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A novel series of quinolone imidazoles as new type of antimicrobial agents were synthesized. Most compounds exhibited good bioactivities especially against MRSA even superior to reference drugs. They induced bacterial resistance more slowly than clinical drugs and gave low cytotoxicity to human cells. The pKa values of these compounds showed appropriate ranges to pharmacokinetic behaviors. The interactions between compound 8b, Cu(2+) ion, and MRSA DNA revealed that compound 8b could intercalate into DNA through copper ion bridge to form a steady 8b-Cu(2+) -DNA ternary complex which might further block DNA replication to exert the powerful bioactivities. Study of compound 8b with human serum albumin indicated that compound 8b could be effectively stored and carried by human serum albumin., (© 2015 John Wiley & Sons A/S.)

A series of 2-(2-(4-fluorobenzyl)-6-(substituted phenyl) pyrimidin-4-yl)-3H-benzo[f]chromen-3-one derivatives (1a-1o) were selectively prepared in high yields under microwave irradiation. The synthesized compounds were characterized by elemental and spectroscopic analysis; in addition the structures of compound (1a), (1b) and (1j) were elucidated by the X-ray diffraction technique. Compounds (1a-1o) were evaluated for their in-vitro antitubercular activity while the most active compounds were further subjected for their cytotoxicity and DNA cleavage study. Results revealed that most of the tested compounds displayed potent antitubercular activity with MIC in the range 0.05-2.81 μg/mL. Among them, compound (1b) possessed excellent activity (MIC 0.05 μg/mL) against M.tb H37Rv strain and exhibited low level of cytotoxicity against Vero cells, which suggested compound (1b) is a promising lead for subsequent investigation in search of new antitubercular agents. DNA cleavage by gel electrophoresis method revealed that compounds (1b, 1g, 1k and 1n) were found to cleave the DNA completely., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Ventilator-associated pneumonia (VAP) is a life-threatening disease that is associated with high rates of morbidity and likely mortality, placing a heavy burden on an individual and society. Currently available diagnostic and therapeutic approaches for VAP treatment are limited, and the prognosis of VAP is poor. The present study aimed to reveal and discriminate the identification of the full spectrum of the pathogens in patients with VAP using high-throughput sequencing approach and analyze the species richness and complexity via alpha and beta diversity analysis. The bronchoalveolar lavage fluid samples were collected from 27 patients with VAP in intensive care unit. The polymerase chain reaction products of the hypervariable regions of 16S rDNA gene in these 27 samples of VAP were sequenced using the 454 GS FLX system. A total of 103,856 pyrosequencing reads and 638 operational taxonomic units were obtained from these 27 samples. There were four dominant phyla, including Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. There were 90 different genera, of which 12 genera occurred in over ten different samples. The top five dominant genera were Streptococcus, Acinetobacter, Limnohabitans, Neisseria, and Corynebacterium, and the most widely distributed genera were Streptococcus, Limnohabitans, and Acinetobacter in these 27 samples. Of note, the mixed profile of causative pathogens was observed. Taken together, the results show that the high-throughput sequencing approach facilitates the characterization of the pathogens in bronchoalveolar lavage fluid samples and the determination of the profile for bacteria in the bronchoalveolar lavage fluid samples of the patients with VAP. This study can provide useful information of pathogens in VAP and assist clinicians to make rational and effective therapeutic decisions.

An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We identified antibiotic-resistant bacterial isolates from the surface waters of Jiulong River basin in southern China and determined their extent of resistance, as well as the prevalence and characterization of three classes of integrons. A phylogenetic analysis of 16S ribosomal DNA (rDNA) sequences showed that 20 genera were sampled from a total of 191 strains and the most common genus was Acinetobacter. Antimicrobial susceptibility testing revealed that the 191 isolates were all multiresistant and there were high levels of resistance to 19 antimicrobials that were tested, particularly the β-lactam, sulfonamide, amphenicol, macrolide, and rifamycin classes. Moreover, class 1 integrons were ubiquitous while only five out of 191 strains harbored class 2 integrons and no class 3 integrons were detected. The variable region of the class 1 integrons contained 30 different gene cassette arrays. Nine novel arrays were found in 65 strains, and seven strains had empty integrons. Among these 30 arrays, there were 34 different gene cassettes that included 25 resistance genes, six genes with unknown functions, two mutant transposase genes, and a new gene. The unique array dfrA1-sat2-aadA1 was detected in all five isolates carrying the class 2 integron. We found that antibiotic-resistant bacterial isolates from Jiulong River were diverse and antibiotic resistance genes associated with integrons were widespread.

Background: The current guidelines recommend a combination of ceftriaxone and azithromycin as a first-line treatment of gonorrhea in the United States and Europe. Despite not being recommended as a first-line regimen in Japan, an oral 2-g dose of azithromycin did become available for gonococcal infections in 2009. Recently, the emergence of azithromycin-resistant Neisseria gonorrhoeae isolates has been reported in several countries, including Japan., Methods: Antimicrobial susceptibility testing was performed on a total of 677 clinical isolates of N. gonorrhoeae obtained from January 2010 to December 2013 in Fukuoka, Japan. A molecular analysis by N. gonorrhoeae multiantigen sequence typing was conducted on the azithromycin-resistant isolates., Results: The proportion of azithromycin-resistant isolates (minimum inhibitory concentration > 0.5 μg/mL) increased significantly from 1.8% in 2010 to 22.6% in 2013 (P < 0.001). Among 50 azithromycin-resistant isolates, 30 (60%) exhibited a resistant phenotype to multiple drugs including cefixime. The 2 predominant sequence types (STs) identified by N. gonorrhoeae multiantigen sequence typing were ST6798 (por allele 4033 and tbpB allele 110) and ST1407 (por allele 908 and tbpB allele 110) at 40.0% (20/50) and 12.0% (6/50), respectively. There was a statistically significant increase of the proportion of ST6798 from 0% (0/19) in 2010-2012 to 64.5% (20/31) in 2013 (P < 0.001)., Conclusions: Over the previous 4 years, an increasing prevalence of azithromycin-resistant N. gonorrhoeae isolates with a multidrug-resistant phenotype was observed. Furthermore, the azithromycin-resistant isolates seemed to belong to 2 predominant STs. As a result, continued surveillance of gonococci resistant to antimicrobial agents, including azithromycin in Fukuoka, Japan, is necessary.

