of novel heterologous streptomycete hosts producing nosokomycin A2 (one of the members of Mm family) and determine their potential for antibiotic production. The rpoB point mutation in the model strain of Streptomyces coelicolor (strain M1152) significantly improved nosokomycin A2 production compared to parental strains (M145 and M512), while double rpoBrpsL mutation in the same species (strain M1154) decreased it. Our results point to the previously unanticipated epistatic interactions between mutations that individually are known to be highly beneficial for antibiotic production. We also showed for the first time that facultative chemolitotrophic streptomycete S. thermospinosisporus and chloramphenicol producer S. venezuelae can be used as the hosts for moe genes.

[Genes for biosynthesis of butenolide-like signalling molecules in Streptomyces ghanaensis, their role in moenomycin production].

Moenomycins (Mm)--phosphoglycolipid compounds produced by Streptomyces ghanaensis ATCC14672--are considered a promising model for development of novel-class of antibiotics. In this regard it is important to generate Mm overproducing strains which would be a basis for economically justified production of this antibiotic. In this work a set of genes for synthesis and reception of low-molecularweight signaling molecules (LSM) in ATCC14672 were described and their significance for Mm production was studied. The ATCC 14672 genome carries structural and regulatory genes for production of LSMs of avenolide and γ-butyrolacone families. Additional copies of LSM biosynthetic genes ssfg_07848 and ssfg_07725 did not alter the Mm production level. ATCC14672 LSMs are not capable of restoring the sporulation of butyrolactone-nonpro-ucing mutant ofS . griseus. Likewise, while the heterologous host S. lividans 1326 produced Mm, its mutant M707 (deficient in the butyrolactone synthase gene scbA) did not. Thus, while the natural level of LSMs pro-uction by ATCC 14672 does not limit Mm synthesis, the former is essential for the synthesis of moenomycins.

[Characterization of Streptomyces globisporus 1912 lnd-cluster region containing lndY, lndYR, lndW2 and lndW genes].

Streptomyces globisporus 1912 lnd-cluster region which flanks structural lndZ5-lndZ6 genes contains four open reading frames lndW, lndW2, lndYR and lndY. The latter one encodes putative proteinase which can regulate landomycin production and morphogenesis like LndYR. However, results of lndY overexpression and gene knockout showed that lndY did not participate in regulation of landomycin production and morphogenesis of Streptomyces globisporus 1912. Using transcriptional fusion of promoter lndWp to catechol dioxygenase reporter gene xylE the temporal character of interaction of promoter lndWp and repressor LndYR was studied.