“Doubly Selective” Antimicrobial Polymers: How Do They Differentiate between Bacteria?

We have investigated how doubly selective synthetic mimics of antimicrobial peptides (SMAMPs), which can differentiate not only between bacteria and mammalian cells, but also between Gram‐negative and Gram‐positive bacteria, make the latter distinction. By dye‐leakage experiments on model vesicles and complementary experiments on bacteria, we were able to relate the Gram selectivity to structural differences of these bacteria types. We showed that the double membrane of E. coli rather than the difference in lipid composition between E. coli and S. aureus was responsible for Gram selectivity. The molecular‐weight‐dependent antimicrobial activity of the SMAMPs was shown to be a sieving effect: while the 3000 g mol^?1 SMAMP was able to penetrate the peptidoglycan layer of the Gram‐positive S. aureus bacteria, the 50000 g mol^?1 SMAMP got stuck and consequently did not have antimicrobial activity.     

Chemical Evolution of a Bacterial Proteome

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L ‐β‐(thieno[3,2‐b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L ‐tryptophan (Trp). A long‐term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp→[3,2]Tpa substitutions in their proteomes in response to the 20 899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new‐to‐nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.     

Fabrication of an Electrochemical E. coli Biosensor in Biowells Using Bimetallic Nanoparticle‐Labelled Antibodies

An electrochemical biosensor was developed for the determination of Escherichia coli (E. coli) in water. For this purpose, silver‐gold core‐shell (Ag@Au) bioconjugates and anti‐E. coli modified PS‐microwells were designed in a sandwich‐type format in order to obtain higher sensitivity and selectivity. Ag@Au bimetallic nanoparticles were synthesized by co‐reduction method. The core‐shell formation was analyzed by using UV‐Vis spectroscopy and transmission electron microscopy. Biotin labeled anti‐E. coli antibodies were coupled with Ag@Au nanoparticles to form bioconjugates. The electrochemical immunosensor was prepared by immobilizing anti‐E. coli on polystyrene (PS)‐microwells via chemical bonding. These modified microwells were identified with X‐ray photoelectron spectroscopy and surface enhanced Raman spectroscopy. E. coli was sandwiched between Ag@Au bioconjugates and anti‐E. coli on PS‐microwells at different concentrations. The relationship between the E. coli concentration and stripping current of gold ions (Au³⁺) were investigated by square wave anodic stripping voltammetry at pencil graphite electrode. The proposed method can provide some advantages such as lower detection limit and shorter detection time. The electrochemical response for the immunosensor was linear with the concentration of the E. coli in the range of 10¹ and 10⁵ cfu/mL with a limit of detection 3 cfu/mL. The procedure maintains good sensitivity and repeatability and also offers utility in the fields of environmental monitoring and clinical diagnosis.     

Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA

Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions. Primer extension reactions are commonly used for the assay of such reaction events. However, current assay protocols generally rely on radiolabeling, fluorescence reporter labeling, or removal of specific deoxynucleotide triphosphate in the reaction mixture. Herein we report on the design of two novel assay protocols that utilize a dideoxynucleotide‐terminated template strand and a phosphorothiolate‐modified deoxynucleotide‐terminated template strand. We designed and synthesized a deoxyuridine triphosphate analogue (dU*TP) containing 2‐bromoisobutyryl group and demonstrated that it could be well recognized by ?29DNA polymerase, E. coli DNA polymerase I Klenow Fragment, Bst DNA polymerase Large Fragment, and E. coli DNA polymerase I Klenow Fragment (exo⁽), which translated to effective incorporation of dU*TP into DNA. dU*TP was also successfully incorporated into extremely long single‐stranded DNA at high‐density using ?29 DNA polymerase by rolling circle amplification.     

A Multi‐component All‐DNA Biosensing System Controlled by a DNAzyme

We report on a programmable all‐DNA biosensing system that centers on the use of a 4‐way junction (4WJ) to transduce a DNAzyme reaction into an amplified signal output. A target acts as a primary input to activate an RNA‐cleaving DNAzyme, which then cleaves an RNA‐containing DNA substrate that is designed to be a component of a 4WJ. The formation of the 4WJ controls the release of a DNA output that becomes an input to initiate catalytic hairpin assembly (CHA), which produces a second DNA output that controls assembly of a split G‐quadruplex as a fluorescence signal generator. The 4WJ can be configured to produce either a turn‐off or turn‐on switch to control the degree of CHA, allowing target concentration to be determined in a quantitative manner. We demonstrate this approach by creating a sensor for E. coli that could detect as low as 50 E. coli cells mL^?1 within 85 min and offers an amplified bacterial detection method that does not require a protein enzyme.     

