Stat3 promotes mitochondrial transcription and oxidative respiration during maintenance and induction of naive pluripotency.

Transcription factor Stat3 directs self-renewal of pluripotent mouse embryonic stem (ES) cells downstream of the cytokine leukemia inhibitory factor (LIF). Stat3 upregulates pivotal transcription factors in the ES cell gene regulatory network to sustain naïve identity. Stat3 also contributes to the rapid proliferation of ES cells. Here, we show that Stat3 increases the expression of mitochondrial-encoded transcripts and enhances oxidative metabolism. Chromatin immunoprecipitation reveals that Stat3 binds to the mitochondrial genome, consistent with direct transcriptional regulation. An engineered form of Stat3 that localizes predominantly to mitochondria is sufficient to support enhanced proliferation of ES cells, but not to maintain their undifferentiated phenotype. Furthermore, during reprogramming from primed to naïve states of pluripotency, Stat3 similarly upregulates mitochondrial transcripts and facilitates metabolic resetting. These findings suggest that the potent stimulation of naïve pluripotency by LIF/Stat3 is attributable to parallel and synergistic induction of both mitochondrial respiration and nuclear transcription factors.GM’s laboratory is supported by grants from Armenise-Harvard Foundation and Telethon Foundation (TCP13013). The Cambridge Stem Cell Institute receives core funding from the Wellcome Trust and Medical Research Council. GM was supported by a Human Frontier Science Program Fellowship. AS is a Medical Research Professor.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.15252/embj.20159262

Adherence to dietary guidelines (DG) and body weight change (BWC) in early-stage breast cancer (EBC): A prospective trial in patients submitted to nutrition evidence-based educational intervention

n/

Evidence-based tailored nutrition educational intervention improves adherence to dietary guidelines, anthropometric measures and serum metabolic biomarkers in early-stage breast cancer patients: A prospective interventional study

Purpose: The impact of the adherence to dietary guidelines of early-stage breast cancer (EBC) patients on body composition changes during treatment is not entirely defined. This study aimed to evaluate the role of an evidence-based nutrition educational intervention, according to adherence to dietary guidelines, in EBC patients. Methods: This prospective study included EBC patients, candidates for neoadjuvant/adjuvant therapy. Patients received an evidence-based tailored nutrition educational intervention. The adherence to dietary guidelines, anthropometric and dietary assessments, and blood glucose and lipid profile tests were evaluated at baseline and after a 12-months nutritional intervention. Results: Two hundred and forty-three patients were enrolled. At baseline, 38.3% and 23.9% of patients were overweight and obese, weight gain ≥5% (compared to 6-months before enrollment) and central obesity were observed in 47.3% and 52.7% of patients, respectively. Adherence to dietary guidelines was low (median Med-Diet score: 6 [IQR 4-8]). After the nutritional intervention (median follow-up: 22 months [range 12-45]), adherence to dietary guidelines significantly increased (median Med-Diet score: 12 [IQR 8-13]), p < 0.0001). High adherence to dietary guidelines (defines as Med-Diet score ≥10) significantly correlated with: 1) overall weight loss ≥5% (21.8% vs. 2.5%, p = 0.003); 2) median BMI drop (from 25.6 kg/m2 to 24.4 kg/m2, p = 0.003); 3) lower prevalence of central obesity (38.2% vs. 7.2%, p = 0.01); 4) improvement in blood glucose levels and lipid profile. Conclusion: This study suggests that an evidence-based tailored nutrition educational intervention during treatment for EBC significantly increases overall adherence to dietary guidelines, and it improves both anthropometric measures and serum metabolic biomarkers in patients with high adherence

Belle and Aurora A are novel components of the circadian machinery in Drosophila melanogaster