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.

Vancomycin-resistant Enterococci (VRE) is a serious concern for public health. Serious infections with VRE have very limited effective antimicrobial therapy, and alternative treatment approaches are highly desirable. One promising approach might be the photodynamic antimicrobial chemotherapy. In the present study, we investigated the photodynamic inactivation (PDI) of two VRE strains mediated by 5-aminolevulinic acid (5-ALA) and its derivative 5-ALA methyl ester (MAL). The photodynamic damages to bacteria on the level of genomic DNA, the leakage of cell components, and the changes of membrane structure were investigated. After treated with 10 mM 5-ALA and irradiated by the 633 ± 10 nm LED for 60 min, 5.37 and 5.22 log10 reductions in bacterial survival were achieved for the clinical isolate of VRE and E. faecalis (ATCC 51299), respectively. After treated with 10 mM MAL and irradiated by the LED for 60 min, 5.02 and 4.91 log10 reductions in bacterial survival were observed for the two VRE strains, respectively. In addition, the photocleavage on genomic DNA and the rapid release of intracellular biopolymers were detected in PDI-treated bacteria. The intensely denatured cytoplasm and the aggregated ribosomes were also found in PDI-treated bacteria by transmission electron microscopy. Although 5-ALA and MAL-mediated PDI could induce the photocleavage on genomic DNA, the PDI of the two VRE strains might be predominantly attributed to the envelope injury, the intracellular biopolymers leakage, and the cytoplasm denature.

Japanese cedar (JC) pollinosis is caused by Japanese cedar pollen (JCP) and most common seasonal allergic disease in Japan. Subcutaneous immunotherapy (SCIT) with allergen extract of JCP (JCP-allergen extract) is well established for JC pollinosis treatment with improvement of symptoms. However, major drawbacks for SCIT are repeated painful injections, frequent hospital visits and anaphylactic risk. Currently, sublingual immunotherapy (SLIT) has received much attention as an advanced alternative application with lower incidence of systemic reactions because the liquid or tablet form of allergen is placed under the tongue. The aim of this study was safety evaluation of standardized JCP-allergen extract currently developed for SLIT in JC pollinosis. JCP-allergen extract showed no potential genotoxicity. No systemic effects were observed in rats administered JCP-allergen extract orally for 26 weeks followed by 4-week recovery period. Mild local reactions such as hyperplasia and increased globule leukocytes resulting from vehicle (glycerin)-induced irritation were observed in stomach. No-observed-adverse-effect level was greater than 10,000 JAU/kg/day for systemic toxicity, equivalent to 300-fold the human dose. No local irritation was found in rabbits oral mucosae by 7-day sublingual administration. These results demonstrate the safe profile of standardized JCP-allergen extract, suggesting it is suitable for SLIT in JC pollinosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The root of Polygala tenuifolia Willdenow has been used for the treatment against insomnia, amnesia, depression, palpitations with anxiety, and memory improvement. However, there is no sufficient background information on toxicological evaluation of the root to given an assurance of safety for developing dietary supplements and functional foods. As part of a safety evaluation, the potential genotoxicity of the root extract of P. tenuifolia was evaluated using a standard battery of tests (bacterial reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay). In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, the extract did not increase the number of revertant colonies in any tester strain with or without metabolic activation by S9 mix, and did not cause chromosomal aberration in short-period test with the S9 mix or in the continuous (24h) test. A bone marrow micronucleus test in ICR mice dosed by oral gavage at doses up to 2000 mg/kg/day showed no significant or dose dependent increase in the frequency of micronucleated polychromatic erythrocytes (PCE). These results indicate that ingesting the rot extract P. tenuifolia is not genotoxic at the proper dose., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Tobacco smoke from a combustible cigarette contains more than 6000 constituents; approximately 150 of these are identified as toxicants. Technologies that modify the tobacco blend to reduce toxicant emissions have been developed. These include tobacco sheet substitute to dilute toxicants in smoke and blend treated tobacco to reduce the levels of nitrogenous precursors and some polyphenols. Filter additives to reduce gas (vapour) phase constituents have also been developed. In this study, both tobacco blend and filter technologies were combined into an experimental cigarette and smoked to International Organisation on Standardisation and Health Canada puffing parameters. The resulting particulate matter was subjected to a battery of in vitro genotoxicity and cytotoxicity assays - the Ames test, mouse lymphoma assay, the in vitro micronucleus test and the Neutral Red Uptake assay. The results indicate that cigarettes containing toxicant reducing technologies may be developed without observing new additional genotoxic hazards as assessed by the assays specified. In addition, reductions in bacterial mutagenicity and mammalian genotoxicity of the experimental cigarette were observed relative to the control cigarettes. There were no significant differences in cytotoxicity relative to the control cigarettes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Nitromezuril (NZL) is a novel triazine compound that exhibits remarkable anticoccidial activity. However, mutagenicity and genotoxicity of NZL have not been evaluated to date. This study evaluated the potential risks of NZL by testing for bacterial reverse mutation (Ames), mouse sperm abnormality (SA), bone marrow micronucleus (MN) and chromosomal aberration (CA). Mice were orally administered with NZL at 385, 192 and 96 mg/kg, corresponding to 0.5 ×, 0.25 × and 0.125 × the LD50 of NZL, respectively. No significant increases in SA and CA were found in mice treated with NZL for 5d and 3d, respectively (P>0.05). NZL at 96-385 mg/kg did not have significant influence on micronucleated polychromatic erythrocyte counts (P>0.05). These results suggest that NZL is not genotoxic. However, Ames test results were positive both with and without the S9 system for Salmonella typhimurium TA98 and TA100, suggesting that NZL may be mutagenic. The mutagenic effects of NZL were different in in vitro and in vivo assays. Further studies should be conducted to confirm the safety of using and developing NZL as a novel anticoccidial drug., (Copyright © 2015. Published by Elsevier Inc.)

