1,720,964 research outputs found
Production and characterization of therapeutic proteins/peptides: Human Recombinant FSH-beta subunit expressed in Plant Cells and Chemical Synthesis of Human Osteocalcin and Neuritogenic Peptides
Full text linkSince 1980s proteins have emerged as a major new class of pharmaceuticals with over 350 marketed products that are mainly therapeutics with a small number of diagnostics and vaccines. In 2015 among the 10 best-selling drugs in the world, 7 are recombinant proteins or monoclonal antibodies. There is still considerable confusion about what therapeutic proteins are. Regulatory authorities (U S Food and Drug Administration and European Medicines Agency) provided different definitions describing them as Biological medicinal products, Biopharmaceuticals, Biological or Biologic depending on production system, biological source and pharmaceutical category. The European Medicines Agency (EMA) provided an actual definition describing biological medicinal products as “a protein or nucleic acid–based pharmaceutical substance used for therapeutic or in vivo diagnostic purposes, which is produced by means other than direct extraction from a native (non-engineered) biological source”. This definition suggests that even small polypeptides and proteins obtained by chemical synthesis can be considered as therapeutic proteins.
Nowadays, most of approved therapeutic proteins are produced by DNA recombinant technologies. Recombinant proteins are mainly restricted to mammalian cells (CHO) as bioreactors which carry out post-translational modifications that significantly enhance the protein bioactivity. Recently, plants are emerging as an attractive alternative to conventional expression systems, due to its practical, economic and safety advantages, correct folding and similar glycosylation pattern like eukaryotes. In 2013 U S Food and Drug Administration approved the first plant-made pharmaceutical Elelyso® (Protalix and Pfizer) to treat Gaucher’s Disease.
In this scenario, Active Botanicals Research (Brendola, VI, Italy) proposes suspension cell lines of N. benthamiana as alternative expression platform for recombinant protein and in particular for recombinant human follicle stimulating hormone beta subunit. Follicle Stimulating Hormone (FSH) is a gonadotropin that stimulates steroidogenesis and gametogenesis in the gonads in order to support and regulate the ovarian follicular maturation in women and sperm production in men. FSH is clinically used for controlled ovarian stimulation in women treated with assisted reproductive technologies, the treatment of anovulatory infertility in women and hypogonadotropic hypogonadism in men. FSH is most often administered in one of two forms: recombinant FSH expressed in CHO system (Gonal-F® (Merck Serono) and Puregon® (Merck Sharp and Dohme)) or highly purified human menopausal gonadotropin (Menopur® (Ferring) and Merional® (Pharmasure)). The first chapter of this thesis focuses on extraction, purification and characterization of recombinant human follicle stimulating hormone beta subunit expressed in suspension cell lines of N. benthamiana. Extraction is based on KDEL-strategy, a retention signal which locks the protein in the endoplasmatic reticulum preventing the release into the cytoplasm. Purified recombinant human Follicle Stimulating Hormone beta subunit (rhFSHβ) was obtained with three consecutive chromatographic steps: IMAC chromatography, size-exclusion chromatography and ion-exchange chromatography.
Two purified isoforms of recombinant protein were chemically characterized by enzymatic deglycosylation with PNGase F and tryptic digestion, covering the total amino acid sequence while the assignment of disulphide bridges pattern (six internal disulphide bridges) confirmed the correct folding of protein.
It is known that FSH evokes the physiological response as a heterodimer. In all glycoproteins the common alpha subunit is non-covalently associated whit the beta subunit, which is structurally unique in its peptide sequence to each member of the family. However, some studies suggest a possible biological activity about the single beta subunit and different FSH beta subunit preparations are on market declaring its biological activity (FSHβ subunit ab191730 Abcam).
Purified rhFSHβfrom ABResearch was tested on isolated Sertoli cells from pubertal porcine, unfortunately, preliminary data suggested no biological activity of the recombinant monomer. Waiting for recombinant human FSH alpha subunit from competent cell lines of N. Benthamiana, the biological activity of reconstituted heterodimer (rhFSHβfrom ABResearch + Native Human Chorionic Gonadotropin) was evaluated and compared with commercial FSH Gonal-F®. Data showed equal biochemical responses suggesting the great potential of the new expression system for active recombinant therapeutic proteins.
The process has been improved to obtain 4.5/5 mg of purified rhFSHβ for kg of cells, exceeding abundantly average yields in plant expression system.
