Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork

A Novel NHE1-Centered Signaling Cassette Drives Epidermal Growth Factor Receptor–Dependent Pancreatic Tumor Metastasis and Is a Target for Combination Therapy

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers principally because of early invasion and metastasis. The epidermal growth factor receptor (EGFR) is essential for PDAC development even in the presence of Kras, but its inhibition with erlotinib gives only a modest clinical response, making the discovery of novel EGFR targets of critical interest. Here, we revealed by mining a human pancreatic gene expression database that the metastasis promoter Na+/H+ exchanger (NHE1) associates with the EGFR in PDAC. In human PDAC cell lines, we confirmed that NHE1 drives both basal and EGF-stimulated three-dimensional growth and early invasion via invadopodial extracellular matrix digestion. EGF promoted the complexing of EGFR with NHE1 via the scaffolding protein Na +/H + exchanger regulatory factor 1, engaging EGFR in a negative transregulatory loop that controls the extent and duration of EGFR oncogenic signaling and stimulates NHE1. The specificity of NHE1 for growth or invasion depends on the segregation of the transient EGFR/Na +/H + exchanger regulatory factor 1/NHE1 signaling complex into dimeric subcomplexes in different lipid raftlike membrane domains. This signaling complex was also found in tumors developed in orthotopic mice. Importantly, the specific NHE1 inhibitor cariporide reduced both three-dimensional growth and invasion independently of PDAC subtype and synergistically sensitized these behaviors to low doses of erlotinib

Processos de escrita e autoria sobre a ação docente enquanto prática formativa

Este artigo apresenta os processos de escrita e autoria enquanto prática formativa. O objetivo é analisar os significados e sentidos atribuídos pelos professores ao trabalho de escrita e autoria sobre sua ação docente. Para isso, analisa a correspondência eletrônica que foi trocada entre professores da Educação Básica e professores-pesquisadores da Universidade envolvidos em um projeto de pesquisa-ação colaborativa. A análise evidencia a escrita como ato solitário e fruto de um trabalho reflexivo caracterizado como árduo, semelhante à gestação e ao trabalho de parto. Embora descrito como penoso, o processo de escrita e autoria sobre a atividade docente é entendido como estratégia eficaz para o processo de formação continuada e como potência para a aprendizagem da docência

Sigh in patients with acute hypoxemic respiratory failure and acute respiratory distress syndrome: the PROTECTION pilot randomized clinical trial

Background: Sigh is a cyclic brief recruitment manoeuvre: previous physiological studies showed that its use could be an interesting addition to pressure support ventilation to improve lung elastance, decrease regional heterogeneity and increase release of surfactant. Research question: Is the clinical application of sigh during pressure support ventilation (PSV) feasible? Study design and methods: We conducted a multi-center non-inferiority randomized clinical trial on adult intubated patients with acute hypoxemic respiratory failure or acute respiratory distress syndrome undergoing PSV. Patients were randomized to the No Sigh group and treated by PSV alone, or to the Sigh group, treated by PSV plus sigh (increase of airway pressure to 30 cmH2Ofor 3 seconds once per minute) until day 28 or death or successful spontaneous breathing trial. The primary endpoint of the study was feasibility, assessed as non-inferiority (5% tolerance) in the proportion of patients failing assisted ventilation. Secondary outcomes included safety, physiological parameters in the first week from randomization, 28-day mortality and ventilator-free days. Results: Two-hundred fifty-eight patients (31% women; median age 65 [54-75] years) were enrolled. In the Sigh group, 23% of patients failed to remain on assisted ventilation vs. 30% in the No Sigh group (absolute difference -7%, 95%CI -18% to 4%; p=0.015 for non-inferiority). Adverse events occurred in 12% vs. 13% in Sigh vs. No Sigh (p=0.852). Oxygenation was improved while tidal volume, respiratory rate and corrected minute ventilation were lower over the first 7 days from randomization in Sigh vs. No Sigh. There was no significant difference in terms of mortality (16% vs. 21%, p=0.342) and ventilator-free days (22 [7-26] vs. 22 [3-25] days, p=0.300) for Sigh vs. No Sigh. Interpretation: Among hypoxemic intubated ICU patients, application of sigh was feasible and without increased risk

