Probability of double-strand breaks in genome-sized DNA by γ-ray decreases markedly as the DNA concentration increases.

By use of the single-molecule observation, we count the number of DNA double-strand breaks caused by γ-ray irradiation with genome-sized DNA molecules (166 kbp). We find that P1, the number of double-strand breaks (DSBs) per base pair per unit Gy, is nearly inversely proportional to the DNA concentration above a certain threshold DNA concentration. The inverse relationship implies that the total number of DSBs remains essentially constant. We give a theoretical interpretation of our experimental results in terms of attack of reactive species upon DNA molecules, indicating the significance of the characteristics of genome-sized giant DNA as semiflexible polymers for the efficiency of DSBs.

Involvement of type I and type II mechanisms on the photoinactivation of non-enveloped DNA and RNA bacteriophages.

Microbial photodynamic inactivation (PDI), involving the use of a photosensitizer (PS), light and molecular oxygen, with the subsequent production of reactive oxygen species (ROS), has been considered a promising and effective technology for viral inactivation. Although singlet oxygen is generally accepted as the main damaging species in PDI, ROS like free radicals may also be involved in the process, inducing damages to proteins, lipids, nucleic acids and other molecular structures. In this study, the relative importance of each mechanism (type I and type II) on the photoinactivation of non-enveloped DNA (T4-like phage) and RNA (Qß phage) viruses was evaluated. For this purpose, two cationic porphyrins (Tri-Py(+)-Me-PF and Tetra-Py(+)-Me) and four different ROS scavengers were used. The scavenging effect of sodium azide and L-histidine (singlet oxygen quenchers) and of D-mannitol and L-cysteine (free radical scavengers) was assessed by exposure of both phages (T4-like and Qß) to each cationic porphyrin (5.0µM for T4-like phage and 0.5µM for Qß phage) and white light (40Wm(-2)) in the presence of different concentrations of the scavengers (5, 10, 50 and 100mM). Sodium azide and L-histidine gave the best protection, reducing the phototoxic effect of Tri-Py(+)-Me-PF on T4-like phage respectively by 80% and 72% and in the presence of Tetra-Py(+)-Me by 90% and 78%. Free radical scavengers D-mannitol and L-cysteine did not significantly reduce the rate of T4-like phage photoinactivation (around 20% protection, for both PS). The sodium azide protection on Qß phage photoinactivation, in the presence of Tri-Py(+)-Me-PF, was lower (39%) when compared with T4-like phage. D-mannitol did not exert on Qß phage any protective effect after 90min of irradiation. The effect of the simultaneous presence of singlet oxygen and free radicals scavengers at 100mM confirmed that singlet oxygen (type II mechanism) is clearly the main ROS involved in T4-like and Qß phages photoinactivation by these two cationic PS. As RNA-type phages are more easily photoinactivated when compared with DNA-type ones, the protection conferred by the scavengers during the PDI process is lower and this should be taken into account when the main mechanism involved in PDI of different viruses is to be studied.

Photocatalytic inactivation of bacteriophages by TiO2-coated glass plates under low-intensity, long-wavelength UV irradiation.

The International Organization for Standardization (ISO) was used to evaluate antibacterial activity by titanium dioxide (TiO(2)) photocatalysis since 2006. We evaluated photocatalytic inactivation of Qß and T4 bacteriophages induced by low-intensity, long-wavelength ultraviolet A (UVA; 0.1 mW cm(-2) and 0.001 mW cm(-2)) irradiation on a TiO(2)-coated glass plate using the ISO methodology. The results indicated that both bacteriophages were inactivated at 0.001 mW cm(-2) UVA. The intensity of UV light, including long-wavelength light (UVA), is very low in an actual indoor environment. Thus, TiO(2) photocatalysis can be beneficial for inactivating viruses in an indoor environment. Experiments using qPCR and bovine serum albumin degradation assume that viral inactivation is caused by outer viral protein disorder and not by viral RNA reduction by reactive oxygen species produced during TiO(2) photocatalysis. Furthermore, we showed that the ISO methodology for standard testing of antibacterial activity by TiO(2) photocatalysis can be applied to assess antiviral activity.

Functional cationic nanomagnet-porphyrin hybrids for the photoinactivation of microorganisms.

Cationic nanomagnet-porphyrin hybrids were synthesized and their photodynamic therapy capabilities were investigated against the Gram (-) Escherichia coli bacteria, the Gram (+) Enterococcus faecalis bacteria and T4-like phage. The synthesis, structural characterization, photophysical properties, and antimicrobial activity of these new materials are discussed. The results show that these new multicharged nanomagnet-porphyrin hybrids are very stable in water and highly effective in the photoinactivation of bacteria and phages. Their remarkable antimicrobial activity, associated with their easy recovery, just by applying a magnetic field, makes these materials novel photosensitizers for water or wastewater disinfection.

