Isolation of a bacterial consortium able to degrade the fungicide thiabendazole: the key role of a Sphingomonas phylotype

Thiabendazole (TBZ) is a fungicide used in fruit-packaging plants. Its application leads to the production of wastewaters requiring detoxification. In the absence of efficient treatment methods, biological depuration of these effluents could be a viable alternative. However, nothing is known regarding the microbial degradation of the recalcitrant and toxic to aquatics TBZ. We report the isolation, via enrichment cultures from a polluted soil, of the first bacterial consortium able to rapidly degrade TBZ and use it as a carbon source. Repeated efforts using various culture-dependent approaches failed to isolate TBZ-degrading bacteria in axenic cultures. Denaturating gradient gel electrophoresis (DGGE) and cloning showed that the consortium was composed of α-, β- and γ-Proteobacteria. Culture-independent methods including antibiotics-driven selection with DNA/RNA-DGGE, q-PCR and stable isotope probing (SIP)-DGGE identified a Sphingomonas phylotype (B13) as the key degrading member. Cross-feeding studies with structurally related chemicals showed that ring substituents of the benzimidazole moiety (thiazole or furan rings) favoured the cleavage of the imidazole moiety. LC-MS/MS analysis verified that TBZ degradation proceeds via cleavage of the imidazole moiety releasing thiazole-4-carboxamidine, which was not further transformed, and the benzoyl moiety, possibly as catechol, which was eventually consumed by the bacterial consortium as suggested by SIP-DGGE.

     

Factors Influencing Non-<Emphasis Type="Italic">albicans</Emphasis> Candidemia: A Case–Case–Control Study

The study identified factors predisposing to non-albicans candidemia with special interest to prior antimicrobial treatment. A retrospective, case–case–control study was performed at the University Hospital of Heraklion, Greece, from November 2007 through September 2011 including adult patients. The study had three groups. The first included 58 patients with non-albicans candidemia, the second 48 with C. albicans candidemia, while the third (control) 104 without candidemia. Each of the two candidemia groups was compared with the control using multivariate logistic regression model. The mean (SD) age of the non-albicans, the albicans and the control patients was 67 (12), 67 (18) and 59 (19) years, respectively. The most common non-albicans Candida spp. isolated were C. parapsilosis in 19 patients (33%), C. glabrata in 17 (29%) and C. tropicalis in 15 (26%). Independent risk factors for non-albicans candidemia were prior treatment with quinolones (p < 0.001), b-lactam-b-lactamase inhibitors (p = 0.011) and presence of central venous catheter (p = 0.05), while for C. albicans candidemia were prior treatment with quinolones (p < 0.001), carbapenems (p = 0.003) along with cardiac disease (p < 0.001). Neither duration of hospitalization nor in-hospital mortality [41% for the non-albicans vs 29% for C. albicans group (p = 0.192)] was significantly different between the two candidemia groups. The study reveals the role of antimicrobial exposure as a risk factor for candidemia caused by different species. Prior treatment with b-lactam-b-lactamase inhibitors was associated with non-albicans, while with carbapenems with C. albicans candidemia. Prior use of quinolones was associated with candidemia in general.     

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor

Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.     

Internal arsenite bioassay calibration using multiple bioreporter cell lines

Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error‐prone. A new approach to control assay variations and improve application ease could be an internal calibration based on the use of multiple bioreporter cell lines with drastically different reporter protein outputs at a given analyte concentration. To test this concept, different Escherichia coli‐based bioreporter strains expressing either cytochrome c peroxidase (CCP, or CCP mutants) or β‐galactosidase upon induction with arsenite were constructed. The reporter strains differed either in the catalytic activity of the reporter protein (for CCP) or in the rates of reporter protein synthesis (for β‐galactosidase), which, indeed, resulted in output signals with different intensities at the same arsenite concentration. Hence, it was possible to use combinations of these cell lines to define arsenite concentration ranges at which none, one or more cell lines gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of arsenite in drinking water (10 µg l^?1).     