Antimicrobial peptide modification of biomaterials using supramolecular additives

Biomaterials based on non‐active polymers functionalized with antimicrobial agents by covalent modification or mixing are currently regarded as high potential solutions to prevent biomaterial associated infections that are major causes of biomedical device failure. Herewith a strategy is proposed in which antimicrobial materials are prepared by simply mixing‐and‐matching of ureido‐pyrimidinone (UPy) based supramolecular polymers with antimicrobial peptides (AMPs) modified with the same UPy‐moiety. The N‐terminus of the AMPs was coupled in solution to an UPy‐carboxylic acid synthon resulting in formation of a new amidic bond. The UPy‐functionalization of the AMPs did not affect their secondary structure, as proved by circular dichroism spectroscopy. The antimicrobial activity of the UPy‐AMPs in solution was also retained. In addition, the incorporation of UPy‐AMPs into an UPy‐polymer was stable and the final material was biocompatible. The addition of 4 mol % of UPy‐AMPs in the UPy‐polymer material protected against colonization by Escherichia coli, and methicillin‐sensitive and ‐resistant strains of Staphylococcus aureus. This modular approach enables a stable but dynamic incorporation of the antimicrobial agents, allowing at the same time for the possibility to change the nature of the polymer, as well as the use of AMPs with different activity spectra. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 1926–1934     

A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.     

Synthesis,Characterization, and Repair of a Flexible O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine Intrastrand Cross‐Link

Oligonucleotides tethered by an alkylene linkage between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross‐linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene‐ and heptylene‐linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B‐form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O⁶‐alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada‐C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair.     

Amide Moieties Modulate the Antimicrobial Activities of Conjugated Oligoelectrolytes against Gram-negative Bacteria

Cationic conjugated oligoelectrolytes (COEs) are a class of compounds that can be tailored to achieve relevant in vitro antimicrobial properties with relatively low cytotoxicity against mammalian cells. Three distyrylbenzene-based COEs were designed containing amide functional groups on the side chains. Their properties were compared to two representative COEs with only quaternary ammonium groups. The optimal compound, COE2−3C−C3-Apropyl , has an antimicrobial efficacy against Escherichia coli with an MIC=2 μg mL⁻¹, even in the presence of human serum albumin low cytotoxicity (IC₅₀=740 μg mL⁻¹) and minimal hemolytic activity. Moreover, we find that amide groups increase interactions between COEs and a bacterial lipid mimic based on calcein leakage assay and allow COEs to readily permeabilize the cytoplasmic membrane of E. coli. These findings suggest that hydrogen bond forming moieties can be further applied in the molecular design of antimicrobial COEs to further improve their selectivity towards bacteria.     

Biomimetic design of amphiphilic polycations and surface grafting onto polycarbonate urethane film as effective antibacterial agents with controlled hemocompatibility

The synthetic polycations are ideal candidates as antimicrobial agents, because they resemble natural antimicrobial peptides, but to render hemocompatibility to these materials is a great challenge. Herein, we used 2‐(tert‐butyl‐aminoethyl) methacrylate (TBAEMA), to synthesize its homopolymer and pegylated random and diblock copolymers with polyethyleneglycol methacrylate (PEGMA, M_n = 360 Da) by single‐electron transfer–living radical polymerization (SET‐LRP). In the second step, the secondary amino groups in the precursor polymers were quaternized with iodomethane and bromohexane, to obtain three series of quaternized polymers. The antimicrobial properties of these quaternized polymers were evaluated against Escherichia coli (E. coli), by studying the minimum inhibitory concentrations (MICs) which ranged between 32 and 200 mg L^?1 and showed higher values for the quaternized random than the diblock copolymers. In addition to, we have also demonstrated the grafting of these polycations onto polycarbonate urethane film surfaces, which showed good killing efficacy against E. coli. Furthermore, the hemolysis of these materials was investigated against human red blood cells, which indicated that except the quaternized homopolymers that showed highest hemolysis, all other amphiphilic polycations exhibited very low hemolytic activity. Therefore, our designed materials with controlled structures and functionality, synthesized from cheaply available resources could serve as useful agents in the field of biomedicines and implantable materials. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3166–3176     