Photocatalytic treatment of bioaerosols: impact of the reactor design.

Comparing the UV-A photocatalytic treatment of bioaerosols contaminated with different airborne microorganisms such as L. pneumophila bacteria, T2 bacteriophage viruses and B. atrophaeus bacterial spores, pointed out a decontamination sensitivity following the bacteria > virus > bacterial spore ranking order, differing from that obtained for liquid-phase or surface UV-A photocatalytic disinfection. First-principles CFD investigation applied to a model annular photoreactor evidenced that larger the microorganism size, higher the hit probability with the photocatalytic surfaces. Applied to a commercial photocatalytic purifier case-study, the CFD calculations showed that the performances of the studied purifier could strongly benefit from rational reactor design engineering. The results obtained highlighted the required necessity to specifically investigate the removal of airborne microorganisms in terms of reactor design, and not to simply transpose the results obtained from studies performed toward chemical pollutants, especially for a successful commercial implementation of air decontamination photoreactors. This illustrated the importance of the aerodynamics in air decontamination, directly resulting from the microorganism morphology.

Potential to use ultraviolet-treated bacteriophages to control foodborne pathogens.

The use of replication-deficient UV-treated bacteriophages, or phages, presents an alternative to viable phages for food biocontrol applications. Nontransducing UV-treated phages, if used correctly, are unlikely to produce viable progeny phages, which might otherwise mediate undesirable horizontal gene transfer events. Phage T4 and Escherichia coli were used as a model system to examine this possibility. UV-treated phages were able to cause a reduction in the optical density of outer membrane-free cell suspensions and they also killed host cells under conditions not permitting their multiplication, that is, 24 degrees C for 2 h and 37 degrees C for 15 min. Host cell reductions were also demonstrated in broth and on meat at 5 degrees C when high concentrations of phages of 2.3 x 10(9) PFU mL(-1) and 1.8 x 10(8) PFU cm(-2), respectively, were used. At 24 degrees C and 37 degrees C, "lysis from without" was likely to be the mechanism responsible for the reduction in host cell concentrations, but at 5 degrees C this may not have been the case.

[Comparative study of the resistance of bacteriophageT4, PhiX174D, MS2 and f2 to gamma radiation].

OBJECTIVE: To screen the suitable bacteriophage as virus indicator in irradiation sterilization. METHODS: Suspensions of bacteriophage T4, phiX174D, MS2, and f2, Escherichia coli 8099, and Bacillus subtilis var.niger.sp. ATCC9372 were irradiated with (60)Co-gamma ray. The mean log(10) inactivation value (LIV) and killing log value (KL) were calculated. RESULTS: (1) Under 100 Gy of gamma-radiation, the LIV levels of the bacteriophage T4, PhiX174, f2, and MS2 were 6.31, 6.92, 5.74, and 4.46 log(10) respectively, all reaching the disinfection level (LIV >/= 4.00 log(10)), (2) Under the same absorbed dose, the KL of Escherichia coli 8099 was > 7.97 log(10); (3) Under the same absorbed dose, the KL of the Bacillus subtilis var.niger.sp. ATCC9372 was 1.61 log(10). CONCLUSION: The order of resistance of the above six microorganisms to gamma-radiation from the biggest to the smallest is as follows: Bacillus subtilis var. niger. sp. > bacteriophage MS2 > bacteriophage f2 > bacteriophage T4 > bacteriophage phiX 174D > E. coli.

Removal of particle-associated bacteriophages by dual-media filtration at different filter cycle stages and impacts on subsequent UV disinfection.

This bench-scale study investigated the passage of particle-associated bacteriophage through a dual-media (anthracite-sand) filter over a complete filter cycle and the effect on subsequent ultraviolet (UV) disinfection. Two model viruses, bacteriophages MS2 and T4, were considered. The water matrix was de-chlorinated tap water with either kaolin or Aldrich humic acid (AHA) added and coagulated with alum to form floc before filtration. The turbidity of the influent flocculated water was 6.4+/-1.5 NTU. Influent and filter effluent turbidity and particle counts were measured as well as headloss across the filter media. Filter effluent samples were collected for phage enumeration during three filter cycle stages: (i) filter ripening; (ii) stable operation; and (iii) end of filter cycle. Stable filter operation was defined according to a filter effluent turbidity goal of <0.3 NTU. Influent and filter effluent samples were subsequently exposed to UV light (254 nm) at 40 mJ/cm(2) using a low pressure UV collimated beam. The study found statistically significant differences (alpha=0.05) in the quantity of particle-associated phage present in the filter effluent during the three stages of filtration. There was reduced UV disinfection efficiency due to the presence of particle-associated phage in the filter effluent in trials with bacteriophage MS2 and humic acid floc. Unfiltered influent water samples also resulted in reduced UV inactivation of phage relative to particle-free control conditions for both phages. Trends in filter effluent turbidity corresponded with breakthrough of particle-associated phage in the filter effluent. The results therefore suggest that maintenance of optimum filtration conditions upstream of UV disinfection is a critical barrier to particle-associated viruses.