Decorin binding proteins A and B (DbpA and B) of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.

Remnant specimens from 601 women obtained in a cross-sectional study from rural South Africa were tested for Mycoplasma genitalium. Overall, 10.8% of women were infected with M. genitalium either in the vagina or in the rectum. Macrolide resistance, although of low prevalence, in M. genitalium is described for the first time in Sub-Saharan Africa.

Bacillus subtilis contains two vegetative diadenylate cyclases, DisA and CdaA, which produce cyclic di-AMP (c-di-AMP), and one phosphodiesterase, GdpP, that degrades it into a linear di-AMP. We report here that DisA and CdaA contribute to elicit repair of DNA damage generated by alkyl groups and H2O2, respectively, during vegetative growth. disA forms an operon with radA (also termed sms) that encodes a protein distantly related to RecA. Among different DNA damage agents tested, only methyl methane sulfonate (MMS) affected disA null strain viability, while radA showed sensitivity to all of them. A strain lacking both disA and radA was as sensitive to MMS as the most sensitive single parent (epistasis). Low c-di-AMP levels (e.g. by over-expressing GdpP) decreased the ability of cells to repair DNA damage caused by MMS and in less extent by H2O2, while high levels of c-di-AMP (absence of GdpP or expression of sporulation-specific diadenylate cyclase, CdaS) increased cell survival. Taken together, our results support the idea that c-di-AMP is a crucial signalling molecule involved in DNA repair with DisA and CdaA contributing to modulate different DNA damage responses during exponential growth., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We report the results of an investigation of the activity of a series of amidine and bisamidine compounds against Staphylococcus aureus and Escherichia coli. The most active compounds bound to an AT-rich DNA dodecamer (CGCGAATTCGCG)2 and using DSC were found to increase the melting transition by up to 24 °C. Several compounds also inhibited undecaprenyl diphosphate synthase (UPPS) with IC50 values of 100-500 nM, and we found good correlations (R(2) = 0.89, S. aureus; R(2) = 0.79, E. coli) between experimental and predicted cell growth inhibition by using DNA ΔTm and UPPS IC50 experimental results together with one computed descriptor. We also solved the structures of three bisamidines binding to DNA as well as three UPPS structures. Overall, the results are of general interest in the context of the development of resistance-resistant antibiotics that involve multitargeting.

Background: In intensive pig husbandry systems, antibiotics are frequently administrated during early life stages to prevent respiratory and gastro-intestinal tract infections, often in combination with stressful handlings. The immediate effects of these treatments on microbial colonization and immune development have been described recently. Here we studied whether the early life administration of antibiotics has long-lasting effects on the pig's intestinal microbial community and on gut functionality., Methodology/principal Findings: To investigate the long-lasting effect of early-life treatment, piglets were divided into three different groups receiving the following treatments: 1) no antibiotics and no stress, 2) antibiotics and no stress, and 3) antibiotics and stress. All treatments were applied at day four after birth. Sampling of jejunal content for community scale microbiota analysis, and jejunal and ileal tissue for genome-wide transcription profiling, was performed at day 55 (~8 weeks) and day 176 (~25 weeks) after birth. Antibiotic treatment in combination with or without exposure to stress was found to have long-lasting effects on host intestinal gene expression involved in a multitude of processes, including immune related processes., Conclusions/significance: The results obtained in this study indicate that early life (day 4 after birth) perturbations have long-lasting effects on the gut system, both in gene expression (day 55) as well as on microbiota composition (day 176). At day 55 high variance was observed in the microbiota data, but no significant differences between treatment groups, which is most probably due to the newly acquired microbiota during and right after weaning (day 28). Based on the observed difference in gene expression at day 55, it is hypothesized that due to the difference in immune programming during early life, the systems respond differently to the post-weaning newly acquired microbiota. As a consequence, the gut systems of the treatment groups develop into different homeostasis.

The analysis was implemented concerning application of biological chips technique developed by the V A. Engelgardt institute of molecular biology--"TB-Biochip" designed for detecting DNA agent of tuberculosis in diagnostic samples of human respiratory organs and establishing its medicinal sensitivity to rifampicin and isoniazid. It is demonstrated that "TB-Biochip" is a sensitive and highly specific method of detecting medicinal sensitivity to main anti-tuberculosis pharmaceuticals. At that, analysis time amount to less than 72 hours. The comparative analysis was carried out concerning results of detection of medicinal sensitivity of mycobacteria by two express techniques--TB-Biochip (Biochip-IMB) and BACTECTM MGITTM 960 ("Becton Dickonson"). The data obtained by two techniques matched in 97.6%for rifampicin and in 92.1% for isoniazid.