The circadian clock is a self-sustained system that synchronizes the physiology and behaviour of most organisms with the light-dark (LD) cycles. In humans, several pathological conditions, including cancer, are related to the deregulation of the circadian clock. Drosophila melanogaster is one of the most important models for the study of the circadian mechanism. The proteins Timeless (Tim) and Period (Per) are two main components of the master clock, which is located in the brain. The synchronization of the circadian clock to environmental changes is mostly achieved by blue light-dependent degradation of Tim, via the photoreceptor Cryptochrome (Cry). Cry mediates the daily resetting of the clock by light, binding to Tim and targeting it for degradation via proteasome. The oscillator is able to keep circadian time even in the absence of external stimuli and consists of a network of genes that function in transcriptional/translational feedback loops. Among the post-translational modification that regulate clock proteins amount, localization and activity, phosphorylation is likely the most important one. We have initiated a study in order to identify new molecular partners for Cry, both in light and dark. 1) The search for new partners of Cry by co-IP and mass spectrometry led to the identification of Belle, an ATP-dependent RNA helicase. Belle is expressed in the glial cells in fly adult brain, where also Cry is expressed. Moreover, the expression of this RNA-helicase oscillates in light-dark cycles (LD) and constant dark conditions (DD) in wild-type flies. Moreover, flies mutant for this gene do not show the typical rhythmic activity profile and show an impairment in Per expression in a subset of circadian neurons. Thereafter the downregulation of the gene causes reduced photophobicity in 3rd instar larvae and significant defects in the locomotor activity, suggesting an impairment of the circadian clock. Our data suggest that Belle is a novel “clock” protein involved in the circadian machinery of Drosophila, where it could act in the post-transcriptional control of circadian components. 2) It has been shown that the C-terminus of Cry contains putative targets for phosphorylation that play a role in the modulation of protein activity. Cry activation by light could be due to a conformational change or a release of a repressor. Phosphorylation could play an important role in the light dependent activation of the protein. In fact, AtCry1 and AtCry2 in plants (Arabidopsis thaliana) and mCry2 in mammals are phosphorylated after light exposure. in vitro experiments, performed in our lab, showed that Drosophila Cry is a putative target of the Serine-Threonine kinase Aurora A. Moreover, in silico analyses revealed that Cry C-terminus seems to contain consensus motifs which could be a target for this kinase. In particular, two Serine residues belonging to the SLIT and SNEE kinase consensus motifs in the C-terminal region result specifically phosphorylated. We have conducted a study in order to verify the in vivo role for Aurora A in Cry regulation. Analysis of single and double transgenic mutants for the residues target of phosphorylation seem to exclude a role for this sites in mediating Cry degradation after light exposure. However, experiments performed in flies over-expressing Aurora A show modifications in the circadian control of their rhythmic behaviour and display an increased degradation of Cry after light exposure. Taken together, these data suggest a putative implication of Aurora A in the regulation of Cry activity.La maggior parte degli organismi viventi sincronizza le proprie attività fisiologiche e comportamentali con l’alternanza di cicli luce-buio, che si ripetono con un periodo di circa 24 ore per effetto della rotazione terrestre. Ciò è permesso grazie ad un orologio endogeno, detto circadiano (dal latino circa diem): esso rappresenta l’esempio più significativo del controllo temporale dei processi biologici. Nei mammiferi controlla l’alternanza di sonno e veglia, la secrezione di ormoni e le fluttuazioni della temperatura corporea; nelle piante la fioritura e le attività foto sintetiche; nei cianobatteri i processi di fissazione dell’azoto e del metabolismo di alcuni amminoacidi; nei funghi la regolazione dei fenomeni di sporulazione; nel moscerino della frutta, Drosophila melanogaster, l’attività locomotoria e la schiusa pupale. L’orologio circadiano è capace di percepire alterazioni di variabili ambientali e può sincronizzarsi in risposta a queste ultime, tuttavia è anche un sistema in grado di “autosostenersi” essendo attivo anche in assenza di stimoli esterni. In Drosophila, evento fondamentale per la sincronizzazione dell’orologio in risposta alle variazioni ambientali è la degradazione in presenza di luce della proteina Timeless (Tim), questa è mediata dalla sua interazione con il fotorecettore della luce blu Cryptochrome (Cry). In seguito a fotoattivazione, Cry interagisce con Tim che viene fosforilata, ubiquitinata e degradata dal proteasoma. Successivamente, anche Cry viene degradata via proteasoma dopo esposizione alla luce ma il meccanismo che regola questo processo rimane ancora largamente sconosciuto. Il lavoro qui presentato si divide in 2 parti, la prima è volta allo studio di un interattore molecolare di Cry e della sua caratterizzazione come componente dell’orologio circadiano, la seconda è volta all’analisi del ruolo di una chinasi nella regolazione e nella degradazione di Cry dopo esposizione alla luce. Parte 1 Uno studio condotto nel nostro laboratorio volto alla ricerca di nuovi partner molecolari di Cry ha portato all’identificazione di Belle, una RNA elicasi ATP-dipendente. Questa proteina è espressa nelle cellule gliali del cervello di Drosophila, dove anche la proteina Cry è espressa, avvalorando l’ipotesi che vi sia un’interazione fisica tra le due. Esperimenti di Real-time PCR condotti su estratti di teste di adulti wild-type hanno evidenziato che l’espressione di questa RNA elicasi oscilla nelle 24 ore sia in alternanza di luce-buio (LD) sia in assenza di stimolo luminoso (DD). Inoltre, individui mutanti per il gene belle, mostrano un profilo locomotorio aritmico e difetti nell’espressione della proteina orologio Per in alcuni neuroni fondamentali per la generazione della ritmicità. Oltre a ciò, la caratterizzazione di linee in cui l’espressione del gene belle è ridotta ha evidenziato sia una riduzione della ritmicità circadiana negli adulti, sia un deficit a livello di risposta fotofobica dopo esposizione alla luce nelle larve terzo stadio, suggerendo che Belle sia richiesto per il normale funzionamento dell’orologio molecolare. Nel complesso, i risultati ottenuti avvalorano l’ipotesi che la proteina Belle non sia solo un interattore del fotorecettore Cry ma anche un nuovo componente dell’orologio centrale. Parte 2 È stato osservato che l’estremità C-terminale di Cry ha un ruolo predominante nel regolare le risposte alla luce in Drosophila. Infatti la rimozione di questo dominio sembra eliminare la necessità di luce per l’interazione con le proteine orologio Tim e Per. Mediante saggi in vitro condotti nel nostro laboratorio, è stato osservato che Cry viene fosforilata da Aurora A, una serin treonin chinasi coinvolta nel ciclo cellulare. Mediante spettrometria di massa sono stati individuati due siti di fosforilazione che corrispondono alla Serina 529, nella sequenza consenso SLIT, e della Serina 540, nella sequenza consenso SNEE, entrambi localizzati nella regione C-terminale. Allo scopo di verificare l’effettivo ruolo della chinasi Aurora A nella regolazione dell’attività di Cry, è stata condotta un’analisi su linee linee transgeniche di Drosophila in cui i due residui sono stati mutati singolarmente o contemporaneamente. In queste linee è stata valutata la sensibilità alla luce in confronto alla linea selvatica. I risultati ottenuti non hanno evidenziato una sostanziale differenza a livello di sensibilità alla luce e ciò può essere dovuto al fatto che la variante mutata di Cry è espressa a livelli molto bassi se confrontata con la forma selvatica. Tuttavia, esperimenti condotti su linee che over-esprimono la chinasi Aurora A hanno evidenziato sia un incremento nella degradazione di Cry dopo esposizione alla luce continua, sia un deficit nella risposta circadiana a “pulse” di luce somministrati durante la notte soggettiva. Questi risultati suggeriscono che Aurora A potrebbe avere un ruolo nell’orologio circadiano ed in particolare nella regolazione dell’attività di Cry in seguito ad esposizione alla luce

