A Novel N-Halamine Biocidal Nanofibrous Membrane for Chlorine Rechargeable Rapid Water Disinfection Applications.

Disinfecting pathogenic contaminated water rapidly and effectively on sites is one of the critical challenges at point-of-use (POU) situations. Currently available technologies are still suffering from irreversible depletion of disinfectants, generation of toxic by-products, and potential biofouling problems. Herein, we developed a chlorine rechargeable biocidal nanofibrous membrane, poly(acrylonitrile-co-5-methyl-5-(4'-vinylphenyl)imidazolidine-2,4-dione) (P(AN-VAPH)), via a combination of a free radical copolymerization reaction and electrospun technology. The copolymer exhibits good electrospinnability and desirable mechanical properties. Also, the 5-methyl-5-(4'-vinylphenyl)imidazolidine-2,4-dione (VAPH) moieties containing unique hydantoin structures are able to be chlorinated and converted to halamine structures, enabling the P(AN-VAPH) nanofibrous membrane with rapid and durable biocidal activity. The chlorinated P(AN-VAPH) nanofibrous membranes showed intriguing features of unique 3D morphological structures with large specific surface area, good mechanical performance, rechargeable chlorination capacity (>5000 ppm), long-term durability, and desirable biocidal activity against both bacteria and viruses (>99.9999% within 2 min of contact). With these attributes, the chlorinated P(AN-VAPH) membranes demonstrated promising disinfecting efficiency against concentrated bacteria-contaminated water during direct filtration applications with superior killing capacity and high flowing flux (5000 L m-2 h-1).

Daylight-Induced Antibacterial and Antiviral Nanofibrous Membranes Containing Vitamin K Derivatives for Personal Protective Equipment.

During the development of antibacterial and antiviral materials for personal protective equipment (PPE), daylight active functional polymeric materials containing vitamin K compounds (VKs) and impacts of polymer structures to the functions were investigated. As examples, hydrophobic polyacrylonitrile (PAN) and hydrophilic poly(vinyl alcohol-co-ethylene) (PVA-co-PE) polymers were directly blended with three VK compounds and electrospun into VK-containing nanofibrous membranes (VNFMs). The prepared VNFMs exhibited robust photoactivity in generating reactive oxygen species (ROS) under both daylight (D65, 300-800 nm) and ultraviolet A (UVA, 365 nm) irradiation, resulting in high antimicrobial and antiviral efficiency (>99.9%) within a short exposure time (<90 min). Interestingly, the PVA-co-PE/VK3 VNFM showed higher ROS production rates and better biocidal functions than those of the PAN/VK3 VNFM under the same photoirradiation conditions, indicating that PVA-co-PE is a better matrix polymer material for these functions. Moreover, the prepared PVA-co-PE/VK3 VNFM maintains its powerful microbicidal function even after five times of repeated exposures to bacteria and viruses, showing the stability and reusability of the antimicrobial materials. The fabrication of photoinduced antimicrobial VNFMs may provide new insights into the development of non-toxic and reusable photoinduced antimicrobial materials that could be applied in personal protective equipment with improved biological protections.

Identifying the cellular targets of natural products using T7 phage display.

Covering: up to the end of 2015While Nature continues to deliver a myriad of potent and structurally diverse biologically active small molecules, the cellular targets and modes of action of these natural products are rarely identified, significantly hindering their development as new chemotherapeutic agents. This article provides an introductory tutorial on the use of T7 phage display as a tool to rapidly identify the cellular targets of natural products and is aimed specifically at natural products chemists who may have only limited experience in molecular biology. A brief overview of T7 phage display is provided, including its strengths, weaknesses, and the type of problems that can and cannot be tackled with this technology. Affinity probe construction is reviewed, including linker design and natural product derivatisation strategies. A detailed description of the T7 phage biopanning procedure is provided, with valuable tips for optimising each step in the process, as well as advice for identifying and avoiding the most commonly encountered challenges and pitfalls along the way. Finally, a brief discussion is provided on techniques for validating the cellular targets identified using T7 phage display.

Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine.

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.

Evolution at a high imposed mutation rate: adaptation obscures the load in phage T7.

