Modifiche longitudinali del metaboloma urinario e plasmatico di neonati con asfissia perinatale sottoposti ad ipotermia terapeutica.

Introduzione: l’asfissia perinatale è una condizione di compromissione degli scambi gassosi feto-placentari conseguenti all’interruzione del flusso placentare. Tale patologia è ancora ad oggi una delle maggiori cause di mortalità e morbidità in epoca neonatale. La sua complicanza più temibile è l’encefalopatia ipossico ischemica che può influenzare in maniera importante la prognosi a breve e a lungo termine del neonato. Ad oggi l’unica terapia che si è dimostrata in grado di migliorare la prognosi di questi neonati è l’ipotermia terapeutica (IT). La candidabilità del neonato alla terapia si basa principalmente su criteri clinici e strumentali; ad oggi, non esistono biomarcatori che possano guidarne la scelta. La metabolomica è un campo di ricerca che si sta dimostrando estremamente promettente in ambito di asfissia perinatale e che potrebbe permettere di identificare metaboliti o pathway metaboliche in grado di cambiare in futuro l’approccio clinico in questa patologia. Scopo dello studio: l'obiettivo primario dello studio era descrivere le modificazioni dinamiche nel tempo del metaboloma urinario e plasmatico in una stessa coorte di neonati asfittici. Obiettivo secondario è stato ricercare potenziali corrispondenze tra le modificazioni plasmatiche ed urinarie del metaboloma durante le fasi di pre-IT (T0), le 72 ore di IT (IPO) e di post-riscaldamento (PT). Materiali e metodi: sono stati arruolati neonati con più di 35 settimane gestazionali, con segni e sintomi di asfissia perinatale alla nascita e sottoposti ad IT. Sono stati raccolti campioni di urine e plasma prima dell’avvio dell’IT, durante le 72h di IT e dopo l’IT. I campioni sono stati analizzati con tecniche di metabolomica untargeted e targeted utilizzando un sistema di cromatografia liquida ad alte prestazioni accoppiato ad uno spettrometro di massa. I dati derivati sono stati analizzati con tecniche di analisi statistica univariate e multivariate, nonché metodiche di over-representation pathway analysis. Un gruppo di 22 neonati sani sono stati arruolati per effettuare un paragone del metaboloma a T0 tra malati e non malati. Risultati: per 12 pazienti sono stati raccolti i campioni di urina prima dell’ipotermia (T0), durante l’ipotermia (IPO) e dopo l’ipotermia (PT) e di 19 pazienti è stato ottenuto un campione di plasma per ognuna delle tre fasi. La combinazione di analisi univariata e multivariata ha messo in luce 31 metaboliti urinari e 34 metaboliti plasmatici rilevanti nel descrivere le modificazioni nelle tre fasi. Le pathway analysis hanno permesso di individuare le vie metaboliche perturbate nelle tre fasi. Nei campioni di urine sono state identificate le pathway di steroidogenesi, di degradazione della lisina e di sintesi della carnitina (β-ossidazione), mentre nel plasma le analisi targeted hanno individuato come alterate le pathway relative al metabolismo del triptofano, della glicina e della serina, della metionina, del riciclo dell'ammoniaca, della biosintesi della spermidina e della spermina e della sintesi della carnitina. Conclusioni: il nostro studio ci ha permesso di descrivere le modifiche dinamiche del metaboloma urinario e plasmatico dei neonati asfittici nel tempo, nonché di determinare quali sono le vie metaboliche che vengono significativamente alterate durante l'asfissia perinatale. In particolare da osservare che la via metabolica della β-ossidazione è costantemente influenzata sia nelle urine che nel plasma. Saranno necessari futuri studi randomizzati controllati per valutare il razionale di trattamenti potenzialmente di supporto basati sulle variazioni metaboliche rilevate nel tempo di questi pazienti.Background: perinatal asphyxia is a condition of impaired fetal-placental gas exchange resulting from the interruption of placental flow. This pathology is still today one of the major causes of neonatal mortality and morbidity. Its most fearsome complication is hypoxic-ischemic encephalopathy, which can significantly influence short and long-term prognosis of patients. To date, the only therapy that has been shown to improve the prognosis of these newborns is therapeutic hypothermia (TH). Infant's candidacy for this therapy is based mainly on clinical and instrumental criteria; unfortunately, no early biomarkers that can guide this choice are currently available. Metabolomics is a science that is proving extremely promising in the field of perinatal asphyxia and could allow the identification of metabolites or metabolic pathways that may be able to change the future clinical approach to this pathology. Aims: the primary objective of the study was to describe the dynamic changes over time of the urinary and plasma metabolome in the same cohort of asphyxiated newborns. A secondary objective was to search for potential correspondences between the plasma and urinary changes of the metabolome during three phases: pre-TH (T0), the 72 hours of TH (IPO) and the rewarming phase (PT). Materials and methods: newborns were enrolled if older than 35 gestational weeks, with signs and symptoms of perinatal asphyxia at birth and undergoing TH. Urine and plasma samples were collected before starting TH, during the 72h of TH and after TH. Samples were analyzed with untargeted and targeted metabolomics analysis using a high performance liquid chromatography system coupled to a mass spectrometer. The obtained data were analyzed with univariate and multivariate statistical analysis techniques and over-representation pathway analysis methods. A group of 22 healthy subjects was also enrolled to make a comparison at T0 between ill and normal newborns. Results: urine samples were collected for 12 patients before (T0), during (IPO) and after hypothermia (PT); in addition, 19 patients had plasma samples collected at these three time points. The combination of the univariate and multivariate analysis revealed that 31 urinary metabolites and 34 plasma metabolites are relevant in describing the changes in the three phases. Pathway analysis made it possible to identify the perturbed metabolic pathways in the three phases. In urine samples pathways of steroidogenesis, lysine degradation and carnitine synthesis (β-oxidation) were identified; as for targeted analysis on plasmatic samples, pathways related to the metabolism of tryptophan, glycine and serine, methionine, ammonia recycling, spermidine and spermine biosynthesis and carnitine synthesis (β-oxidation) were significantly affected. Conclusions: our study allowed us to describe the dynamic changes in the urinary and plasma metabolome of asphyxiated infants over time, as well as to determine which metabolic pathways are significantly altered during perinatal asphyxia. In particular, it should be noted that the metabolic pathway of β-oxidation is constantly influenced in both urine and plasma. Future randomized controlled trials are needed to assess the rationale of potentially supportive treatments directly targeting the detected metabolic variations over time of these patients

