185 research outputs found
Capturing and Amplifying Impurities from Recombinant Therapeutic Proteins Via Combinatorial Peptide Libraries: A Proteomic Approach
No full textA review. The technique of combinatorial peptide ligand libraries (CPLL), for capturing and amplifying low-abundance proteins in r-DNA products as well as in a no. of other biol. systems, is here analyzed in depth and reviewed. This methodol. is based on a creation of several millions of bio-specific ligands composed of hexapeptides produced in a combinatorial way. When acting on an overloading and satn. principle, high-abundance species are captured in limited amts., whereas low-abundance ones keep being concd. on their bio-specific ligand till substantial harvesting from soln. (the capture process occurring in general from ca. 50% up to 90% efficiency). Examples are given on tracking host-cell impurities present in, e.g., recombinant albumin or monoclonal antibodies. Addnl., other examples of detecting traces of additives and fining agents in such beverages as white and red wines are presented. The unique mechanisms underlying the protein capture in the CPLL methodol., as opposed to capture by homogeneous beads, as represented by ion-exchangers and by hydrophobic resins, are discussed in depth
Combinatorial peptide ligand libraries: The conquest of the 'hidden proteome' advances at great strides
No full textA review. The combinatorial peptide ligand library (CPLL) is compared here with the immuno-depletion method for evaluating their resp. abilities in digging deeper and deeper into the low-abundance proteome. A recent report suggested in fact that immuno-subtraction for biomarkers discovery in sera does not perform so well, since it results in a meagre 25% increase in identified proteins compared with unfractionated plasma, leaving little capacity to sample lower abundance proteins. On the contrary, CPLLs permit from 300 to 600% increments in detection abilities, as amply demonstrated in several reports. Moreover, when dealing with large sample vols., an amplification factor of up to four orders of magnitude for trace proteins could be demonstrated, with 80% capture efficiencies even in large (up to 1 L) sample vols. At present, the lower detection ability of CPLLs has been evaluated at 1 ng/mL (traces of casein additives in white wines
In taberna quando sumus: a drunkard's cakewalk through wine proteomics
No full textA review. Anal. of white and red wine trace proteomes via capture with combinatorial peptide ligand libraries (CPLL) is reported here. Most of the alc. beverages tested (all of Italian origin) were found to contain only traces of casein (on av. from 20 to 60 Î1⁄4g/L, with a detectability of as low as 1 Î1⁄4g/L) and not any grape protein any longer, as they had been fined with bovine casein (surprisingly also red wines for which the typical fining agent is egg albumin). However, anal. of untreated white wine (Recite, from Garganega grapes in the Veneto region) via CPLL capture indeed permitted to detect close to 100 unique gene products from the grapes, suggesting the possibility of proteotyping grand crus, i.e. those aged, high quality wines that should not be treated with fining agents. Thus the CPLL technique could become a formidable tool for traceability of beverages in particular and of foodstuff in general. For trace protein anal., a new, most powerful CPLL methodol. emerges: capture at pH=2.2 in 0.1 % trifluoroacetic acid (TFA) under the conditions mimicking reversed-phase mechanisms of adsorption
The proteome buccaneers: how to unearth your treasure chest via combinatorial peptide ligand libraries
No full textLiver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy.
No full textMitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35-55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9±0.5. ELISA estimated TP content as 0.5±0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients. © 2014 Boschetti et al
Plucking, pillaging and plundering proteomes with combinatorial peptide ligand libraries
No full textRecent developments in the technique of combinatorial peptide ligand libraries, for enhancing the visibility of the low-abundance proteome, are reviewed here. Novel en bloc elution systems, allowing essentially complete proteome recovery in a single step, are reported here, particularly, en bloc elution with 3–5% boiling sodium dodecyl sulphate (SDS) or in urea–thiourea–CHAPS added with either 40 mM formic acid or 25 mM cysteic acid. Novel capturing systems are also discussed: in particular, although capturing at pH 7.2 in physiological saline has always been recommended, it is shown that capturing also at acidic (pH 3.8) and alkaline (pH 9.5) values substantially increments the total captured protein population. Some examples of detection of novel proteins by the described methodology are also discussed. In particular, in the case of venom proteins, where essentially all components had been detected and fully described by conventional means, the application of the ligand library technology allowed the discovery of two, previously unreported, trace enzymes necessary for the maintenance of the native structure of venom components, namely peroxiredoxin and glutaminyl cyclase. In the case of the red blood cell (RBC) cytoplasmic proteome, where a grand total of 1570 components of the 2% minority proteomes have been identified, these findings allowed to unravel the genetic defect of a rare RBC disease, called congenital dyserythropoietic anemia type II. The mutations are located in the SEC23B gene coding for the SEC23B protein, detected for the first time in the RBC proteome thanks to the peptide capturing technolog
BOURDIEU'S WORK ON LITERATURE. CONTEXTS, STAKES AND PERSPECTIVES
