38,334 research outputs found
Raman spectroscopy determination of the mineral characteristics of microcalcifications in breast cancer: a way towards an improved screening approach
No full textDEVELOPMENT OF A NOVEL NANOSENSOR FOR THE STUDY OF BIOMOLECULAR INTERACTIONS
No full textThe evaluation and quantification of molecular interactions is of paramount importance in modern biology and molecular medicine. Therefore, there is a continuous exploration for new methodologies capable to detect and to measure binding affinities during reversible
molecular interactions. This work is devoted to explore a new tool based on the high
sensitivity that the measurement of the scattered light intensity offers when the binding
occurs on the surface of index-matched colloids.
Static light scattering is not a traditional technique to study molecular association because
the binding of insulated ligands and receptors in dilute solutions produce negligible
increment of the scattered light, while mesoscopic particles hosting multiple receptors or
ligands, including real bacteria, typically scatter too much light compared with the
contributions due to molecular adhesion on their surface. This difficulty can be overcome
by supporting the receptors on nano-scale latex spheres whose refractive index closely
matches the one of water.
As “Phantom Nanoparticles” (PNPs), we have used highly hydrophobic monodisperse
spherical fluoroelastomer colloids, with radius R = 39 Å} 1 nm, and whose refractive index,
at our working temperature (30ÅãC) and wavelength (633 nm), is np0 = 1.3248 (under the
same conditions the refractive index of water is nW = 1.3319). Surfactants added to a PNP
dispersion readily adsorb on their hydrophobic surfaces, generating a self-assembled
monolayer which can be easily equipped with molecular hydrophilic end groups of various
kind, including well-known receptors and/or ligands.
This label-free method has been assessed through the precise determination of the binding
constant of the antibiotic vancomycin with the tripeptide L-Lys-D-Ala-D-Ala and of the
vancomycin dimerization constant. We have enlightened the role of bidentate effect and
molecular hindrance in the activity of this glycopeptide.
After this success first result, an accurate determination of the optimal properties of
nanoparticles employed has been performed by comparative experiments and through
theoretical evaluation (CHAPETR 3). The effects of size, refractive index, electric charge,
and dilution on the reliability and accuracy of the method has been evaluated. Quite
surprisingly, perfect index matching and minimal size (i.e., maximum surface), which is
almost attained in one of the colloids here employed, do not represent the ideal conditions.
Rather, we show that a nanoparticle radius of 100 nm and a refractive index slightly below
that of water yields the best signal/background amplitude. We also show that repulsive
interactions can lead to artifacts in the adsorption isotherm, thus indicating that
electrostatic stabilization should be kept at a minimum.
Successively, the particles, already optically phantom, have also been made biologically
“invisible” through PEG coating and decorated by interacting proteins, thus providing a
mean to investigate the biological properties of proteins (CHAPETR 4). Avidin decorated
Phantom Nanoparticles have been prepared and were employed to detect interactions
between different kinds of biotinylated proteins. Using this approach, biotinylated protein
A was anchored on the surface of the nanoparticles, and were exploited as a functional
probe for the rapid, quantitative, picomolar detection of human IgG antibodies.
We used Phantom Nanoparticles to evaluate substrate recognition by Streptomyces PMF
Phospholipase D inactivated mutants (CHAPETR 5). The use of this specific technique
seems to have some peculiar advantages over other methods in the case of phospholipidsacting
enzymes. In fact, the substrate (or a substrate analog) can be organized onto the
surface of Phantom Nanoparticles at a desired concentration with optimal display of the
polar head group, while the hydrophobic chains result packed into the surfactant
monolayer, thus limiting the occurrence of non-specific interactions.
The last part of this work is focused on an attempt toward the stable functionalization of
Phantom Nanoparticles (CHAPETR 6). Diacetylene surfactants spontaneously adsorb on the
nanoparticles and then they are polymerized by exposure to UV light. So far, the stability
of the amphiphilic coating around the nanoparticle solely depended on weak hydrophobic
interactions. The attachment of the polymer to the particle surface, because of the
numerous contact points, is highly stable and can be improved further by crosslinking of
the polymer shell. Huns generating a quantifiable number of functional groups suitable for
covalent receptor anchorage.
All these observations, demonstrate the feasibility of this new technique, which makes it
possible to easily generate different synthetic receptors, and highlight this technique as a
versatile novel method to study, both qualitatively and quantitatively, of molecular
recognition processes.
The work described in this thesis is partially published in:
Morasso C., Bellini T., Monti D., Bassi M., Prosperi D., Riva S.:
Dispersed phantom scatterer technique reveals subtle differences in
substrate recognition by phospholipase D inactive mutant;
ChemBioChem. Submitted, currently under review
Prosperi D., Morasso C., Tortora P., Monti D., Bellini T.: Avidin decorated
core-shell nanoparticles for biorecognition studies by elastic light
scattering; ChemBioChem. 2007 (8): 1021-1028 (Impact factor: 3,446).
Prosperi D., Morasso C., Mantegazza F., Buscaglia M., Houg L., Bellini
T.: Phantom nanoparticles as probes of biomolecular interaction; Small.
2006 (8-9):1060-1067 (Impact factor: 6,408)
Erratum to: Effect of moderate red wine intake on cardiac prognosis after recent acute myocardial infarction of subjects with Type 2 diabetes mellitus (Diabetic Medicine, (2006), 23, 9, (974-981), 10.1111/j.1464-5491.2006.01886.x)
No full textIn an article by Marfella et al, the author name C. Saron is incorrect and should be listed as C. Sardu. Therefore the correct author list is: R. Marfella, F. Cacciapuoti, M. Siniscalchi, F. C. Sasso, F. Marchese, F. Cinone, E. Musacchio, M. A. Marfella, L. Ruggiero, G. Chiorazzo, D. Liberti, G. Chiorazzo, G. F. Nicoletti, C. Sardu, F. D'Andrea, C. Ammendola, M. Verza and L. Coppola.In an article by Marfella et al, the author name C. Saron is incorrect and should be listed as C. Sardu. Therefore the correct author list is: R. Marfella, F. Cacciapuoti, M. Siniscalchi, F. C. Sasso, F. Marchese, F. Cinone, E. Musacchio, M. A. Marfella, L. Ruggiero, G. Chiorazzo, D. Liberti, G. Chiorazzo, G. F. Nicoletti, C. Sardu, F. D'Andrea, C. Ammendola, M. Verza and L. Coppola
Raman analysis of microcalcifications in male breast cancer
No full textMicrocalcifications (MCs) are important disease markers for breast cancer. Many studies were conducted on their characterization in female breast cancer (FBC), but no information is available on their composition in male breast cancer (MBC). Raman spectroscopy (RS) is a molecular spectroscopy that can rapidly explore the biochemical composition of MCs without requiring any staining protocol. In this study, we optimized an algorithm to identify the mineral components present in MCs from Raman images. The algorithm was then used to study and compare MCs identified on breast cancer pieces from male and female patients. In total, we analyzed 41 MCs from 5 invasive MBC patients and 149 MCs from 13 invasive FBC patients. Results show that hydroxyapatite is the most abundant type of calcium both in MBC and FBC. However, some differences in the amount and distribution of calcium minerals are present between the two groups. Besides, we observed that MCs in MBC have a higher amount of organic material (collagen) than FBC. To the best of our knowledge, this study provides the first overview of the composition of MCs present in MBC patients; and suggests that these patients have specific features that differentiate them from the previously studied FBC. Our result support thus the need for studies designed explicitly to the understanding of MBC
Plasma fabrication and SERS functionality of gold crowned silicon submicrometer pillars
No full textSequential plasma processes combined with specific lithographic methods allow for the fabrication of advanced material structures. In the present work, we used self-assembled colloidal monolayers as lithographic structures for the conformation of ordered Si submicrometer pillars by reactive ion etching. We explored different discharge conditions to optimize the Si pillar geometry. Selected structures were further decorated with gold by conventional sputtering, prior to colloidal monolayer lift-off. The resulting structures consist of a gold crown, that is, a cylindrical coating on the edge of the Si pillar and a cavity on top. We analysed the Au structures in terms of electronic properties by using X-ray absorption spectroscopy (XAS) prior to and after post-processing with thermal annealing at 300 °C and/or interaction with a gold etchant solution (KI). The angular dependent analysis of the plasmonic properties was studied with Fourier transformed UV-vis measurements. Certain conditions were selected to perform a surface enhanced Raman spectroscopy (SERS) evaluation of these platforms with two model dyes, prior to confirming the potential interest for a well-resolved analysis of filtered blood plasma
Preoperative Systemic Inflammatory Biomarkers Are Independent Predictors of Disease Recurrence in ER+ HER2- Early Breast Cancer
No full textThe host’s immune system plays a crucial role in determining the clinical outcome of many cancers, including breast cancer. Peripheral blood neutrophils and lymphocytes counts may be surrogate markers of systemic inflammation and potentially reflect survival outcomes. The aim of the present study is to assess the role of preoperative systemic inflammatory biomarkers to predict local or distant relapse in breast cancer. In particular we investigated ER+ HER2- early breast cancer, considering its challenging risk stratification. A total of 1,763 breast cancer patients treated at tertiary referral Breast Unit were reviewed. Neutrophil-to-lymphocyte (NLR), platelet-to-lymphocyte (PLR) and lymphocyte-to-monocyte (LMR) ratios were assessed from the preoperative blood counts. Multivariate analyses for 5-years locoregional recurrence-free (LRRFS), distant metastases-free (DMFS) and disease-free survivals (DFS) were performed, taking into account both blood inflammatory biomarkers and clinical-pathological variables. Low NLR and high LMR were independent predictors of longer LRRFS, DMFS and DFS, and low PLR was predictive of better LRRFS and DMFS in the study population. In 999 ER+ HER2- early breast cancers, high PLR was predictive of worse LRRFS (HR 0.42, p=0.009), while high LMR was predictive of improved LRRFS (HR 2.20, p=0.02) and DFS (HR 2.10, p=0.01). NLR was not an independent factor of 5-years survival in this patients’ subset. Inflammatory blood biomarkers and current clinical assessment of the disease were not in agreement in terms of estimate of relapse risk (K-Cohen from -0.03 to 0.02). In conclusion, preoperative lymphocyte ratios, in particular PLR and LMR, showed prognostic relevance in ER+ HER2- early breast cancer. Therefore, they may be used in risk stratification and therapy escalation/de-escalation in patients with this type of tumor
Raman imaging as a tool for the chemical and spatial characterization of breast microcalcifications to improve lesion assessment
No full textBackground: microcalcifications (MC) are common findings on screening mammography and are among the earliest signs of breast cancer. At the same time, from the use of well-known radiographic risk score systems, which include MC assessment, such as Breast Imaging-Reporting and Data System (BI-RADS), only 20% of screened patients are further associated with malignancy. This leads to repeated biopsies and to unnecessary surgeries with discomfort for patients and increased costs for the healthcare system. Furthermore, the definitive diagnosis by histological and immunohistological evaluations is still laborious and time-consuming.
Raman Spectroscopy (RS) is a photonic approach capable to provide detailed chemical information of analysed samples without complex tissue preparation or staining. RS has a proven ability to distinguish different crystal structures, including those commonly present in MC. In this context some studies based on RS suggested a correlation between MC chemical features and pathology. On the other hand, previous Raman-based studies mainly investigated the overall MC chemical composition (by single-point scans) while an extensive MC characterization by Raman imaging approaches (mapping) for diagnostic purposes is still lacking.
The aim of this study is to assess the usefulness of Raman imaging as a quick and accurate tool for a complete spatial characterization of MC detected on screening mammography and sampled by breast biopsy in order to better distinguish malignant vs. benign lesions.
Method: 30 patients with breast calcifications detected on mammography with radiological classification BI-RAD 3-5 where selected and evaluated by core biopsy. 10 μm formalin-fixed paraffin-embedded (FFPE) histological sections obtained from biopsies were dewaxed with a specifically developed protocol that allows the removal of paraffin in less than 15 minutes. All MC present in each tissue section (usually from 2 to >10 per section) were then characterized by a Raman microscope thus obtaining Raman maps with lateral resolution between 5 and 10 μm. After pre-processing steps the Raman maps were analysed by both clustering and multivariate analysis approaches used to produce false-colour images and to perform automated features identification.
Results: Our results confirm that hydroxyapatite is the prevalent form of calcium phosphate in MC and that MC composition correlates with lesion malignancy. In addition, thanks to the Raman imaging approach used here, we report for the first time that hydroxyapatite is more homogeneously distributed in malignant lesions and that, on the contrary, benign lesions show a heterogeneous distribution of hydroxyapatite, whitlockite and calcium-carbonate, inside the lesion and in the surrounding tissue.
Conclusion: These evidences suggest that the characterization of MC by Raman imaging is a potential tool for the definition of new diagnostic signatures of breast cancer, especially if we consider that these evaluations can be performed by the simple and relative fast scanning of dewaxed slices, without altering the clinical workflow and without the need of staining or antibodies. Further studies with a larger cohort will be done to validate these results
- …