Mitochondrial functions in the modulation of immunometabolism: physiological and pathological aspects

Recenti studi sul metabolismo delle cellule dell’immunità innata ed adattiva hanno messo in luce lo stretto legame esistente tra stato metabolico e fenotipo di queste cellule. Secondo un concetto emergente, cambiamenti nel profilo metabolico non soltanto sostengono la risposta immunitaria come conseguenza dell’attivazione di specifiche vie di segnale intracellulari, ma possono anche indurre l’acquisizione di determinati immunofenotipi agendo sul signaling intracellulare. Alla luce di queste osservazioni, il metabolismo mitocondriale rappresenta un interessante bersaglio terapeutico per la modulazione della risposta immunitaria in stati infiammatori patologici. In questo studio sono stati utilizzati due modelli in vitro di cellule della linea mieloide stimolate fisicamente e biologicamente per determinare i check points metabolici responsabili dell’interazione tra infiammazione e metabolismo, al fine di identificare targets farmacologici per impedire il priming metabolico di cellule dell’immunità innata, quali macrofagi e cellule dendritiche. Nella prima parte di questo studio è stata utilizzata la linea cellulare di macrofagi murini RAW 264.7 come modello in vitro per studiare le conseguenze dell’attivazione mediata dal Lipopolisaccaride (LPS) sul metabolismo mitocondriale e gli effetti su quest’ultimo del para- Idrossifenilpiruvato (pHPP), un intermedio del catabolismo di Tirosina e Fenilalanina che oltre a fornire substrati al Ciclo di Krebs (Fumarato e Acetil- Coenzima A), presenta attività antiossidante ed anti-infiammatoria. I risultati ottenuti mostrano che il trattamento delle RAW 264.7 con una concentrazione 2 di LPS paragonabile a quella rilevata nel siero di pazienti settici induce il rilascio della citochina pro-infiammatoria Interleuchina 6 (IL-6), un marcato incremento dell’espressione della Ossido Nitrico Sintasi inducibile (iNOS) ed un conseguente aumento della produzione di Ossido Nitrico (NO). L’analisi respirometrica e del flusso metabolico delle RAW 264.7 trattate con LPS ha rivelato un netto shift metabolico consistente nella riduzione della fosforilazione ossidativa mitocondriale (OxPhos), accompagnata da un aumento dell’attività glicolitica. È stata infatti riscontrata una significativa riduzione di circa il 50% della respirazione mitocondriale e delle specifiche attività del Complesso I e del Complesso IV della catena respiratoria, compensate da un incremento della glicolisi ed accompagnate da un’aumentata produzione di specie reattive dell’ossigeno. L’inibizione dell’attività respiratoria è stata anche osservata in seguito all’incubazione di fibroblasti umani neonatali (NHDF-neo) con siero di pazienti settici ed il pHPP è risultato in grado di inibire tutte le alterazioni finora descritte nelle sia RAW 264.7 stimolate con LPS che nelle NHDF-neo incubate con siero settico, grazie alla combinazione delle sue dirette attività antiossidanti e bio-energizzanti. Per quanto concerne la seconda parte di questo progetto di ricerca, sono state utilizzate cellule dendritiche derivanti da monociti (MoDCs) sottoposte ad un breve (3h) shock termico (39°C) per simulare uno stato febbrile, al fine di approfondire il suo diretto effetto sul metabolismo delle cellule dendritiche. È noto infatti che la febbre sia un sintomo altamente conservato a livello evolutivo, poiché in grado di stimolare la risposta immunitaria innata ed adattativa inducendo l’acquisizione di specifici immunofenotipi e profili di espressione genica. Considerando inoltre che il metabolismo mitocondriale gioca un ruolo fondamentale nel contesto della risposta immunitaria non solo per fornire l’energia necessaria ai molteplici processi cellulari ma anche come piattaforma di segnale, abbiamo ipotizzato che lo stato febbrile potesse causare alterazioni nel metabolismo ossidativo mitocondriale delle cellule dendritiche. I risultati ottenuti mostrano chiaramente che un blando stress termico è in grado 3 di stimolare il rilascio di TNF-α da parte delle cellule dendritiche e che questa attivazione immunitaria è accompagnata da una riprogrammazione del metabolismo ossidativo, che si traduce in una diminuzione dell’attività respiratoria mitocondriale, in un’aumentata produzione di specie reattive dell’azoto e dell’ossigeno e nell’accumulo di Ca2+ nei mitocondri. L’inibizione dell’attività respiratoria mitocondriale indotta dall’ipertermia è un processo irreversibile, dal momento che il ricondizionamento delle cellule in normotermia a 37°C non permette un recupero dell’attività della catena respiratoria, ma può essere prevenuta dal cotrattamento con Rosso Rutenio, inibitore dell’uniporto mitocondriale per il Ca2+. Inoltre l’effetto inibitorio dell’ipertermia sulla catena respiratoria può essere mimato esponendo cellule in normotermia al medium condizionato da cellule precedentemente sottoposte ad ipertermia, suggerendo così il coinvolgimento di mediatori rilasciati nel terreno di coltura cellulare. Queste osservazioni combinate con un’analisi di gene expression possono essere spiegate tramite un modello basato su un sistema di signaling autocrino mediato dalle heat shock proteins via recettori toll-like e dall’attivazione termica dei canali di tipo TRPV (transient receptor potential cation). In conclusione, i risultati presentati in questo studio supportano la nozione emergente secondo la quale la risposta immunitaria indotta da stimoli sia fisici che biologici coinvolge e richiede specifiche riprogrammazioni del metabolismo delle cellule immuno-competenti. Pertanto, in casi di risposta immunitaria disregolata la modulazione dello switch metabolico in specifici check points può rappresentare un’efficace strategia farmacologica per prevenire il priming metabolico pro-infiammatorio delle cellule immunocompetenti, attenuando nel contempo i sintomi indotti dalla endotossiemia così come da temperature febbrili