Impact of iron particles in groundwater on the UV inactivation of bacteriophages MS2 and T4.

AIMS: To investigate the impact of iron particles in groundwater on the inactivation of two model viruses, bacteriophages MS2 and T4, by 254-nm ultraviolet (UV) light. METHODS AND RESULTS: One-litre samples of groundwater with high iron content (from the Indianapolis Water Company, mean dissolved iron concentration 1.3 mg l(-1)) were stirred vigorously while exposed to air, which oxidized and precipitated the dissolved iron. In parallel samples, ethylenediaminetetra-acetic acid (EDTA) was added to chelate the iron and prevent formation of iron precipitate. The average turbidity in the samples without EDTA (called the 'raw' samples) after 210 min of stirring was 2.7 +/- 0.1 NTU while the average turbidity of the samples containing EDTA (called the 'preserved' samples) was 1.0 +/- 0.1 NTU. 'Raw' and 'preserved' samples containing bacteriophage MS2 were exposed to 254-nm UV light at doses of 20, 40, or 60 mJ (cm(2))(-1), while samples containing bacteriophage T4 were exposed to 2 or 5 mJ (cm(2))(-1), using a low pressure UV collimated beam. The UV inactivation of both phages in the 'raw' groundwater was lower than in the EDTA-'preserved' groundwater to a statistically significant degree (alpha = 0.05), due to the association of phage with the UV-absorbing iron precipitate particles. A phage elution technique confirmed that a large fraction of the phage that survived the UV exposures were particle-associated. CONCLUSIONS: Phages that are associated with iron oxide particles in groundwater are shielded from UV light to a measurable and statistically significant degree at a turbidity level of 2.7 NTU when the phage particle association is induced under experimental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: While the particle association of the phage in this study was induced experimentally, the findings provide further evidence that certain particles in natural waters and wastewaters (e.g. iron oxide particles) may have the potential to shield viruses from UV light.

Inactivation of particle-associated viral surrogates by ultraviolet light.

This study investigated whether colloid-sized particles can enmesh and protect viruses from 254-nm ultraviolet (UV) light and sought to determine the particle characteristics (e.g. size, chemical composition) that are most relevant in causing a protective effect. Two viral surrogates (MS2 coliphage and bacteriophage T4), three types of particles (kaolin clay, humic acid powder, and activated sludge), two coagulants (alum and ferric chloride), two filtration conditions (none and 0.45 microm), and two UV doses (40 and 80 mJ/cm2 for MS2 coliphage; 2 and 7 mJ/cm2 for bacteriophage T4) were considered in a series of bench-scale UV collimated beam experiments. Transmission electron microscopy was used to qualitatively confirm the phage particle-association after coagulation. Humic acid and activated sludge floc particles shielded both viral surrogates to a statistically significant degree (with >99% confidence) relative to particle-free control conditions, while the kaolin clay particles provided no significant protection. The results of the study suggest that particles <2 microm in diameter are large enough to protect viruses from UV light and that particulate chemical composition (e.g. UV-absorbing organic content) may be a critical factor in the survival of particle-associated viruses during UV disinfection.

Mutations in bacteriophage T4 genome.

Bacteriophage (phage) T4 belonging to T-even phages is one of the best known phages with a completely deciphered genome sequence. As a model of living systems, T4 phage has many technical advantages. It can be very easily grown in large quantities, manipulated by classical genetics, and engineered by site-directed mutagenesis. Many substances have been first tested for mutagenicity in T-even phages. The results of these tests were very often applicable to higher organisms due to similar mechanisms of mutagenesis. T4 phage is also important in phage therapy, which represents an alternative treatment of bacterial infections since the bacterial resistance to antibiotics has become a serious medical problem. The site-directed mutagenesis is a method that enables to introduce mutations which can influence phage affinity to bacteria and can be a practical technique for enriching phage collections and for widening specificity of phages for new bacterial strains now insensitive to phage therapy.

DNA damage and biological effects induced by photosensitization with new N(1)-unsubstituted furo[2,3-h]quinolin-2(1H)-ones.

