An in vitro model of nociceptors in human trigeminal nerves

Background: The oro-facial region is densely innervated by the trigeminal nerve, which when stimulated can induce noxious pain sensation and contribute to neurogenic inflammation in local tissues. Recent research on the expression of specialised ion channels on the trigeminal nerve has highlighted the need to undertake more extensive studies on ion channel expression/functionality with the aim of elucidating their role in pain sensations. A major family of such ion channels is the transient receptor potential (TRP) channels which are activated by a wide variety of thermal, mechanical or chemical stimuli and merit investigation as possible druggable targets for future analgesics.
Objective: Study of TRP channel expression and regulation in oro-facial tissues is hindered by the fact that the cell bodies of neurons innervating these tissues are located in the trigeminal ganglion. Using dental pulp stem cells differentiated towards peripheral neuronal equivalents (PNEs), we sought to determine TRP channel expression, functionality and potential modulation by cytokines in this novel model.
Method: Dental pulp stem cells (DPSCs) were grown on substrate-coated tissue culture plates and differentiated towards a neuronal phenotype using neuronal induction media. Quantitative polymerase chain reaction (qPCR) was performed on PNEs +/-cytokine treatment. Ion channel functionality was investigated using whole cell patch clamping.
Result: qPCR analysis showed that PNEs expressed the TRP channels TRPA1, TRPV1, TRPV4 and TRPM8. TRPA1 was the most abundantly expressed TRP channel studied whereas TRPM8 was lowly expressed. TRP channel expression was shown to be regulated by treatment with inflammatory cytokines. Patch clamp studies using specific agonists and antagonists for TRPA1 and TRPV1 showed these channels were functional.
Conclusion: PNEs differentiated from DPSCs provide a suitable model for TRP channel expression, regulation, and sensistisation in oro-facial tissues. This human neuronal model has potential for use in pre-clinical studies of novel analgesics.

Root caries development in older adults- a prospective cohort study

Objectives: To identify factors associated with root caries development during a two year period in a population of independently living older adults. Methods: A prospective cohort study was carried out with 334 independently living volunteers aged 65 and older. At baseline (t0), each participant completed a questionnaire which recorded age, gender, medical history, fluoride exposure, oral and denture hygiene practices, smoking and alcohol consumption, diet information, and socio economic information. Clinical examinations were performed and stimulated saliva samples were collected. Patients were reviewed 12(t1) and 24(t2) months later to determine the root caries increment. Results: 307 adults were assessed at t1 and 280 were assessed at t2 with 83.8% of participants examined at 24 months. Incidence of root caries in this cohort was 17.4% at t1 and 21.6% at t2. The mean root caries increment was 0.43 (SD 1.45) surfaces at t1 and 0.70 (SD 1.86) surfaces at t2. Age >70 years, completing education at primary level, poor oral hygiene, xerostomia, coronal decay at baseline, higher root caries index at baseline and number of exposed root surfaces showed a statistically significant association (P < 0.05) with root caries development. Conclusion: Root caries is a substantive dental health problem for our older population. Root caries prevention strategies should be targeted at older adults who have poor plaque control and high levels of caries experience. In particular patients with xerostomia should be targeted with preventive measures.

Risk indicators associated with root decay in independently living older adults

Objective: To determine the risk indicators associated with root caries experience in a cohort of independently living older adults in Ireland.
Methods: The data reported in the present study were obtained from a prospective longitudinal study conducted on the risk factors associated with root caries incidence in a cohort of independently living older adults (n=334). Each subject underwent an oral examination, performed by a single calibrated examiner, to determine the root caries index and other clinical variables. Questionnaires were used to collect data on oral hygiene habits, diet, smoking and alcohol habits and education level. A regression analysis with the outcome variable of root caries experience (no/yes) was conducted.
Results: A total of 334 older adults with a mean age of 69.1 years were examined. 53.3% had at least one filled or decayed root surface. The median root caries index was 3.13 (IQR 0.00, 13.92). The results from the multivariate regression analysis indicated that individuals with poor plaque control (OR 9.59, 95%CI 3.84-24.00), xerostomia (OR 18.49, 95%CI 2.00-172.80), two or more teeth with coronal decay (OR 4.50, 95% CI 2.02-10.02) and 37 or more exposed root surfaces (OR 5.48, 95% CI 2.49-12.01) were more likely to have been affected by root caries.
Conclusions: The prevalence of root caries was high in this cohort. This study suggests a correlation between root caries and the variables poor plaque control, xerostomia, coronal decay (≥2 teeth affected) and exposed root surfaces (≥37). The significance of these risk indicators and the resulting prediction model should be further evaluated in a prospective study of root caries incidence.
Clinical Significance: Identification of risk indicators for root caries in independently living older adults would facilitate dental practitioners to identify those who would benefit most from interventions aimed at prevention.

