Domenico Franco

Phage-AgNPs complex as SERS probe for U937 cell identification.

The early diagnosis of malignancy is the most critical factor for patient survival and the treatment of cancer. In particular, leukemic cells are highly heterogeneous, and there is a need to develop new rapid and accurate detection systems for early diagnosis and monitoring of minimal residual disease. This study reports the utilization of molecular networks consisting of entire bacteriophage structure, displaying specific peptides, directly assembled with silver nanoparticles as a new Surface Enhanced Raman Spectroscopy (SERS) probe for U937 cells identification in vitro. A 9-mer pVIII M13 phage display library is screened against U937 to identify peptides that selectively recognize these cells. Then, phage clone is assembled with silver nanoparticles and the resulting network is used to obtain a SERS signal on cell-type specific molecular targets. The proposed strategy could be a very sensitive tool for the design of biosensors for highly specific and selective identification of hematological cancer cells and for detection of minimal residual disease in a significant proportion of human blood malignancy.

A micro-Raman spectroscopic investigation of leukemic U-937 cells in aged cultures

Recently it has been shown that micro-Raman spectroscopy combined with multivariate analysis is able to discriminate among different types of tissues and tumoral cells by the detection of significant alterations and/or reorganizations of complex biological molecules, such as nucleic acids, lipids and proteins. Moreover, its use, being in principle a non-invasive technique, appears an interesting clinical tool for the evaluation of the therapeutical effects and of the disease progression. In this work we analyzed molecular changes in aged cultures of leukemia model U937 cells with respect to fresh cultures of the same cell line. In fact, structural variations of individual neoplastic cells on aging may lead to a heterogeneous data set, therefore falsifying confidence intervals, increasing error levels of analysis and consequently limiting the use of Raman spectroscopy analysis. We found that the observed morphological changes of U937 cells corresponded to well defined modifications of the Raman contributions in selected spectral regions, where markers of specific functional groups, useful to characterize the cell state, are present. A detailed subcellular analysis showed a change in cellular organization as a function of time, and correlated to a significant increase of apoptosis levels. Besides the aforementioned study, Raman spectra were used as input for principal component analysis (PCA) in order to detect and classify spectral changes among U937 cells.

Raman spectroscopy differentiates between sensitive and resistant multiple myeloma cell lines

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific and biologically perturbing. Here, we show that single-cell micro-Raman spectroscopy can be used to discriminate between resistant and sensitive multiple myeloma cell lines based on their highly reproducible biomolecular spectral signatures. In order to demonstrate robustness of the proposed approach, we used two different cell lines of multiple myeloma, namely MM.1S and U266B1, and their counterparts MM.1R and U266/BTZ-R subtypes, resistant to dexamethasone and bortezomib, respectively. Then, micro-Raman spectroscopy provides an easily accurate and noninvasive method for cancer detection for both research and clinical environments. Characteristic peaks, mostly due to different DNA/RNA ratio, nucleic acids, lipids and protein concentrations, allow for discerning the sensitive and resistant subtypes. We also explored principal component analysis (PCA) for resistant cell identification and classification. Sensitive and resistant cells form distinct clusters that can be defined using just two principal components. The identification of drug-resistant cells by confocal micro-Raman spectroscopy is thus proposed as a clinical tool to assess the development of resistance to glucocorticoids and proteasome inhibitors in myeloma cells.

Matrix-assisted laser desorption/ionization time-of-flight vs. fast-atom bombardment and electrospray ionization mass spectrometry in the structural characterization of bacterial poly(3-hydroxyalkanoates).

RATIONALE Bacterial poly(3-hydroxyalkanoates) (PHAs) are an emergent class of plastic materials available from renewable resources. Their properties are strictly correlated with the comonomeric composition and sequence, which may be determined by various mass spectrometry approaches. In this paper we compare fast-atom bombardment (FAB) and electrospray ionization (ESI) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of partially pyrolyzed samples. METHODS We determined the compositions and sequences of the medium-chain-length PHAs (mcl-PHAs) prepared by bacterial fermentation of Pseudomonas aeruginosa ATCC 27853 cultured in media containing fatty acids with 8, 12, 14, 18, and 20 carbon atoms as carbon sources by means of MALDI-TOFMS of pyrolyzates, and compared the results with those obtained by FAB- and ESI-MS in previous studies. MALDI matrices used were 9-aminoacridine (9-AA) and indoleacrylic acid (IAA). RESULTS MALDI-TOFMS was carried out in negative ion mode when using 9-AA as a matrix, giving a semi-quantitative estimation of the 3-hydroxyacids constituting the PHAs, and in positive mode when using IAA, allowing us, through statistical analysis of the relative intensity of the oligomers generated by pyrolysis, to establish that the polymers obtained are true random copolyesters and not a mixture of homopolymers or copolymers. CONCLUSIONS MALDI-TOFMS in 9-AA and IAA of partial pyrolyzates of mcl-PHAs represents a powerful method for the structural analysis of these materials. In comparison with FAB and ESI, MALDI provided an extended mass range with better sensitivity at higher mass and a faster method of analysis.

The role of glutamine in Pseudomonas mediterranea in biotechnological processes.

In this work, in order to study the effect of glutamine as co-feeder on growth kinetics, biomass and PHA production in Pseudomonas mediterranea, different co-metabolic strategies were employed. Unrelated (glycerol and glucose) and related (sodium octanoate) carbon sources both in presence and absence of glutamine have been tested. For each cultural condition, we (i) evaluated growth kinetics and measured the cell metabolic activity by MTT assay, (ii) monitored PHA production and (iii) estimated the expression of phaC1 and phaC2 genes through RT-PCR. Our results show that the use of glutamine as co-feeder in P. mediterranea led to an improvement of the specific growth rate and cell metabolic activity and enhanced the uptake of all the carbon sources assayed. Moreover, the use of glutamine reduced significantly the time required for PHA production and increased biopolymer yield, as consequence of an early activation of phaC1 and phaC2.

Phage-coated paramagnetic beads as selective and specific capture system for biosensor applications

M13 bacteriophage, engineered to express specific and selective peptides on its capsid proteins, can be used as a good substitute for antibodies in recognition of specific targets showing at the same time thermal and mechanical resistance, strength and stability in the presence of different organic solvents and compounds. Magnetic beads are useful tools to identify, separate and concentrate specific target in solution. These are usually coated either with antibodies, which are sensitive to chemical and physical changes, or peptides that are laborious and expensive to produce. In this work we proposed a phage-based biosensor model for capture and identification of specific bacteria in a liquid sample. Commercial fluorescent paramagnetic beads were functionalized with the entire 9mer-M13 phage clone selected towards the pathogen Pseudomonas aeruginosa. Phage probes covalently linked to beads were capable to capture bacteria in suspension. Complexes of phage-coated beads and bacteria were specifically identified using an optical detection system based on the double coloring of non-specifically stained bacteria as well as of beads own fluorescence.