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Objective: Multidrug-resistant, highly pathogenic Escherichia coli strains are the primary causative agents of intestinal and extraintestinal human diseases. The extensive utilization of antibiotics for farm animals has been identified as a contributing factor to the emergence and dissemination of E. coli strains that exhibit multidrug resistance and possess high pathogenic potential. Consequently, a significant research objective is to examine the genetic diversity of pathogenic E. coli strains and to identify those that may pose a threat to human health. Methods: In this study, we present the results of genome sequencing and analysis, as well as the physiological characterization of E. coli strain APEC 36, which was isolated from the liver of a broiler chicken with generalized colibacillosis. Results: We found that APEC 36 possess a number of mechanisms of antibiotic resistance, including antibiotic efflux, antibiotic inactivation, and antibiotic target alteration/replacement/protection. The most widely represented group among these mechanisms was that of antibiotic efflux. This finding is consistent with the strain's documented resistance to multiple antibiotics. APEC 36 has an extremely rare variant of the beta-lactamase CTX-M-169. Notwithstanding the multitude of systems for interfering with foreign DNA present in the strain, seven plasmids have been identified, three of which may possess novel replication origins. Additionally, qnrS1 , which confers resistance to fluoroquinolones, was found to be encoded in the genome rather than in the plasmid. This suggests that the determinants of antibiotic resistance may be captured in the genome and stably transmitted from generation to generation. Conclusions: The APEC 36 strain has genes for toxins, adhesins, protectins, and an iron uptake system. The obtained set of genetic and physiological characteristics allowed us to assume that this strain has a high pathogenic potential for humans.

The physics of nonlinear optical materials is incredibly versatile, with the design of novel materials and structures offering numerous degrees of freedom. Nevertheless, weak inherent nonlinearity of conventional optical materials continues to hinder the progress of a number of important applications. In this study, we delve into the realm of broadband enhancement of nonlinearity within one-dimensional (1d) plasmonic metamaterials, exploring its intricate connection with nonlocality. Specifically, we introduce a phenomenological framework for quantifying the effective third-order nonlinear susceptibility of 1d multiphase plasmonic nanostructures, utilizing heavily doped semiconductors, and subsequently applying this approach using realistic material parameters. Both direct and inverse problems of nonlinearity enhancement have been addressed. Our findings demonstrate a remarkable capability to significantly augment the third-order nonlinear susceptibility across a defined frequency range, while concurrently gauging the impact of nonlocality on this enhancement.

2-Ethyl-6-methyl-3-hydroxypyridine succinate (EMHPS, Mexidol) is an original antioxidant and an anti-ischemic drug with the possibility of wide applications in the complex therapy of diseases, accompanied by the development of oxidative stress and ischemia; for example, ischemic stroke, chronic cerebral ischemia, and chronic heart failure. The use of EMHPS in the complex therapy of the above diseases may cause the development of drug-drug interactions, particularly pharmacokinetic interactions at the level of transporter proteins. In the present study, we evaluated the interaction of EMHPS with ABCB1 and SLCO1B1. In Caco-2 cells, it was shown that EMHPS is not a substrate of ABCB1 and that it does not affect its expression, but at the same time, it inhibits the activity of this transporter. Its inhibitory activity was inferior to verapamil-a classic inhibitor of ABCB1. In HEK293 and HEK293-SLCO1B1 cells, it was shown that EMHPS is not a substrate of SLCO1B1 either, but that it inhibited the activity of the transporter. However, its inhibitory activity was inferior to the classic inhibitor of SLCO1B1-rifampicin. Furthermore, it was found out that EMHPS does not affect SLCO1B1 expression in HepG2 cells. The approach proposed by the FDA (2020) and the International Transporter Consortium (2010) was used to assess the clinical significance of the study results. The effect of EMHPS on SLCO1B1 and the systemic inhibition of ABCB1 by EMPHS are not clinically significant, but ABCB1 inhibition by EMHPS in the gastrointestinal tract should be tested in vivo through clinical trials.

Cholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin β-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB in E. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow E. coli culture in the synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium using Ni 2+ -chelate affinity chromatography techniques. Forty-eight hours after induction of CtxB expression, concentration of the target product could be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure during expression and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level production of recombinant CtxB for medical and research purposes.

The solute carrier organic anion transporter family member, OATP1B1, is one of the most important transporter proteins, which mediate penetration of many endogenous substances and xenobiotics into hepatocytes. A model system providing expression of the functional protein is needed to assess interaction of OATP1B1 with various substances. Based on the HEK293 cells, we obtained the HEK293-OATP1B1 cell line, constitutively expressing the SLCO1B1 gene encoding the OATP1B1 transporter. Expression of the SLCO1B1 gene was confirmed by real-time PCR analysis and Western blotting. Functionality of the transporter was assessed by the transport of atorvastatin, which is a substrate of OATP1B1. Cells of the resulting cell line, which selectively express the functionally active recombinant OATP1B1 transporter, can be used to study functions of the protein and to test drugs for being substrates, inducers, and inhibitors of OATP1B1, and to assess the risks of drug interactions.

Efficient simulation methods for taking nonlocal effects in nanostructures into account have been developed, but they are usually computationally expensive or provide little insight into underlying physics. A multipolar expansion approach, among others, holds promise to properly describe electromagnetic interactions in complex nanosystems. Conventionally, the electric dipole dominates in plasmonic nanostructures, while higher order multipoles, especially the magnetic dipole, electric quadrupole, magnetic quadrupole, and electric octopole, can be responsible for many optical phenomena. The higher order multipoles not only result in specific optical resonances, but they are also involved in the cross-multipole coupling, thus giving rise to new effects. In this work, we introduce a simple yet accurate simulation modeling technique, based on the transfer-matrix method, to compute higher-order nonlocal corrections to the effective permittivity of 1d plasmonic periodic nanostructures. In particular, we show how to specify the material parameters and the arrangement of the nanolayers in order to maximize or minimize various nonlocal corrections. The obtained results provide a framework for guiding and interpreting experiments, as well as for designing metamaterials with desired dielectric and optical properties.

Tuberculosis is one of the most threatening infectious diseases and represents an important and significant reason for mortality in high-burden regions. The only licensed vaccine, BCG, is hardly capable of establishing long-term tuberculosis protection and is highly variable in its effectiveness. Even after 100 years of BCG use and research, we still cannot unequivocally answer the question of which immune correlates of protection are crucial to prevent Mycobacterium tuberculosis (Mtb) infection or the progression of the disease. The development of a new vaccine against tuberculosis arises a nontrivial scientific challenge caused by several specific features of the intracellular lifestyle of Mtb and the ability of the pathogen to manipulate host immunity. The purpose of this review is to discuss promising strategies and the possibilities of creating a new vaccine that could replace BCG and provide greater protection. The considered approaches include supplementing mycobacterial strains with immunodominant antigens and genetic engineering aimed at altering the interaction between the bacterium and the host cell, such as the exit from the phagosome. Improved new vaccine strains based on BCG and Mtb undergoing clinical evaluation are also overviewed.

The study of diseases of the central nervous system (CNS) at the molecular level is challenging because of the complexity of neural circuits and the huge number of specialized cell types. Moreover, genomic association studies have revealed the complex genetic architecture of schizophrenia and other genetically determined mental disorders. Investigating such complex genetic architecture to decipher the molecular basis of CNS pathologies requires the use of high-throughput models such as cells and their derivatives. The time is coming for high-throughput genetic technologies based on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas systems to manipulate multiple genomic targets. CRISPR/Cas systems provide the desired complexity, versatility, and flexibility to create novel genetic tools capable of both altering the DNA sequence and affecting its function at higher levels of genetic information flow. CRISPR/Cas tools make it possible to find and investigate the intricate relationship between the genotype and phenotype of neuronal cells. The purpose of this review is to discuss innovative CRISPR-based approaches for studying the molecular mechanisms of CNS pathologies using cellular models.

Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.

The review is devoted to the use of the CRISPR/Cas system for obtaining knockdowns of target bacterial genes by CRISPR-mediated interference (CRISPRi). CRISPRi is based on the preservation of the ability of the inactivated dCas nuclease in complex with guide RNA to bind a target, which leads to reversible repression of the selected genes. The review describes the principle of operation of CRISPR/Cas and CRIS-PRi/dCas and provides examples of various approaches to the use of CRISPRi with the most popular inactivated nucleases dCas9 and dCas12a. Also, attention is paid to the use of CRISPRi screening for genome-wide studies and the modular system for identifying many important patterns at the Mobile-CRISPRi genome level. In addition, we discuss the use of CRISPRi to optimize biotechnological production, such as the synthesis of malonyl-CoA, L-lysine, L-glutamate, and other significant products.

Broadband light absorption is a basis for the proper functionality of various materials, microstructures, and devices. Despite numerous studies, however, many aspects of broadband absorption remain uncovered. In this paper, we demonstrate an inverse-problem approach to designing nanostructures with a very low optical reflection and high absorption through a frequency band. Particular emphasis is made on a subwavelength transparent film as a top layer and anisotropic substrate. The polarization-dependent metamaterial absorber based on a subwavelenth semiconductor multicomponent multilayer structure is proposed and numerically investigated. For an illustration, we consider a four-component heavily doped silicon lattice with a thin undoped silicon top layer. The dielectric response of the structure is engineered by controlling the free carrier density and filling factor of each layer. A simulation study reveals a power law dependence of the bandwidth on the maximum reflectivity within the band.

Approximately 1/6 of humanity is at high risk of experiencing cholera epidemics. The development of effective and safe vaccines against Vibrio cholerae, the primary cause of cholera, is part of the public health measures to prevent cholera epidemics. Natural nontoxigenic V. cholerae isolates represent a source of new genetically improved and relatively safe vaccine strains. However, the genomic engineering of wild-type V. cholerae strains is difficult, and these strains are genetically unstable due to their high homologous recombination activity. We comprehensively characterized two V. cholerae isolates using genome sequencing, bioinformatic analysis, and microscopic, physiological, and biochemical tests. Genetic constructs were Gibson assembled and electrotransformed into V. cholerae . Bacterial colonies were assessed using standard microbiological and immunological techniques. As a result, we created a synthetic chromoprotein-expressing reporter operon. This operon was used to improve the V. cholerae genome engineering approach and monitor the stability of the genetic constructs. Finally, we created a stable candidate V. cholerae vaccine strain bearing a recA deletion and expressing the β-subunit of cholera toxin. Thus, we developed a strategy for the rapid creation of genetically stable and relatively safe candidate vaccine strains. This strategy can be applied not only to V. cholerae but also to other important human bacterial pathogens.

This work describes a modification of the pRH2521 vector of the pRH2502/pRH2521 system for CRISPR-dCas9-mediated RNA interference. The modification enabled an increase in the cloning efficiency of guide RNA spacers. The ability of the modified pRH2502/pRH2521 system to suppress the transcription of certain genes was evaluated with the use of genes of Mycobacterium tuberculosis adenylate cyclases. The results revealed the limitations of the pRH2502/pRH2521 system for CRISPR interference associated with the probability of the detection of a protospacer adjacent motif (PAM) in the gene promoter region., Competing Interests: The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors., (© Pleiades Publishing, Inc. 2021, ISSN 0003-6838, Applied Biochemistry and Microbiology, 2021, Vol. 57, No. 4, pp. 421–425. © Pleiades Publishing, Inc., 2021.Russian Text © The Author(s), 2021, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2021, Vol. 57, No. 4, pp. 326–331.)

