Valutazione delle variazioni di importanti parametri funzionali mitocondriali in relazione all'invecchiamento ed all'esposizione cronica a farmaci antinfiammatori non steroidei con diverso grado di selettività verso la COX-2.

L'invecchiamento è una condizione associata ad un graduale decadimento della funzione fisiologica cellulare e alla perdita di integrità della cellula stessa. I mitocondri, organelli strettamente coinvolti nel metabolismo energetico cellulare e nell’omeostasi della cellula, sembrano svolgere un ruolo essenziale nei processi di invecchiamento e di danno cellulare, associato a stati patologici. La principale funzione fisiologica mitocondriale è la produzione di adenosina trifosfato (ATP), attraverso il processo della fosforilazione ossidativa. Tale funzionalità può essere valutata attraverso la misurazione del potenziale di membrana e l’osservazione dei parametri di respirazione mitocondriale. Durante la senescenza, diversi meccanismi concorrono a determinare una riduzione dell’efficienza della respirazione mitocondriale. Infatti, mitocondri epatici isolati da ratti vecchi mostrano una riduzione di circa il 25% della velocità della fosforilazione ossidativa, rispetto ai mitocondri isolati da ratti giovani. Risultati analoghi si osservano in cellule senescenti appartenenti anche a tessuti diversi. Attraverso la misurazione del potenziale di membrana di mitocondri isolati da un’ampia varietà di cellule, appartenenti anche a specie differenti, si evidenzia una significativa dissipazione del potenziale di membrana mitocondriale, con un decremento di circa 10 mV del potenziale basale, rispetto ai mitocondri di cellule giovani. Il primo obiettivo della mia tesi è stato quello di mettere a punto un metodo per monitorare i parametri di respirazione mitocondriale, che insieme alla registrazione del potenziale di membrana mitocondriale, ci permettesse di valutare con accuratezza la funzionalità mitocondriale e l’efficienza della fosforilazione ossidativa in mitocondri epatici isolati da ratti senescenti, con riferimento ai dati ottenuti per i ratti giovani e ai dati già presenti in letteratura. La respirazione mitocondriale è stata valutata attraverso l’utilizzo dell’elettrodo di Clark, sensibile all’ossigeno. Con tale approccio è stato possibile monitorare il consumo di ossigeno da parte dei mitocondri isolati, facendo riferimento ad importanti parametri, quali il rapporto del controllo della respirazione (RCR), l’indice di efficacia della respirazione (rapporto ATP/O) ed il rapporto FaseIII/FaseUC. Il potenziale basale di membrana mitocondriale e la sua variazione in seguito all’aggiunta di agenti disaccoppianti sono stati registrati con un’ analisi potenziometrica, attraverso l’utilizzo di un mini-elettrodo sensibile al catione tetrafenilfosfonio (TPP+). Il secondo obiettivo della mia tesi è stato valutare eventuali disfunzioni mitocondriali, associate al trattamento farmacologico per due settimane, di ratti senescenti con farmaci antinfiammatori, in particolare inibitori non selettivi delle ciclossigenasi, quali indometacina e diclofenac, ed inibitori COX-2 selettivi, quali celecoxib ed etoricoxib, comunemente utilizzati nel trattamento dell’artrosi. L’utilizzo cronico di inibitori non selettivi delle ciclossigenasi è in grado di indurre una serie di effetti dannosi a livello della mucosa gastrointestinale. Nonostante rimangano ancora poco chiari i meccanismi biochimici alla base di tali effetti, le alterazioni nel metabolismo energetico mitocondriale sembrano essere strettamente coinvolte nel processo di danno cellulare. Numerosi studi rilevano la capacità di tali farmaci di inibire o disaccoppiare la respirazione mitocondriale. A causa dell’eccessiva difficoltà nell’isolare mitocondri in rese soddisfacenti alle necessità sperimentali, nonché “respiranti”, dal tessuto intestinale, ho utilizzato nel mio studio mitocondri epatici. Tuttavia, è comunemente accettato che non ci siano differenze significative tra i mitocondri isolati da organi differenti, nella loro risposta ad agenti disaccoppianti o ad inibitori del trasporto di elettroni nella catena respiratoria. Il potenziale basale di membrana mitocondriale, osservato nei ratti giovani, è di circa -170 mV, in accordo con la letteratura. I mitocondri del ratto vecchio mostrano un decremento del potenziale (circa -150 mV), indicando una capacità energetica inferiore. I mitocondri epatici ottenuti dai ratti giovani mostrano un indice dell’efficacia della fosforilazione (ATP/O) ed un rapporto del controllo della respirazione (RCR) in accordo con la letteratura. Gli stessi parametri risultano, invece, ridotti nei mitocondri epatici isolati dai ratti senescenti; confermando che il processo di invecchiamento è in grado di alterare la funzionalità bioenergetica mitocondriale

Save Antibiotics: a call for action of the World Alliance Against Antibiotic Resistance (WAAAR)

