The non-proteic extrusive secondary metabolites in ciliated protists

The non-proteic extrusive secondary metabolites in ciliated protists F. Buonanno1, A. Anesi2, G. Guella2, E. Marcantoni3, S. Giorgi3, C. Ortenzi1 1Laboratory of Protistology and Biology Education, University of Macerata, 62100 Macerata, Italy 2Bioorganic Chemistry Laboratory, Department of Physics, University of Trento, 38050 Povo, Trento, Italy 3School of Sciences and Technologies, Section of Chemistry, University of Camerino, 62032 Camerino, Macerata, Italy Extrusomes are membrane-bound ejectable organelles widely distributed in protists. They are usually localized in the cell cortex and attached to the cell membrane, and they are able to discharge their contents to the outside of the cell in response to mechanical or chemical stimuli. Notably, cells that discharge their extrusomes remains intact and functional. The chemical nature of protists\u2019 extrusive compounds characterized to date is extremely variable, including proteins, carbohydrates, lipids, and dozens of additional classes of secondary metabolites. However an increasing set of data are now available for particular group of protists, the ciliated protozoa. Many of non-proteic extrusive secondary metabolites in ciliates function for chemical offense or defense in prey-predator interactions against unicellular or/and multicellular organisms. It is worthy of note that at least some of these secondary metabolites have been demonstrated to show antibiotic, anti-cancer and pro-apoptotic properties in addition to their physiological functions. Among these compounds, euplotin C produced by the ciliate Euplotes crassus, and climacostol produced by Climacostomum virens, have been shown to activate programmed cell death by impairing mitochondrial membrane potential and inducing ROS generation in mammalian tumor cell lines. Interestingly, an antimicrobial activity against Gram-positive bacteria and fungal pathogens was also demonstrated for climacostol. Overall, in addition to the understanding of their physiological and ecological functions, the study of non-proteic secondary metabolites of ciliated protozoa may set the basis for the development of a novel series of antitumor and antimicrobial agents

Leptin counteracts the hypoxia-induced inhibition of spontaneously firing hippocampal neurons: a microelectrode array study.

Besides regulating energy balance and reducing body-weight, the adipokine leptin has been recently shown to be neuroprotective and antiapoptotic by promoting neuronal survival after excitotoxic and oxidative insults. Here, we investigated the firing properties of mouse hippocampal neurons and the effects of leptin pretreatment on hypoxic damage (2 hours, 3% O(2)). Experiments were carried out by means of the microelectrode array (MEA) technology, monitoring hippocampal neurons activity from 11 to 18 days in vitro (DIV). Under normoxic conditions, hippocampal neurons were spontaneously firing, either with prevailing isolated and randomly distributed spikes (11 DIV), or with patterns characterized by synchronized bursts (18 DIV). Exposure to hypoxia severely impaired the spontaneous activity of hippocampal neurons, reducing their firing frequency by 54% and 69%, at 11 and 18 DIV respectively, and synchronized their firing activity. Pretreatment with 50 nM leptin reduced the firing frequency of normoxic neurons and contrasted the hypoxia-induced depressive action, either by limiting the firing frequency reduction (at both ages) or by increasing it to 126% (in younger neurons). In order to find out whether leptin exerts its effect by activating large conductance Ca(2+)-activated K(+) channels (BK), as shown on rat hippocampal neurons, we applied the BK channel blocker paxilline (1 µM). Our data show that paxilline reversed the effects of leptin, both on normoxic and hypoxic neurons, suggesting that the adipokine counteracts hypoxia through BK channels activation in mouse hippocampal neurons

Two firing modes and well-resolved Na+, K+, and Ca2+ currents at the cell-microelectrode junction of spontaneously active rat chromaffin cell on MEAs

We recorded spontaneous extracellular action potentials (eAPs) from rat chromaffin cells (CCs) at 37 °C using microelectrode arrays (MEAs) and compared them with intracellularly recorded APs (iAPs) through conventional patch clamp recordings at 22 °C. We show the existence of two distinct firing modes on MEAs: a ~ 4 Hz irregular continuous firing and a frequent intermittent firing mode where periods of high-intraburst frequency (~ 8 Hz) of ~ 7 s duration are interrupted by silent periods of ~ 12 s. eAPs occurred either as negative- or positive-going signals depending on the contact between cell and microelectrode: either predominantly controlled by junction-membrane ion channels (negative-going) or capacitive/ohmic coupling (positive-going). Negative-going eAPs were found to represent the trajectory of the Na(+), Ca(2+), and K(+) currents passing through the cell area in tight contact with the microelectrode during an AP (point-contact junction). The inward Nav component of eAPs was blocked by TTX in a dose-dependent manner (IC(50) ~ 10 nM) while the outward component was strongly attenuated by the BK channel blocker paxilline (200 nM) or TEA (5 mM). The SK channel blocker apamin (200 nM) had no effect on eAPs. Inward Nav and Cav currents were well-resolved after block of Kv and BK channels or in cells showing no evident outward K(+) currents. Unexpectedly, on the same type of cells, we could also resolve inward L-type currents after adding nifedipine (3 μM). In conclusion, MEAs provide a direct way to record different firing modes of rat CCs and to estimate the Na(+), Ca(2+), and K(+) currents that sustain cell firing and spontaneous catecholamines secretion

Education and training among Italian postgraduate medical schools in public health: a comparative analysis