Bacillus subtilis bacteria play an important role in veterinary medicine, medicine, and biotechnology, and the permanently growing demand for biotechnological products fuels the improvement of the properties of biotechnological strains. B. subtilis strains with improved characteristics maybe obtained by rational design and the directed evolution technologies, or be found among newly described strains. In the course of the long-term microbiome composition studies in the Russian segment of the International Space Station, the B. subtilis 20 strain was isolated, this strain shows the capacity for rapid growth and considerable biomass accumulation, as well as increased resistance to acidification of the environment in comparison to the "terrestrial" B. subtilis 168 strain. What is more, B. subtilis 20 is hyperresistant to the DNA and protein damaging factors that are linked to the overexpression of the genes controlling DNA repair, hydrogen sulfide production, and reactive oxygen species neutralization. The described properties of B. subtilis 20 are indicative of its considerable potential as a promising producer of biologically active compounds.

Despite the efforts of the global medical and scientific community, tuberculosis remains the leading cause of death from infectious diseases. The expectation of success associated with the development of new anti-TB drugs was not justified, and the attention of researchers was largely drawn to the creation of new mycobacterial strains for vaccination against tuberculosis. The proposed review contains current information on the existing vaccine strains and the development of new, genetically engineered strains for the prevention of tuberculosis and the prevention and treatment of other diseases. The review includes relevant information on the correlation between BCG vaccination and the frequency and severity of COVID-19 infection., Competing Interests: The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors., (© Pleiades Publishing, Inc. 2020.)

We investigated the proliferation and osteogenic differentiation of mesenchymal stem cells cultured on fibroin microcarriers. Effective cell proliferation on the surface of the microcarriers, determined by the large surface area, and the contribution of microcarrier mineralization to the stimulation of the osteogenic differentiation of mesenchymal stem cells was revealed.

Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis ( Mtb ), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro , the AC-overexpressing pMind Rv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo , AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMind Rv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo .

Regenerative properties of fibroin implant vitalized with allogeneic bone marrow cells were assessed. The study was performed using the experimental model of rat jejunum wall damage. Three weeks after surgery, we observed recovery of all layers of the jejunum wall at the site of injury and complete degradation of the implant material.

It has been shown that inactivation of the VapC toxin from the VapВС toxin-antitoxin system prevents mycobacterial cells from transitioning to an ovoid state that meets the criteria of dormancy. The results indicate a potential target for medicines that prevent the development of latent tuberculosis infection and provide a basis to obtain bacterial cells for the testing of compounds that are active towards dormant forms of mycobacteria.

3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds leads to an increase in the expression of alkaline phosphatase, an early marker of bone formation. Increased expression is associated with the actin cytoskeleton reorganization under the influence of 3D cultivation and osteogenic calcium phosphate component of the microcarrier.

The study of the stimulating effect of the microgels (MGs) based on recombinant 1F9 spidroin on the regeneration of the deep skin wound in mice was carried out. The use of spidroin MGs was shown to increase significantly the quality of healing compared to the control. The introduction of the MG in the wound edges led to recovery of all the structural elements of the skin: the epidermis, the dermis, including vascular and nervous network, in the periphery of the wound underlying muscles, and skin appendages (sebaceous and sweat glands and hair follicles) was revealed.

We show how to achieve a giant permittivity combined with negligible losses in both the visible and the near-IR for composites made of alternating layers of plasmonic and gain materials as the electric field is directed normally to the layers. The effects of nonlocality are taken into account that makes the method quite realistic. Solving the dispersion equation for eigenmodes of an infinite layered composite, we show that both propagating and nonpropagating modes can be excited, that leads to the realization of a giant nonlocal permittivity. Both phase and group velocities for the propagating eigenmode have been calculated showing that slow light can be achieved in the system under study. The results obtained open new possibilities for designing nanolaser, slow-light, superresolution imaging devices, etc.

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50-150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.