This study demonstrated berberine to be a potential natural compound against Actinobacillus pleuropneumoniae. Liquid doubling dilution, transmission electron microscopy (TEM), SDS-PAGE and 4',6-diamidino-2-phenylindole (DAPI) staining were employed to elucidate the antibacterial activity and mechanism of berberine. The minimal inhibitory concentration of berberine was 0.3125 mg/mL, and time-kill curves showed concentration and time dependence. The TEM micrographs displayed damaged cell wall, concentrated cytoplasm, cytoplasmic content leakage and cell death. SDS-PAGE and DAPI assays revealed that berberine can restrain DNA and protein syntheses. Berberine inhibited the synthesis of proteins associated with the growth and cleavage of bacteria and then blocked the division and development of bacteria. The compound ultimately induced cytoplasm pyknosis and bacterial death.

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4-5), generate reactive oxygen species (ROS) and H2O2, and have a high oxidation-reduction potential. To isolate the role of pH in the antibacterial activity of CB clay mixtures, we exposed three different strains of Escherichia coli O157:H7 to 10% clay suspensions. The clay suspension completely killed acid-sensitive and acid-tolerant E. coli O157:H7 strains, whereas incubation in a low-pH buffer resulted in a minimal decrease in viability, demonstrating that low pH alone does not mediate antibacterial activity. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to the antibacterial leachate. Further, supplementation with numerous ROS scavengers eliminated lipid peroxidation, but did not rescue the cells from CB-L-mediated killing. In contrast, supplementing CB-L with EDTA, a broad-spectrum metal chelator, reduced killing. Finally, CB-L was equally lethal to cells in an anoxic environment as compared to the aerobic environment. Thus, ROS were not required for lethal activity and did not contribute to toxicity of CB-L. We conclude that clay-mediated killing was not due to oxidative damage, but rather, was due to toxicity associated directly with released metal ions.

Silver nanoparticles are known to have antimicrobial properties and have been used extensively in medicine, although the mechanism(s) of action have not yet been clearly established. In the present study, the findings suggest a novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli, namely, the induction of a bacterial apoptosis-like response. We propose a possible mechanism for the bacterial apoptosis-like response that includes the following: accumulation of reactive oxygen species (ROS) (detected with H2DCFDA staining), increased intracellular calcium levels (detected with Fura-2 AM), phosphatidylserine exposure in the outer membrane (detected with Annexin V) which is the hallmarks of early apoptosis, disruption of the membrane potential [detected with DiBAC4(3)], activation of a bacterial caspase-like protein (detected by FITC-VAD-FMK staining) and DNA degradation (detected with TUNEL assay) which is the hallmarks of late apoptosis in bacterial cells treated with silver nanoparticles. We also performed RecA expression assay with western blotting and observed activation of SOS response to repair the damaged DNA. To summarize, silver nanoparticles are involved in the apoptosis-like response in E. coli and the novel mechanisms which were identified in this study, suggest that silver nanoparticles may be an effective antimicrobial agent with far lower propensity for inducing microbial resistance than antibiotics.

The effects of doxycycline-streptomycin-rifampin versus a standard doxycycline-streptomycin regimen on residual Brucella DNA were compared in 36 acute brucellosis patients. At admission, all patients given triple (n = 22) and double (n = 14) regimens had detectable Brucella DNA with similar mean loads (P = 0.982). At follow-up, 14 to 20 months postpresentation, significantly more patients receiving triple than double regimens had undetectable Brucella DNA (P = 0.026). The doxycycline-streptomycin-rifampin regimen eliminates Brucella DNA more efficiently than doxycycline-streptomycin, which may result in superior long-term clearance of Brucella., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Antimicrobial screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. These compounds were evaluated for antimicrobial activity against a panel of bacterial and fungal strains. The strains were treated with these benzothiazole derivatives at varying concentrations, and MIC's were calculated. Structures of these compounds have been determined by spectroscopic studies viz., FT-IR, 1H NMR, 13C NMR and elemental analysis. Significant antimicrobial activity was observed for some members of the series, and compounds viz. 3-(4-(benzo[d]thiazol-2-yl) phenyl-2-(4-methoxyphenyl)thiazolidin-4-one and 3-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxy phenyl)thiazolidin-4-one were found to be the most active against E.coli and C. albicans with MIC values in the range of 15.6-125 microg/ml. Preliminary study of the structure-activity relationship revealed that electron donating groups associated with thiazolidine bearing benzothiazole rings had a great effect on the antimicrobial activity of these compounds and contributes positively for the action. DNA cleavage experiments gave valuable hints with supporting evidence for describing the mechanism of action and hence showed a good correlation between their calculated MIC's and its lethality.

The tri-hybrid peptide-LHP7 has the potent activity against Gram-positive and Gram-negative as well as fungi, but its mechanism of action has remained elusive. The effluences of LHP7 on the Staphylococcus aureus cell membrane and targets of intracellular action were investigated. LHP7 exhibited an inhibitory effect on the S. aureus growth, similar to those achieved by plectasin, vancomycin and gramicidin. The membrane integrity studies confirmed that LHP7 disrupted the cell membrane, indicating a membrane permeabilizing killing action. A marginal decline in the intensity fluorescence indicated no significant depolarization of the membrane potential following LHP7 treatment. Furthermore, electron microscopy showed that cell shrinkage, cell wall thickening, cellular content leakage, and cell disruption were observed in the cells treated with LHP7. A gel retardation assay showed that LHP7 bound to the genomic DNA of S. aureus or plasmid DNA at a mass ratio of 2.5–10 (peptide/DNA). Circular dichroism indicated that LHP7 inserted into the groove of DNA. The cell cycle analysis showed that after the treatment with LHP7 for 30 and 60 min, the proportion of cells in I-phase increased from 8.71 to 12.09 % and from 8.71 to 15.68 %, indicating that LHP7 induced arrest of cells in the I-phase. These results would conduce to elucidate its underlying antibacterial mechanism.

Engineering of enzymes to more efficiently activate genotoxic prodrugs holds great potential for improving anticancer gene or antibody therapies. We report the development of a new, GFP-based, high-throughput screening platform to enable engineering of prodrug-activating enzymes by directed evolution. By fusing an inducible SOS promoter to an engineered GFP reporter gene, we were able to measure levels of DNA damage in intact Escherichia coli and separate cell populations by fluorescence activating cell sorting (FACS). In two FACS iterations, we were able to achieve a 90,000-fold enrichment of a functional prodrug-activating nitroreductase from a null library background., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Indolicidin (IR13), a 13-residue antimicrobial peptide from the cathelicidin family, is known to exhibit a broad spectrum of antimicrobial activity against various microorganisms. This peptide inhibits bacterial DNA synthesis resulting in cell filamentation. However, the precise mechanism remains unclear and requires further investigation. The central PWWP motif of IR13 provides a unique structural element that can wrap around, and thus stabilize, duplex B-type DNA structures. Replacements of the central Trp-Trp pair with Ala-Ala, His-His, or Phe-Phe residues in the PxxP motif significantly affects the ability of the peptide to stabilize duplex DNA. Results of microscopy studies in conjunction with spectroscopic data confirm that the DNA duplex is stabilized by IR13, thereby inhibiting DNA replication and transcription. In this study we provide high-resolution structural information on the interaction between indolicidin and DNA, which will be beneficial for the design of novel therapeutic antibiotics based on peptide scaffolds., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of dual-targeting, alcohol-containing benzothiazoles has been identified with superior antibacterial activity and drug-like properties. Early lead benzothiazoles containing carboxylic acid moieties showed efficacy in a well-established in vivo model, but inferior drug-like properties demanded modifications of functionality capable of demonstrating superior efficacy. Eliminating the acid group in favor of hydrophilic alcohol moieties at C(5), as well as incorporating solubilizing groups at the C(7) position of the core ring provided potent, broad-spectrum Gram-positive antibacterial activity, lower protein binding, and markedly improved efficacy in vivo., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Extracts of Echinodorus grandiflorus obtained from dried leaves by three different techniques were evaluated by bacterial lysogenic induction assay (Inductest) in relation to their genotoxic properties. Before being added to test cultures, extracts were sterilized either by steam sterilization or ultraviolet light. Only the extracts prepared by infusion and steam sterilized have shown genotoxic activity. The phytochemical analysis revealed the presence of the flavonoids isovitexin, isoorientin, swertisin and swertiajaponin, isolated from a genotoxic fraction. They were assayed separately and tested negative in the Inductest protocol. The development of browning color and sweet smell in extracts submitted to heat, prompted further chemical analysis in search for Maillard's reaction precursors. Several aminoacids and reducing sugars were cast in the extract. The presence of characteristic Maillard's melanoidins products was determined by spectrophotometry in the visible region and the inhibition of this reaction was observed when its characteristic inhibitor, sodium bisulfite, was added prior to heating. Remarkably, this is the first paper reporting on the appearance of such compounds in a phytomedicine preparation under a current phytopharmaceutical procedure. The genotoxic activity of such heat-prepared infusions imply in some risk of developing degenerative diseases for patients in long-term, uncontrolled use of such phytomedicines.

Objective: To detect Mycoplasma genitalium in urine samples of infertile men and men without any signs of infection in order to investigate whether M. genitalium and other genital mycoplasmas (Mycoplasma hominis and Ureaplasma spp) are found more often in urine samples of infertile men than in asymptomatic controls and to determine resistance to macrolides., Methods: The study included first void urine samples taken from 145 infertile men and 49 men with no symptoms of urethritis. M. genitalium, Chlamydia trachomatis and Neisseria gonorrhoeae were detected by commercial PCR. Trichomonas vaginalis was detected by microscopy and culture. M. hominis and Ureaplasma spp were detected by culture. M. genitalium was detected by in-house conventional and real-time PCR., Results: Two M. genitalium positive samples were found among samples obtained from infertile men. All asymptomatic men were M. genitalium negative. Macrolide resistance was not found in either of the two positive samples., Conclusions: In comparison with reported data, an unusually low prevalence of M. genitalium was found in infertile men. The reasons for this unexpected result are not known; possibly, local demographic and social characteristics of the population influenced the result. Further studies to investigate M. genitalium in infertile and other groups of patients are needed., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other β-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations.

The bacterial sliding clamp (SC), also known as the DNA polymerase III β subunit, is an emerging antibacterial target that plays a central role in DNA replication, serving as a protein-protein interaction hub with a common binding pocket to recognize linear motifs in the partner proteins. Here, fragment-based screening using X-ray crystallography produced four hits bound in the linear-motif-binding pocket of the Escherichia coli SC. Compounds structurally related to the hits were identified that inhibited the E. coli SC and SC-mediated DNA replication in vitro. A tetrahydrocarbazole derivative emerged as a promising lead whose methyl and ethyl ester prodrug forms showed minimum inhibitory concentrations in the range of 21-43 μg/mL against representative Gram-negative and Gram-positive bacteria species. The work demonstrates the utility of a fragment-based approach for identifying bacterial sliding clamp inhibitors as lead compounds with broad-spectrum antibacterial activity.

