Indolamine accumulation and TDC/T5H expression profiles reveal the complex and dynamic regulation of serotonin biosynthesis in tomato (Solanum lycopersicum L.)

Tryptamine and serotonin are indolamines that fulfill diverse biological functions in all kingdoms of life. Plants convert l-tryptophan into tryptamine and then serotonin via consecutive decarboxylation and hydroxylation reactions catalyzed by the enzymes tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H). Tryptamine and serotonin accumulate to high levels in the edible fruits and seeds of many plant species, but their biological roles in reproductive organs remain unclear and the metabolic pathways have not been characterized in detail. We identified three TDC genes and a single T5H gene in tomato (Solanum lycopersicum L.) by homology-based screening and confirmed their activity by heterologous expression in Nicotiana benthamiana. The co-analysis of targeted metabolomics and gene expression data revealed complex spatiotemporal gene expression and metabolite accumulation patterns that suggest the involvement of the serotonin pathway in multiple biological processes. Our data support a model in which SlTDC1 allows tryptamine to accumulate in fruits, SlTDC2 causes serotonin to accumulate in aerial vegetative organs, and SlTDC3 works with SlT5H to convert tryptamine into serotonin in the roots and fruits

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required

Optimization of a Sustainable Protocol for the Extraction of Anthocyanins as Textile Dyes from Plant Materials

Anthocyanins are the largest group of polyphenolic pigments in the plant kingdom. These non-toxic, water-soluble compounds are responsible for the pink, red, purple, violet, and blue colors of fruits, vegetables, and flowers. Anthocyanins are widely used in the production of food, cosmetic and textile products, in the latter case to replace synthetic dyes with natural and sustainable alternatives. Here, we describe an environmentally benign method for the extraction of anthocyanins from red chicory and their characterization by HPLC-DAD and UPLC-MS. The protocol does not require hazardous solvents or chemicals and relies on a simple and scalable procedure that can be applied to red chicory waste streams for anthocyanin extraction. The extracted anthocyanins were characterized for stability over time and for their textile dyeing properties, achieving good values for washing fastness and, as expected, a pink-to-green color change that is reversible and can therefore be exploited in the fashion industry

Expression and purification of the mutated form of human GAD65 in different biological systems