Febrile temperature reprograms by redox-mediated signaling the mitochondrial metabolic phenotype in monocyte-derived dendritic cells

Fever-like hyperthermia is known to stimulate innate and adaptive immune responses. Hyperthermia-induced immune stimulation is also accompanied with, and likely conditioned by, changes in the cell metabolism and, in particular, mitochondrial metabolism is now recognized to play a pivotal role in this context, both as energy supplier and as signaling platform. In this study we asked if challenging human monocyte-derived dendritic cells with a relatively short-time thermal shock in the fever-range, typically observed in humans, caused alterations in the mitochondrial oxidative metabolism. We found that following hyperthermic stress (3 h exposure at 39 °C) TNF-α-releasing dendritic cells undergo rewiring of the oxidative metabolism hallmarked by decrease of the mitochondrial respiratory activity and of the oxidative phosphorylation and increase of lactate production. Moreover, enhanced production of reactive oxygen and nitrogen species and accumulation of mitochondrial Ca2+was consistently observed in hyperthermia-conditioned dendritic cells and exhibited a reciprocal interplay. The hyperthermia-induced impairment of the mitochondrial respiratory activity was (i) irreversible following re-conditioning of cells to normothermia, (ii) mimicked by exposing normothermic cells to the conditioned medium of the hyperthermia-challenged cells, (iii) largely prevented by antioxidant and inhibitors of the nitric oxide synthase and of the mitochondrial calcium porter, which also inhibited release of TNF-α. These observations combined with gene expression analysis support a model based on a thermally induced autocrine signaling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells

Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control

Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control

Clock-genes and mitochondrial respiratory activity: evidence of a reciprocal interplay

In the past few years mounting evidences have highlighted the tight correlation between circadian rhythms and metabolism. Although at the organismal level the central timekeeper is constituted by the hypothalamic suprachiasmatic nuclei practically all the peripheral tissues are equipped with autonomous oscillators made up by common molecular clockworks represented by circuits of gene expression that are organized in interconnected positive and negative feed-back loops. In this study we exploited a well-established in vitro synchronization model to investigate specifically the linkage between clock gene expression and the mitochondrial oxidative phosphorylation (OxPhos). Here we show that synchronized cells exhibit an autonomous ultradian mitochondrial respiratory activity which is abrogated by silencing the master clock gene ARNTL/BMAL1. Surprisingly, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the rhythmic clock-gene expression and a similar result was attained with mtDNA depleted cells (Rho0). Our findings provide a novel level of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork