Spatial expression of DNA topoisomerase I genes during cell proliferation in Daucus carota

The spatial expression of carrot (Daucus carota L.) top1 genes encoding the two isoforms of the enzyme DNA topoisomerase I (EC 5.99.1.2) was investigated. In situ hybridization analysis performed with a probe recognizing both top1 transcripts provided evidence that in explanted hypocotyls induced to proliferate in vitro by the addition of the growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), the mRNA accumulation parallels the proliferation of provascular cells of the stelar cylinder. During somatic embryogenesis, the histological distribution of top1 transcripts was strongly evident at the stage of torpedo-shaped embryos, but gene expression was not only restricted to meristematic regions. When the spatial localization was extended to carrot vegetative apices and the investigation was carried out with specific probes for top1a and top1b, both transcripts preferentially accumulated in tissues having mitotic activity

DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity

Background and Aims The germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help monitoring seed deterioration. Methods Dry seeds were subjected to artificial aging and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (Random Amplified Polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin Ciocalteu reagent method. Electron Paramagnetic Resonance (EPR) provided profiles of free radicals. Quantitative RealTime-Polymerase Chain Reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (Type 2 Metallothionein) and SOD (Superoxide Dismutase). A modified QRT-PCR protocol was used to determine telomere length. Key Results The RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial aging. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterised by a lower antioxidant specific activity. Significant up-regulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species. Conclusions Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species

PSMA-PET Guided Treatment in Prostate Cancer Patients with Oligorecurrent Progression after Previous Salvage Treatment

Background: Prostate Specific Membrane Antigen-Positron Emission Tomography (PSMA-PET) is used to select recurrent prostate cancer (PCa) patients for metastases-directed therapy (MDT). We aimed to evaluate the oncologic outcomes of second-line PSMA-guided MDT in oligo-recurrent PCa patients. Methods: we performed a retrospective analysis of 113 recurrent PCa after previous radical prostatectomy and salvage therapies with oligorecurrent disease at PSMA-PET (≤3 lesions in N1/M1a-b) in three high-volume European centres. Patients underwent second-line salvage treatments: MDT targeted to PSMA (including surgery and/or radiotherapy), and the conventional approach (observation or Androgen Deprivation Therapy [ADT]). Patients were stratified according to treatments (MDT vs. conventional approach). Patients who underwent MDT were stratified according to stage in PSMA-PET (N1 vs. M1a-b). The primary outcome of the study was Progression-free survival (PFS). Secondary outcomes were Metastases-free survival (MFS) and Castration Resistant PCa free survival (CRPC-FS). Kaplan-Meier analyses assessed PFS, MFS and CRPC-FS. Multivariable Cox regression models identified predictors of progression and metastatic disease. Results: Overall, 91 (80%) and 22 (20%) patients were treated with MDT and the conventional approach, respectively. The median follow-up after PSMA-PET was 31 months. Patients who underwent MDT had a similar PFS compared to the conventional approach (p = 0.3). Individuals referred to MDT had significantly higher MFS and CRPC-FS compared to those who were treated with the conventional approach (73.5% and 94.7% vs. 30.5% and 79.5%; all p ≤ 0.001). In patients undergoing MDT, no significant differences were found for PFS and MFS according to N1 vs. M1a-b disease, while CRPC-FS estimates were significantly higher in patients with N1 vs. M1a-b (100% vs. 86.1%; p = 0.02). At multivariable analyses, age (HR = 0.96) and ADT during second line salvage treatment (HR = 0.5) were independent predictors of PFS; MDT (HR 0.27) was the only independent predictor of MFS (all p ≤ 0.04) Conclusion: Patients who underwent second-line PSMA-guided MDT experienced higher MFS and CRPC-FS compared to men who received conventional management

Increased Resistance of Bt Aspens to Phratora vitellinae (Coleoptera) Leads to Increased Plant Growth under Experimental Conditions

