Impiego dell'elettroforesicapillare per la caratterizzazione di microrganismi: l'analisi di ceppi di Saccharomyces cerevisiae

La possibilità di analizzare e determinare microrganismi, quali batteri e lieviti, in maniera rapida e riproducibile, suscita notevole interesse scientifico data la loro versatilità nel campo della ricerca e dell’industria. Rispetto alle classiche tecniche impiegate in microbiologia, i metodi basati sull’elettroforesi capillare appaiono a tutt'oggi alquanto promettenti poiché in grado non solo di separare con elevata efficienza molecole, ma anche particelle colloidali e quindi cellule. In particolari condizioni, infatti, le cellule microbiche evidenziano in superficie una propria carica netta, grazie alla composizione della loro parete cellulare, e conseguentemente si comportano alla stregua dei colloidi [1,2] il ché rende possibile la loro analisi elettroforetica [2-4]. Nella presente attività di ricerca è stato messo a punto un originale metodo di analisi basato su elettroforesi capillare con rivelazione a diode array applicato alla caratterizzazione di ceppi di lievito Saccharomyces cerevisiae. L'implementazione della metodica ha previsto l'ottimizzazione di una serie di parametri elettroforetici quali ad es. la viscosità del tampone di corsa, il volume di campione e l’intensità del campo elettrico applicato. Successivamente si è focalizzato l’interesse sullo studio del comportamento elettroforetico di diversi ceppi di Saccharomyces cerevisiae cresciuti in terreno liquido YPD (Yeast extract, Peptone, D-glucose). Lo studio ha evidenziato una variabilità significativa dei profili elettroforetici, imputata alle differenti velocità di crescita di ciascun ceppo. In particolare, la crescita del ceppo selvaggio 4LBI3 ha evidenziato, inoltre, che esiste una relazione tra il profilo elettroforetico e la fase di crescita in cui si trova la popolazione cellulare esaminata. L’analisi delle dimensioni cellulari mediante microscopia ottica ha evidenziato infine una correlazione tra il profilo elettroforetico del campione e l’effettiva eterogeneità dimensionale della popolazione cellulare analizzata. I risultati sperimentali ottenuti dimostrano la validità dell’elettroforesi capillare per lo studio delle dinamiche di crescita dei lieviti e ne incoraggiano una futura applicazione per analisi quantitative anche di altre specie microbiche. [1] Radko S.P., Chrambach A., Electrophoresis, (2002) 23: pp. 1957-1972 [2] Rodriguez M.A., Armstrong D.W., J. of Chromatography B, 800, 7 (2004). [3] Armstrong D.W., Girod L.H., Rodriguez M.A. et al., Analy. Chem., 74, 5523 (2002). [4] Armstrong D.W., Schulte G., Schneiderheinze J.M., Westenberg D.J., Anal. Chem., 71, 5465 (1999)

BRAF Inhibitors in Thyroid Cancer: Clinical Impact, Mechanisms of Resistance and Future Perspectives

The Kirsten rat sarcoma viral oncogene homolog (RAS)/v-raf-1 murine leukemia viral oncogene homolog 1 (RAF)/mitogen-activated protein kinase 1 (MAPK) signaling cascade is the most important oncogenic pathway in human cancers. Tumors leading mutations in the gene encoding for v-raf murine sarcoma viral oncogene homolog B (BRAF) serine-threonine kinase are reliant on the MAPK signaling pathway for their growth and survival. Indeed, the constitutive activation of MAPK pathway results in continuous stimulation of cell proliferation, enhancement of the apoptotic threshold and induction of a migratory and metastatic phenotype. In a clinical perspective, this scenario opens to the possibility of targeting BRAF pathway for therapy. Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 29–83% of human thyroid malignancies and, differently from melanomas, are less sensitive to BRAF inhibitors and develop primary or acquired resistance due to mutational events or activation of alternative signaling pathways able to reactivate ERK signaling. In this review, we provide an overview on the current knowledge concerning the mechanisms leading to resistance to BRAF inhibitors in human thyroid carcinomas and discuss the potential therapeutic strategies, including combinations of BRAF inhibitors with other targeted agents, which might be employed to overcome drug resistance and potentiate the activity of single agent BRAF inhibitors

Quantification of L-lysine in cheese by a novel amperometric biosensor

Lysine quantification in cheese by a novel, highly selective amperometric biosensor is reported. Based on L-lysine-a-oxidase immobilized by co-crosslinking onto Platinum (Pt) electrodes modified by overoxidized polypyrrole, the sensor proved almost specific to lysine, sensitive and stable. The pure enzymatic nature of current signals was confirmed by a control electrode modified without enzyme. The precision of the method showed relative standard deviations of 4.7% and 9.2% respectively for Parmigiano Reggiano and Grana Padano cheese (n = 5). The recovery data on various cheese, spiked with lysine at 50–100% of the measured content, ranged from 85% to 99%. Different types of cheese were analysed showing lysine concentrations related to the ripening time and the manufacture technology, in agreement with literature data. Within dairy products, no appreciable lysine was detected in yogurts. The method adopted revealed suited to satisfy the demands for precise and sensitive detection of lysine with minimal sample preparation and clean-up

Heat shock proteins in cancer stem cell maintenance: a potential therapeutic target?

Cancer stem cells (CSCs) are a subpopulation of tumor cells with unlimited self-renewal capability, multilineage differentiation potential and long-term tumor repopulation capacity. CSCs reside in anatomically distinct regions within the tumor microenvironment, called niches, and this favors the maintenance of CSC properties and preserves their phenotypic plasticity. Indeed, CSCs are characterized by a flexible state based on their capacity to interconvert between a differentiated and a stem-like phenotype, and this depends on the activation of adaptive mechanisms in response to different environmental conditions. Heat Shock Proteins (HSPs) are molecular chaperones, upregulated upon cell exposure to several stress conditions and are responsible for normal maturation, localization and activity of intra and extracellular proteins. Noteworthy, HSPs play a central role in several cellular processes involved in tumor initiation and progression (i.e. cell viability, resistance to apoptosis, stress conditions and drug therapy, EMT, bioenergetics, invasiveness, metastasis formation) and, thus, are widely considered potential molecular targets. Furthermore, much evidence suggests a key regulatory function for HSPs in CSC maintenance and their upregulation has been proposed as a mechanism used by CSCs to adapt to unfavorable environmental conditions, such as nutrient deprivation, hypoxia, inflammation. This review discusses the relevance of HSPs in CSC biology, highlighting their role as novel potential molecular targets to develop anticancer strategies aimed at CSC targeting

Capillary electrophoresis of Escherichia coli: a first attempt

In the last few years, capillary electrophoresis have been used to characterize and separate microorganisms on the bases of their electrophoretic mobility [1]. Indeed microorganisms have an external structure, the “cell wall”, with a characteristic molecular composition which distinguishes microbial species and strains from each other (yeasts, bacteria, viruses). More importantly, the cell wall contains several ionizable groups that in particular conditions give rise to a superficial charge causing them to migrate with a typical migration time under an applied electric field [2,3]. Our previous works on backer’s yeast [4] have demonstrated that the characteristic electropherogram of this species shows two peaks ascribed to neutral and negative charged cells. Relevant microscopic studies have further pointed out that the electrophoretic profile also reflects the dimensional distribution of cells in the analyzed sample. In the following work, our interest has been focused on the electrophoretic behavior of Escherichia coli, a microorganism of significant interest for its role in several infectious diseases and its importance in biotechnological industries. As a first attempt, it was necessary to optimize the electrophoretic conditions for the identification and efficient separation of this microorganism by capillary electrophoresis; accordingly, the effects of the running buffer, pH, the separation voltage and the microbial aggregation on the electrophoretic profile of Escherichia coli were studied. Once typical electropherogram of that bacteria was identified, a sample containing Saccharomyces cerevisiae and Escherichia coli was analyzed using capillary electrophoresis and the experimental results have demonstrated the power of the technique in detecting the bacterial contamination of fungal sample. Thus capillary electrophoresis is able to replace traditional method of microbial identification because it permits rapid, easy and highly sensitive microbial analysis and diagnoses at low costs on several biological samples. [1] E.Kłodzińska, B.Buszewski, Analytical Chemistry, 81, 2009, 8-15. [2] A.T.Poortinga, R.Bos, W.Norde, H.J.Busscher, Surface Science Report. 47, 2002, 1-32. [3] S.P.Radko, A.Chrambach, Electrophoresis, 23, 2002, 1957-1972. [4] F.Crispo, A.Capece, R.Ciriello, P.Romano, A.Guerrieri, paper under submission

Assessment of the genetic polymorphism and physiological characterization of indigenous Oenococcus oeni strains isolated from Aglianico del Vulture red wine

The aim of this study was a reliable intra-species discrimination and strain biodiversity in Oenococcus oeni populations of two different Aglianico wineries by molecular, biochemical, and physiological characterization. Pulsed field gel electrophoresis (PFGE) analysis revealed a high polymorphism related to the origin (winery) of strains, while differential display PCR (DD-PCR) allowed a further discrimination of strains from the same winery. Moreover, the heterogeneity of these natural populations was investigated by capillary electrophoresis and enzymatic assays. A variability related to a different surface charge distribution was observed among strains, linked to their origin. Malolactic activity study evidenced strain-specific differences in malic acid degradation, and then, only the presence of L(-)-malic acid in the medium induced the mle gene. This study provided evidences on the importance of intra-species biodiversity ofmalolactic bacterial populations in wine ecosystems, as eachwine possess peculiar winemaking conditions and physical–chemical properties which make specific the bacterial survival and growth. This study highlighted a great biodiversity among O. oeni strains that can be also winery specific. Such biodiversity within a certain winery and winemaking area is important for selecting malolactic starters, and strain-specific trait identification is especially important to match individual strains to specific industrial process

Flow cytometry and capillary electrophoresis analyses in ethanol-stressed Oenococcus oeni strains and changes assessment of membrane fatty acid composition

Aims: This study aimed to investigate the dynamics and physiological heterogeneity of Oenococcus oeni under different conditions, cell membrane fluidity and permeability variations, and assessment of changes in cell surface charging rates. Methods and Results: Flow cytometry, membrane fatty acid analysis and capillary electrophoresis were performed to study ethanol-induced variations. Different physiological states were assessed, revealing cell subpopulations able to adapt and withstand to environmental stress, in order to recover their functionality. Moreover, total results demonstrated changes in cell surface and membrane fatty acid redistribution with a saturation degree and an unsaturated/saturated fatty acid ratio fairly steady in control and in different ethanol stresses. Conclusions: This study revealed a great variability among O. oeni strains and the importance to investigate the mechanisms by a multiparametric approach based on the structural and physiological bacterial adjustments in different stresses tolerance. Significance and Impact of the Study: Intermediate physiological state assessment in O. oeni with recovery possibility could be an important criterion for potential starter culture application. The flow cytometry application with changes in monitoring membrane fatty acid composition and in surface charging rates allowed the characterization of sorted subpopulations that may contribute to further understanding of diversity and heterogeneity in physiology of bacterial populations

BRAF Inhibitors in Thyroid Cancer: Clinical Impact, Mechanisms of Resistance and Future Perspectives

The Kirsten rat sarcoma viral oncogene homolog (RAS)/v-raf-1 murine leukemia viral oncogene homolog 1 (RAF)/mitogen-activated protein kinase 1 (MAPK) signaling cascade is the most important oncogenic pathway in human cancers. Tumors leading mutations in the gene encoding for v-raf murine sarcoma viral oncogene homolog B (BRAF) serine-threonine kinase are reliant on the MAPK signaling pathway for their growth and survival. Indeed, the constitutive activation of MAPK pathway results in continuous stimulation of cell proliferation, enhancement of the apoptotic threshold and induction of a migratory and metastatic phenotype. In a clinical perspective, this scenario opens to the possibility of targeting BRAF pathway for therapy. Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 29–83% of human thyroid malignancies and, differently from melanomas, are less sensitive to BRAF inhibitors and develop primary or acquired resistance due to mutational events or activation of alternative signaling pathways able to reactivate ERK signaling. In this review, we provide an overview on the current knowledge concerning the mechanisms leading to resistance to BRAF inhibitors in human thyroid carcinomas and discuss the potential therapeutic strategies, including combinations of BRAF inhibitors with other targeted agents, which might be employed to overcome drug resistance and potentiate the activity of single agent BRAF inhibitors

Amperometric biosensor based on Laccase immobilized onto a screen-printed electrode by Matrix Assisted Pulsed Laser Evaporation

A Laccase-based biosensor for the determination of phenolic compounds was developed by using Matrix Assisted Pulsed Laser Evaporation as an innovative enzyme immobilization technique. and the deriving biosensor was characterized and applied for the first time. Laccase was immobilized onto different substrates including screen printed carbon electrodes and spectroscopic, morphologic and electrochemical characterizations were carried out. A linear range from 1 to 60 \u3bcM was achieved working at 5.5 pH and 120.2 V detection potential vs Ag pseudoreference. The limits of detection and quantification were found to be 1 and 5 \u3bcM, respectively. A good fabrication reproducibility, stability of response and selectivity toward interferents were also found The potential of the developed biosensor was tested in the determination of total polyphenol content in real matrices (tea infusion, ethanolic extract from Muscari comosum bulbs and aqueous solution of a food supplement from black radish root and artichoke leaves) and the results were compared with those obtained by using the Folin\u2013Ciocalteu method