Riassunto Il diabete insulino-dipendente di tipo 1 (T1D), il quale colpisce lo 0.03-0.04% della popolazione, \ue8 causato dalla distruzione autoimmune delle cellule beta del pancreas deputate alla produzione di insulina, rendendo necessaria una terapia sostitutiva continua con questo ormone (Gepts, 1965). Questo forma di diabete \ue8 considerata una malattia cronica con un forte impatto sociale, a causa della comparsa di complicazioni legate al progredire della malattia ed alla giovane et\ue0 dei pazienti essendo la pi\uf9 diffusa malattia cronica in bambini e ragazzi al di sotto dei 14 anni di et\ue0. Attualmente non esiste una cura della malattia ed il trattamento con insulina rappresenta semplicemente una terapia sostitutiva continua. L\u2019induzione della tolleranza mediante l\u2019utilizzo di auto antigeni \ue8 considerata una strategia utile per la prevenzione o il rallentamento della progressione delle malattie autoimmuni (Harrison, 2005). In particolare, la GAD65 \ue8 considerata un possibile vaccino per il diabete di tipo 1 e, di conseguenza, sono stati effettuati diversi studi di immunoterapia antigene-specifica (ASI) utilizzando questo auto antigene per testare la sua efficacia nell\u2019induzione della tolleranza. I risultati ottenuti nel modello animale, rappresentato dai topi NOD (Non-Obese Diabetic mouse), dimostrano la possibilit\ue0 di fornire un trattamento preventivo contro il diabete mediante la somministrazione di GAD65. Recentemente, un\u2019azienda svedese, \u201cDiamyd Medical\u201d, ha condotto gli studi clinici di Fase II e III in uomo. Nel primo caso, \ue8 stato dimostrato che due iniezioni subcutanee da 20 \ub5g ciascuna del farmaco in esame (GAD-alum) pu\uf2 revocare il progredire del T1D in pazienti di et\ue0 compresa fra 10 e 18 anni in cui la malattia \ue8 insorta da breve tempo e fornire protezione contro di essa (Ludvigsson J. et al., 2008), mentre le prove cliniche di Fase III sono fallite. Sono stati proposti ulteriori studi clinici per testare la possibilit\ue0 di intervento a tempi diversi rispetto all\u2019insorgenza e sviluppo della malattia, tipi diversi di terapia e nuove modi di somministrazione dell\u2019autoantigene. Le attuali proposte includono terapie preventive in individui ad alto rischio (attualmente in Fase II di sperimentazione clinica), terapie di combinazione che sfruttano l\u2019associazione dell\u2019immunoterapia non antigene-specifica (NASI) mediante un immunosoppressore, con l\u2019immunoterapia antigene-specifica (ASI), utilizzando per esempio la GAD65, e ASI con auto antigeni multipli (Lernmark and Larsson, 2011; Larsson and Lernmark, 2011). Inoltre, la modalit\ue0 di somministrazione necessita di ulteriori studi, quali, per esempio, l\u2019induzione di tolleranza orale sfruttando la GAD65, come \ue8 stato fatto mediante la somministrazione orale dell\u2019insulina. Viste le prove cliniche in corso o previste in futuro con l\u2019obiettivo di testare diverse formulazioni del vaccino basate su auto antigeni del T1D, attualmente, la produzione di GAD65 umana \ue8 un punto cruciale per la pianificazione delle strategie future di prevenzione della malattia. Fino ad ora, la GAD65 umana (hGAD65) \ue8 stata ottenuta da diversi sistemi omologi ed eterologi, ma, le attuali piattaforme di produzione sono troppo costose ed incapaci di fornire quantit\ue0 di auto antigene sufficienti a soddisfare la richiesta per i trattamenti di immunoterapia. Le due forme della GAD65 umana, hGAD65 e hGAD65mut, sono state, quindi, espresse nel sistema eterologo Escherichia coli per stabilire se entrambe le molecole vengono accumulate come proteine insolubili, come precedentemente gi\ue0 osservato nel caso della hGAD65 (Mauch L. et al., 1993), e se la produzione di hGAD65mut risulta maggiore di quella della forma nativa dell\u2019enzima, come precedentemente descritto in piante di Nicotiana tabacum var. Sr1 (Avesani L. et al., 2010). E stato dimostrato che entrambe le forme della GAD65 umana sono accumulate in corpi di inclusione insolubili e vengono solubilizzate solo in presenza di concentrazioni denaturanti di urea. Inoltre, \ue8 stato provato che anche nel sistema batterico la hGAD65mut \ue8 prodotta in quantit\ue0 maggiori rispetto alla hGAD65. I sistemi di produzione vegetali possono offrire vantaggi dal punto di vista economico e della produzione su larga scala di proteine ampiamente utilizzate in ambito terapeutico (Ma J.C.K. et al., 2005 a, b; Barasan and Rodriguez-Cerezo, 2008). Di conseguenza, il principale obiettivo del progetto di ricerca \ue8 la valutazione dell\u2019idoneit\ue0 del sistema vegetale per la produzione della GAD65 umana. La GAD65 umana \ue8 stata precedentemente espressa in piante di tabacco transgeniche, ma i livelli di produzione si sono rivelati deludenti raggiungendo valori massimi pari allo 0.25% delle proteine solubili totali (Total Soluble Proteins, TSP) (Porceddu A. et