Epidemiology of Overweight and Obesity in a Greek Adult Population: the ATTICA Study

Objective: To evaluate the status of overweight and obesity in a Greek random sample. Research Methods and Procedures: From 2001 to 2002, 1514 men (20 to 87 years old) and 1528 women (20 to 89 years old) were enrolled into the study. Among several sociodemographic, lifestyle, and bioclinical factors, anthropometric characteristics were also recorded. Overweight and obesity were defined according to the World Health Organization classification. Results: The prevalences of overweight and obesity were 53% and 20% in men and 31% and 15% in women (p for gender differences < 0.05). The age‐adjusted peak prevalence of obesity was observed in men older than 40 years old and women between 50 and 59 years old (Bonferonni α < 0.001). Central obesity prevailed in 36% of men and 43% of women (p for gender differences < 0.001). Obesity varied from 10% in rural to 25% in urban areas, but this difference was explained mainly by differences in occupational status (p = 0.9). Moreover, obese and overweight participants were older, less educated, more frequently sedentary, consumed higher quantities of alcoholic beverages, and were devoted to an unhealthier diet as compared with those of normal weight (all p < 0.05). A positive association was also observed between BMI and diastolic and systolic blood pressures, total cholesterol, triglycerides, and glucose levels (all p < 0.001). Discussion: Overweight and obesity seem to be a great health problem in the Greek population, especially in middle‐aged and older adults. Unfavorable lifestyle habits, low education, and the classical cardiovascular risk factors were associated with the prevalence of these health conditions.     

Adeno-associated virus type 12 (AAV12): a novel AAV serotype with sialic acid- and heparan sulfate proteoglycan-independent transduction activity

Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy. Recent isolations of novel AAV serotypes have led to significant advances by broadening the tropism and increasing the efficiency of gene transfer to the desired target cell. However, a major concern that remains is the strong preexisting immune responses to several vectors. In this paper, we describe the isolation and characterization of AAV12, an AAV serotype with unique biological and immunological properties. In contrast to those of all other reported AAVs, AAV12 cell attachment and transduction do not require cell surface sialic acids or heparan sulfate proteoglycans. Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. The feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications.     

Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal-contaminated bulk and rhizosphere soil

Bacterial diversity in 16S ribosomal DNA and reverse-transcribed 16S rRNA clone libraries originating from the heavy metal-contaminated rhizosphere of the metal-hyperaccumulating plant Thlaspi caerulescens was analysed and compared with that of contaminated bulk soil. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, alpha-Proteobacteria, beta-Proteobacteria, Acidobacteria and the Planctomycetales. Only 14.7% of all phylotypes (sequences with similarities> 97%), but 45% of all clones, were common in the rhizosphere and the bulk soil clone libraries. The combined use of rDNA and rRNA libraries indicated which taxa might be metabolically active in this soil. All dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries compared with the rDNA libraries. Clones belonging to the Verrucomicrobiales, Firmicutes, Cytophaga-Flavobacterium-Bacteroides and OP10 were found only in rDNA clone libraries, indicating that they might not represent active constituents in our samples. The most remarkable result was that sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA (50% and 60% of all phylotypes respectively). Seventy per cent of these clone sequences were related to the Rubrobacteria subgroups 2 and 3, thus providing for the first time evidence that this group of bacteria is probably metabolically active in heavy metal-contaminated soil.     

Differences in circular dichroism and fluorescence between the DNA complexes formed with 3-amino- and 3,8- diaminophenanthridinium derivatives

The conformation of DNA complexes formed with various 3-amino- and 3,8-diamino-phenanthridinium derivatives were examined by CD and fluorescence methods. The CD of these complexes is characterized by major bands in the 300–350-nm and the 400–550-nm regions. The CD properties of the complexes formed with diaminophenanthridinium derivatives suggest that the structure of such complexes is well represented by the intercalation complex formed between DNA and ethidium bromide. The substantial and regular increases in ellipticities near 308 nm that occur with increasing DNA-bound diaminophenanthridinium to DNA phosphate ratios (r) may result from direct interactions between molecules intercalated in neighbouring binding sites. In contrast, the changes in the shape of the CD of DNA complexes of monoaminophenanthridinium derivatives with r and the much lower maximum ellipticities attained suggest that near-neighbor interactions among intercalated monoaminophenanthridinium derivatives occur much less efficiently than in the corresponding diamino complexes, if at all. Although alternative explanations for the differences in the optical properties between the mono- and diaminophenanthridinium complexes of DNA may be offered, such results seem to indicate that complexes formed with monoaminophenanthridinium derivatives are characterized by a conformation which is quite distinct and different from that of the DNA–diaminophenanthridinium complexes. This conclusion is further supported by the considerable increase in fluorescence that accompanies the binding of the diaminophenanthridinium derivatives to DNA as compared to the minor increases, which occur upon the binding of the monoaminophenanthridinium compounds. The importance of conformation as a factor influencing template, function, especially with respect to the RNA polymerase-catalyzed synthesis of RNA, is now well appreciated. Therefore, methods which could provide information readily about changes in the conformation of a template, i.e., as a result of dye intercalation, are expected to facilitate our understanding of the effects of conformational change on the function and activity of templates.