Analisi comparativa dei percorsi formativi offerti dalle Scuole di specializzazione di Igiene e Medicina Preventiva in Italia Background: Il percorso formativo dello specialista in Igiene e Medicina Preventiva dovrebbe garantire adeguate conoscenze tecnico-scientifiche e professionali nei campi della medicina preventiva, della promozione della salute e della programmazione dei servizi sanitari secondo quanto indicato anche dal DM 285/2005. La Consulta degli Specializzandi, da sempre coinvolta in attività di monitoraggio della formazione a livello nazionale, si prefigge l’obiettivo di valutare l’omogeneità delle proposte formative tra le diverse sedi italiane, non solo per segnalare le criticità, ma anche per evidenziarne le opportunità. Metodi: Lo studio, di tipo cross-sectional, è stato condotto mediante la somministrazione di un questionario semi-strutturato inviato per la compilazione ai rappresentanti delle 32 le Scuole di Igiene e Medicina Preventiva italiane. Lo strumento di valutazione è costituito da quattro sezioni: informazioni generali, attività formativa universitaria, attività formativa extra-universitaria, attività formativa intersettoriale. L’indagine è stata svolta nel periodo tra marzo e maggio 2013 ed è stata prodotta un’analisi descrittiva dei dati ottenuti. Risultati: Il questionario è stato compilato da 28 Scuole su 32 (tasso di risposta 88%), distribuite su tutto il territorio nazionale. Il numero di medici in formazione varia tra 7 e 31 e il rapporto tra docenti del settore scientifico-disciplinare di interesse e i discenti è compreso tra 0,2 e 2. Per quanto riguarda la didattica, solo in 4 Scuole si effettuano tutti i corsi previsti dal DM. La maggior parte delle sedi svolge almeno il 75% dei corsi previsti, ma esistono sedi in cui il numero di corsi è inferiore al 50%. La maggior parte delle Scuole svolge più del 60% delle attività professionalizzanti essenziali secondo il decreto, ma 2 Scuole non arrivano al 50%. Tutte le Scuole prevedono un tirocinio di 6-12 mesi in ASL, affiancando principalmente attività del Dipartimento di Prevenzione. Ovunque è previsto un periodo in Direzione Medica Ospedaliera, mentre le Strutture Riabilitative rientrano raramente nella rete formativa. Nella maggioranza delle Scuole è possibile frequentare aziende con rischio biologico oppure seguire simili attività nei Servizi dedicati della ASL. Molte Scuole, infine, consentono di frequentare diverse strutture territoriali (Agenzia di Controllo delle Acque), regionali (Assessorati) o nazionali (Ministero, Istituto Superiore di Sanità); in alcuni casi si tratta di Università gemellate e Istituti di Ricerca. Conclusioni: Nonostante il DM 285/2005 indichi quali siano le fondamenta della sanità pubblica, la flessibilità nella scelta formativa è vista come requisito essenziale per ottimizzare le risorse e contestualizzare l’adeguata formazione del medico in formazione specialistica in Igiene e Medicina Preventiva. La maggior parte delle Scuole di Specializzazione italiane dovrebbe però prevedere lo svolgimento della quasi totalità delle attività formative previste, al fine di non creare disuguaglianze formative tra gli specializzandi. Infine, considerato che la sanità pubblica è una disciplina in continuo divenire, il DM del 2005 andrebbe rivisitato tenendo in considerazione la flessibilità della formazione ed i continui cambiamenti dei bisogni di salute essenziali della popolazione. Inoltre, nel processo di rivisitazione dei bisogni formativi dei medici in formazione specialistica, dovrebbero essere coinvolti anche i discenti al fine di rafforzare il potere e l’efficacia dell’insegnamento.Background The postgraduate medical Schools in Public Health (locally known as School of Hygiene and Preventive Medicine) should ensure adequate scientific and technical knowledge and professional skills in preventive medicine, health promotion and healthcare planning as provided by Ministerial Decree 285/2005. The Italian Committee of Medical Residents in Hygiene, Preventive Medicine and Public Health of the Italian Society of Hygiene, Public Health and Preventive Medicine - S.It.I. (Consulta Nazionale dei medici in formazione specialistica S.It.I.) has always been engaged in monitoring activities on public health teaching, guaranteeing the homogeneity of educational proposals among all national Schools in Public Health. The purpose of this study is to provide a 'snapshot' of public health education and training in Italy and to identify the improvement actions needed for implementing an innovative and homogeneous public health training. Methods: A cross-sectional study was carried out over a period of three months (March to May 2013). A self-administered questionnaire was e-mailed to local Committee’s delegates of all 32 postgraduate medical Schools in Public Health in Italy. The questionnaire was structured in four sections: general information, University education and training, extra-University training, interdisciplinary activities. The majority of local Committee’s delegates have agreed to be enrolled in the survey. Results: A total of 28 questionnaires were returned (88% response rate). The number of residents in each Italian School in Public Health ranged from 7 to 31. The distribution of professors in relation to residents is not similar for each University Schools. The ratio professors/residents spanning from 0.2 to 2. About teaching, only 4 University Schools offered all courses requested by Ministerial Decree 285/2005. Most of them offered at least 75% of the requested courses, but there were Schools in which the courses were less than 50%. The vast majority of schools held more than 60% of the qualifying activities considered essential according to the Decree, while 2 Schools were below 50%. All Schools required an internship of 6-12 months in local health authority offices (ASL), mainly concerning the Department of Prevention activities. In all Schools a period of stay in a Hospital Medical Direction was scheduled, while professional activities at Residential care homes were very rarely included in training programmes. Many Schools allowed residents to attend companies with biological hazard or to follow similar activities in dedicated services of ASL. Finally, in the majority of Schools, a training period in various local (Service for Water Control), regional (Departments) or national (Ministry, National Institute of Health) health facilities was contemplated and, in some cases, also in other Universities or Research Institutes. Conclusions: Although the Ministerial Decree indicates the essential milestones of the public health education, flexibility is seen as an important element in order to optimize resources and contextualize the adequate education of residents. In any case, at least regarding public health courses, the majority of University education and extra-University training activities should be carried out by all Schools. In order to obtain shared knowledge and skills, the Ministerial Decree should be revised taking into account flexibility and changing as intrinsic characteristics of public health profession and learners should be involved in the reform to strengthening the role of public health teachin

Logistic regression of attitudes and coverage for influenza vaccination among Italian Public Health medical residents