One main aim with genetic modification (GM) of trees is to produce plants that are resistant to various types of pests. The effectiveness of GM-introduced toxins against specific pest species on trees has been shown in the laboratory. However, few attempts have been made to determine if the production of these toxins and reduced herbivory will translate into increased tree productivity. We established an experiment with two lines of potted aspens (Populus tremula×Populus tremuloides) which express Bt (Bacillus thuringiensis) toxins and the isogenic wildtype (Wt) in the lab. The goal was to explore how experimentally controlled levels of a targeted leaf beetle Phratora vitellinae (Coleoptera; Chrysomelidae) influenced leaf damage severity, leaf beetle performance and the growth of aspen. Four patterns emerged. Firstly, we found clear evidence that Bt toxins reduce leaf damage. The damage on the Bt lines was significantly lower than for the Wt line in high and low herbivory treatment, respectively. Secondly, Bt toxins had a significant negative effect on leaf beetle survival. Thirdly, the significant decrease in height of the Wt line with increasing herbivory and the relative increase in height of one of the Bt lines compared with the Wt line in the presence of herbivores suggest that this also might translate into increased biomass production of Bt trees. This realized benefit was context-dependent and is likely to be manifested only if herbivore pressure is sufficiently high. However, these herbivore induced patterns did not translate into significant affect on biomass, instead one Bt line overall produced less biomass than the Wt. Fourthly, compiled results suggest that the growth reduction in one Bt line as indicated here is likely due to events in the transformation process and that a hypothesized cost of producing Bt toxins is of subordinate significance

Vascular risk factors in glaucoma: the results of a national survey

Background The role of vascular risk factors in glaucoma is still being debated. To assess the importance of vascular risk factors in patients with primary open-angle glaucoma (POAG), data from the medical history of 2,879 POAG patients and 973 age-matched controls were collected and analyzed. Methods Design: observational survey. Setting: 35 Italian academic centers. Study population: POAG patients and age-matched controls. In order to reduce bias consecutive patients were included. Observation procedures: data concerning vascular risk factors were collected for all patients with a detailed questionnaire. A complete ophthalmological examination with assessment of intraocular pressure (IOP), visual field, optic disc, and systemic blood pressure was performed. Main outcome measures: the ESH-ESC (European Society of Hypertension-European Society of Cardiology) guidelines were used to calculate the level of cardiovascular risk. Crude and adjusted estimates of the odds ratios (OR) were calculated for all cardiovascular risk factors in POAG and controls. Results The study included 2,879 POAG patients and 973 controls. POAG cases had a significantly higher systolic and diastolic blood pressure (p=0.001) and systolic perfusion pressure (p=0.02) as compared with controls. Also mean IOP was significantly higher in the POAG group (p=0.01), while diastolic perfusion pressure was not significantly different in the two groups. Myopia was more prevalent in the POAG group (23 vs 18%, p=0.005) as well as a positive family history for glaucoma (26 vs 12%, p= 0.004). POAG patients tended to have a higher cardiovascular risk than controls: 63% of glaucoma cases vs 55% of controls (OR: 1.38, p=0.005) had a “high” or “very high” cardiovascular risk. Conclusions The level of cardiovascular risk was significantly higher in glaucoma patients than in controls

Different approaches for bladder neck dissection during robot-assisted radical prostatectomy: the Aalst technique

ABSTRACT Introduction: Bladder neck dissection is one of the most delicate surgical steps of robotic-assisted radical prostatectomy (RARP) [1, 2], and it may affect surgical margins rate and functional outcomes [3, 4]. Given the relationship between outcomes and surgical experience [5–7], it is crucial to implement a step-by-step approach for each surgical step of the procedure, especially in the most challenging part of the intervention. In this video compilation, we described the techniques for bladder neck dissection utilized at OLV Hospital (Aalst, Belgium). Surgical Technique: We illustrated five different techniques for bladder neck dissection during RARP. The anterior technique tackles the bladder neck from above until the urethral catheter is visualized, and then the dissection is completed posteriorly. The lateral and postero-lateral approaches involve the identification of a weakness point at the prostate-vesical junction and aim to develop the posterior plane – virtually until the seminal vesicles – prior to the opening of the urethra anteriorly. Finally, we described our techniques for bladder neck dissection in more challenging cases such as in patients with bulky middle lobes and prior surgery for benign prostatic hyperplasia. All approaches follow anatomic landmarks to minimize positive surgical margins and aim to preserve the bladder neck in order to promote optimal functional recovery. All procedures were performed with DaVinci robotic platforms using a 3-instruments configuration (scissors, fenestrated bipolar, and needle driver). As standard protocol at our Institution, urinary catheter was removed on postoperative day two [8]. Conclusions: Five different approaches for bladder neck dissection during RARP were described in this video compilation. We believe that the technical details provided here might be of help for clinicians who are starting their practice with this surgical intervention

Effects of Heavy Metals and Arbuscular Mycorrhiza on the Leaf Proteome of a Selected Poplar Clone: A Time Course Analysis

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with the roots of most plant species. While receiving photosynthates, they improve the mineral nutrition of the plant and can also increase its tolerance towards some pollutants, like heavy metals. Although the fungal symbionts exclusively colonize the plant roots, some plant responses can be systemic. Therefore, in this work a clone of Populus alba L., previously selected for its tolerance to copper and zinc, was used to investigate the effects of the symbiosis with the AM fungus Glomus intraradices on the leaf protein expression. Poplar leaf samples were collected from plants maintained in a glasshouse on polluted (copper and zinc contaminated) or unpolluted soil, after four, six and sixteen months of growth. For each harvest, about 450 proteins were reproducibly separated on 2DE maps. At the first harvest the most relevant effect on protein modulation was exerted by the AM fungi, at the second one by the metals, and at the last one by both treatments. This work demonstrates how importantly the time of sampling affects the proteome responses in perennial plants. In addition, it underlines the ability of a proteomic approach, targeted on protein identification, to depict changes in a specific pattern of protein expression, while being still far from elucidating the biological function of each protein

Phytoremediation of heavy metal-contaminated sites: Eco-environmental concerns, field studies, sustainability issues and future prospects

Environmental contamination due to heavy metals (HMs) is of serious ecotoxicological concern worldwide because of their increasing use at industries. Due to non-biodegradable and persistent nature, HMs cause serious soil/water pollution and severe health hazards in living beings upon exposure. HMs can be genotoxic, carcinogenic, mutagenic, and teratogenic in nature even at low concentration. They may also act as endocrine disruptors and induce developmental as well as neurological disorders and thus, their removal from our natural environment is crucial for the rehabilitation of contaminated sites. To cope with HM pollution, phytoremediation has emerged as a low-cost and eco-sustainable solution to conventional physico-chemical cleanup methods that require high capital investment and labor alter soil properties and disturb soil microflora. Phytoremediation is a green technology wherein plants and associated microbes are used to remediate HM-contaminated sites to safeguard the environment and protect public health. Hence, in view of the above, the present paper aims to examine the feasibility of phytoremediation as a sustainable remediation technology for the management of metals-contaminated sites. Therefore, this paper provides an in-depth review on both the conventional and novel phytoremediation approaches, evaluate their efficacy to remove toxic metals from our natural environment, explore current scientific progresses, field experiences and sustainability issues and revise world over trends in phytoremediation research for its wider recognition and public acceptance as a sustainable remediation technology for the management of contaminated sites in 21st century

Deep lamellar keratoplasty with trypan blue intrastromal staining.

This technique was developed to facilitate removal of the deep stromal layers during deep lamellar keratoplasty. A trypan blue 0.02% solution is injected into the stromal fibers, enabling the surgeon to visualize and remove the posterior stromal layers. This decreases the risk of perforation of Descemet’s membrane and the endothelium