两种希夫碱对大肠杆菌抗菌活性的微量热法研究

The microcalorimetric method was used to study the antibacterial activity of two newly synthesized Schiff base compounds （H2L3＇ and H2L3） on Escherichia coli, trying to obtain the action on both of multiplying bacteria and non-multiplying bacteria at one experiment. The metabolic power-time curves of the bacteria treated with the compounds were obtained, and the thermokinetic parameters were analyzed, from which the antibacterial activities of these compounds were evaluated. The results showed that both of the two compounds have good activity on aerobic multiplying metabolism of E. coli, with the value of ICso 75.8 and 168.8 mg/L respectively, but have not effective action on fermentation metabolism of E. coli. The action of the compounds on the non-multiplying metabolism was investigated by taking the heat output of E. coli in the stationary phase as the guideline of the activity. The value of MSCso （minimum stationary-cidal concentration 50） of them is 118 and 187.5 mg/L, respectively. So, H2L^3 has stronger antibacterial action on E. coli than H2L^3 either for multiplying bacteria or non-multiplying bacteria, and their activity on the aerobic multiplying bacteria of E. coil is mainly shown. It does strongly suggest that the calorimetric method should play an important role in the fight against the drug-resistant bacteria.     

EFFECTS OF ACRIDINE PLUS NEAR ULTRAVIOLET LIGHT ON ESCHERICHIA COLI MEMBRANES AND DNA IN VIVO

Results from a variety of experiments indicate that photodynamic damage to E. coli treated with the hydrophobic photosensitizer acridine plus near-UV light involves both cell membranes and DNA. Split-dose survival experiments with various E. coli mutants reveal that cells defective in rec A, uvr A, or pol A functions are all capable of recovery from photodynamic damage. Alkaline sucrose gradient analysis of DNA from control and treated cells revealed that acridine plus near-UV light treatment converts normal DNA into a more slowly sedimenting form. However, the normal DNA sedimentation properties are not restored under conditions where split-dose recovery is effective. Several lines of evidence suggest that membrane damage may be important in the inactivation of cells by acridine plus near-UV light. These include (a) a strong dependence of sensitivity on the fatty acid composition of the membranes; (b) a strong dependence of sensitivity on the osmolarity of the external medium; and (c) the extreme sensitivity of an E. coli mutant having a defect in its outer membrane barrier properties. Direct evidence that acridine plus near-UV light damages cell membranes was provided by the observations that (a) the plasma membrane becomes permeable to o-nitrophenyl-ß-D-galactopyranoside and (b) the outer membrane becomes permeable to lysozyme after treatment. A notable result was that cells previously sensitized to lysozyme by exposure to acridine plus near-UV light lose that sensitivity upon subsequent incubation. This strongly suggests that E. coli cells are capable of repairing damage localized in the outer membrane.     

Synthesis and Antimicrobial Evaluation of Novel Polyfused Heterocycles‐Based Quinolone

Syntheses of some new heterocyclic compounds incorporating quinolone moieties were achieved via reaction of 4‐hydroxy‐7‐methoxyquinolin‐2(1H)‐one ( 1 ) or 3‐bromo‐4‐hydroxy‐7‐methoxyquinolin‐2(1H)‐one ( 2 ) with binucleophilic reagents. The newly synthesized compounds were characterized by elemental analyses and spectral data (IR, ¹H‐NMR and mass spectra). The newly synthesized compounds were screened for their antibacterial activity against Gram‐positive bacteria (Bacillus thuringiensis) and Gram‐negative bacteria (Escherichia coli). The results showed clearly that compounds 1 and 3 are the more potent antibacterial agents against E. coli, compounds 4 , 5 , 6 and 8 , 9 , 10 , 11 , 12 , 13 exhibited moderate activities against E. coli strain, and compounds 7 and 11 exhibited weak activities compared with Gentamicin as a well known standard drug.     

Antibacterial Activities of Tellurium Nanomaterials

We prepared four differently shaped Te nanomaterials (NMs) as antibacterial reagents against Escherichia coli. By controlling the concentrations of hydrazine (N₂H₄) as reducing agent, NaCl, and temperature, we prepared Te nanowires, nanopencils, nanorices, and nanocubes. These four Te NMs resulted in a live/dead ratio of E. coli cells of less than 0.1, which is smaller than that of Ag nanoparticles. The order of antibacterial activity against E. coli is nanocubes ≈ nanorices > nanopencils ≈ nanowires. This is in good agreement with the concentration order of tellurite (TeO₃²⁻) ions released from Te NMs in E. coli cells, revealing that TeO₃²⁻ ions account for the antibacterial activity of the four Te NMs. We found that spherical Te nanoparticles (32 nm in diameter) with TeO₃²⁻ ions were formed in the E. coli cells. Compared to Ag nanoparticles that are commonly used as antibacterial reagents, Te NMs have higher antibacterial activity and lower toxicity. Thus, Te NMs hold great practical potential as a new and efficient antibacterial agent.     