Evolution at high mutation rates is expected to reduce population fitness deterministically by the accumulation of deleterious mutations. A high enough rate should even cause extinction (lethal mutagenesis), a principle motivating the clinical use of mutagenic drugs to treat viral infections. The impact of a high mutation rate on long-term viral fitness was tested here. A large population of the DNA bacteriophage T7 was grown with a mutagen, producing a genomic rate of 4 nonlethal mutations per generation, two to three orders of magnitude above the baseline rate. Fitness-viral growth rate in the mutagenic environment-was predicted to decline substantially; after 200 generations, fitness had increased, rejecting the model. A high mutation load was nonetheless evident from (i) many low- to moderate-frequency mutations in the population (averaging 245 per genome) and (ii) an 80% drop in average burst size. Twenty-eight mutations reached high frequency and were thus presumably adaptive, clustered mostly in DNA metabolism genes, chiefly DNA polymerase. Yet blocking DNA polymerase evolution failed to yield a fitness decrease after 100 generations. Although mutagenic drugs have caused viral extinction in vitro under some conditions, this study is the first to match theory and fitness evolution at a high mutation rate. Failure of the theory challenges the quantitative basis of lethal mutagenesis and highlights the potential for adaptive evolution at high mutation rates.

Viral resistance evolution fully escapes a rationally designed lethal inhibitor.

Viruses are notoriously capable of evolving resistance to drugs. However, if the endpoint of resistance evolution is only partial escape, a feasible strategy should be to stack drugs, so the combined effect of partial inhibition by several drugs results in net inhibition. Assessing the feasibility of this approach requires quantitative data on viral fitness before and after evolution of resistance to a drug, as done here with bacteriophage T7. An inhibitory gene expressed from a phage promoter aborts wild-type T7 infections. The effect is so severe that the phage population declines when exposed to the inhibitor but expands a billion-fold per hour in its absence. In prior work, T7 evolved modest resistance to this inhibitor, an expected result. Given the nature of the inhibitor, that it used the phage's own promoter to target the phage's destruction, we anticipated that resistance evolution would be limited as the phage may need to evolve a new regulatory system, with simultaneous changes in its RNA polymerase (RNAP) and many of its promoters to fully escape inhibition. We show here that further adaptation of the partially resistant phage led to complete resistance. Resistance evolution was due to three mutations in the RNAP gene and two other genes; unexpectedly, no changes were observed in promoters. Consideration of other mechanisms of T7 inhibition leaves hope that permanent inhibition of viral growth with drugs can in principle be achieved.

DNA recognition by the DNA primase of bacteriophage T7: a structure-function study of the zinc-binding domain.

Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.

Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation.

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4-N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.

Near-ultraviolet photolysis of beta-phenylpyruvic acid generates free radicals and results in DNA damage.

Ultraviolet A (UVA) light (315-400 nm) is ubiquitously found in our environment and constitutes about 95% of the total solar UV; all UVC and most UVB being absorbed by the stratospheric ozone layer. Compared with UVB and C, UVA does not show any direct effect on biological systems. Indirect effects of UVA, however, have been recognised overwhelmingly and this includes photosensitization of biological and non-biological compounds and production of free radicals many of which include oxygen and are hence known as reactive oxygen species or ROS. Several types of free radicals have been identified although their impacts on various macro- and micro-biomolecules are yet to be fully elucidated. beta-Phenylpyruvic acid is ubiquitously found in eukaryotic cells as a metabolite of phenylalanine, which is subsequently converted to phenyllactate and/or to 2-hydroxyphenylacetate and mandelate. In patients suffering from phenylketonuria the hydroxylation of phenylalanine to tyrosine is defective due to lack of phenylalanine hydroxylase. These result in accumulation and excretion of this compound in the urine. Here we present evidence that photolysis of beta-phenylpyruvic acid by a skin tanning lamp, emitting 99% UVA (315-400 nm) and 1% UVB (290-315 nm) generates carboxyl radicals (CO(2)(*)) and also possibly causes direct electron transfer (or type 1) reactions. Electron paramagnetic resonance was used to detect the free radicals. To determine the biological effects of this photolytic reaction, T7 was exposed to these photolytic reactive agents and found to lead to high levels of phage inactivation. Damage to DNA and/or components such as tail fibre proteins may be involved in T7 inactivation. In addition, our unpublished data suggest that certain phenylketonuria cell lines are more sensitive to PPA+NUV, lending importance to photolytic studies of this agent.