We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.

Hyaluronic acid capsule is one of the most important virulence factors of group A Streptococcus (GAS). Over-production of capsule has been thought to enhance GAS virulence during infections. However, although the increased of capsule expression associates with increased bacterial virulence and invasive ability, over-production of capsule has not often been observed among clinical isolates. In the present study, we identified two mucoid emm12 type isolates that can convert to the hypermucoid morphology under both in vitro and in vivo conditions. Consistent with previous studies, hypermucoid variants are more invasive in the mouse air-pouch infection model. However, one of the hypermucoid variants showed a growth-defective phenotype in regular broth culture conditions and is significantly more susceptible to various DNA-damaging treatments when compared with the mucoid variant. These properties of the hypermucoid variant may be adverse factors inhibiting its adaptation to the host environment during infections., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

The Brazilian anti-tuberculosis vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG) BCG Moreau is unique in having a deletion of 7608 bp (RD16) that results in the truncation of a putative TetR transcriptional regulator, the ortholog of Mycobacterium tuberculosis rv3405c, BCG&#95;M3439c. We investigated the effect of this truncation on the expression of the rv3406 ortholog (BCG&#95;M3440), lying 81 bp downstream in the opposite orientation. RT-PCR and western blot experiments show that rv3406 mRNA and Rv3406 accumulate in BCG Moreau but not in BCG Pasteur (strain that bears an intact rv3405c), suggesting this to be a result of rv3405c truncation. Recombinant Rv3405c forms a complex with the rv3405c-rv3406 intergenic region, which contains a characteristic transcription factor binding site, showing it to have DNA binding activity. Complementation of M. bovis BCG Moreau with an intact copy of rv3405c abolishes Rv3406 accumulation. These results show that Rv3405c is a DNA binding protein that acts as a transcriptional repressor of rv3406., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol of E. coli require several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intact E. coli cells that survive phage exposure. Escherichia coli O157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P < 0.01) phage efficacy as cell surface-attached phage particles lysed E. coli O157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol of E. coli O157:H7.

Predictive models are beneficial tools for researchers to use in prioritizing nanoparticles (NPs) for toxicological tests, but experimental evaluation can be time-consuming and expensive, and thus, priority should be given to tests that identify the NPs most likely to be harmful. For characterization of NPs, the physical binding of NPs to DNA molecules is important to measure, as interference with DNA function may be one cause of toxicity. Here, we determined the interaction energy between 12 types of NPs and DNA based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) model and then predicted the affinity of the NPs for DNA. Using the single-molecule imaging technique known as atomic force microscopy (AFM), we experimentally determined the binding affinity of those NPs for DNA. Theoretical predictions and experimental observations of the binding affinity agreed well. Furthermore, the effect of NPs on DNA replication in vitro was investigated with the polymerase chain reaction (PCR) technique. The results showed that NPs with a high affinity for DNA strongly inhibited DNA replication, whereas NPs with low affinity had no or minimal effects on DNA replication. The methodology here is expected to benefit the genotoxicological testing of NPs as well as the design of safe NPs.

A rapid and simple method for the detection of drug-resistant Mycobacterium tuberculosis is critical for the efficient treatment and control of this pathogen in developing country. Here we developed a single multiplex amplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer and temperature switch PCR (TSP) strategy were included. Using this method, we detected rifampin resistance-associated mutations at codons 511, 516, 526 and 531 in the rifampin resistance-determining region of rpoB gene. The performance of M-ARMS-PCR assay was evaluated with 135 cultured isolates of M. tuberculosis. The sensitivity and specificity were 94.2% and 100%, respectively, compared with direct DNA sequencing, and 86.67% and 89.71%, respectively, compared with culture-based phenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCR method is useful and efficient with an intended application in provincial Centers for Disease Control and Prevention for rapid detection of rifampin resistance-associated mutations., (© 2013.)

An iminothiadiazolo-pyrimidinone derivative, 0002-04-KK, harboring a furan moiety, acts as an antimicrobial agent with a minimum inhibitory concentration (MIC) against Staphylococcus aureus of 25 μg ml(-1). Several derivatives of 0002-04-KK were synthesized and among them 0026-59-KK, harboring a nitrofuran moiety, had the most potent antimicrobial activity with an MIC of 6 μg ml(-1). Both 0002-04-KK and 0026-59-KK inhibited the biosynthesis of DNA, RNA and proteins. Peptidoglycan biosynthesis was inhibited by 0026-59-KK, and slightly inhibited by 0002-04-KK. Derivative 0002-04-KK showed bactericidal activity in contrast to the bacteriostatic activity of 0002-04-KK. Derivative 0002-04-KK had less toxicity in silkworms (lethal dose fifty (LD50): >230 μg g(-1)) than 0002-04-KK (LD50: 100 μg g(-1)). The bactericidal activity against S. aureus was because of the nitrofuran moiety. These findings suggest that iminothiadiazolo-pyrimidinone compounds could be used as lead molecules to develop antimicrobial agents.