Elucidation of the Ammonium Major Facilitator (AMF) Family in Plants

The discovery of the Ammonium Major Facilitator (AMF) family in plants and yeast by overexpression of the soybean transcription factor GmbHLHm1 in yeast has opened up new insights into the transport of NH4+ in eukaryotic systems. Using both yeast and Xenopus oocyte expression systems, ScAMF1 and a plant homolog (GmAMF3) were shown to transport NH4+ and the toxic ammonium analogue methylammonium (MA). The AMF family is conserved in most plants including Arabidopsis thaliana where three homologs exist, these being AtAMF1 (At2g22730), AtAMF2 (At5g64500) and AtAMF3 (At5g65687). All three AMF genes are expressed throughout the plant with noticeable expression in senescing shoot tissues. Transient expression in Nicotiana benthamiana leaves indicated AtAMF1, AtAMF2, and AtAMF3 proteins are located on the ER, tonoplast and plasma membrane, respectively. Functional testing in an ammonium transport deficient yeast strain (31019b) did not result in activities which rescued growth of yeast cells grown on low ammonium concentrations. However, like GmAMF1;3 and ScAMF1, AtAMF2 was capable of inducing an increased sensitivity to the toxic ammonium analogue methylammonium. These alternative protein locations and activities may reflect a distinct function of each protein in cellular NH₄+ transport and homeostasis. A disruption of both the high (Trk1) and low (Trk2) affinity K+ transport proteins in the yeast strain CY162 (trk1D; trk2D) was found to inhibit growth on high concentrations of NH₄+, a process potentially linked to the efflux of amino acids out of yeast cells. The overexpression of the tonoplast-localised AMF protein, AtAMF2, was shown to rescue CY162 when grown at high NH₄+ concentrations while continuing to induce a toxic phenotype to high concentrations of MA. The data suggests that AtAMF2 may participate in the release of acid-trapped NH4+ from the vacuole into the cytoplasm, a process required to supply nutrients to support cellular growth in NH4+ grown but amino acid-starved cells. In contrast, when MA is supplied, vacuole-localised MA+ is released by AtAMF2 into the cytoplasm inducing a toxicity phenotype. This process was enhanced when cells were supplied with only proline or limited concentrations of amino acids. Collectively these data suggest the tonoplast-localised AtAMF2 is a functional NH₄+ efflux protein that can support cellular growth when limited by available nitrogen (NH₄+ and or amino acid) resources. Individual amf T-DNA knockouts in Arabidopsis were identified and used to create multiple amf mutations through selected crosses. The amf1 mutant displayed an increased rate of unidirectional 15NH₄+ influx into Arabidopsis roots that was not present in either amf2 or amf3. The growth of mutants with amf1 background (amf1, amf1amf2 or amf1amf3) on low NO₃- (0.05 mM) and adequate K+ (3.75 mM) were found to be sensitive to 20 mM MA. In the presence of low or high NO₃- (0.05 and 7.5 mM, respectively) and low K+ (<0.1 mM), root growth in the amf2amf3 mutant was significantly inhibited by 2 mM NH₄+, a phenotype that could be rescued with the provision of external K+. These data demonstrated that AtAMF1 might have a dominant role in NH₄+ toxicity tolerance when supplied low concentrations of NO₃- while AtAMF2 and AtAMF3 aid in the management of NH₄+ toxicity in a K+-dependent manner. Collectively with experiments in yeast, the in-planta experiments confirm the importance of K+ availability in mitigating NH4+ toxicity in Arabidopsis, a process which appears to involve members of the AMF family.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201

Additional file 1: of Membrane potential independent transport of NH3 in the absence of ammonium permeases in Saccharomyces cerevisiae

Contains details on strain construction and confirmation, additional details on calculations and additional metabolome and proteome measurements. (PDF 1095 kb