Clasmatodendrosis and ß-amyloidosis in aging hippocampus.

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as ß-amyloid (Aß) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aß autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aß aggregation (+300 and +800% of aggregated Aß peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aß clearance (-45% of Aß deposits on APJs, and +33% of Aß deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aß deposits and are involved in novel Aß-mediated adhesions between neurons. These data define the effects of Aß deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and ß-amyloidosis in aging hippocampus.

In vitro assessment of antibody-conjugated gold nanorods for systemic injections.

BACKGROUND: The interest for gold nanorods in biomedical optics is driven by their intense absorbance of near infrared light, their biocompatibility and their potential to reach tumors after systemic administration. Examples of applications include the photoacoustic imaging and the photothermal ablation of cancer. In spite of great current efforts, the selective delivery of gold nanorods to tumors through the bloodstream remains a formidable challenge. Their bio-conjugation with targeting units, and in particular with antibodies, is perceived as a hopeful solution, but the complexity of living organisms complicates the identification of possible obstacles along the way to tumors. RESULTS: Here, we present a new model of gold nanorods conjugated with anti-cancer antigen 125 (CA125) antibodies, which exhibit high specificity for ovarian cancer cells. We implement a battery of tests in vitro, in order to simulate major nuisances and predict the feasibility of these particles for intravenous injections. We show that parameters like the competition of free CA125 in the bloodstream, which could saturate the probe before arriving at the tumors, the matrix effect and the interference with erythrocytes and phagocytes are uncritical. CONCLUSIONS: Although some deterioration is detectable, anti-CA125-conjugated gold nanorods retain their functional features after interaction with blood tissue and so represent a powerful candidate to hit ovarian cancer cells.

Supercontinuum source tuned by an on-axis monochromator for fluorescence lifetime imaging.

We report on the application of an optically tunable coherent white light source, based on the supercontinuum generation from microstructured optical fibres, to fluorescence lifetime imaging (FLIM) microscopy and Foerster resonance energy transfer (FRET). A prototype lens based on monotonic longitudinal chromatic aberration is used for tuning the supercontinuum wavelengths from 400 to 1000 nm and acts as an axial monochromator, suitable for fibre delivery in confocal microscopy.

Collagen ultrastructural symmetry and its malignant alterations in human breast cancer revealed by polarization-resolved second-harmonic generation microscopy.

Several specific alterations of the extracellular matrix can be considered a distinctive hallmark of cancer. In particular, a different morphology of the collagen scaffold is frequently found within the peritumoural environment. In this study, we report about a significant difference in the ultrastructural organization of collagen at the supra-molecular level between the perilesional scaffold and the tumour area in human breast carcinoma samples. In particular, we demonstrated that polarization-resolved second-harmonic generation (P-SHG) microscopy is able to link the altered collagen architecture at the ultrastructural level found in perilesional tissue with a different organization of collagen fibrils at the molecular level.

Intramolecular photo-switching and intermolecular energy transfer as primary photoevents in photoreceptive processes: the case of Euglena gracilis.

In this paper we report the results of measurements performed by FLIM on the photoreceptor of Euglenagracilis. This organelle consists of optically bistable proteins, characterized by two thermally stable isomeric forms: A(498,) non fluorescent and B(462), fluorescent. Our data indicate that the primary photoevent of Euglena photoreception upon photon absorption consists of two contemporaneous different phenomena: an intramolecular photo-switch (i.e., A(498) becomes B(462)), and a intermolecular and unidirectional Forster-type energy transfer. During the FRET process, the fluorescent B(462) form acts as donor for the non-fluorescent A(498) form of the protein nearby, which acts as acceptor. We hypothesize that in nature these phenomena follow each other with a domino progression along the orderly organized and closely packed proteins in the photoreceptor layer(s), modulating the isomeric composition of the photoreceptive protein pool. This mechanism guarantees that few photons are sufficient to produce a signal detectable by the cell.

Morpho-mechanics of human collagen superstructures revealed by all-optical correlative micro-spectroscopies.

In every biological tissue, morphological and topological properties strongly affect its mechanical features and behaviour, so that ultrastructure, composition and mechanical parameters are intimately connected. Overall, it is their correct interplay that guarantees the tissue functionality. The development of experimental methods able to correlate these properties would open new opportunities both in the biological and the biomedical fields. Here, we report a correlative study intended to map supramolecular morphology, biochemical composition and viscoelastic parameters of collagen by all-optical microscopies. In particular, using human corneal tissue as a benchmark, we correlate Second-Harmonic Generation maps with mechanical and biochemical imaging obtained by Brillouin and Raman micro-spectroscopy. The study highlights how subtle variations in supramolecular organization originate the peculiar mechanical behavior of different subtypes of corneal lamellae. The presented methodology paves the way to the non-invasive assessment of tissue morpho-mechanics in biological as well as synthetic materials.

Three-dimensional mapping of the orientation of collagen corneal lamellae in healthy and keratoconic human corneas using SHG microscopy.

Keratoconus is an eye disorder that causes the cornea to take an abnormal conical shape, thus impairing its refractive functions and causing blindness. The late diagnosis of keratoconus is among the principal reasons for corneal surgical transplantation. This pathology is characterized by a reduced corneal stiffness in the region immediately below Bowman's membrane, probably due to a different lamellar organization, as suggested by previous studies. Here, the lamellar organization in this corneal region is characterized in three dimensions by means of second-harmonic generation (SHG) microscopy. In particular, a method based on a three-dimensional correlation analysis allows to probe the orientation of sutural lamellae close to the Bowman's membrane, finding statistical differences between healthy and keratoconic samples. This method is demonstrated also in combination with an epi-detection scheme, paving the way for a potential clinical ophthalmic application of SHG microscopy for the early diagnosis of keratoconus. SHG image acquired with sagittal optical sectioning (A) of a healthy cornea and (B) of a keratoconic cornea. Scale bars: 30 µm.

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression.

Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-go-go-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the ß1 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the ß1 integrin-hERG1 interaction. Although open hERG1 channels did not interact as strongly with ß1 integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/ß1 integrin interaction was disrupted. We conclude that the interaction of ß1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.

A molecular imaging analysis of Cx43 association with Cdo during skeletal myoblast differentiation.

Cell-to-cell contacts are crucial for cell differentiation. The promyogenic cell surface protein, Cdo, functions as a component of multiprotein clusters to mediate cell adhesion signaling. Connexin 43, the main connexin forming gap junctions, also plays a key role in myogenesis. At least part of its effects is independent of the intercellular channel function, but the mechanisms underlying are unknown. Here, using multiple optical approaches, we provided the first evidence that Cx43 physically interacts with Cdo to form dynamic complexes during myoblast differentiation, offering clues for considering this interaction a structural basis of the channel-independent function of Cx43.

Quantitative readout of optically encoded gold nanorods using an ordinary dark-field microscope.

In this paper we report on a new use for dark-field microscopy in order to retrieve two-dimensional maps of optical parameters of a thin sample such as a cryptograph, a histological section, or a cell monolayer. In particular, we discuss the construction of quantitative charts of light absorbance and scattering coefficients of a polyvinyl alcohol film that was embedded with gold nanorods and then etched using a focused mode-locked Ti:Sapphire oscillator. Individual pulses from this laser excite plasmonic oscillations of the gold nanorods, thus triggering plastic deformations of the particles and their environment, which are confined within a few hundred nm of the light focus. In turn, these deformations modify the light absorbance and scattering landscape, which can be measured with optical resolution in a dark-field microscope equipped with an objective of tuneable numerical aperture. This technique may prove to be valuable for various applications, such as the fast readout of optically encoded data or to model functional interactions between light and biological tissue at the level of cellular organelles, including the photothermolysis of cancer.

Dermal matrix scaffold engineered with adult mesenchymal stem cells and platelet-rich plasma as a potential tool for tissue repair and regeneration.

The purpose of this study was to investigate the efficacy of Integra, an artificial dermal matrix used as a dermal template for skin regeneration, to form a multifunctional scaffold with human bone marrow-derived mesenchymal stem cells (hMSCs) and platelet-rich plasma (PRP) for tissue engineering and regenerative technology. First, we showed that PRP, used as a supplement for growth medium, represented an optimal substitute for animal serum as well as a source of multiple growth factors, was able to satisfactorily support cell viability and cell proliferation and influence stemness gene expression in hMSCs. Moreover, Integra appeared to be a suitable substrate for hMSCs colonization, as judged by two-photon microscopy combined with fluorescence lifetime imaging (FLIM) and confocal analysis. The cells were then seeded on Integra + PRP for 24 and 48 h. Notably, in these conditions, the seeded cells exhibited a greater aptitude to colonize the scaffold, showed improved cell adhesion and spreading as compared with those cultured on Integra alone, and acquired a fibroblast-like phenotype, indicating that the bioengineered scaffold provided an appropriate environment for cellular growth and differentiation. In conclusion, these results, although preliminary, provide clues for the design of new therapeutic strategies for skin regeneration, consisting in the combination of mesenchymal stem cells with engineered biomaterials.

Hypergravity speeds up the development of T-lymphocyte motility.

The effect of altered gravity on single cells has been reported in a number of studies. From the investigation of the immune system response to spaceflight conditions, interest has focused on the influence of gravity on single lymphocytes. Microgravity has been shown to decrease lymphocyte activation and to influence motility. On the other hand, the effect of hypergravity on lymphocyte motility has not been explored. We studied the migration of human peripheral blood T lymphocytes cultured in vitro in a hypergravity environment (10g). After hypergravity culture for 1-11 days, T cells were seeded on a fibronectin-coated glass surface, observed by time-lapse bright-field microscopy, and tracked by a computer program. We found that T cells, activated and then cultured in hypergravity, become motile earlier than cells cultured at normal gravity. These results suggest that hypergravity stimulates T cell migration.