al., 1999; Ma S. et al., 2004; Wang et al., 2008; Avesani L. et al., 2003). Recentemente, una forma mutata e cataliticamente inattiva della GAD65 umana, hGAD65mut, \ue8 stata espressa in piante transgeniche di Nicotiana tabacum var. Sr1. Le piante pi\uf9 alte esprimenti hanno accumulato livelli di proteina ricombinante dieci volte maggiori (2.2% TSP) rispetto a quelli ottenuti nelle piante pi\uf9 alte esprimenti trasformate con la hGAD65 (Avesani L. et al., 2010). Tale sistema di produzione vegetale necessita di essere ulteriormente caratterizzato in modo da poter verificare l\u2019ipotesi secondo cui le propriet\ue0 catalitiche della forma nativa della GAD65 umana possono contribuire ai bassi livelli di produzione. In studi precedenti, infatti, \ue8 stato dimostrato l\u2019assenza di attivit\ue0 enzimatica in vitro per la forma mutata della GAD65. Di conseguenza, si \ue8 effettuato un saggio enzimatico in vivo con il quale \ue8 stata confermata l\u2019assenza di attivit\ue0 enzimatica della stessa molecola anche nel sistema eterologo vegetale. Un secondo sistema vegetale \ue8 stato testato per la produzione di entrambe le forme della GAD65 umana. Nicotiana tabacum var. Maryland mammoth \ue8 una variet\ue0 di tabacco che produce una maggiore quantit\ue0 di biomassa della pi\uf9 diffusa Sr1 e, quindi, potrebbe essere un eccellente candidato per la produzione di proteine ricombinanti ampiamente utilizzate in ambito terapeutico. Per questo, entrambe le forme della GAD65 umana, hGAD65 e hGAD65mut, sono state espresse in piante di tabacco transgeniche della variet\ue0 Maryland mammoth, dimostrando che, anche in tale sistema vegetale, la produzione di hGAD65mut \ue8 maggiore di quella della forma nativa dell\u2019enzima, come precedentemente descritto in piante di tabacco transgeniche della variet\ue0 Sr1 (Avesani L. et al., 2010). L\u20191% delle proteine solubili totali \ue8 generalmente il livello di espressione minimo richiesto per considerare l\u2019estrazione di proteine ad uso farmaceutico derivate da pianta economicamente vantaggiosa (Ma J.C.K. et al., 2003). Visto che tale valore \ue8 stato raggiunto e superato gi\ue0 nella prima generazione di piante di Nicotiana tabacum var. Sr1, si \ue8 proceduto alla messa a punto del downstream processing della proteina ricombinante dal sistema vegetale. I risultati ottenuti durante il processo di selezione della popolazione di piante esprimenti la hGAD65mut in modo stabile ed omogeneo e di studio e valutazione del protocollo di estrazione e purificazione della proteina dal tessuto fogliare sono riportati e discussi.Abstract Type 1 insulin-dependent diabetes (T1D) which afflicts 0.03-0.04% of population is caused by autoimmune destruction of insulin-secreting beta cells, leading to an insulin deficiency (Gepts, 1965). It is considered a chronic disease with a strong social impact because of high prevalence of late-onset complications and the young age of affected patients being the most frequent chronic disease in children younger than 14-years old. Until now there are no possibilities to cure it and insulin treatment is only a life-long replacement therapy. Tolerance induction through autoantigen administration is one of the strategies useful to prevent or to slow down autoimmune diseases (Harrison, 2005). In particular, for T1D GAD65 has been evaluated as a good candidate vaccine and different Antigen-Specific Immunotherapy (ASI) studies using this autoantigen have been done to test its efficacy in tolerance induction. Results obtained in the non-obese diabetic (NOD) mouse models indicate the potential of GAD65 administration to provide a preventive treatment for diabetes. Recently, Diamyd Medical, a Swedish company, has conducted phase II and phase III clinical trials in humans: in the first case it was demonstrated that two subcutaneous injections of 20\ub5g of alum-formulated GAD65 can reverse the progress of recent-onset T1D in 10 to 18-years-old patients and give protection against it (Ludvigsson et al., 2008), while phase III trials failed. New clinical studies have been proposed including new challenges in timing, different types of therapies and new administration routes. Current proposals include preventive therapies in high-risk individuals (current Phase II trial), combination therapies exploiting the combination of Non-Antigen Specific Immunotherapy (NASI) through an immunosoppressor, together with Antigen-Specific Immunotherapy (ASI), using for example GAD65, and ASI using multiple autoantigens (Lernmark and Larsson, 2011; Larsson and Lernmark, 2011). In addition the route of administration needs further studies, such as oral tolerance induction through GAD65, as it has been done using oral insulin. At present, human GAD65 production is a central point for planning future T1D prevention strategies because of the undergoing and future trials using different vaccine preparations based on