Aim: To assess whether silk fibroin-based microvehicles (MVs) may be used to grow fibroblasts (FBs) and keratinocytes (KCs), key cellular components in skin regeneration after injury., Subjects and Methods: Cryogrinding was applied to derive MVs from fibroin-based and fibroin- and 30% gelatin-containing composite matrices. To examine the structure of the matrices and MVs, confocal microscopy was used to conjugate the polymer with the dye tetramethylrhodamine isothiocyanate. Microparticle size distribution was estimated by granulometric analysis. 3T3 mouse FBs and cultured primary mouse KCs expressing green fluorescent protein (GFP) were used to study whether fibroin-based MVs might be suitable for growing the cells involved in skin regeneration. KC growth was analyzed by confocal laser scanning microscopy from cellular GFP expression. The proliferation rate of FBs and KCs was estimated by a MTT assay., Results: There were two derived MV types: fibroin-based and fibroin and 30% gelatin-containing composite ones. On day 1, 3T3 mouse FBs on the fibroin-based gelatin-free MVs actively proliferated and the presence of gelatin in MVs diminished the proliferation of these cells. Fibroin-based MVs were shown to be suitable for the effective in vitro growth of KCs expressing cytokeratins 5 and 14, the major markers of KCs in the basal layer. Gelatin did not give rise to accelerated KC growth. The investigation has demonstrated that is possible to regulate FB proliferation on MVs, which is of great importance in delivering the cells into the site of injury since intensive proliferation of FBs may lead to the development of fibrosis and the formation of scar tissue. Balanced FB growth is essential to the creation of optimal conditions for KC growth in composite tissue-engineering constructions., Conclusion: The use of fibroin-based MVs is promising for the design of novel therapeutic materials and injectable cell therapy for different diseases.

We derive approximate analytical expressions for the effective permittivity tensor of two-phase metamaterials whose geometry is close to one-dimensional (quasi-one-dimensional metamaterials). Specifically, we consider the metamaterial made of parallel slabs with width given by a linear or parabolic function. Using our approach, the design of epsilon-near-zero, ultra-low and high refractive index metallodielectric metamaterials with extended bandwidth has been demonstrated. In addition, generalizations to the three-dimensional case and some limitations of the presented technique are briefly considered.

The structure of various toxin-antitoxin (TA) families and the principles of their action are reviewed. TA loci are widely distributed in the genomes of eubacteria and archaea. Most TA systems are two-component and function in a similar way: a stable toxin alters vitally important cell functions and can be inactivated by a labile antitoxin.

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) - an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) - most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82-92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes'', well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.

Four major families of bacterial histone-like proteins (HU, IHF, H-NS, FIS), united on the basis of structural similarity and performing specific structural and regulatory functions in the cell, are discussed. Histone-like proteins perform topological modification of the chromosome (twisting, bending, and folding) and directly regulate the functioning of promoters of individual operons. Histone-like proteins are critical for the regulation of cell metabolism, are involved in the response to environmental changes, and play a key role in the transition to and maintenance of the quiescent state of bacteria.

The role of histone-like protein (Hlp) in the development of a dormant state in long-incubated stationary-phase Mycobacterium smegmatis cells was studied in two models: (1) adoption of 'nonculturable' (NC) state, which is reversible due to resuscitation with proteinaceous resuscitation-promoting factor (Rpf) and (2) the formation of morphologically distinct, ovoid resting forms. In the first model, inactivation of the hlp gene resulted in prolongation of culturability of starved cells followed by irreversible nonculturability when mycobacterial cells were unresponsive to resuscitation with Rpf. In the second model, M. smegmatis strain with the inactivated hlp gene was able to form dormant ovoid cells, but they were less resistant to heating and UV radiation than those of wild-type strain. The susceptibility of ovoid cells produced by Delta hlp mutant to these damaging factors was probably due to a less condensed state of DNA, as revealed by fluorescent microscopy and DAPI staining. Evidently, Hlp is essential for cell viability at a later stage of NC dormancy or provides a greater stability of specialized dormant forms.