Photosensitized inactivation of T7 phage as surrogate of non-enveloped DNA viruses: efficiency and mechanism of action.

We investigated the efficiency and the mechanism of action of a tetraphenyl porphyrin derivative in its photoreaction with T7 phage as surrogate of non-enveloped DNA viruses. TPFP was able to sensitize the photoinactivation of T7 phage in spite of the lack of its binding to the nucleoprotein complex. The efficiency of TPFP photosensitization was limited by the aggregation and by the photobleaching of porphyrin molecules. Addition of sodium azide or 1,3-dimethyl-2-thiourea (DMTU) to the reaction mixture moderated T7 inactivation, however, neither of them inhibited T7 inactivation completely. This result suggests that both Type I and Type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated nucleoprotein complex. Polymerase chain reaction (PCR) also failed to demonstrate any DNA damage. Circular dichroism (CD) spectra of photosensitized nucleoprotein complex indicated changes in the secondary structure of both the DNA and proteins. We suggest that damages in the protein capsid and/or loosening of protein-DNA interaction can be responsible for the photodynamic inactivation of T7 phage. The alterations in DNA secondary structure might be the result of photochemical damage in phage capsid proteins.

[Photodynamic inactivation of porphyrin sensitized T7 phages: efficiency and mechanism of action]. / Porfirin származékok alkalmazása T7 fág fotodinamikus inaktivációjában: a reakció hatékonysága és mechanizmusa.

We investigated the efficiency and the mechanism of action of two glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 bacteriophage. Both types of porphyrins sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different. Our result suggests that both type I and type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photo-chemically treated nucleoprotein complex. Polymerase chain reaction (PCR) analysis failed to demonstrate any DNA damage.

Stringent regulation and high-level expression of heterologous genes in Escherichia coli using T7 system controllable by the araBAD promoter.

The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to induce protein production when cells entered the late log growth phase. As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337-fold increase over that achieved on a shake-flask scale. Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.

High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system.

The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system. We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter. As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock. In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C. Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case. Using this approach, the recombinant protein accounted for 20% of total protein content of the cell. These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability. In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow. By applying the two-step temperature change. a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained. This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale. Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.

Photoinduced inactivation of T7 phage sensitized by symmetrically and asymmetrically substituted tetraphenyl porphyrin: comparison of efficiency and mechanism of action.

We investigated the efficiency and the mechanism of action of two--one symmetrically and one asymmetrically substituted--glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 phage as a model of nucleoprotein (NP) complexes. A correlation was found between the dark inactivation of T7 and the binding of porphyrins determined by fluorescence spectroscopy. Both types of porphyrin sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different. There was no correlation between the dark binding and the photosensitizing efficacy of the two derivatives. Inactivation was moderated by 1,3-diphenylisobenzofuran and 1,3-dimethyl-2-thiourea; however, neither of them inhibited T7 inactivation completely. This result suggests that both Type-I and Type-II reactions play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated NP complex. Polymerase chain reaction analysis of a 555 bp segment of gene 1 and a 3826 bp segment of genes 3 and 4 failed to demonstrate any DNA damage.

Photochemical and photobiological properties of new bispsoralen derivatives (Bis[PsCn]PIP, n = 4, 6, 8).

Bispsoralen derivatives possessing two psoralens and one piperazine molecule, 1,4-bis[n'-(8-psoralenoxy) alkyl] piperazine (Bis[PsCn]PIP, n = 4, 6, 8), show high water solubility, efficient intercalation into DNA and good photocrosslinking efficiency of DNA. Bis(PsC4)PIP shows high lethality on bacteriophage T7 and can effectively inhibit the amplification of DNA by stopping the polymerase chain reactions in a short period of irradiation time.

