Omniphobic Photoresist-Assisted Patterning of Porous Polymethacrylate Films

Patterning of various surface properties, including roughness, wettability, adhesiveness, and mechanical properties, can markedly enhance the functionality of test systems. Thus, porous polymethacrylates prepared by polymerization-induced phase separation (PIPS) represent a promising class of functional materials for the construction of miniaturized test systems. Different porosity, surface chemistry, and wettability are achieved in porous polymethacrylates with different precursor compositions. Nevertheless, only wettability microstructuring has been highlighted for these materials thus far. Here, the study presents a novel method for the direct and selective deposition of porous polymethacrylate films with different surface chemistry and porosity. The selective adhesion of omniphobic-omniphilic wettability patterns is used to facilitate the polymer pattern formation. The feasibility of patterning with different monomers and porogenic solvents is demonstrated. The topological study confirms the selective application of polymer structures with different thickness and roughness. The wettability characterization of the omniphobic material shows no significant changes caused by the operations performed. Thus, a new pattern with a greater difference in the wettability of the areas is produced in the process. Discontinuous dewetting of different liquids is performed. The use of poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) modified patterns for precise living cell patterning is also demonstrated. Herein, the authors introduce a new method for selective deposition of porous polymethacrylate films by utilizing omniphobic-omniphilic wettability patterns to facilitate selective adhesion. Patterning for precursor compositions with different monomers and porogenic solvents is showcased. Discontinuous dewetting of various liquids is demonstrated on polymethacrylate patterns. Additionally, the use of HEMA-EDMA modified patterns for precise living cell patterning is shown

Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents

Activitat antibiòtica; Ribosoma bacterià; Simulacions de dinàmica molecularActividad antibiótica; Ribosoma bacteriano; Simulaciones de dinámica molecularAntibiotic activity; Bacterial ribosome; Molecular dynamics simulationsIn the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.This research was funded by RFBR [grants 20-04-00873 to N.V.S. (synthesis of analogs, binding assays, in vitro translation), 20-015-00537 to P.A.N. (potential measurement, screening of TolC-containing transporters), and 20-54-76002 to I.A.O. (toeprinting and in vitro translation)], President grant MD 2626.2021.1.4 to I.A.O. (bacteria inhibition assays), grants from the Instituto de Salud Carlos III: PI17/02087 to A.L. (cancer cell proliferation assays) by the Ministry of Science and Higher Education of the Russian Federation [grant FENU-2020-0019 to G.I.M. (molecular dynamics simulations)] and by the Government of the Russian Federation [No. AAAA-A17-117120570004-6 to A.A.B.]

Abstract OR-4: New Antibiotic Binding Site on the 30S Ribosomal Subunit

Background: Antibiotic resistance becomes one of the main problems of modern medicine; therefore, the development of new antibacterial compounds is absolutely necessary. The ribosome is the target for a lot of different antibiotics; there are several main binding sites on the ribosome – decoding center, peptidyl-transferase center, and ribosome exit tunnel. Modification or mutation of nucleotides in these sites could make cells resistant to structurally different antibiotics. Methods: pDualrep2 reporter system was used for detection of the protein synthesis inhibitors in cultural broths of new soil bacteria. By means of a cell-free translation system, the inhibitory activity and mechanism of action of Auraplanin were estimated. CryoEM data collection was performed on a Titan Krios operated at 300 kV, equipped with a Falcon II direct electron detector. Results: In this work, we have found a new inhibitor of protein synthesis, which binds in a completely new binding site. This compound is produced by Actinoplanes sp. VKM Ac-2862 and by Cryo-EM study of its complex with E.coli ribosome, it was shown, that it binds close to 560 loop of 30S ribosomal subunit. The new compound is a derivative of tetramic acid and we called it Auraplanin, because of bright orange color of the producer strain. Structural data are in good agreement with genetic results – resistant mutations were located close determined binding site. Substitutions C564G, G558U, and G566A significantly increase minimal inhibitory concentration, all these mutations were not detected previously. We also observed resistant mutation in ribosomal protein S4, this mutation was previously identified as error-prone. Interestingly, ribosomal ambiguity mutations, G299A and G347U, also increased resistance to Auraplanin. Conclusion: On the basis of the genetic, structural and biochemical studies we hypothesized that Auraplanin acts prevent the transfer from an open to a closed conformation of 30S subunit, in contrast to streptomycin, which promotes the formation of a closed state