Introduction. A few number of literature specifically addresses vaccination uptake among Public Health Residents (PHRs). Influ- enza vaccine attitudes and risk perceptions of PHRs across Italy were studied, contributing to literature on influenza vaccination uptake predictors, in particular among young physicians. Methods. An online survey was conducted in 25 Schools of Public Health in Italy in 2011-2012. Results were analysed using preva- lence and logistic regression methods. Results. A total of 365 Italian public health residents were included in the study. Vaccination uptake was confirmed by 22.2 and 33.2% of PHRs in 2010-2011 and 2011-2012, respectively. For the 2010-2011 influenza season, vaccination was associated with male sex (adj-OR 3.43; 95%CI = 1.5-7.84) and vaccination history (adj-OR 29.44; 95%CI = 6.4-135.04). For the 2011-2012 season, vaccination was significantly associated with having had between one and three influenza vaccinations in the previ- ous five years (adj-OR 11.56; 95%CI = 6.44-20.75) or more than three (adj-OR 136.43; 95%CI = 30.8-604.7) and with individual participation in general population vaccination campaigns (adj- OR 1.85; 95%CI = 1.01-3.41). Discussion. Italian residents in public health have no confidence and a low personal risk perception about vaccinations therefore taking no measures to protect patients, general population and themselves. Annual influenza vaccination acceptance is associ- ated with influenza vaccine uptake in the previous years and per- sonal involvement in general population vaccination campaigns. These factors should be considered for the design of future cam- paigns targeting public health residents

DNA binding and oxidative DNA damage induced by climacostol\u2013copper(II) complexes: Implications for anticancer properties

Climacostol is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids. Climacostol exerts a potent antimicrobial activity against a panel of bacterial and fungal pathogens. In addition it inhibits the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death via intrinsic pathway. In this work, we investigated the possibility that climacostol exerts a prooxidant effect, inducing plasmid DNA strand breakage and eukaryotic DNA damage in presence of Cu(II) ions. Inhibition of DNA breakage using SOD, catalase and neocuproine confirmed the involvement of reactive oxygen species and Cu(I) ions in the DNA damage. UV\u2013visible absorption changes and mass spectrometric analysis identified a product of reaction as a deprotonated form of climacostol. Study of the interaction with DNA, using fluorescence spectroscopic techniques, showed that climacostol binds with DNA. Given the structure\u2013activity relationship of this compound and the mechanism of its prooxidant effect, we propose that the Cu(II)-mediated oxidative DNA damage by climacostol could explain its antimicrobial and antiproliferative activity

Alpha-synuclein oligomers alter the spontaneous firing discharge of cultured midbrain neurons

The aim of this work was to monitor the effects of extracellular α-synuclein on the firing activity of midbrain neurons dissociated from substantia nigra TH-GFP mice embryos and cultured on microelectrode arrays (MEA). We monitored the spontaneous firing discharge of the network for 21 days after plating and the role of glutamatergic and GABAergic inputs in regulating burst generation and network synchronism. Addition of GABAA, AMPA and NMDA antagonists did not suppress the spontaneous activity but allowed to identify three types of neurons that exhibited different modalities of firing and response to applied L-DOPA: high-rate (HR) neurons, low-rate pacemaking (LR-p), and low-rate non-pacemaking (LR-np) neurons. Most HR neurons were insensitive to L-DOPA, while the majority of LR- p neurons responded with a decrease of the firing discharge; less defined was the response of LR-np neurons. The effect of exogenous α-synuclein (α-syn) on the firing discharge of midbrain neurons was then studied by varying the exposure time (0–48 h) and the α-syn concentration (0.3–70 μM), while the formation of α-syn oligomers was monitored by means of AFM. Independently of the applied concentration, acute exposure to α-syn monomers did not exert any effect on the spontaneous firing rate of HR, LR-p, and LR-np neurons. On the contrary, after 48 h exposure, the firing activity was drastically altered at late developmental stages (14 days in vitro, DIV, neurons): α-syn oligomers progressively reduced the spontaneous firing discharge (IC50 = 1.03 μM), impaired burst generation and network synchronism, proportionally to the increased oligomer/monomer ratio. Different effects were found on early-stage developed neurons (9 DIV), whose firing discharge remained unaltered, regardless of the applied α-syn concentration and the exposure time. Our findings unravel, for the first time, the variable effects of exogenous α-syn at different stages of midbrain network development and provide new evidence for the early detection of neuronal function impairment associated to aggregated forms of α-syn