No full textLITERATURE HOLDS a very important place in Pierre Bourdieu’swork: his notion of field was first applied to literature, while the mostcomplete and systematic presentation of his theory of fields is to be found in The Rules of Art, a work devoted to art and literature. What expla-nation can be given for the relevance of these objects to Bourdieu’s theor-etical work? His interest in art and literature may appear somewhat surprising and improbable at first sight when one considers that for Bourdieu the sociology of culture, in the strictest sense of the term, was just one aspect of a much vaster project: a general theory of the ‘social world’. In order to explain Bourdieu’s choice of object, in the first part of this article I will consider the national and international context within which his theory has been built. I will show that in the French intellectual space, particularly in that period, literature was a central theoretical object. In the international context, the attention paid to literature was justified by the importance given to the symbolic phenomena in the main contemporary theoretical traditions. Bourdieu himself stated, referring to Gaston Bachelard, that ‘epistem-ology is always conjunctural: its propositions and thrust are determined by the principal scientific threat of the moment ’ (Bourdieu and Wacquant, 1992: 174). Moreover, against the errors that can result from an ahistorical and derealizing reading, he argued that the proper understanding of any given author requires a double contextualization. On the one hand, in order to appreciate the singularity and difficulty of his theoretical acquisition, it is necessary to bring to light the effort involved in reaching such an achieve-ment by reconstructing the mental space of the author, the problems that he attempted to resolve, the theoretical possibilities with regard to which his hypotheses were defined, and the position that the author held in his field of production at the time in which they were formulated. On the other hand
The “Dark Side” of Food Stuff Proteomics: The CPLL-Marshals Investigate
No full textThe present review deals with analysis of the proteome of animal and plant-derived food stuff, as well as of non-alcoholic and alcoholic beverages. The survey is limited to those systems investigated with the help of combinatorial peptide ligand libraries, a most powerful technique allowing access to low- to very-low-abundance proteins, i.e., to those proteins that might characterize univocally a given biological system and, in the case of commercial food preparations, attest their genuineness or adulteration. Among animal foods the analysis of cow’s and donkey’s milk is reported, together with the proteomic composition of egg white and yolk, as well as of honey, considered as a hybrid between floral and animal origin. In terms of plant and fruits, a survey is offered of spinach, artichoke, banana, avocado, mango and lemon proteomics, considered as recalcitrant tissues in that small amounts of proteins are dispersed into a large body of plant polymers and metabolites. As examples of non-alcoholic beverages, ginger ale, coconut milk, a cola drink, almond milk and orgeat syrup are analyzed. Finally, the trace proteome of white and red wines, beer and aperitifs is reported, with the aim of tracing the industrial manipulations and herbal usage prior to their commercialization
PREVENIRE LA CORRUZIONE: NUOVE STRATEGIE REGOLATORIE TRA PUBBLICO E PRIVATO
No full textL’obiettivo della ricerca è quello di studiare le contaminazioni tra diversi livelli di governance nel settore dell’anticorruzione. Anzitutto, ci si soffermerà sugli effetti che le convenzioni internazionali e più in generale gli strumenti prodotti nella sfera sovranazionale hanno determinato sul diritto anticorruzione italiano. A ben vedere, questa contaminazione tra diversi livelli di regolazione si realizza non solo attraverso strumenti cogenti ma anche, e soprattutto, attraverso strumenti flessibili come raccomandazioni e linee guida. Il diritto anticorruzione rappresenta un laboratorio di contaminazioni non solo a livello verticale tra sfera sovranazionale e nazionale, ma anche a livello orizzontale. Alla delimitazione del campo della ricerca alle interazioni tra pubblico e privato corrisponde il capitolo II: in esso si dà conto del fatto che, in materia di prevenzione del rischio di corruzione, i confini tra sfera pubblica e sfera privata sono sempre più sfumati. Infine, nel capitolo III, si cerca di trovare un filo conduttore tra le varie forme di contaminazioni regolatorie analizzate, inserendole entro un più ampio ciclo in cui si susseguono regulation e enforcement. Ci si chiede se e in che misura questo ciclo sia effettivamente virtuoso: in particolare, se un processo di law-making improntato ai criteri della flessibilità, della partecipazione dei soggetti interessati e della continua valutazione della sua efficacia, sia effettivamente idoneo a prevenire fenomeni corruttivi.The aim of the research is to study interaction between different levels of governance in the anti-corruption sector. Firstly, the domestic law, especially in the anti-corruption field is strongly influenced by international instruments. The Italian system, for example, starts out from the Anti-Corruption Law, Law No. 190 (the so called "Severino Law", approved by Parliament on 6 November 2012, entering into force on 28 November 2012, and implemented by several regulations). This Law was adopted to implement the UN Convention against Corruption. Secondly, the research aims to study interaction between public and private sector: fundamental concepts for corruption prevention, such as risk management, have been borrowed from one sector to another. Finally, there many players in the anti-corruption sector who are able to create rules (lawmakers, stakeholders, Authorities): the question is if they are part of a virtuous circle in which regulation and enforcement are closely linked. In order to prevent corruption, the law-making process must be based on specific criteria such as flexibility, stakeholder participation and ongoing evaluation of its effectiveness
pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries
No full text- …