[Effect of chitosan derivatives on the reproduction of Coliphages T2 and T7]. / Vliianie proizvodnykh khitozana na reproduktsiiu kolifagov T2 i T7.

The effect of chitosan derivatives with different degrees of polymerization and deamination, as well as of chitosan 6-O-sulfate and chitosan N-succinate-6-O-sulfate, on the reproduction of coliphages T2 and T7 in Escherichia coli and on the growth of this bacterium was studied. Chitosan derivatives decreased the yield of coliphages and exhibited bactericidal activity. The efficiency of inhibition of viral infection and the bactericidal activity of chitosan were found to be dependent on the degree of its polymerization. At the same time, there was no correlation between the degree of chitosan deamination and the extent of inhibition of viral infection. Anionic chitosan derivatives virtually did not possess antiviral or bactericidal activity. It is assumed that chitosan blocks some stages of phage reproduction. The decrease in the phage-producing ability of E. coli may also be due to the bactericidal effect of chitosan.

Toward antiviral strategies that resist viral escape.

We studied the effect on viral growth of drugs targeting different virus functions using a computer simulation for the intracellular growth of bacteriophage T7. We found that drugs targeting components of negative-feedback loops gain effectiveness against mutant viruses that attenuate the drug-target interaction. The greater inhibition of such mutants than of the wild type suggests a drug design strategy that would hinder the development of drug resistance.

Generation of reactive oxygen species from the photolysis of histidine by near-ultraviolet light: effects on T7 as a model biological system.

Near-ultraviolet (NUV) light (280-400 nm) has a variety of effects on biological systems; these effects are increased, often synergistically, in the presence of sensitizers. A variety of both man-made and naturally occurring sensitizers have been identified, but their precise roles and relative contributions to cellular damage are not yet fully established. DNA seems to be a major target and a variety of types of damage have been observed. In this report we present evidence that histidine can also act as a sensitizer of NUV. Upon NUV photolysis a variety of reactive oxygen species, including superoxide anions, hydroxyl radicals and hydrogen peroxide, are produced as determined by the effects of various scavengers. pH influences the reaction, alkaline media being most effective, as has previously been reported for the photolysis of H2O2, tyrosine, phenylalanine and tryptophan. Exposure of phage T7 to a combination of histidine and NUV leads to synergistic inactivation and scavengers of O2.-, .OH and H2O2 reduce this effect. These results point to a possible involvement of sunlight-induced histidine photolysis in cellular damage. The fact that photolysis is maximal at high pH indicates that biological effects are likely to be highly localized, e.g., at enzyme active sites.

On the mechanism of inhibition of phage T7 RNA polymerase by lac repressor.

We study here the effect on phage T7 RNA polymerase activity of lac repressor bound downstream of the T7 promoter. When repressor binds in vitro at an operator centered at +13 or +15 with respect to transcription start, it does not prevent initiation, though the transcript yield is reduced. However, the processivity of the polymerase is depressed and transcript extension is blocked at positions +4 and +6, respectively. These results indicate that repressor and polymerase do not simply exclude each other from the promoter. Rather, they would come into steric conflict and compete for establishment or retention of interactions with the same segment of DNA, without this leading to the immediate displacement of either polymerase or repressor. The resulting destabilization of the transcription complex would depress both initiation rate and enzyme processivity. In contrast to the above results, little reduction in runoff transcription is observed when operator is centered at +47. The decreased sensitivity of polymerase to repressor bound at +47 versus +13 or +15 is likely to be due to the higher stability of the elongation complex during the transcription of downstream regions in comparison with the first transcribed nucleotides. We also show that under conditions of leaky repression and with operator centered at +13, a mutant T7 RNA polymerase showing normal promoter affinity but a slower elongation rate is more sensitive to repression than the wild-type enzyme, both in vitro and in vivo. In vitro, this higher sensitivity is largely due to a reduced ability of the mutant to overcome the elongation block at position +4. The parallel between the in vitro and in vivo data suggests that in vivo the repressor also does not prevent polymerase from binding to promoter, but interferes with subsequent steps in initiation and transcript extension, in this case presumably largely extension beyond +4.