The new model of obtaining of ovoid resting forms Mycobacterium smegmatis, which are morphologically different from vegetative (rod-like) cells, was developed. Ovoid forms were characterized by a drastically decreased level of metabolic activity, an increased stability to heat processing and antibiotics action, and also by prolonged (more than 2 months) storage time preserving colony-forming ability. Obtained resting forms of mycobacteria may be used in test-systems for checking efficiency of new medical agents against latent forms of tuberculosis and determination of role of these of those genes in entering rest state.

Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in "spent culture medium." Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.

Background: Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs., Methodology/principal Findings: Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC(50) 1-7 microg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC(50). The candidate compounds suppressed resuscitation of dormant ("non-culturable") cells of M. smegmatis at 1 microg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 microg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations., Conclusions/significance: NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.

To work out a test system for studying the inhibition of isoprenoid biosynthesis through non-mevalonate pathway, the conversion of 2-(14)C-methylerythritol 2,4-cyclodiphosphate (14C-MEC) into isoprenoids of three bacteria species and into plastids of 11 higher plant species was studied. The highest efficiency (up to 63%) of the process was observed with chromoplasts from narcissus (Narcissus pseudonarcissus L.) and celandine (Chelidonium majus L.). Twenty five percent of added 14C-MEC was recovered in an isoprenoid fraction of red pepper (Capsicum annuum L.) chromoplasts. Thus, these three models can be used to test antibiotic inhibitors of isoprenoid biosynthesis.

We suggest an extension of the spectral density function approach to describe the complex dielectric response of suspensions of arbitrarily shaped particles having a thin shell, in particular, biological cells. The approach is shown to give analytical results in some simple but practically important cases. In the general case, for the 3-phase systems it reduces to determination of the spectral density function for the suspension of a certain kind. Prospects and limitations of the approach, as well as practical examples, are also considered.

We consider a simple analytical model for the electric near field of a semi-infinite conical probe and apply it to study the incident angle dependence of the field for the case of side illumination by both the plane wave and the Gaussian beam. The electric near field is shown to peak when approaching the grazing incidence. In some cases, a peak can also occur at an incident angle somewhat below 90 degrees . The results obtained are in qualitative agreement with those for a thin semi-infinite wire and previously published results for the finite-size conical probes.

2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate (MEC), an intermediate of the biosynthesis of isoprenoid compounds in bacteria, was found to be capable of exerting a resuscitating effect on resting Mycobacterium smegmatis cells. The introduction of an additional copy of the ispE gene encoding cytidyl-methylerythritol kinase, an enzyme involved in MEC synthesis in M. smegmatis, resulted in the emergence of a capacity for spontaneous reactivation of "nonculturable" M. smegmatis cells, which is not characteristic of the wild-type cells of this species. The involvement of MEC in the transition from the "nonculturable" state to the state of active growth is indicative of a previously unknown function of MEC, assumed to consist in regulation of the bacterial genome activity.

Finite-difference time-domain (FDTD) technique simulations are performed to study the near-field resonance properties of a silver conical nano-tip with a rounded end. Varying the tip geometry, we have computed the electric field distribution, as well as the electric field enhancement factor in the immediate vicinity of the tip apex. The aim of this study is to find optimal geometric parameters of the conical tip, such as its angle and length, in order to maximize the electric field enhancement factor. The increase of the tip length is shown to result in a redshift of the tip resonance wavelength. In addition, some subsidiary (non-dipole) peaks appear for relatively long tips. The peak enhancement values for the small-angle tips increase with the tip length while those for the large-angle ones decrease with it. At the same time, the dependencies of the field enhancement on the cone angle exhibit non-monotonic behavior. In other words, an optimal angle exists allowing one to maximize the electric near field. Finally, the effect of the supporting dielectric medium on the electric field near the tip apex is discussed. In the approximation used, the effect is shown to leave the main conclusions unchanged.