Bioprospecting for the soil-derived actinobacteria and bioactive secondary metabolites on the Western Qinghai-Tibet Plateau

IntroductionThe increase in incidence of multidrug-resistant bacteria and the inadequacy of new antimicrobial drugs have led to a widespread outbreak of bacterial antimicrobial resistance. To discover new antibiotics, biodiversity, and novelty of culturable actinobacteria dwelled in soil of the Western Qinghai-Tibet Plateau were investigated. By integrating antibacterial assay with omics tools, Amycolatopsis sp. A133, a rare actinobacterial strain and its secondary metabolites were further studied.MethodCulture-dependent method was used to obtain actinobacterial strains from two soil samples collected from Ali region in Qinghai-Tibet Plateau. The cultural extractions of representative strains were assayed against “ESKAPE” pathogens by paper-disk diffusion method and the double fluorescent protein reporter “pDualrep2” system. An Amycolatopsis strain coded as A133 was prioritized and its secondary metabolites were further analyzed and annotated by omics tools including antiSMASH and GNPS (Global Natural Social Molecular Networking). The predicted rifamycin analogs produced by Amycolatopsis sp. A133 were isolated and identified by chromatographic separation, such as Sephadex LH-20 and HPLC, and spectral analysis, such as NMR and UPLC-HRESI-MS/MS, respectively.ResultsA total of 406 actinobacteria strains affiliated to 36 genera in 17 families of 9 orders were isolated. Out of 152 representative strains, 63 isolates exhibited antagonistic activity against at least one of the tested pathogens. Among them, 7 positive strains were identified by the “pDualrep2” system as either an inhibitor of protein translation or DNA biosynthesis. The cultural broth of Amycolatopsis sp. A133 exhibited a broader antimicrobial activity and can induce expression of TurboRFP. The secondary metabolites produced by strain A133 was annotated as rifamycins and zampanolides by antiSMASH and GNPS analysis. Five members of rifamycins, including rifamycin W, protorifamycin I, rifamycin W-M1, proansamycin B, and rifamycin S, were purified and identified. Rifamycin W-M1, was found as a new member of the naturally occurring rifamycin group of antibiotics.DiscussionAssisted by omics tools, the successful and highly efficient discovery of rifamycins, a group of clinically used antibiotics from actinobacteria in Ali area encouraged us to devote more energy to explore new antibiotics from the soils on the Western Tibetan Plateau

Dispirooxindole-β-Lactams: Synthesis via Staudinger Ketene-Imine Cycloaddition and Biological Evaluation

In this work, we present the first synthesis of dispirooxindole-β-lactams employing optimized methodology of one-pot Staudinger ketene-imine cycloaddition with N-aryl-2-oxo-pyrrolidine-3-carboxylic acids as the ketene source. Spiroconjugation of indoline-2-one with β-lactams ring is considered to be able to provide stabilization and wide scope of functionalization to resulting scaffolds. The dispipooxindoles obtained demonstrated medium cytotoxicity in the MTT test on A549, MCF7, HEK293, and VA13 cell lines, and one of the compounds demonstrated antibacterial activity against E. coli strain LPTD

A New Albomycin-Producing Strain of <i>Streptomyces globisporus</i> subsp. <i>globisporus</i> May Provide Protection for Ants <i>Messor structor</i>

There are several well-studied examples of protective symbiosis between insect host and symbiotic actinobacteria, producing antimicrobial metabolites to inhibit host pathogens. These mutualistic relationships are best described for some wasps and leaf-cutting ants, while a huge variety of insect species still remain poorly explored. For the first time, we isolated actinobacteria from the harvester ant Messor structor and evaluated the isolates’ potential as antimicrobial producers. All isolates could be divided into two morphotypes of single and mycelial cells. We found that the most common mycelial morphotype was observed among soldiers and least common among larvae in the studied laboratory colony. The representative of this morphotype was identified as Streptomyces globisporus subsp. globisporus 4-3 by a polyphasic approach. It was established using a E. coli JW5503 pDualRep2 system that crude broths of mycelial isolates inhibited protein synthesis in reporter strains, but it did not disrupt the in vitro synthesis of proteins in cell-free extracts. An active compound was extracted, purified and identified as albomycin δ2. The pronounced ability of albomycin to inhibit the growth of entomopathogens suggests that Streptomyces globisporus subsp. globisporus may be involved in defensive symbiosis with the Messor structor ant against infections