Colistin is an old antibiotic which has been used as a therapeutic option for carbapenem- and multidrug-resistant Gram-negative bacteria, like Acinetobacter baumannii. This pathogen produces life-threatening infections, mainly in patients admitted to intensive care units. Rapid detection of resistance to colistin may improve patient outcomes and prevent the spread of resistance. For this purpose, Micromax technology was evaluated in four isogenic A. baumannii strains with known mechanisms of resistance to colistin and in 66 isolates (50 susceptible and 16 resistant). Two parameters were determined, DNA fragmentation and cell wall damage. To assess DNA fragmentation, cells trapped in a microgel were incubated with a lysing solution to remove the cell wall, and the released nucleoids were visualized under fluorescence microscopy. Fragmented DNA was observed as spots that diffuse from the nucleoid. To assess cell wall integrity, cells were incubated with a lysis solution which removes only weakened cell walls, resulting in nucleoid release exclusively in affected cells. A dose-response relationship was demonstrated between colistin concentrations and the percentages of bacteria with DNA fragmentation and cell wall damage, antibiotic effects that were delayed and less frequent in resistant strains. Receiver operating characteristic (ROC) curves demonstrated that both DNA fragmentation and cell wall damage were excellent parameters for identifying resistant strains. Obtaining ≤11% of bacteria with cell wall damage after incubation with 0.5 μg/ml colistin identified resistant strains of A. baumannii with 100% sensitivity and 96% specificity. Results were obtained in 3 h 30 min. This is a simple, rapid, and accurate assay for detecting colistin resistance in A. baumannii, with strong potential value in critical clinical situations.

Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations-MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms' "fingerprint" (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.

Sanguinarine showed strong inhibitory effect against Microcystis aeruginosa, a typical water bloom-forming and microcystins-producing cyanobacterium. The EC50 of sanguinarine against the growth of M. aeruginosa NIES-843 was 34.54±1.17 μg/L. Results of chlorophyll fluorescence transient analysis indicated that all the electron donating side, accepting side, and the reaction center of the Photosystem II (PS II) were the targets of sanguinarine against M. aeruginosa NIES-843. The elevation of reactive oxygen species (ROS) level in the cells of M. aeruginosa NIES-843 upon exposure indicated that sanguinarine induced oxidative stress in the active growing cells of M. aeruginosa NIES-843. Further results of gene expression analysis indicated that DNA damage and cell division inhibition were also involved in the inhibitory action mechanism of sanguinarine against M. aeruginosa NIES-843. The inhibitory characteristics of sanguinarine against M. aeruginosa suggest that the ecological- and public health-risks need to be evaluated before its application in cyanobacterial bloom control to avoid devastating events irreversibly., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Three square-planar complexes of nickel(II) with the tridentate condensation derivative of 2-(diphenylphosphino)benzaldehyde and ethyl carbazate, and monodentate pseudohalides, have been synthesized. Their crystal structures have been determined. All the complexes showed a significant antifungal activity, while only the azido complex displayed antibacterial activity. All the complexes were cytotoxic to a panel of six tumor cell lines, the azido complex showing a similar activity as cisplatin to leukemia cell line K562 and lower toxicity to normal MRC-5 cells than that anticancer agent. The complexes interfered with cell cycle of tumor cells and induced plasmid DNA cleavage., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Cold atmospheric-pressure plasmas are currently in use in medicine as surgical tools and are being evaluated for new applications, including wound treatment and cosmetic care. The disinfecting properties of plasmas are of particular interest, given the threat of antibiotic resistance to modern medicine. Plasma effluents comprise (V)UV photons and various reactive particles, such as accelerated ions and radicals, that modify biomolecules; however, a full understanding of the molecular mechanisms that underlie plasma-based disinfection has been lacking. Here, we investigate the antibacterial mechanisms of plasma, including the separate, additive and synergistic effects of plasma-generated (V)UV photons and particles at the cellular and molecular levels. Using scanning electron microscopy, we show that plasma-emitted particles cause physical damage to the cell envelope, whereas UV radiation does not. The lethal effects of the plasma effluent exceed the zone of physical damage. We demonstrate that both plasma-generated particles and (V)UV photons modify DNA nucleobases. The particles also induce breaks in the DNA backbone. The plasma effluent, and particularly the plasma-generated particles, also rapidly inactivate proteins in the cellular milieu. Thus, in addition to physical damage to the cellular envelope, modifications to DNA and proteins contribute to the bactericidal properties of cold atmospheric-pressure plasma.

Strain characteristics of 51 Shigella sonnei isolates obtained from children seeking medical care (MC) and 48 isolates recovered during a prospective diarrhoea birth cohort (BC) study were compared. Biochemical characterization and antibiotic susceptibility testing determined that all S. sonnei isolates were biotype g and multidrug-resistant. Plasmid profiling identified 15 closely related patterns and XbaI pulsed-field gel electrophoresis confirmed the high degree of genetic similarity between isolates. All S. sonnei isolates harboured ipaH and class II integrase genes and 84∙3 and 80% of the MC and BC isolates, respectively carried the sen gene. Neither the class I integrase nor the set gene was detected. Our results indicate that S. sonnei isolates associated with severe diarrhoea were indistinguishable from those associated with mild diarrhoea. Additional genetic tests with greater discrimination might offer an opportunity to determine genetic differences within the globally disseminating biotype g clone.

Growth curve of bacteria treated by the rare earth elements and calmodulin inhibitor (chlorpromazine) was studied, and the effect of REE on calf thymus-DNA and intracellular DNA in bacteria was studied by fluorescence spectrum. The results showed that the logarithmic phase of growth and fission of bacteria can be advanced by REE, so they grew faster than contrast. At the same time, Bacillus subtilis was found bigger by SEM. But growth and fission was inhibited by chlorpromazine, indicating that the mechanism about growth and fission of bacteria by REE was connected with calmodulin inhibitor. Fluorescence of ct-DNA can be enhanced by REE. But intracellular DNA in bacteria was different, it can be enhanced by Ce(NO3)3, quenched by La(NO3)3. So we think that the function of REE to biological materials was very complicated.

