CYP72A67 catalyses a key oxidative step in Medicago truncatula hemolytic saponin biosynthesis

In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethylmethanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstreamof oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis

A Structural View on the Stereospecificity of Plant Borneol‐Type Dehydrogenases

The development of sustainable processes for the valorization of byproducts and other waste streams remains an ongoing challenge in the field of catalysis. Racemic borneol, isoborneol and camphor are currently produced from alpha-pinene, a side product from the production of cellulose. The pure enantiomers of these monoterpenoids have numerous applications in cosmetics and act as reagents for asymmetric synthesis, making an enzymatic route for their separation into optically pure enantiomers a desirable goal. Known short-chain borneol-type dehydrogenases (BDHs) from plants and bacteria lack the required specificity, stability or activity for industrial utilization. Prompted by reports on the presence of pure (-)-borneol and (-)-camphor in essential oils from rosemary, we set out to investigate dehydrogenases from the genus Salvia and discovered a dehydrogenase with high specificity (E>120) and high specific activity (>0.02 U mg(-1)) for borneol and isoborneol. Compared to other specific dehydrogenases, the one reported here shows remarkably higher stability, which was exploited to obtain the first three-dimensional structure of an enantiospecific borneol-type short-chain dehydrogenase. This, together with docking studies, led to the identification of a hydrophobic pocket in the enzyme that plays a crucial role in the stereo discrimination of bornane-type monoterpenoids. The kinetic resolution of borneol and isoborneol can be easily integrated into the existing synthetic route from alpha-pinene to camphor thereby allowing the facile synthesis of optically pure monoterpenols from an abundant renewable source

Ottimizzazione del processo di riscaldo per tempra di grandi fucinati mediante analisi FEM

In questo lavoro è presentata la simulazione del processo di riscaldo per austenitizzazione di grandi fucinatiin acciaio destinati alla tempra differenziale. In ogni trattamento termico, le grandezze controllabili a livelloindustriale sono il tempo e la temperatura. È quindi agendo su queste che si può ottimizzare il processo perottenere le proprietà e le microstrutture desiderate per l’acciaio esaminato, e in definitiva massimizzare laqualità del pezzo finale. I prodotti analizzati sono i cilindri di laminazione fucinati, la cui produzione richiede unattento ciclo termico successivo alla deformazione plastica. Infatti, dopo un ciclo termico preliminare di ricotturacon lo scopo di facilitare le lavorazioni di sgrossatura alle macchine utensili, questi pezzi vengono sottopostiad una tempra differenziale per incrementare la durezza dello strato di lavoro, il cui spessore varia in funzionedelle specifiche richieste dal cliente. L’ottimizzazione di un processo può essere implementata utilizzando leggiempiriche ed il proprio know-how in prove sperimentali. La simulazione numerica rappresenta uno strumentodi previsione che potrebbe favorire un approccio più sistematico e scientifico alla risoluzione, in fase diprogetto, di tali problematiche industriali, specialmente quando il rapporto di scala tra il provino sperimentaleed il componente reale è molto elevato, come nel caso oggetto del presente lavoro. Lo strumento numericotipicamente adottato per simulare tale processo è la modellazione e l’analisi agli elementi finiti (FEM). Avendocome possibili parametri il livello di suddivisione del modello (meshatura), le caratteristiche chimico-fisiche delmateriale ed i parametri di scambio termico (entrambe differenziabili topologicamente all’interno del modellostesso) l’approccio FEA consente di simulare sia la forgiatura che i trattamenti termici ed in particolare le fasi diriscaldo, mantenimento e raffreddamento. I risultati ottenibili sono il campo termico, la distribuzione delle variefasi ed i valori di tensioni residue derivanti dal trattamento termico stesso. I risultati sono monitorabili nell’interoarco temporale del processo. Questo tipo di analisi agli elementi finiti consente di prevedere le proprietà delpezzo nell’intero arco temporale del processo: una volta caratterizzato il materiale ed i forni di riscaldo, si haa disposizione uno strumento capace di prevedere in maniera affidabile i risultati di un trattamento termico. Ilsoftware utilizzato in questo studio è il codice FEM commerciale Forge

Prediction of Resting Energy Expenditure in Children: May Artificial Neural Networks Improve Our Accuracy?

The inaccuracy of resting energy expenditure (REE) prediction formulae to calculate energy metabolism in children may lead to either under- or overestimated real caloric needs with clinical consequences. The aim of this paper was to apply artificial neural networks algorithms (ANNs) to REE prediction. We enrolled 561 healthy children (2-17 years). Nutritional status was classified according to World Health Organization (WHO) criteria, and 113 were obese. REE was measured using indirect calorimetry and estimated with WHO, Harris-Benedict, Schofield, and Oxford formulae. The ANNs considered specific anthropometric data to model REE. The mean absolute error (mean \ub1 SD) of the prediction was 95.8 \ub1 80.8 and was strongly correlated with REE values (R2 = 0.88). The performance of ANNs was higher in the subgroup of obese children (101 \ub1 91.8) with a lower grade of imprecision (5.4%). ANNs as a novel approach may give valuable information regarding energy requirements and weight management in children