New furoquinolinones unsubstituted at the N(1) position were prepared and their photobiological activities were studied in comparison with 4,6,8,9-tetramethylfuro[2,3-h]quinolin-2(1H)-one (HFQ) and 8-MOP. The anti-proliferative activity of furoquinolinones 3a-f was tested upon UVA irradiation in mammalian cells, studying DNA synthesis and clonal growth capacity, and in micro-organisms, evaluating T2 infectivity. Almost all compounds appeared to be more active than 8-MOP, and free of any mutagenic activity and skin phototoxicity. Among them, compound 3b was the most effective one. Similarly to HFQ, compound 3b appeared to be very active also in DNA damaging, forming monoadducts and DPC(L=0), but no ISC and DPC(L>0), both responsible for furocoumarin genotoxicity and phototoxicity. Moreover, Ehrlich ascites cells, photoinactivated by the new furoquinolinone 3b and injected into recipient mice, proved to be capable of inducing protection against a successive challenge performed with the same tumor cells. For all these features, 3b seemed to be a new promising potential drug for PUVA therapy and photopheresis.

DNA helicase mutants of bacteriophage T4 that are defective in DNA recombination.

We previously isolated a plasmid-borne, recombination-deficient mutant derivative of the bacteriophage T4 DNA helicase gene 41. We have now transferred this 41rrh1 mutation into the phage genome in order to characterize its mutational effects further. The mutation impairs a recombination pathway that is distinct from the pathway involving uvsX, which is essential for strand transfer, and it also eliminates most homologous recombination between a plasmid and the T4 genome. Although 41rrh1 does not affect T4 DNA replication from some origins, it does inactivate plasmid replication that is dependent on ori(uvs Y) and ori(34), as well as recombination-dependent DNA replication. Combination of 41rrh1 with some uvsX alleles is lethal. Based on these results, we propose that gene 41 contributes to DNA recombination through its role in DNA replication.

RBE-LET relationships in mutagenesis by ionizing radiation.

The paper considers the relationship between the quality of radiation and biological lesions produced by ionizing radiation. The paper provides a brief review of the modelling of induction of strand breakage, chromosome aberration, revertant mutation in bacteria and Drosophila melanogaster. Experimental data are presented for the relative biological effectiveness of helium ions and alpha-particles for mutation induction and genome lethality in Escherichia coli. The paper examines the relationship between the mutational events and LET. The RBE-LET values for T4 phage, E. coli WP2 and mwh (multiple wing hair) show dependency on LET while the wi (white-ivory) allele mutants show no dependency.

Mathematical description of synergistic interaction of hyperthermia and ionizing radiation.

A simple mathematical model of simultaneous combined action of ionizing radiation and hyperthermia has been proposed. The model suggests that the synergistic interaction of ionizing radiation and hyperthermia is expected to result from the additional lethal damage arising from the interaction of sublesions induced by both agents. These sublesions are considered nonlethal after each agent taken alone. The model was applied to the quantitative analysis of the simultaneous action of hyperthermia and ionizing radiation. It predicts the dependence of synergistic interaction of the ratio of lethal events produced by every agent used, as well as the maximal value of the synergistic effect, conditions at which the maximal interactive effect can be achieved, and the dependence of synergistic effect on dose rate. The predictions of the model have been tested with four experimental data sets reported in the literature. The theory appears to be appropriate and the conclusions valid.

Involvement of a replicative DNA helicase of bacteriophage T4 in DNA recombination.

Bacteriophage T4 gene 41 encodes a replicative DNA helicase that is a subunit of the primosome which is essential for lagging-strand DNA synthesis. A mutation, rrh, was generated and selected in the helicase gene on the basis of limited DNA replication that ceases early. The survival of ultraviolet-irradiated phage and the frequency of genetic recombination are reduced by rrh. In addition, rrh diminishes the production of concatemeric DNA. These results strongly suggest that the gene 41 replicative helicase participates in DNA recombination.

The effect of alpha particles on bacteriophage T4Br+.

It is generally accepted that heavy charged particles play an important part in generating the secondary flux of nuclear particles formed by the interaction of space hadrons with nuclei. It is assumed that these particles are responsible for the high biological efficiency of space hadrons in causing cellular damage by their strong interactions. To examine this assumption we investigated the effects of 5.3 MeV alpha particles on bacteriophage T4. This energy provides a LET value of 88.6 KeV/micrometer lying in the range of the highest biological efficiency.

Effect of HZE particles and space hadrons on bacteriophages.

The effect of high energy (HZE) particles and high energy hadrons on T4Br+ bacteriophage was analyzed. The experiments were done in orbital flight, on high mountains, on an accelerator, and with an alpha particle source. We studied the survival rate of the bacteriophage, the mutation frequency, the mutation spectrum and the revertability under the action of chemical mutagens with a known mechanism of action on DNA. It was found that the biological efficiency of HZE particles and high energy hadrons is greater than that of gamma radiation. The spectra of mutations produced by these mutations and the mechanisms of their action are also different. These effects were local, because of the mode of interaction of the radiant energy with biological objects, and depended on the linear energy transfer (LET). The modes have now been experimentally defined.