In the second chapter of this work, chemical synthesis of human osteocalcin (OC) 1-49 by solid-phase synthesis (SPPS) with Fmoc strategy is described. Human OC is a small protein of 49 amino acid residues mainly produced and secreted by osteoblasts, it represents one of the most abundant (10-20%) non-collagenous proteins in the bone tissue of vertebrates with a highly conserved primary structure. It has long been known that OC acquires high affinity for calcium ions through the post-translational modification of glutamate residues by carboxylation with a vitamin-K-dependent γ-glutamyl carboxylase (GGCX). Carboxylated residues, known as Gla residues, lead to a conformational change, stabilize the alpha-helical structure and confer a greater affinity for Ca2+ and hydroxyapatite. OC is involved in different diagnostic fields due to its crucial roles in several physiological processes including remodeling of bone tissue, insulin production and regulation, testosterone secretion from testes and regulation of neurotransmitter levels in the brain. Recently osteocalcin has been proposed as potential therapeutic protein in regulation of androgen activity in a non-steroidal manner.
Nowadays purified human OC 1-49 is obtained by extraction from human bones or solid-phase synthesis with the Boc strategy. Due to limited supply of human osteocalcin from bone, the solid-phase synthesis represents the most accessible strategy.
In this study, SPPS Fmoc chemistry was proposed as alternative strategy due to the greater yields of synthesized peptides, minor side reactions during cleavage and high safety compared to Boc approach. Chemical synthesis was optimized getting final yield of 80/85% exceeding average yields of traditional chemical synthesis. The purified protein is subjected to the disulfide bond-forming reaction and subsequently it was chemically characterized by RP-HPLC, mass spectrometry analysis and enzymatic digestion. Conformational characterization and study of binding with Ca2+ were performed by spectroscopic measurements, hydrogen/deuterium exchange and ITC titration analysis comparing the new synthesized human osteocalcin with the commercial one (Bachem).
Peptides as therapeutics are the focus of the third chapter.
Peptides perform crucial roles in human physiology as growth factors, hormones, ion channel ligands, neurotransmitters and they are recognized for being highly selective and efficacious signaling molecules with attractive pharmacological profile. Peptides represent the new attractive perspective for therapeutics due to high safety, tolerability and efficacy properties. Lower production complexity and lower cost also increase interest in peptide drugs research and development compared with protein therapeutics.
Nowadays synthesized peptides are used in regenerative medicine due to their potential as guidance cues for neurite elongation and thus to activate intracellular pathways leading to cell differentiation.
Ten novel peptides derived from cell adhesion molecules and extracellular matrix proteins families (CHL1, Neurofascin, NrCAM, DCC, ROBO2 and 3, LINGO2, Contactin 1, 2 and 5) are designed to mimic guidance cues from the neural environment.
Peptides are synthesized with SPPS Fmoc strategy and characterized by RP-HPLC, mass spectrometry and circular dichroism analyses. According to previous experiments with L1-A and LINGO1-A, all synthetic peptides are tested on human neuroblastoma cell lines to evaluate their effect on neuronal differentiation and especially in neurite outgrowth and elongation.
Preliminary data suggest a prototype for the development of implants for long-term neuronal growth and differentiation.Dagli anni 80, le proteine terapeutiche sono emerse come la classe di farmaci più promettenti con più di 350 prodotti in commercio tra terapeutici, diagnostici e vaccini. Nel 2015 tra i 10 farmaci più venduti al mondo, 7 sono proteine ricombinanti od anticorpi monoclonali.
C’è ancora confusione relativamente al concetto di proteina terapeutica. Le autorità regolatorie come FDA ed EMA hanno dato diverse definizioni sulla base dei sistemi di produzione, delle fonti biologiche e della categoria farmaceutica di appartenenza.
L’Agenzia Europea dei Medicinali (EMA) descrive i prodotti medicinali biologici come sostanze farmaceutiche di natura proteica (o acidi nucleici) utilizzate in vivo per scopi terapeutici o diagnostici, le quali sono prodotte con metodologie diverse dalla diretta estrazione da fonti biologiche native (non ingegnerizzate geneticamente). Questa definizione suggerisce che anche le piccole proteine e peptidi prodotti chimicamente siano considerate proteine terapeutiche.
Oggigiorno, la maggior parte delle proteine approvate per scopi terapeutici sono prodotte con la tecnologia del DNA ricombinante. Le cellule ovariche di criceto cinese (CHO) sono considerate i bioreattori per eccellenza poiché sono in grado di apportare le modifiche post-traduzionali adatte e necessarie per una corretta attività biologica. Recentemente le piante sono emerse come un’attraente alternativa ai normali sistemi di espressione grazie ai vantaggi di tipo economico, di sicurezza, del corretto folding della proteina e del pattern di glicosilazione simile a quello umano. Nel 2013 l’autorità regolatoria americana ha approvato il primo farmaco proteico prodotto interamente in pianta contro la malattia di Gaucher.
In questo scenario, Active Botanicals Research (Brendola, VI, Italia), ha proposto linee cellulari in sospensione da N. benthamiana come piattaforma alternativa per l’espressione di proteine ricombinanti ed in particolare per la sub unità beta dell’ ormone follicolo stimolante (FSH). L’ormone follicolo stimolante è una gonadotropina che stimola la produzione di steroidi e dei gameti nelle gonadi per supportare e regolare la maturazione follicolare nelle donne e degli spermatozoi nei maschi. FSH è utilizzato clinicamente per controllare la stimolazione ovarica nella fecondazione assistita, nei casi d’infertilità femminile e ipogonadismo maschile.
FSH è somministrato in due forme: la proteina ricombinante espressa in CHO (Gonal-F® (Merck Serono) and Puregon® (Merck Sharp and Dohme)) o purificata dall’urina di donne in menopausa (Menopur® (Ferring) and Merional® (Pharmasure)).
Il primo capitolo di questa tesi affronta il processo di estrazione, purificazione e caratterizzazione della sub unità beta dell’ormone follicolo stimolante espresso in cellule in sospensione di N. benthamiana. L’estrazione sfrutta il KDEL: un segnale di ritenzione che è aggiunto all’estremità C-terminale della proteina e che permette di bloccarla all’interno del reticolo endoplasmatico. Il prodotto purificato è stato ottenuto in seguito a tre step cromatografici consecutivi: cromatografia di affinità (IMAC), ad esclusione molecolare e in fine a scambio ionico.
Due isoforme della stessa proteina ricombinante sono state caratterizzate chimicamente mediante deglicosilazione enzimatica con PNGase F e digestione triptica in modo da coprire totalmente la sequenza aminoacidica e in secondo luogo assegnare tutti e sei i ponti disolfuro dimostrando il corretto folding della proteina.
E’ risaputo che l’ormone follicolo stimolante svolge la sua funzione come etero dimero. In tutte le glicoproteine, la comune subunità alpha è legata in modo non covalente alla subunità beta, la quale è unica nella sequenza e struttura per ciascun membro della famiglia. Tuttavia, alcuni studi suggeriscono una possibile attività biologica da parte della singola subunità beta paragonabile all’intero etero dimero. In commercio è presente una preparazione con dichiarata attività biologica (FSHβ subunit ab191730 Abcam).
La specie purificata da ABResearch è stata testata su cellule isolate del Sertoli da testicolo di maiale. I dati preliminari hanno dimostrato l’assenza di attività biologica del singolo monomero. Aspettando la produzione della subunità alpha, l’etero dimero tra la subunità beta di ABR e la subunità alpha commerciale è stato costituito e testato dimostrando un’attività paragonabile al prodotto commerciale.
I dati sperimentali sostengono quindi le potenzialità di questo nuovo sistema di espressione di proteine terapeutiche, ottenendo inoltre delle rese che superano notevolmente le rese medie ottenute dai classici sistemi vegetali (4.5/5 mg per Kg di cellule).
Nel secondo capitolo di questo lavoro è descritta la sintesi chimica di osteocalcina umana 1-49 (OC) mediante la sintesi di peptidi su fase solida con strategia Fmoc.
Osteocalcina umana è una piccola proteina di 49 amino acidi prodotta principalmente dagli osteoblasti e rappresenta una delle proteine più abbondanti nel tessuto osseo dei vertebrati con un altissimo grado di conservazione della struttura primaria. E’ risaputo che osteocalcina acquisisce la sua caratteristica affinità per gli ioni calcio in seguito a specifiche modifiche post traduzionali da parte dell’enzima vitamina K dipendente glutamil-carbossilasi a livello di specifici residui di acido glutammico. Il binding con gli ioni calcio stabilizza la struttura alpha elica della proteina.
OC è coinvolta in diversi campi diagnostici a causa del suo ruolo cruciale in diversi processi fisiologici che comprendono il rimodellamento osseo, produzione e regolazione di insulina, secrezione di testosterone a livello testicolare e regolazione di neuro trasmettitori. Recentemente è stata proposta come proteina capace di regolare l’attività androgena in modo non steroideo.
Oggigiorno OC è ottenuta mediante estrazione da ossa di defunti o mediante sintesi chimica su fase solida con strategia di Boc. In seguito alla scarsa disponibilità di tessuto osseo e agli ovvi problemi etici, la sintesi chimica rappresenta la principale via di approvvigionamento.
In questa tesi si propone una strategia alternativa (Fmoc chemistry) conosciuta per le maggiori rese di reazione, vantaggi economici, maggiore sicurezza e minore contributo di reazioni collaterali rispetto alla classica strategia di Boc.
La sintesi chimica è stata ottimizzata fino a ottenere una resa del 80/85% superando largamente le rese medie della classica sintesi chimica. La proteina purificata è stata soggetta al processo ossidativo per la costituzione del ponte disolfuro ed ottenere così la proteina nella forma nativa. In seguito alla caratterizzazione chimica mediante RP-HPLC e spettrometria di massa, OC è stata caratterizzata da un punto di vista conformazionale mediante tecniche spettroscopiche come dicroismo circolare nel lontano UV e fluorescenza. Studi di binding al calcio sono stati condotti mediante l’utilizzo di tecniche spettroscopiche paragonando OC sintetizzata con quella commerciale (Bachem).
Il terzo capitolo riguarda i peptidi come prodotti terapeutici.
I peptidi ricoprono ruoli fisiologici cruciali come fattori di crescita, ormoni, ligandi di canali ionici, neurotrasmettitori e sono riconosciuti per la loro alta selettività ed efficacia come segnali molecolari con un attraente profilo farmacologico. I peptidi rappresentano una nuova attraente prospettiva nel settore farmaceutico grazie al loro grado di sicurezza, tollerabilità ed efficacia. La minor complessità di produzione e prezzi notevolmente più contenuti ha attirato l’interesse della ricerca scientifica sui peptidi come potenziali farmaci rispetto alle proteine terapeutiche.
Oggigiorno i peptidi sintetizzati vengono utilizzati nella medicina rigenerativa grazie al loro potenziale nella trasmissione del segnale, elongazione di neuriti e differenziazione cellulare.
Dieci nuovi peptidi derivanti da molecole di adesione cellulare e proteine della matrice cellulare (CHL1, Neurofascin, NrCAM, DCC, ROBO2 and 3, LINGO2, Contactin 1, 2 and 5) sono stati progettati per mimare i segnali guida nell’ambiente neurale. I peptidi sono stati sintetizzati su fase solida e purificati mediante RP-HPLC, analizzati mediante spettrometria di massa e dicroismo circolare. In accordo con i dati ottenuti con i peptidi L1-A e LINGO1-A, tutti e dieci i peptidi testati su linee cellulari di neuroblastoma umano hanno dimostrato di essere efficaci nella differenziazione neuronale ed in particolare nella crescita ed elongazione dei neuriti.
Questi dati preliminari suggeriscono un prototipo per lo sviluppo di impianti per la crescita e differenziazione neuronale a lungo termine
Enhanced neuronal cell differentiation combining biomimetic peptides and a carbon nanotube-polymer scaffold
No full textCarbon nanotubes are attractive candidates for the development of scaffolds able to support neuronal growth and differentiation thanks to their ability to conduct electrical stimuli, to interface with cells and to mimic the neural environment. We developed a biocompatible composite scaffold, consisting of multi-walled carbon nanotubes dispersed in a poly-L-lactic acid matrix able to support growth and differentiation of human neuronal cells. Moreover, to mimic guidance cues from the neural environment, we also designed synthetic peptides, derived from L1 and LINGO1 proteins. Such peptides could positively modulate neuronal differentiation, which is synergistically improved by the combination of the nanocomposite scaffold and the peptides, thus suggesting a prototype for the development of implants for long-term neuronal growth and differentiation.
The study describes the design and preparation of nanocomposite scaffolds with multi-walled carbon nanotubes in a poly-L-lactic acid matrix. This compound used in combination with peptides leads to synergistic effects in supporting neuronal cell growth and differentiation
Molecular Mapping of α -Thrombin( α T)/ β 2-Glycoprotein I(β2GpI) Interaction Reveals How β2GpI Affects α T Functions
No full text: β2-Glycoprotein I (β2GpI) is the major autoantigen in the antiphospholipid syndrome, a thrombotic autoimmune disease. Nonetheless, the physiological role of β2GpI is still unclear. In a recent work, we have shown that β2GpI selectively inhibits the procoagulant functions of human a-thrombin (αT) (i.e. prolongs fibrin clotting time, tc, and inhibits αT-induced platelets aggregation) without affecting the unique anticoagulant activity of the protease, i.e. the proteolytic generation of the anticoagulant protein C (PC) from the PC zymogen, which interacts with αT exclusively at the protease catalytic site. Here we used several different biochemical/biophysical techniques and molecular probes for mapping the binding sites in αT-β2GpI complex. Our results indicate that αT exploits the highly electropositive exosite-II, which is also responsible for anchoring αT on the platelet GpIbα receptor, for binding to a continuous negative region on β2GpI structure, spanning domain IV and (part of) domain V, while the protease active site and exosite-I (i.e. the fibrinogen binding site) remain accessible for substrate/ligand binding. Furthermore, we provided evidence that the apparent increase in tc, previously observed with β2GpI, is more likely caused by alteration of the ensuing fibrin structure rather than by inhibition of fibrinogen hydrolysis. Finally, we produced a theoretical docking model of αT-β2GpI interaction, which was in agreement with the experimental results. Altogether, these findings help to understand how β2GpI affects αT interactions and suggest that β2GpI may function as a scavenger of αT for binding to GpIbα receptor, thus impairing platelets aggregation while enabling normal cleavage of fibrinogen and PC
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Uncarboxylated Osteocalcin Stimulates 25-Hydroxy Vitamin D Production in Leydig Cell Line Through a GPRC6a-Dependent Pathway
Full text linkRecent studies disclosed a cross talk between testis and bone. By the action of LH, Leydig cells are able to modulate bone metabolism through testosterone and insulin-like factor 3. Moreover, LH modulates the Leydig expression of CYP2R1, the key enzyme involved in vitamin D (Vit D) 25-hydroxylation. However, pathways regulating CYP2R1 expression have been poorly investigated. The cross talk from the bone to the testis of the vitamin D 25-hydroxylase CYP2R1 involves osteocalcin (OC), which is produced by the osteoblasts and stimulates the production of testosterone by the Leydig cells through its putative receptor GPRC6A, a cation-sensing G-protein-coupled receptor. The aim of this study was to investigate the possible action of OC on CYP2R1 expression and 25-hydroxy Vit D (25-OH Vit D) production in a mouse Leydig cell line (MA-10). After confirmation of the expression of GPRC6A by MA-10, we found that stimulation with either human chorionic gonadotropin or uncarboxylated-OC (ucOC) increases CYP2R1 protein expression in a dose-dependent manner and, in turn, increases the release of 25-OH Vit D in culture medium. This effect was abolished by receptor blockade with, respectively, anti-LH receptor and anti-GPRC6A antibodies. Moreover, both agonists converged to phosphorylation of Erk1/2 by a likely differential action on second messengers. Human chorionic gonadotropin induced slow "tonic" increase of intercellular calcium and accumulation of cAMP, whereas ucOC mainly induced phasic increase of cell calcium. Supporting these findings, we found that serum ucOC positively correlated with 25-OH Vit D levels in 40 overweight male patients and 21 controls. Altogether, our results suggest that OC contributes with LH to 25-OH Vit D production by Leydig cells
Detection of Cholesterol and its Oxidized Derivatives in Human Sperm Membranes through a Fast and Reliable LC-MS Method
Full text linkThe trafficking of membrane cholesterol (Chol) in mammals represents key processes in cells function, particularly in sperm physiology since Chol removal is strictly associated with motility gain. Moreover, the involvement of oxidized Chol derivatives, known as oxysterols, has been recently summoned in the regulation of sperm function. The study of sterols dynamic in human sperm is largely hampered by the low sample availability and the inadequate sensitivity of current methods based on chromatographic techniques. In this study we aimed to develop a robust and reliable method based on liquid chromatography-mass spectrometry (LC-MS) to quantify the sperm levels of Chol and major oxysterols 7β-OH-Cholesterol (7β -OHC) and 7-keto-Cholesterol (7-KC). In particular, by finely tuning the reverse-phase chromatography parameters, the unambiguous identification of Chol, 7β -OHC and 7-KC in biological samples was allowed on the bases of their different retention times, accurate m/z determination and natural isotope abundance pattern. Finally, by applying the method on real sperm samples from 12 semen donors, we documented that normozoospermic subjects (total sperm count >39 × 106 cells/ejaculate) showed an underrepresentation of all steroids compared to subjects with oligozoospermia (total sperm count ≤ 39 × 106 cells/ejaculate; respectively P=0.048 for Chol, P=0.006 for 7β-OHC and P=0.001 for 7-KC). These preliminary data suggest further investigation about the impact of disorders of the spermatogenic process on the composition and function of the sperm membrane
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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