Nusinersen treatment and cerebrospinal fluid neurofilaments : An explorative study on Spinal Muscular Atrophy type 3 patients

The antisense oligonucleotide Nusinersen has been recently licensed to treat spinal muscular atrophy (SMA). Since SMA type 3 is characterized by variable phenotype and milder progression, biomarkers of early treatment response are urgently needed. We investigated the cerebrospinal fluid (CSF) concentration of neurofilaments in SMA type 3 patients treated with Nusinersen as a potential biomarker of treatment efficacy. The concentration of phosphorylated neurofilaments heavy chain (pNfH) and light chain (NfL) in the CSF of SMA type 3 patients was evaluated before and after six months since the first Nusinersen administration, performed with commercially available enzyme-linked immunosorbent assay (ELISA) kits. Clinical evaluation of SMA patients was performed with standardized motor function scales. Baseline neurofilament levels in patients were comparable to controls, but significantly decreased after six months of treatment, while motor functions were only marginally ameliorated. No significant correlation was observed between the change in motor functions and that of neurofilaments over time. The reduction of neurofilament levels suggests a possible early biochemical effect of treatment on axonal degeneration, which may precede changes in motor performance. Our study mandates further investigations to assess neurofilaments as a marker of treatment response

Severe diaphragmatic dysfunction with preserved activity of accessory respiratory muscles in a critically ill child : a case report of failure of neurally adjusted ventilatory assist (NAVA) and successful support with pressure support ventilation (PSV)

BACKGROUND: Neurally adjusted ventilatory assist (NAVA) is an alternative to pressure support ventilation (PSV) potentially improving patient-ventilator interaction. During NAVA, diaphragmatic electrical activity (EAdi) is used to trigger the ventilator and perform a proportional respiratory assistance. We present a case in which the presence of severe bilateral diaphragmatic dysfunction led to a failure of NAVA. On the contrary, the preserved activity of the accessory inspiratory muscles allowed a successful respiratory assistance using PSV. CASE PRESENTATION: A 10-year-old girl developed quadriplegia after neurological surgery. Initially, no spontaneous breathing activity was present and volume controlled ventilation was necessary. Two months later spontaneous inspiratory efforts were observed and a maximal negative inspiratory force of -\u200920 cmH2O was recorded. In addition, a NAVA nasogastric tube was placed. The recorded EAdi signal, despite showing a phasic activity, had a very low amplitude (1-2\u2009\u3bcV). Two brief (15\u2009min) breathing trials to compare PSV (pressure support\u2009=\u20098 cmH2O) with NAVA (Gain\u2009=\u20095 cmH2O/\u3bcV, inspiratory trigger\u2009=\u20090.3\u2009\u3bcV) were performed. On PSV, the patient was well adapted with stable tidal volumes, respiratory rates, minute ventilation, end-tidal and venous carbon dioxide levels. When switched to NAVA, her breathing pattern became irregular and she showed clear sign of increased work of breathing and distress: tidal volume dropped and respiratory rate rose, leading to an increase in total minute ventilation. Nevertheless, end-tidal and venous carbon dioxide rapidly increased (from 49 to 55\u2009mmHg and from 52 to 57\u2009mmHg, respectively). An electromyographic study documented an impairment of the diaphragm with preserved activity of the accessory inspiratory muscles. CONCLUSIONS: We document the failure of mechanical assistance performed with NAVA due to bilateral diaphragmatic dysfunction in a critically ill child. The preserved activity of some accessory respiratory muscles allowed to support the patient effectively with pressure support ventilation, i.e. by applying a pneumatic trigger. The present case underlines (i) the importance of the integrity of the respiratory centers, phrenic nerves and diaphragm in order to perform NAVA and (ii) the possible diagnostic role of EAdi monitoring in complex cases of weaning failure

Analisi del processo di deformazione a caldo e dell’evoluzione microstrutturale di un acciaio al 3% Cr mediante prove di torsione

Lo studio dell’evoluzione della microstruttura durante il processo di deformazione plastica di acciai rivestefondamentale importanza. In questo lavoro è stato analizzato il comportamento a deformazione a caldo di unacciaio 3% Cr tramite prove di torsione eseguite nell’intervallo di temperatura 1000?1200°C e con e.compresatra 0.01 e 1.00s-1. L’analisi condotta ha consentito di determinare le costanti della legge di potenza che regolail processo di deformazione a caldo (A0 , Q ed n ) tramite le quali è possibile ottimizzare i processi industriali.L’evoluzione microstrutturale durante la deformazione a caldo è stata valutata in termini di grado di percentualedi fase ricristallizzata ed è analizzata attraverso microscopia elettronica a trasmissione (TEM) e microscopiaad orientazione di immagine-diffrazione da retrodiffusione elettronica (OIM?EBSD). In particolare attraversoquesta ultima tecnica viene stimata la frazione volumetrica di ricristallizzato in termini di parametro GOS (GrainOrientation Spread), che meglio sembra interpretare l’evoluzione microstrutturale dopo deformazione a caldo inmicrostrutture martensitiche