Functional analysis of String/CDC25 phosphatase in post-mitotic neurons

Cell cycle and differentiation are two highly coordinated processes during organ development. Recent studies have demonstrated that core cell cycle regulators also play cell cycle-independent functions in post-mitotic neurons, and are essential for the maintenance of neuronal homeostasis. CDC25 phosphatases are well-established CDK activators and their activity is mainly associated to proliferating tissues. The expression and activity of mammalian CDC25s has been reported in adult brains. However, their physiological relevance and the potential substrates in a non-proliferative context have never been addressed. string (stg) encodes the Drosophila CDC25 homolog. Previous studies from our group showed that stg is expressed in photoreceptors (PRs) and in lamina neurons, which are two differentiated cell types that compose the fly visual system. The aims of this work are to uncover the function of stg and to identify its potential neuronal substrates, using the Drosophila visual system as a model. To gain insight into the function of stg in a non-dividing context we used the GAL4/UAS system to promote downregulation of stg in PR-neurons, through the use of an RNAi transgene. The defects caused by stg loss-of-function were evaluated in the developing eye imaginal disc by immunofluorescence, and during adult stages by scanning electron microscopy. This genetic approach was combined with a specific proteomic method, two-dimensional difference gel electrophoresis (2D-DIGE), to identify the potential substrates in PR-cells. Our results showed that stg downregulation in PRs affects the well-patterned retina organization, inducing the loss of apical maintenance of PR-nuclei on the eye disc, and ommatidia disorganization. We also detected an abnormal accumulation of cytoskeletal proteins and a disruption of the axon structure. As a consequence, the projection of PR-axons into the lamina and medulla neuropils of the optic lobe was impaired. Upon stg downregulation, we also detected that PR-cells accumulate Cyclin B. Although the rough eye phenotype observed upon stg downregulation suggests neurodegeneration, we did not detect neuronal death during larval stages, suggesting that it likely occurs during pupal stages or during adulthood. By 2D-DIGE, we identified seven proteins which were differentially expressed upon stg downregulation, and are potential neuronal substrates of Stg. Altogether, our observations suggest that Stg phosphatase plays an essential role in the Drosophila visual system neurons, regulating several cell components and processes in order to ensure their homeostasis.

Root ferric chelate reductase is regulated by iron and copper in straberry plants

In the present experiment, we studied the interaction between copper (Cu) and iron (Fe) in strawberry plants grown in nutrient solutions containing different concentrations of Fe. Plants grown in the absence of iron (Fe0) had the characteristic symptoms of Fe deficiency, with smaller chlorotic leaves, less biomass, acidification of the nutrient solution, and roots that were smaller and less ramified, while no symptoms of Fe deficiency were observed in plants grown with Fe. A greater amount of Cu was found in roots of chlorotic plants than in those grown with Fe, while plants grown with 20M of Fe (Fe20) in the nutrient solution had a greater amount of Fe compared with plants from the other treatments. Chlorotic plants (Fe0) and plants grown with the greatest level of Fe (Fe20) had a greater root ferric chelate reductase (FC-R; EC 1.16.1.17) activity compared with the other treatments with 5 or 10M Fe in the nutrient solution. The same pattern was obtained for relative FC-R mRNA concentration and for the sum of Fe and Cu contents in shoots (leaves plus crowns). The DNA obtained from amplification of the FC-R mRNA was cloned and several of the inserts analysed by single strand confirmation polymorphism (SSCP). Although there were different SSCP patterns in the Fe20 treatment, all the inserts that were sequenced were very similar, excluding the hypothesis of more than one FC-R mRNA species being present. The results suggest that Cu as well as Fe is involved in FC-R expression and activity, although the mechanism involved in this regulation is unknown so far. Both small contents of Fe and Cu in plants led to an over-expression of the FC-R gene and enhanced FC-R activity in strawberry roots.

Evaluation of the protective effect of Phlomis purpurea against Phytophthora cinnamomi in Fagaceae and of root metabolites involved

Tese de doutoramento, Ciências Agrárias (Proteção de Plantas), Faculdade de Ciência e Tecnologia, Universidade do Algarve, 2014

Root locus of fractional linear systems

In this paper an algorithm for the calculation of the root locus of fractional linear systems is presented. The proposed algorithm takes advantage of present day computational resources and processes directly the characteristic equation, avoiding the limitations revealed by standard methods. The results demonstrate the good performance for different types of expressions.

Molecular regulation of secondary gorwth: searching for SHORT-ROOT function in cork formation

The analysis of molecular regulators involved in controlling the maintenance and function of plant meristems has been the subject of many studies. Some master regulators of these processes have been identified in Arabidopsis benefiting from the array of tools available for genetic and molecular analysis in this model plant. However, aspects such as secondary growth that are more extensively observed in woody plants, have been less studied. Secondary growth is responsible for the enlargement of the plant stems and roots and results from the activity of the lateral (secondary) meristems, vascular cambium and cork cambium (phellogen), which produce two important renewable natural resources, wood and cork, respectively.(...)

Dynamics of serotonergic neurons revealed by fiber photometry

This work was developed in the context of the MIT Portugal Program, area of Bioengineering Systems, in collaboration with the Champalimaud Research Programme, Champalimaud Center for the Unknown, Lisbon, Portugal. The project entitled Dynamics of serotonergic neurons revealed by fiber photometry was carried out at Instituto Gulbenkian de Ciência, Oeiras, Portugal and at the Champalimaud Research Programme, Champalimaud Center for the Unknown, Lisbon, Portugal

Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia.

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Promise for plant pest control: root-associated pseudomonads with insecticidal activities.

Insects are an important and probably the most challenging pest to control in agriculture, in particular when they feed on belowground parts of plants. The application of synthetic pesticides is problematic owing to side effects on the environment, concerns for public health and the rapid development of resistance. Entomopathogenic bacteria, notably Bacillus thuringiensis and Photorhabdus/Xenorhabdus species, are promising alternatives to chemical insecticides, for they are able to efficiently kill insects and are considered to be environmentally sound and harmless to mammals. However, they have the handicap of showing limited environmental persistence or of depending on a nematode vector for insect infection. Intriguingly, certain strains of plant root-colonizing Pseudomonas bacteria display insect pathogenicity and thus could be formulated to extend the present range of bioinsecticides for protection of plants against root-feeding insects. These entomopathogenic pseudomonads belong to a group of plant-beneficial rhizobacteria that have the remarkable ability to suppress soil-borne plant pathogens, promote plant growth, and induce systemic plant defenses. Here we review for the first time the current knowledge about the occurrence and the molecular basis of insecticidal activity in pseudomonads with an emphasis on plant-beneficial and prominent pathogenic species. We discuss how this fascinating Pseudomonas trait may be exploited for novel root-based approaches to insect control in an integrated pest management framework.

The role of the hypothalamic kisspeptin/GPR54 system in the control of GnRH neurons