We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10-100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50-60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Lignosus rhinocerus (Tiger Milk mushroom) is distributed in South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea. In Malaysia, it is the most popular medicinal mushroom used by the indigenous communities to relieve fever, cough, asthma, cancer, food poisoning and as a general tonic. In China, this mushroom is an expensive traditional medicine used to treat liver cancer, chronic hepatitis and gastric ulcers. The sclerotium of the mushroom is the part with medicinal value. This rare mushroom has recently been successfully cultivated making it possible to be fully exploited for its medicinal and functional benefits. The present study was carried out to evaluate the chronic toxicity of the sclerotial powder of Lignosus rhinocerus cultivar (termed TM02), its anti-fertility and teratogenic effects as well as genotoxicity., Materials and Methods: Sprague Dawley rats (10 rats/group/sex) were fed orally with 250, 500 and 1000 mg/kg of sclerotial powder of TM02. The sclerotial powder was orally administered once daily and consecutively for 180 days. At the completion of the oral feeding period, analysis of hematological and clinical biochemical parameters, urine profiles, organ weight as well as histopathological analysis were carried out. The effect of the sclerotial powder on fertility and its possible teratogenicity were examined by feeding rats orally with 100 mg/kg sclerotial powder consecutively for 7-8 weeks. Genotoxicity was evaluated by Ames test using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA., Results: The results showed that oral administration of the sclerotial powder of the Lignosus rhinocerus cultivar at daily dose of up to 1000 mg/kg for 180 days had no adverse effect on the general clinical observations, body weight, hematology, clinical biochemistry, urinalysis, absolute organ weight as well as relative organ weight, nor induced histological changes in the organs. Oral administration of 100 mg/kg sclerotial powder of the Lignosus rhinocerus for 7-8 weeks did not affect the fertility of the rats nor induce teratogenic effect on their offspring. Lignosus rhinocerus sclerotial powder up to 5000 μg/plate in the presence and absence of metabolic activation did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used., Conclusion: Our results showed that the no-observed-adverse-effect level (NOAEL) dose of the sclerotial powder of Lignosus rhinocerus in 180-day chronic toxicity study is more than 1000 mg/kg. Oral feeding of the sclerotial powder at 100mg/kg did not induce adverse effect on rats' fertility nor causing teratogenic effect on their offspring. In the reverse mutation Ames test, the sclerotial powder at all tested concentration did not show any genotoxicity., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Two bisdihydroanthracenone atropodiastereomeric pairs, including homodimeric flavomannin A (1) and the previously unreported flavomannin B (2), two new unsymmetrical dimers (3 and 4), and two new mixed dihydroanthracenone/anthraquinone dimers (5 and 6) were isolated from Talaromyces wortmannii , an endophyte of Aloe vera . The structures of 2-6 were elucidated by extensive NMR and mass spectrometric analyses. The axial chirality of the biaryls was determined using TDDFT ECD and VCD calculations, the combination of which however did not allow the assignment of the central chirality elements of 1. The compounds exhibited antibacterial activity against Staphylococcus aureus , including (multi)drug-resistant clinical isolates. Reporter gene analyses indicated induction of the SOS response for some of the derivatives, suggesting interference with DNA structure or metabolism. Fluorescence microscopy demonstrated defective segregation of the bacterial chromosome and DNA degradation. Notably, the compounds showed no cytotoxic activity, encouraging their further evaluation as potential starting points for antibacterial drug development.

Naturally occurring flavonoids have been reported to possess antimicrobial activity against a wide range of pathogens. However, the antimicrobial action mechanism of these compounds has not yet been elucidated. This study investigated the mechanism underlying the antibacterial activity of four flavonoids: 6,8-diprenyleriodictyol (1), isobavachalcone (2), 6-prenylapigenin (3) and 4-hydroxylonchocarpin (4). In addition, the toxicity of these compounds was evaluated. Determination of the minimum inhibitory concentrations (MICs) was performed by microbroth dilution method. Radiolabeled thymidine, uridine, and methionine were used to evaluate the effect of the compounds on the biosynthesis of DNA, RNA, and proteins while the sensitive cyanine dye DiS-C3-(5) (3,3'-dipropylthiadicarbocyanine iodide) was used for the effect on membrane potential. Bactericidal/bacteriolysis activities were performed by time-kill kinetic method. In the toxicity study, the numbers of survivors was recorded after injection of compounds into the hemolymph of silkworm larvae. Compounds showed significant antibacterial activity against Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) strains with MICs values ranged between 0.5-128 μg/mL. Depolarization of membrane and inhibition of DNA, RNA, and proteins synthesis were observed in S. aureus when treated with those flavonoids. At 5-fold minimum inhibitory concentration, compounds reduced rapidly the bacterial cell density and caused lysis of S. aureus. Compounds 1, 2, and 4 did not show obvious toxic effects in silkworm larvae up to 625 μg/g of body weight. Flavonoids from Dorstenia species, 6,8-diprenyleriodictyol, isobavachalcone, and 4-hydroxylonchocarpin are bactericidal compounds. They cause damage of cell membrane, leading to the inhibition of macromolecular synthesis. Taking into account the in vivo safety and their significant antimicrobial potency, these flavonoids are promising leads for further drug development.