Lisete Fernandes

The evolution of a conjugative plasmid and its ability to increase bacterial fitness

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.

Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase

Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

OsRMC, a negative regulator of salt stress response in rice, is regulated by two AP2/ERF transcription factors

High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5xa0°C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice.

What do we know about the α/β for prostate cancer?

Since last decade, the debate on the parameter which reflects prostate cancer sensitivity to fractionation in a radiotherapy treatment, the α/β, has become extensive. Unlike most tumors, the low labeling indices (LI) and large potential doubling time that characterize the prostate tumor led some authors to consider that it may behave as a late responding tissue. So far, the existing studies with regard to this subject point to a low value of α/β, around 2.7 Gy, which may be considered as a therapeutic gain in relation to surrounding normal tissues by using fewer and larger fractions. The aim of this paper is to review several estimates that have been made in the last few years regarding the prostate cancer α/β both from clinical and experimental data, as well as the set of factors that have potentially influenced these evaluations.

Dosimetric effect of tissue heterogeneity for 125I prostate implants

AIMnTo use Monte Carlo (MC) together with voxel phantoms to analyze the tissue heterogeneity effect in the dose distributions and equivalent uniform dose (EUD) for (125)I prostate implants.nnnBACKGROUNDnDose distribution calculations in low dose-rate brachytherapy are based on the dose deposition around a single source in a water phantom. This formalism does not take into account tissue heterogeneities, interseed attenuation, or finite patient dimensions effects. Tissue composition is especially important due to the photoelectric effect.nnnMATERIALS AND METHODSnThe computed tomographies (CT) of two patients with prostate cancer were used to create voxel phantoms for the MC simulations. An elemental composition and density were assigned to each structure. Densities of the prostate, vesicles, rectum and bladder were determined through the CT electronic densities of 100 patients. The same simulations were performed considering the same phantom as pure water. Results were compared via dose-volume histograms and EUD for the prostate and rectum.nnnRESULTSnThe mean absorbed doses presented deviations of 3.3-4.0% for the prostate and of 2.3-4.9% for the rectum, when comparing calculations in water with calculations in the heterogeneous phantom. In the calculations in water, the prostate D 90 was overestimated by 2.8-3.9% and the rectum D 0.1cc resulted in dose differences of 6-8%. The EUD resulted in an overestimation of 3.5-3.7% for the prostate and of 7.7-8.3% for the rectum.nnnCONCLUSIONSnThe deposited dose was consistently overestimated for the simulation in water. In order to increase the accuracy in the determination of dose distributions, especially around the rectum, the introduction of the model-based algorithms is recommended.

Tissue composition and density impact on the clinical parameters for 125I prostate implants dosimetry

The MCNPX code was used to calculate the TG-43U1 recommended parameters in water and prostate tissue in order to quantify the dosimetric impact in 30 patients treated with (125)I prostate implants when replacing the TG-43U1 formalism parameters calculated in water by a prostate-like medium in the planning system (PS) and to evaluate the uncertainties associated with Monte Carlo (MC) calculations. The prostate density was obtained from the CT of 100 patients with prostate cancer. The deviations between our results for water and the TG-43U1 consensus dataset values werexa0-2.6% for prostate V100,xa0-13.0% for V150, andxa0-5.8% for D90;xa0-2.0% for rectum V100, andxa0-5.1% for D0.1;xa0-5.0% for urethra D10, andxa0-5.1% for D30. The same differences between our water and prostate results were all under 0.3%. Uncertainties estimations were up to 2.9% for the gL(r) function, 13.4% for the F(r,θ) function and 7.0% for Λ, mainly due to seed geometry uncertainties. Uncertainties in extracting the TG-43U1 parameters in the MC simulations as well as in the literature comparison are of the same order of magnitude as the differences between dose distributions computed for water and prostate-like medium. The selection of the parameters for the PS should be done carefully, as it may considerably affect the dose distributions. The seeds internal geometry uncertainties are a major limiting factor in the MC parameters deduction.

Absence of Gim proteins, but not GimC complex, alters stress-induced transcription

Saccharomyces cerevisiae GimC (mammalian Prefoldin) is a hexameric (Gim1-6) cytoplasmic complex involved in the folding pathway of actin/tubulin. In contrast to a shared role in GimC complex, we show that absence of individual Gim proteins results in distinct stress responses. No concomitant alteration in F-actin integrity was observed. Transcription of stress responsive genes is altered in gim2Δ, gim3Δ and gim6Δ mutants: TRX2 gene is induced in these mutants but with a profile diverging from type cells, whereas CTT1 and HSP26 fail to be induced. Remaining gimΔ mutants display stress transcript abundance comparable to wild type cells. No alteration in the nuclear localization of the transcriptional activators for TRX2 (Yap1) and CTT1/HSP26 (Msn2) was observed in gim2Δ. In accordance with TRX2 induction, RNA polymerase II occupancy at TRX2 discriminates the wild type from gim2Δ and gim6Δ. In contrast, RNA polymerase II occupancy at CTT1 is similar in wild type and gim2Δ, but higher in gim6Δ. The absence of active RNA polymerase II at CTT1 in gim2Δ, but not in wild type and gim1Δ, explains the respective CTT1 transcript outputs. Altogether our results put forward the need of Gim2, Gim3 and Gim6 in oxidative and osmotic stress activated transcription; others Gim proteins are dispensable. Consequently, the participation of Gim proteins in activated-transcription is independent from the GimC complex.

EP-1493 ALTERNATIVE REGIMENS FOR PROSTATE CANCER TREATMENT USING RADIATION THERAPY

Purpose/Objective: The purpose of this work was to determine biologically equivalent alternative regimens for the treatment of prostate cancer using External Beam Radiotherapy (EBRT) and Low Dose-Rate Brachytherapy (LDRBT) with 125I implants and to evaluate the sensitivity of these regimens to different sets of radiobiological parameters of the LinearQuadratic (LQ) model. Materials and Methods: The biological effective dose (BED) was used to determinate equivalences of the two modalities assuming that BED values are additive for different treatment techniques. Two sets of parameters were employed considering whether accelerated repopulation of the clonogens during the treatment time occurs: LQ1) α/β=3.1 Gy, α=0.15 Gy-1,μ=2.6 h-1, Tpot=42 days, N=3x106 clonogens, or not, LQ2) α/β=1.5 Gy, α=0.03 Gy