Biological evaluation and spectral characterization of novel tetracenomycin X congener

The aromatic polyketide tetracenomycin X (TcmX) was recently found to be a potent inhibitor of protein synthesis, whose binding site is located in a unique locus within the tunnel of the large ribosomal subunit. The distinct mode of action makes this relatively narrow class of macrolides promising for drug development, in our quest to prevent the spread of drug resistant pathogens. Here we report the isolation and structure elucidation of novel natural tetracenomycin X congener – 6-hydroxytetraceonomycin X (6-OH-TcmX). In contrast to TcmX, 6-OH-TcmX exhibited lower antimicrobial and cytotoxic activity, but comparable in vitro protein synthesis inhibition ability. A survey on spectral properties of tetracenomycins showed profound differences in both UV-absorption and fluorescence spectra of TcmX and 6-OH-TcmX, suggesting the significant influence of 6-hydroxylation on tetracenomycin chromophore. Nonetheless, characteristic spectral properties of tetracenomycins make them suitable candidates as a foundation for semi-synthetic drug development (e.g., for targeted delivery, theranostics or cell imaging)

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL—CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome—PTC and peptide exit tunnel

Mechanism-Based Approach to New Antibiotic Producers Screening among Actinomycetes in the Course of the Citizen Science Project

Since the discovery of streptomycin, actinomycetes have been a useful source for new antibiotics, but there have been diminishing rates of new finds since the 1960s. The decreasing probability of identifying new active agents led to reduced interest in soil bacteria as a source for new antibiotics. At the same time, actinomycetes remain a promising reservoir for new active molecules. In this work, we present several reporter plasmids encoding visible fluorescent protein genes. These plasmids provide primary information about the action mechanism of antimicrobial agents at an early stage of screening. The reporters and the pipeline described have been optimized and designed to employ citizen scientists without specialized skills or equipment with the aim of essentially crowdsourcing the search for new antibiotic producers in the vast natural reservoir of soil bacteria. The combination of mechanism-based approaches and citizen science has proved its effectiveness in practice, revealing a significant increase in the screening rate. As a proof of concept, two new strains, Streptomyces sp. KB-1 and BV113, were found to produce the antibiotics pikromycin and chartreusin, respectively, demonstrating the efficiency of the pipeline

Conjugates of Chloramphenicol Amine and Berberine as Antimicrobial Agents

In order to obtain antimicrobial compounds with improved properties, new conjugates comprising two different biologically active agents within a single chimeric molecule based on chloramphenicol (CHL) and a hydrophobic cation were synthesized and studied. Chloramphenicol amine (CAM), derived from the ribosome-targeting antibiotic CHL, and the plant isoquinoline alkaloid berberine (BER) are connected by alkyl linkers of different lengths in structures of these conjugates. Using competition binding, double reporter system, and toeprinting assays, we showed that synthesized CAM-Cn-BER compounds bound to the bacterial ribosome and inhibited protein synthesis like the parent CHL. The mechanism of action of CAM-C5-BER and CAM-C8-BER on the process of bacterial translations was similar to CHL. Experiments with bacteria demonstrated that CAM-Cn-BERs suppressed the growth of laboratory strains of CHL and macrolides-resistant bacteria. CAM-C8-BER acted against mycobacteria and more selectively inhibited the growth of Gram-positive bacteria than the parent CHL and the berberine derivative lacking the CAM moiety (CH3-C8-BER). Using a potential-sensitive fluorescent probe, we found that CAM-C8-BER significantly reduced the membrane potential in B. subtilis cells. Crystal violet assays were used to demonstrate the absence of induction of biofilm formation under the action of CAM-C8-BER on E. coli bacteria. Thus, we showed that CAM-C8-BER could act both on the ribosome and on the cell membrane of bacteria, with the alkylated berberine fragment of the compound making a significant contribution to the inhibitory effect on bacterial growth. Moreover, we showed that CAM-Cn-BERs did not inhibit eukaryotic translation in vitro and were non-toxic for eukaryotic cells