International audienceResistance to antibiotics has recently increased dramatically worldwide. The pipeline of new classes of antibiotics is dry for at least the next few years. Therefore antibiotic resistance represents one of the most problematic public health issues of our time. Treatment failures already happen in increasing numbers for common community-acquired infections, such as urinary tract infections or intra-abdominal infections. This is due in particular to Enterobacteriaceae harboring extended-spectrum beta-lactamases (ESBL). Enterobacteriaceae harboring carbapenemases are also highly prevalent in many countries. In the future, difficult surgical procedures, transplants, and other immunosuppressive therapies may become very risky. Resistance is mainly due to an excessive usage of antibiotics, in both humans and animals, and to cross-transmission of resistant bacteria. Action is urgently needed. Therefore, a World Alliance Against Antibiotic Resistance (WAAAR) was created in 2011. It includes healthcare professionals, consumers, health managers, and politicians. We present here the main measures proposed by the Alliance, as a result of a strong consensus between the different stakeholders, including general practitioners and veterinarians

Growth of Wild Gilthead Seabream (Sparus aurata L.) Juveniles for Organic Aquaculture

The majority of organic marine fish farms currently begin the production cycle with non-organic juveniles from conventional hatcheries, permitted by the European Regulation on organic fish origin (EC 710/2009) until the end of 2016. Wild juvenile gilthead seabream (Sparus aurata) from coastal lagoons and hatcheries were experimentally reared under organic conditions, in order (1) to investigate differences in fillet lipid content and fatty acids composition, and (2) to propose a possible future source of juveniles destined for organic aquaculture. Wild juveniles were readily distinguishable by their fatty acid signature, showing significantly higher ratio levels of n-3 polyunsaturated fatty acids and n-3/n-6. Fillet lipid composition of organically fed wild S. aurata juveniles was preferable to that from domesticated juveniles. These results seem promising for organic aquaculture, where fish feed is more environmentally sustainable but is of lower nutritional qualit

Rainbow trout (Oncorhynchus mykiss, Walbaum 1792) develop a more robust body shape under organic rearing

Prolonged exposure to captive conditions has led to the development of a rainbow trout ‘farmed’ pheno- type, which is different from that of wild trout. Selec- tion for desirable productive traits in hatcheries has resulted in the development of some morphological traits that are maladaptive in nature. The recent development of organic aquaculture, guided by the well-being of the fish, could potentially produce a new farmed phenotype that would be more adaptive in nature. In this study, rainbow trout reared in intensive and organic farms were compared by means of shape analysis, to detect patterns of shape variation associated with rearing environment. The results of this study highlight a significant effect of the rearing method on rainbow trout shape: organi- cally reared trout showed a higher body profile, in particular in the head and trunk regions, shorter median fins and a deeper caudal peduncle. A combined effect of density and habitat complexity could have contributed to the observed shape differences: in organic rearing systems, lower densi- ties and steady water could increase territoriality and aggressive interactions, promoting body designs more functional in rapid attacks and escapes

An international cross-sectional survey of antimicrobial stewardship programmes in hospitals

Objectives To report the extent and components of global efforts in antimicrobial stewardship (AMS) in hospitals. Methods An Internet-based survey comprising 43 questions was disseminated worldwide in 2012. Results Responses were received from 660 hospitals in 67 countries: Africa, 44; Asia, 50; Europe, 361; North America, 72; Oceania, 30; and South and Central America, 103. National AMS standards existed in 52% of countries, 4% were planning them and 58% had an AMS programme. The main barriers to implementing AMS programmes were perceived to be a lack of funding or personnel, a lack of information technology and prescriber opposition. In hospitals with an existing AMS programme, AMS rounds existed in 64%; 81% restricted antimicrobials (carbapenems, 74.3%; quinolones, 64%; and cephalosporins, 58%); and 85% reported antimicrobial usage, with 55% linking data to resistance rates and 49% linking data to infection rates. Only 20% had electronic prescribing for all patients. A total of 89% of programmes educated their medical, nursing and pharmacy staff on AMS. Of the hospitals, 38% had formally reviewed their AMS programme: reductions were reported by 96% of hospitals for inappropriate prescribing, 86% for broad-spectrum antibiotic use, 80% for expenditure, 71% for healthcare-acquired infections, 65% for length of stay or mortality and 58% for bacterial resistance. Conclusions The worldwide development and implementation of AMS programmes varies considerably. Our results should inform and encourage the further evaluation of this with a view to promoting a worldwide stewardship framework. The prospective measurement of well-defined outcomes of the impact of these programmes remains a significant challeng

Skeletal Anomaly Monitoring in Rainbow Trout (Oncorhynchus mykiss, Walbaum 1792) Reared under Different Conditions

