Identification of ORC1/CDC6-interacting factors in Trypanosoma brucei reveals critical features of origin recognition complex architecture

DNA replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1-6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.     

Does S-metolachlor affect the performance of Pseudomonas sp. strain ADP as bioaugmentation bacterium for atrazine-contaminated soils?

Atrazine (ATZ) and S-metolachlor (S-MET) are two herbicides widely used, often as mixtures. The present work examined whether the presence of S-MET affects the ATZ-biodegradation activity of the bioaugmentation bacterium Pseudomonas sp. strain ADP in a crop soil. S-MET concentrations were selected for their relevance in worst-case scenarios of soil contamination by a commercial formulation containing both herbicides. At concentrations representative of application of high doses of the formulation (up to 50 μg g(-1) of soil, corresponding to a dose approximately 50× higher than the recommended field dose (RD)), the presence of pure S-MET significantly affected neither bacteria survival (~10(7) initial viable cells g(-1) of soil) nor its ATZ-mineralization activity. Consistently, biodegradation experiments, in larger soil microcosms spiked with 20× or 50 × RD of the double formulation and inoculated with the bacterium, revealed ATZ to be rapidly (in up to 5 days) and extensively (>96%) removed from the soil. During the 5 days, concentration of S-MET decreased moderately to about 60% of the initial, both in inoculated and non-inoculated microcosms. Concomitantly, an accumulation of the two metabolites S-MET ethanesulfonic acid and S-MET oxanilic acid was found. Despite the dissipation of almost all the ATZ from the treated soils, the respective eluates were still highly toxic to an aquatic microalgae species, being as toxic as those from the untreated soil. We suggest that this high toxicity may be due to the S-MET and/or its metabolites remaining in the soil.     

Development of mycorrhized vitroplants of Jatropha curcas L. at different rooting stages

In this work, we attempted to assess the effects of inoculation of arbuscular mycorrhizal fungus (AMF), Glomus clarum, on the survival and development of micropropagated Jatropha curcas plantlets at different rooting stages. Elongated shoots (average 3?cm) of J. curcas, maintained for 0, 14, or 21?days on rooting medium in the presence or absence of 1?mg?L^?1 indole-3-butyric acid (IBA), were transferred to a sand:soil:vermiculite (1:1/2:1) (v:v:v) substrate. At the time of transplantation, the plantlets were either inoculated or not inoculated with G. clarum that had been monoaxenically produced in Ri-transformed carrot roots. After a 2-week acclimatization period, 100?% of the plants kept for 0 or 14?days in rooting medium survived. However, those that remained for 21?days in rooting medium displayed post-acclimatization survival rates of 93 and 89?% for plants inoculated and non-inoculated with G. clarum, respectively. Colonization rates ranged from 70 to 93?%, and the stimulatory effects of AMF were evidenced by increased phosphorus uptake by plants and increases in all evaluated growth parameters, except plant height. Plants that were not subjected to the rooting stage showed growth similar to or higher than those subjected to the rooting stage, regardless of the addition of IBA. It can be concluded that stimulatory effects of mycorrhizal fungi were observed, and that the inoculation of J. curcas proved effective during the initial period of the acclimatization phase.     

Neuroprotection afforded by adenosine A2A receptor blockade is modulated by corticotrophin‐releasing factor (CRF) in glutamate injured cortical neurons

In situations of hypoxia, glutamate excitotoxicity induces neuronal death. The release of extracellular adenosine is also triggered and is accompanied by an increase of the stress mediator, corticotrophin‐releasing factor (CRF). Adenosine A_2A receptors contribute to glutamate excitoxicity and their blockade is effective in stress‐induced neuronal deficits, but the involvement of CRF on this effect was never explored. We now evaluated the interaction between A_2A and CRF receptors (CRFR) function, upon glutamate insult. Primary rat cortical neuronal cultures (9 days in vitro) expressing both CRF₁R and CRF₂R were challenged with glutamate (20–1000 μM, 24 h). CRF₁R was found to co‐localize with neuronal markers and CRF₂R to be present in both neuronal and glial cells. The effects of the CRF and A_2A receptors ligands on cell viability were measured using propidium iodide and Syto‐13 fluorescence staining. Glutamate decreased cell viability in a concentration‐dependent manner. Urocortin (10 pM), an agonist of CRF receptors, increased cell survival in the presence of glutamate. This neuroprotective effect was abolished by blocking either CRF₁R or CRF₂R with antalarmin (10 nM) or anti‐Sauvagine‐30 (10 nM), respectively. The blockade of A_2A receptors with a selective antagonist SCH 58261 (50 nM) improved cell viability against the glutamate insult. This effect was dependent on CRF₂R, but not on CRF₁R activation. Overall, these data show a protective role of CRF in cortical neurons, against glutamate‐induced death. The neuroprotection achieved by A_2A receptors blockade requires CRF₂R activation. This interaction between the adenosine and CRF receptors can explain the beneficial effects of using A_2A receptor antagonists against stress‐induced noxious effects.     

Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells

Central nervous system (CNS) disorders remain a formidable challenge for the development of efficient therapies. Cell and gene therapy approaches are promising alternatives that can have a tremendous impact by treating the causes of the disease rather than the symptoms, providing specific targeting and prolonged duration of action. Hampering translation of gene-based therapeutic treatments of neurodegenerative diseases from experimental to clinical gene therapy is the lack of valid and reliable pre-clinical models that can contribute to evaluate feasibility and safety. Herein we describe a robust and reproducible methodology for the generation of 3D in vitro models of the human CNS following a systematic technological approach based on stirred culture systems. We took advantage of human midbrain-derived neural progenitor cells (hmNPCs) capability to differentiate into the various neural phenotypes and of their commitment to the dopaminergic lineage to generate differentiated neurospheres enriched in dopaminergic neurons. Furthermore, we describe a protocol for efficient gene transfer into differentiated neurospheres using CAV-2 viral vectors and stable expression of the transgene for at least 10 days. CAV-2 vectors, derived from canine adenovirus type 2, are promising tools to understand and treat neurodegenerative diseases, in particular Parkinson's disease. CAV-2 vectors preferentially transduce neurons and have an impressive level of axonal retrograde transport in vivo. Our model provides a practical and versatile in vitro approach to study the CNS in a 3D cellular context. With the successful differentiation and subsequent genetic modification of neurospheres we are increasing the collection of tools available for neuroscience research and contributing for the implementation and widespread utilization of 3D cellular CNS models. These can be applied to study neurodegenerative diseases such as Parkinson's disease; to study the interaction of viral vectors of therapeutic potential within human neural cell populations, thus enabling the introduction of specific therapeutic genes for treatment of CNS pathologies; to study the fate and effect of delivered therapeutic genes; to study toxicological effects. Furthermore these methodologies may be extended to other sources of human neural stem cells, such as human pluripotent stem cells, including patient-derived induced pluripotent stem cells.     

A proteomics study of the induction of somatic embryogenesis in Medicago truncatula using 2DE and MALDI-TOF/TOF

Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation.     

Susceptibilities of Candida albicans Mouth Isolates to Antifungal Agents, Essentials Oils and Mouth Rinses

Forty Candida albicans strains isolated from patient's mouth with fixed orthodontic appliances were analyzed to their susceptibilities to antifungal agents, mouth rinses and essential oils. Susceptibility to fluconazole, econazole, miconazole and ketoconazole, amphotericin B and nystatin was assessed by the disk diffusion (DD) method based on the Clinical and Laboratory Standards Institute M44-A protocol, and by Etest (fluconazole and amphotericin B). The susceptibilities to mouth rinses and essential oils were also determined by the DD technique. All isolates tested were susceptible (S) to amphotericin B, nystatin and fluconazole. The overall concordance between the DD and the Etest was 100% for amphotericin and fluconazole. One isolate was resistant to econazole (2.5%) and the other to ketoconazole (2.5%). Econazole and ketoconazole had the highest percentages of susceptible dose dependent (SDD), 55 and 95%, respectively. Regarding to the susceptibility isolates profile, seven phenotypes were detected, and the 3 more represented (90% of the isolates) of them were SDD to one, two or three azoles. The study of mouth rinses showed a high variability of efficacy against C. albicans. The results showed that the isolates susceptibility to essential oils differed (P < 0.05). The profile activity was: cinnamon > laurel > mint > eucalyptus > rosemary > lemon > myrrh > tangerine. The main finding was that the susceptibility to cinnamon and laurel varied among the three more representative antifungal phenotypes (P < 0.05). The susceptibility of econazole-SDD isolates to cinnamon and lemon was higher than those of the econazole-S yeasts (P < 0.05). In contrast, econazole-SDD isolates were less affected by laurel than econazole-S counterparts (P < 0.05).     

The effects of repeated oral gavage on the health of male CD-1 mice

Oral gavage is a widely used method for administering substances to animals in pharmacological and toxicological studies. The authors evaluated whether oral gavage causes behavioral indicators of stress, increased mortality rate, alterations in food and water consumption and body weight or histological lesions in CD-1 mice. Gavage was carried out once per d for 5 d per week over 6 consecutive weeks. The mortality rate of mice in this study was 15%. Mice subjected to gavage did not undergo changes in food or water consumption during the study, and their mean body weights and relative organ weights were similar to those of mice in the control group. Serum cortisol levels at the time of euthanasia in mice in both groups were within the normal range. Histopathology showed acute esophagitis and pleurisy, indicative of perforation of the esophagus, in the two mice that died but no abnormalities in the other mice. The results suggest that animal stress and mortality related to oral gavage can be minimized when the procedure is carried out by an experienced technician.