PKA and cAMP/CNG channels independently regulate the cholinergic Ca2+-response of Drosophila mushroom body neurons

International audienceThe mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca2+-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca2+-activity continuously, over a long time period, and characterized the nicotine-evoked Ca2+-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca2+-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca2+-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca2+-response in the MBs, leading to the conclusion that cAMP modulates the Ca2+-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels

PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca(2+)-Response of Drosophila Mushroom Body Neurons

The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca2+-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca2+-activity continuously, over a long time period, and characterized the nicotine-evoked Ca2+-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca2+-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca2+-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca2+-response in the MBs, leading to the conclusion that cAMP modulates the Ca2+-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels

In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach

AbstractDifferent optical imaging techniques have been developed to study neuronal activity with the goal of deciphering the neural code underlying neurophysiological functions. Because of several constraints inherent in these techniques as well as difficulties interpreting the results, the majority of these studies have been dedicated more to sensory modalities than to the spontaneous activity of the central brain. Recently, a novel bioluminescence approach based on GFP–aequorin (GA) (GFP: Green fluorescent Protein), has been developed, allowing us to functionally record in-vivo neuronal activity. Taking advantage of the particular characteristics of GA, which does not require light excitation, we report that we can record induced and/or the spontaneous Ca2+-activity continuously over long periods. Targeting GA to the mushrooms-bodies (MBs), a structure implicated in learning/memory and sleep, we have shown that GA is sensitive enough to detect odor-induced Ca2+-activity in Kenyon cells (KCs). It has been possible to reveal two particular peaks of spontaneous activity during overnight recording in the MBs. Other peaks of spontaneous activity have been recorded in flies expressing GA pan-neurally. Similarly, expression in the glial cells has revealed that these cells exhibit a cell-autonomous Ca2+-activity. These results demonstrate that bioluminescence imaging is a useful tool for studying Ca2+-activity in neuronal and/or glial cells and for functional mapping of the neurophysiological processes in the fly brain. These findings provide a framework for investigating the biological meaning of spontaneous neuronal activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium

A common molecular logic determines embryonic stem cell self-renewal and reprogramming.

During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single-cell resolution by RT-qPCR Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.S-J.D. is supported by Microsoft Research. G.M.’s laboratory is supported by grants from Giovanni Armenise-Harvard Foundation and Telethon Foundation (TCP13013). A.S. and M.A.L. are funded by the BBSRC. The Cambridge Stem Cell Institute receives core funding from the Wellcome Trust and Medical Research Council. M.A.L. was a Sir Henry Wellcome Postdoctoral fellow and received support from the University of Cambridge Institutional Strategic Support Fund. AS is a Medical Research Council professor