UV mutagenic photoproducts in Escherichia coli and human cells: a molecular genetics perspective on human skin cancer

Abstract— The relevance of photoproducts produced by 254 nm irradiation to human skin cancer is first critically evaluated. Experiments identifying the mutagenic photoproducts at 254 nm are then described. Mutations are primarily due to the(6–4) photoproduct and the cyclobutane pyrimidine dimer, both in E. coli and in human cells. The(6–4) photoproduct may be more important in E. coli and the cyclobutane dimer more important in mammalian cells. In human cells, mutations occur at the C of a TC, CT, or CC cyclobutane dimer, but not at TT cyclobutane dimers, and also appear to occur, less frequently, at the C of TC and CC(6–4) photoproducts. The local structure of DNA is more important in determining the frequency of mutation at a site than is the photoproduct frequency at that site. The effect of DNA structure appears to be due to site-specific lethality.     

Informed Molecular Design of Conjugated Oligoelectrolytes To Increase Cell Affinity and Antimicrobial Activity

Membrane‐intercalating conjugated oligoelectrolytes (COEs) are emerging as potential alternatives to conventional, yet increasingly ineffective, antibiotics. Three readily accessible COEs, belonging to an unreported series containing a stilbene core, namely D4 , D6 , and D8 , were designed and synthesized so that the hydrophobicity increases with increasing side‐chain length. Decreased aqueous solubility correlates with increased uptake by E. coli. The minimum inhibitory concentration (MIC) of D8 is 4 μg mL^?1 against both E. coli and E. faecalis, with an effective uptake of 72 %. In contrast, the MIC value of the shortest COE, D4 , is 128 μg mL^?1 owing to the low cellular uptake of 3 %. These findings demonstrate the application of rational design to generate efficacious antimicrobial COEs that have potential as low‐cost antimicrobial agents.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

Direct demonstration of the monomerization of thymine-containing dimers in U.V.-irradiated DNA by yeast photoreactivating enzyme and light

Abstract— Ultraviolet-irradiated E. coli DNA (³H-thymine-labelled) was mixed with un-irradiated E. coli DNA (¹⁴C-thymine-labelled) and exposed to light in the presence of purified yeast photoreactivating enzyme. As the ³H-thymine-containing cyclobutane dimers disappeared during the photoreactivation, there was a stoichiometric increase of monomeric ³H-thymine as determined from the ³H/¹⁴C ratio in thymine. This is the first direct demonstration that thymine-containing dimers in u.v.-irradiated DNA are monomerized by yeast photoreactivating enzyme in the presence of light.     

DNA polymerase I-mediated repair of 365 nm-induced single-strand breaks in the DNA of Escherichia coli.

Abstract. Irradiation of closed circular phage Λ DNA in vivo at 365 nm results in the induction of single-strand breaks and alkali-labile lesions at rates of 1.1 × 10^-14, and 0.2 × 10^-14/dalton/J/m², respectively. The sum of the induction rates is similar to the rate of induction of single-strand breaks plus alkali-labile lesions (1 × 10^-14/dalton/J/m²) observed in the E. coli genome. Postirradiation incubation of wild-type cells in buffer results in rapid repair of the breaks (up to 80% repaired in 10 min). No repair was observed in a DNA polymerase I-deficient mutant of E. coli.     

Efficient and Selective Carboligation with Whole‐Cell Biocatalysts in Pickering Emulsion

Pickering emulsions (PEs) are particle‐stabilized multiphase systems with promising features for synthetic applications. Described here is a novel, simplified set‐up employing catalytically active whole cells for simultaneous emulsion stabilization and synthetic reaction. In the stereoselective carboligation of benzaldehyde to (R)‐benzoin catalyzed by a benzaldehyde lyase in E. coli, the set‐up yielded maximum substrate conversion within very short time, while economizing material demand and waste. Formation and activity of freshly produced PEs were enhanced when the catalytic whole cells were covered with hydrophobic silicone prior to PE formation. Benchmarked against other easy‐to‐handle whole‐cell biocatalysts in pure organic solvent, neat substrate, an aqueous emulsion in substrate, and a micro‐aquatic system, respectively, the cell‐stabilized PE outperformed all other systems by far.