T1D autoantigens. Until now human GAD65 (hGAD65) has been obtained from different homologous and heterologous platforms. However, actual production platforms are too expensive and unable to provide sufficient quantity of this autoantigen to meet demand for immunotherapy treatments. Both forms of human GAD65, hGAD65 and hGAD65mut, are expressed in E.coli heterologous system to sort out if they are both accumulated as insoluble proteins, as previously described for human GAD65 (Mauch L. et al., 1993), and if hGAD65mut yield is higher than that of the wild type form of the enzyme, as previously reported in Nicotiana tabacum var. Sr1 plants (Avesani L. et al., 2010). They demonstrated to be both accumulated as insoluble inclusion bodies and were solubilized by the use of denaturing concentration of urea. Western and radioimmunoassay analyses demonstrated that hGAD65mut accumulated at higher levels than hGAD65. Plant-based systems may offer advantages in terms of economy and scalability for the large-scale production of therapeutic proteins in high demand (Ma J.C.K. et al., 2005 a, b; Barasan and Rodriguez-Cerezo, 2008). Thus, the principal aim of the PhD project is the evaluation of plant-based platform feasibility for human GAD65 production. Human GAD65 has previously been expressed in transgenic tobacco plants but yields were disappointing (maximum 0.25% of total soluble protein, TSP) (Porceddu et al., 1999; Ma S. et al., 2004; Wang et al., 2008; Avesani L. et al., 2003). In a recent study, a mutated catalytically-inactive form of human GAD65 (hGAD65mut) was expressed in transgenic Nicotiana tabacum var. Sr1 plants. hGAD65mut-highest expressing plants accumulated 10-fold (2.2% TSP) higher levels of recombinant protein than hGAD65-highest expressing plants (Avesani L. et al., 2010). This plant platform production system needs to be characterized thoroughly in order to verify the hypothesis by which the catalytic properties of native hGAD65 could contribute to its poor yields. Since in previous studies it has been demonstrated in vitro the lack of the enzymatic activity for hGAD65mut, an enzymatic assay in vivo is performed in order to demonstrated the absence of enzymatic activity of the mutated form of GAD65 also in the heterologous plant-based system. Results of the assay are discussed. An additional plant-based platform is tested for the production of both forms of human GAD65. In fact, being Nicotiana tabacum var. Maryland mammoth a higher leaf biomass producing variety than the most widely used one, Sr1, it can be, potentially, an excellent candidate for the production of recombinant pharmaceutical proteins with a large demand. Both forms of human GAD65, hGAD65 and hGAD65mut, are expressed in this plant-based platform, demonstrating that also in this system hGAD65mut yield is higher than that of the wild type form of the enzyme, as previously already described in Nicotiana tabacum var. Sr1 plants (Avesani L. et al., 2010). 1% TSP yield is usually regarded as the minimum required to make the extraction of a plant-derived pharmaceutical protein economically feasible (Ma J.K.C. et al., 2003). Since this threshold was exceeded in the first generation of hGAD65mut-transgenic Nicotiana tabacum var. Sr1 plants, a protocol for downstream processing of the recombinant protein from plant systems was investigated. Results obtained during the set up of the extraction and purification protocol are discussed

Plant-Based Systems for Vaccine Production

: Plant systems have been used as biofactories to produce recombinant proteins since 1983. The huge amount of data, collected so far in this framework, suggests that plants display several key advantages over existing traditional platforms when they are intended for therapeutic uses, including safety, scalability, and the speed in obtaining the final product.Here, we describe a method that could be applied for the expression and production of a candidate subunit vaccine in Nicotiana benthamiana plants by transient expression, defining all the protocols starting from plant cultivation to target recombinant protein purification

Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective

In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms

Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective

In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms

Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective

In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plantbased systems over traditional fermenter-based expression platforms

A comparative analysis of recombinant protein expression in different biofactories: bacteria, insect cells and plant systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products. In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable. For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein. Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here