Summary : The hypothalamus represents less than 1 % of the total volume of the brain tissue, yet it plays a crucial role in endocrine regulations. Puberty is defined as a process leading to physical, sexual and psychosocial maturation. The hypothalamus is central to this process, via the activation of GnRH neurons. Pulsatile GnRH secretion, minimal during childhood, increases with the onset of puberty. The primary function of GnRH is to regulate the growth, development and function of testes in boys and ovaries in girls, by stimulating the pituitary gland secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several factors contribute to the timing of puberty, including sex and ethnicity, genetics, dietary intake and energy expenditure. Kisspeptins constitute a family of small peptides arising from the proteolytic cleavage of metastin, a peptide with 54 amino acids initially purified from human placenta. These kisspeptins were the subject of much attention following their discovery because of their antimetastatic properties, but it was more recently that their determining role in the reproductive function was demonstrated. It was shown that kisspeptins are ligands of a receptor, GPR54, whose natural inactivating mutation in humans, or knockout in the mouse, lead to infertility. GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH release and GnRH neuron firing activity, but the neurobiological mechanisms for these actions are unknown. Gprotein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting that both direct and indirect effects are possible. In the first part of my thesis, we investigated a possible connection between the acceleration of sexual development induced by leptin and hypothalamic metastin neurons. However, the data generated by our preliminary experiments confirmed that the commercially available antibodies are non-specific. This finding constituted a major drawback for our studies, which relied heavily upon the neuroanatomical study of the hypothalamic metastinergic pathways to elucidate their sensitivity to exogenous leptin. Therefore, we decided to postpone any further in vivo experiment until a better antibody becomes available, and focused on in vitro studies to better understand the mechanisms of action of kisspeptins in the modulation of the activity of GnRH neurons. We used two GnRH-expressing neuronal cell lines to investigate the cellular and molecular mechanisms of action of metastin in GnRH neurons. We demonstrated that kisspeptin induces an early activation of the MAP kinase intracellular signaling pathway in both cell lines, whereas the SAP/JNK or the Akt pathways were unaffected. Moreover, we found an increase in GnRH mRNA levels after 6h of metastin stimulation. Thus, we can conclude that kisspeptin regulates GnRH neurons both at the secretion and the gene expression levels. The MAPK pathway is the major pathway activated by metastin in GnRH expressing neurons. Taken together, these data provide the first mechanism of action of kisspeptin on GnRH neurons. Résumé : L'hypothalamus est une zone située au centre du cerveau, dont il représente moins de 1 du volume total. La puberté est la période de transition entre l'enfance et l'age adulte, qui s'accompagne de transformations somatiques, psychologiques, métaboliques et hormonales conduisant à la possibilité de procréer. La fonction principale de la GnRH est la régulation de la croissance, du développement et de la fonction des testicules chez les hommes, et des ovaires chez les femmes en stimulant la sécrétion de l'hormone lutéinisante (LH) et de l'hormone folliculostimulante (FSH) par la glande hypophysaire. Plusieurs facteurs contribuent au déclanchement de la puberté, y compris le sexe et l'appartenance ethnique, la génétique, l'apport alimentaire et la dépense énergétique. Les Kisspeptines constituent une famille de peptides résultant de la dissociation proteolytique de la métastine, un peptide de 54 acides aminés initialement purifié à partir de placenta humain. Ces kisspeptines ont fait l'objet de beaucoup d'attention à la suite de leur découverte en raison de leurs propriétés anti-metastatiques, et c'est plus récemment que leur rôle déterminant dans la fonction reproductive a été démontré. Les kisspeptines sont des ligands du récepteur GPR54, dont la mutation inactivatrice chez l'homme, ou le knockout chez la souris, conduisent à l'infertilité par hypogonadisme hypogonadotrope. Les neurones à GnRH jouent un rôle central dans le règlement des fonctions reproductrices et la kisspeptine stimule l'activité des neurones à GnRH et la libération de GnRH par ces neurones. Toutefois, les mécanismes neurobiologiques de ces actions ne sont pas connus. Dans la première partie de ma thèse, nous avons étudié le lien potentiel entre l'accélération du développement sexuel induite par la leptine et les neurones hypothalamiques à metastine. Les données générées dans cette première série d'expériences ont malheureusement confirmé que les anticorps anti-metastine disponibles dans le commerce sont aspécifiques. Ceci a constitué un inconvénient majeur pour nos études, qui devaient fortement s'appuyer sur l' étude neuroanatomique des neurones hypothalamiques à metastine pour évaluer leur sensibilité à la leptine exogène. Nous avons donc décidé de focaliser nos travaux sur une étude in vitro des mécanismes d'action de la kisspeptine pour moduler l'activité des neurones à GnRH. Nous avons utilisé deux lignées de cellules neuronales exprimant la GnRH pour étudier les mécanismes d'action cellulaires et moléculaires de la metastine dans des neurones. Nous avons ainsi pu démontrer que la kisspeptine induit une activation précoce de la voie f de signalisation de la MAP kinase dans les deux lignées cellulaires, alors que nous n'avons observé aucune activation de la voie de signalisation de la P13 Kinase et de la SAP/JNK. Nous avons en outre démontré une augmentation de l'expression de la GnRH par la stimulation avec la Kisspeptine. L'ensemble de ces données contribue à élucider le mécanisme d'action avec lequel la kisspeptine agit dans les neurones à GnRH, en démontrant un effet sur l'expression génique de la GnRH. Nous pouvons également conclure que la voie de la MAPK est la voie principale activée par la metastine dans les neurones exprimant la GnRH.

Distal dendrites of frog motor neurons: a computer-aided electron microscopic study of cobalt-filled cells.

With the aid of the cobalt labelling technique, frog spinal cord motor neuron dendrites of the subpial dendritic plexus have been identified in serial electron micrographs. Computer reconstructions of various lengths (2.5-9.8 micron) of dendritic segments showed the contours of these dendrites to be highly irregular, and to present many thorn-like projections 0.4-1.8 micron long. Number, size and distribution of synaptic contacts were also determined. Almost half of the synapses occurred at the origins of the thorns and these synapses had the largest contact areas. Only 8 out of 54 synapses analysed were found on thorns and these were the smallest. For the total length of reconstructed dendrites there was, on average, one synapse per 1.2 micron, while 4.4% of the total dendritic surface was covered with synaptic contacts. The functional significance of these distal dendrites and their capacity to influence the soma membrane potential is discussed.