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.

Using thermal deposition technique, we have prepared Au/Teflon thin films on glass substrates. To control the film microstructure, both TEM and AFM characterization have been carried out. The visible optical transmission spectra have been measured in-situ. The peak positions and linewidths in the spectra have been identified in terms of the gold nanoparticle shape, size, and arrangement. The results obtained provide evidence for formation of both sphere-like and cylinder-like gold nanoparticles in the films. The fraction of each kind of the particles depends on whether the plasma treatment has been used. The treatment is shown to enhance the fraction of the cylinder-like particles.

Using a simple phenomenological approach, we calculate the percolation threshold for Bruggeman composites having microgeometry of two kinds. Both kinds of composites consist of spheroids whose shape follows the Beta distribution. At the same time, the first one is a mixture of spheroids equally oriented along their revolution axis. In this case the percolation threshold is shown to be the same as for an assembly of equally oriented identical spheroids whose shape corresponds to the most probable shape of the distribution. For such composites the percolation threshold can vary between 0 and 1. The second one is a random mixture of the spheroids. In this case the percolation threshold is expressed in terms of the Gauss hypergeometric function; it is shown to vary between 0 and 1/3. The derived analytical results are supplemented with numerical calculations carried out for different values of the Beta distribution parameters.

We consider generalizations of the classical symmetrical Bruggeman equation based on the concept of shape-distributed particle systems. The use of the Beta distribution for the particle shape is shown to result in some known as well as unknown equations of the effective medium theory. However, these equations yield no percolation threshold. On the other hand, the use of one- and two-dimensional steplike distributions of spheroidal (ellipsoidal) shapes yields a percolation threshold depending on the distribution parameters. The problem of finding the percolation threshold to fit the systems under consideration, as well as the applicability area of the generalized Bruggeman equation and its relation to the Bergman representation, are discussed.

Extraction and purification from the biomass of Corynebacterium ammoniagenes of 2-C-methyl-D-erhythritol 2,4-cyclopyrophosphate (MEC) was associated with its spontaneous transformation into a number of derivatives (which was due to pyrophosphate bond lability and the formation of complexes with metals). These derivatives included 1,2-cyclophospho-4-phosphate, 2,4-diphosphate, 2,3-cyclophosphate, 1,4-diphosphate, and 3,5-diphosphate (identified by 1H, 31P, and 13C NMR spectroscopy) and accounted for about 10% MEC. When added to a solution of DNA in the presence of the Fenton reagent, MEC prevented DNA decomposition. In addition, MEC slowed down the interaction of the reagent with tempol radicals, which indicates that complexation of ferrous ions by MEC attenuates their ability to catalyze the formation of hydroxyl radicals from hydrogen peroxide. In the presence of 0.23 mM MEC, the rate of respiration of rat liver mitochondria increased 1.8 times. At 0.1-1.0 mM, MEC activated in vitro proliferation of human Vgamma9 T-cells. It is suggested that MEC acts as an endogenous stabilizing agent for bacterial cells subjected to oxidative stress and as an immunomodulator for eukaryotic hosts.

Cross reactions between N. meningitidis and M. catarrhalis proteins were studied with the use of a panel of monoclonal antibodies to M. catarrhalis protein antigens. All antigenic preparations under study were shown to give cross reactions between N. meningitidis serotype porin of 39 kD (strain B125) and M. catarrhalis proteins of 40-41 kD. These M. catarrhalis proteins belonged to main proteins of class F and had the function of porins in the cell. In addition, the epitope of 41-kD antigen, detected by monoclonal antibodies 3E10, is common for both N. meningitidis porin and N. meningitidis iron-regulated proteins of 70 and 50 kD. The epitope of M. catarrhalis protein of 67 kD, detected by monoclonal antibodies 1G6, is common for N. meningitidis porin and N. meningitidis iron-regulated proteins of 50 and 55 kD.