The incidence of skeletal anomalies could be used as an indicator of the "quality" of rearing conditions as these anomalies are thought to result from the inability of homeostatic mechanisms to compensate for environmentally-induced stress and/or altered genetic factors. Identification of rearing conditions that lower the rate of anomalies can be an important step toward profitable aquaculture as malformed market-size fish have to be discarded, thus reducing fish farmers' profits. In this study, the occurrence of skeletal anomalies in adult rainbow trout grown under intensive and organic conditions was monitored. As organic aquaculture animal production is in its early stages, organic broodstock is not available in sufficient quantities. Non-organic juveniles could, therefore, be used for on-growing purposes in organic aquaculture production cycle. Thus, the adult fish analysed in this study experienced intensive conditions during juvenile rearing. Significant differences in the pattern of anomalies were detected between organically and intensively-ongrown specimens, although the occurrence of severe, commercially important anomalies, affecting 2-12.5% of individuals, was comparable in the two systems. Thus, organic aquaculture needs to be improved in order to significantly reduce the incidence of severe anomalies in rainbow trout

Monitoring denitrification by means of pH and ORP in continuous-flow conventional activated sludge processes

Indirect signal analysis (pH, ORP and DO) are often used in monitoring and control of SBRs (Sequencing Batch Reactors), where operating conditions can be clearly identified during the various cyclic phases. Only few studies applied this methodology to control continuous flow plants, as it is much more difficult to identify operating conditions because of continually variable inflow characteristics. This work applied indirect signal analysis to control pre-denitrification in continuous-flow activated sludge processes: (i) a laboratory-scale plant, fed with synthetic wastewater, simulating real municipal wastewater and (ii) a pilot-scale plant, fed with real sewage. Three different ranges of ORP values identify three operational conditions of the denitrification process. (1) ORP > 0 mV means that nitrates and/or nitrites are present, possibly due to a low C/N ratio. (2) –50 < ORP < –200 mV is typical of normal operating conditions, that is with a balanced C/N ratio. (3) ORP < –350 mV means that oxidized nitrogen load is too low or that C/N exceeds the stoichiometric ratio. The trend of pH, instead, points out if and how the process is evolving from one to another operating condition. The correlation between pH and ORP signals (as well as their derivatives) allows to restore normal operating conditions by acting on the internal recycle flow-rate. Improved denitrification process ensures lower effluent nitrate concentration, and reduce external carbon dosage to achieve stricter nitrogen limits

Classifying antibiotics in the WHO Essential Medicines List for optimal use—be AWaRe

International audienc

Prioritising research areas for antibiotic stewardship programmes in hospitals: a behavioural perspective consensus paper

SCOPE: Antibiotic stewardship programmes (ASPs) are necessary in hospitals to improve the judicious use of antibiotics. While ASPs require complex change of key behaviours on individual, team, organisation and policy levels, evidence from the behavioural sciences is underutilised in antibiotic stewardship studies across the world, including high-income countries (HICs). A consensus procedure was performed to propose research priority areas for optimising effective implementation of ASPs in hospital settings, using a behavioural perspective. METHODS: A workgroup for behavioural approaches to ASPs was convened in response to the fourth call for leading expert network proposals by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR). Eighteen clinical and academic specialists in antibiotic stewardship, implementation science and behaviour change from four high-income countries with publicly-funded health care systems (that is Canada, Germany, Norway and the UK), met face-to-face to agree on broad research priority areas using a structured consensus method. QUESTION ADDRESSED AND RECOMMENDATIONS: The consensus process on the 10 identified research priority areas resulted in recommendations that need urgent scientific interest and funding to optimise effective implementation of antibiotic stewardship programmes for hospital inpatients in HICs with publicly-funded health care systems. We suggest and detail, behavioural science evidence-guided research efforts in the following areas: 1) Comprehensively identifying barriers and facilitators to implementing antibiotic stewardship programmes and clinical recommendations intended to optimise antibiotic prescribing; 2) Identifying actors ('who') and actions ('what needs to be done') of antibiotic stewardship programmes and clinical teams; 3) Synthesising available evidence to support future research and planning for antibiotic stewardship programmes; 4) Specifying the activities in current antibiotic stewardship programmes with the purpose of defining a 'control group' for comparison with new initiatives; 5) Defining a balanced set of outcomes and measures to evaluate the effects of interventions focused on reducing unnecessary exposure to antibiotics; 6) Conducting robust evaluations of antibiotic stewardship programmes with built-in process evaluations and fidelity assessments; 7) Defining and designing antibiotic stewardship programmes; 8) Establishing the evidence base for impact of antibiotic stewardship programmes on resistance; 9) Investigating the role and impact of government and policy contexts on antibiotic stewardship programmes; and 10) Understanding what matters to patients in antibiotic stewardship programmes in hospitals. Assessment, revisions and updates of our priority-setting exercise should be considered, at intervals of 2 years. To propose research priority areas in low- and medium income countries (LIMCs), the methodology reported here could be applied

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

Background. The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. Results. Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. Conclusions. The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcom