Heterogeneous and self-organizing mineralization of bone matrix promoted by hydroxyapatite nanoparticles.

The mineralization process is crucial to the load-bearing characteristics of the bone extracellular matrix. In this work, we have studied the spatiotemporal dynamics of mineral deposition by human bone marrow mesenchymal stem cells differentiating toward osteoblasts promoted by the presence of exogenous hydroxyapatite nanoparticles. At the molecular level, the added nanoparticles positively modulated the expression of bone-specific markers and enhanced calcified matrix deposition during osteogenic differentiation. The nucleation, growth and spatial arrangement of newly deposited hydroxyapatite nanocrystals have been evaluated using scanning micro X-ray diffraction and scanning micro X-ray fluorescence. As leading results, we have found the emergence of a complex scenario where the spatial organization and temporal evolution of the process exhibit heterogeneous and self-organizing dynamics. At the same time the possibility of controlling the differentiation kinetics, through the addition of synthetic nanoparticles, paves the way to empower the generation of more structured bone scaffolds in tissue engineering and to design new drugs in regenerative medicine.

X-Ray Phase Contrast Tomography Reveals Early Vascular Alterations and Neuronal Loss in a Multiple Sclerosis Model.

The degenerative effects of multiple sclerosis at the level of the vascular and neuronal networks in the central nervous system are currently the object of intensive investigation. Preclinical studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapy in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis, but the neuropathology of specific lesions in EAE and the effects of MSC treatment are under debate. Because conventional imaging techniques entail protocols that alter the tissues, limiting the reliability of the results, we have used non-invasive X-ray phase-contrast tomography to obtain an unprecedented direct 3D characterization of EAE lesions at micro-to-nano scales, with simultaneous imaging of the vascular and neuronal networks. We reveal EAE-mediated alterations down to the capillary network. Our findings shed light on how the disease and MSC treatment affect the tissues, and promote X-ray phase-contrast tomography as a powerful tool for studying neurovascular diseases and monitoring advanced therapies.

Quantitative 3D investigation of Neuronal network in mouse spinal cord model.

The investigation of the neuronal network in mouse spinal cord models represents the basis for the research on neurodegenerative diseases. In this framework, the quantitative analysis of the single elements in different districts is a crucial task. However, conventional 3D imaging techniques do not have enough spatial resolution and contrast to allow for a quantitative investigation of the neuronal network. Exploiting the high coherence and the high flux of synchrotron sources, X-ray Phase-Contrast multiscale-Tomography allows for the 3D investigation of the neuronal microanatomy without any aggressive sample preparation or sectioning. We investigated healthy-mouse neuronal architecture by imaging the 3D distribution of the neuronal-network with a spatial resolution of 640 nm. The high quality of the obtained images enables a quantitative study of the neuronal structure on a subject-by-subject basis. We developed and applied a spatial statistical analysis on the motor neurons to obtain quantitative information on their 3D arrangement in the healthy-mice spinal cord. Then, we compared the obtained results with a mouse model of multiple sclerosis. Our approach paves the way to the creation of a "database" for the characterization of the neuronal network main features for a comparative investigation of neurodegenerative diseases and therapies.

Recent advances in superhydrophobic surfaces and their relevance to biology and medicine.

By mimicking naturally occurring superhydrophobic surfaces, scientists can now realize artificial surfaces on which droplets of a few microliters of water are forced to assume an almost spherical shape and an extremely high contact angle. In recent decades, these surfaces have attracted much attention due to their technological applications for anti-wetting and self-cleaning materials. Very recently, researchers have shifted their interest to investigate whether superhydrophobic surfaces can be exploited to study biological systems. This research effort has stimulated the design and realization of new devices that allow us to actively organize, visualize and manipulate matter at both the microscale and nanoscale levels. Such precise control opens up wide applications in biomedicine, as it allows us to directly manipulate objects at the typical length scale of cells and macromolecules. This progress report focuses on recent biological and medical applications of superhydrophobicity. Particular regard is paid to those applications that involve the detection, manipulation and study of extremely small quantities of molecules, and to those that allow high throughput cell and biomaterial screening.

Inhomogeneity of charge-density-wave order and quenched disorder in a high-Tc superconductor.

It has recently been established that the high-transition-temperature (high-Tc) superconducting state coexists with short-range charge-density-wave order and quenched disorder arising from dopants and strain. This complex, multiscale phase separation invites the development of theories of high-temperature superconductivity that include complexity. The nature of the spatial interplay between charge and dopant order that provides a basis for nanoscale phase separation remains a key open question, because experiments have yet to probe the unknown spatial distribution at both the nanoscale and mesoscale (between atomic and macroscopic scale). Here we report micro X-ray diffraction imaging of the spatial distribution of both short-range charge-density-wave 'puddles' (domains with only a few wavelengths) and quenched disorder in HgBa2CuO4 + y, the single-layer cuprate with the highest Tc, 95 kelvin (refs 26-28). We found that the charge-density-wave puddles, like the steam bubbles in boiling water, have a fat-tailed size distribution that is typical of self-organization near a critical point. However, the quenched disorder, which arises from oxygen interstitials, has a distribution that is contrary to the usually assumed random, uncorrelated distribution. The interstitial-oxygen-rich domains are spatially anticorrelated with the charge-density-wave domains, because higher doping does not favour the stripy charge-density-wave puddles, leading to a complex emergent geometry of the spatial landscape for superconductivity.

Imaging collagen packing dynamics during mineralization of engineered bone tissue.

The structure and organization of the Type I collagen microfibrils during mineral nanoparticle formation appear as the key factor for a deeper understanding of the biomineralization mechanism and for governing the bone tissue physical properties. In this work we investigated the dynamics of collagen packing during ex-vivo mineralization of ceramic porous hydroxyapatite implant scaffolds using synchrotron high resolution X-ray phase contrast micro-tomography (XPCµT) and synchrotron scanning micro X-ray diffraction (SµXRD). While XPCµT provides the direct 3D image of the collagen fibers network organization with micrometer spatial resolution, SµXRD allows to probe the structural statistical fluctuations of the collagen fibrils at nanoscale. In particular we imaged the lateral spacing and orientation of collagen fibrils during the anisotropic growth of mineral nanocrystals. Beyond throwing light on the bone regeneration multiscale process, this approach can provide important information in the characterization of tissue in health, aging and degeneration conditions. STATEMENT OF SIGNIFICANCE: BONE grafts are the most common transplants after the blood transfusions. This makes the bone-tissue regeneration research of pressing scientific and social impact. Bone is a complex hierarchical structure, where the interplay of organic and inorganic mineral phases at different length scale (from micron to atomic scale) affect its functionality and health. Thus, the understanding of bone tissue regeneration requires to image its spatial-temporal evolution (i) with high spatial resolution and (ii) at different length scale. We exploited high spatial resolution X-ray Phase Contrast micro Tomography and Scanning micro X-ray Diffraction in order to get new insight on the engineered tissue formation mechanisms. This approach could open novel routes for the early detection of different degenerative conditions of tissue.

An optimized table-top small-angle X-ray scattering set-up for the nanoscale structural analysis of soft matter.

The paper shows how a table top superbright microfocus laboratory X-ray source and an innovative restoring-data algorithm, used in combination, allow to analyze the super molecular structure of soft matter by means of Small Angle X-ray Scattering ex-situ experiments. The proposed theoretical approach is aimed to restore diffraction features from SAXS profiles collected from low scattering biomaterials or soft tissues, and therefore to deal with extremely noisy diffraction SAXS profiles/maps. As biological test cases we inspected: i) residues of exosomes' drops from healthy epithelial colon cell line and colorectal cancer cells; ii) collagen/human elastin artificial scaffolds developed for vascular tissue engineering applications; iii) apoferritin protein in solution. Our results show how this combination can provide morphological/structural nanoscale information to characterize new artificial biomaterials and/or to get insight into the transition between healthy and pathological tissues during the progression of a disease, or to morphologically characterize nanoscale proteins, based on SAXS data collected in a room-sized laboratory.

Early stage mineralization in tissue engineering mapped by high resolution X-ray microdiffraction.

The specific routes of biomineralization in nature are here explored using a tissue engineering approach in which bone is formed in porous ceramic constructs seeded with bone marrow stromal cells and implanted in vivo. Unlike previous studies this model system reproduces mammalian bone formation, here investigated at high temporal resolution. Different mineralization stages were monitored at different distances from the scaffold interface so that their spatial analysis corresponded to temporal monitoring of the bone growth and mineralization processes. The micrometer spatial resolution achieved by our diffraction technique ensured highly accurate reconstruction of the different temporal mineralization steps and provided some hints to the challenging issue of the mineral deposit first formed at the organic-mineral interface. Our results indicated that in the first stage of biomineralization organic tissue provides bioavailable calcium and phosphate ions, ensuring a constant reservoir of amorphous calcium phosphate (ACP) during hydroxyapatite (HA) nanocrystal formation. In this regard we suggest a new role of ACP in HA formation, with a continuous organic-mineral transition assisted by a dynamic pool of ACP. After HA nanocrystals formed, the scaffold and collagen act as templates for nanocrystal arrangement on the microscopic and nanometric scales, respectively.

A new Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins.

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both alpha2,6- and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing alpha2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the alpha2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-gamma and the tissue inhibitor of metalloproteinases-1. Interferon-gamma purified from the universal host carried 40.4% alpha2,6- and 59.6% alpha2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-gamma produced by normal CHO cells.

Initiation of antiretroviral therapy during primary HIV-1 infection induces rapid stabilization of the T-cell receptor beta chain repertoire and reduces the level of T-cell oligoclonality.

Major T-cell receptor beta chain variable region (TCRBV) repertoire perturbations are temporally associated with the down-regulation of viremia during primary human immunodeficiency virus (HIV) infection and with oligoclonal expansion and clonal exhaustion of HIV-specific cytotoxic T lymphocytes (CTLs). To determine whether initiation of antiretroviral therapy (ART) or highly active antiretroviral therapy (HAART) during primary infection influences the dynamics of T-cell-mediated immune responses, the TCRBV repertoire was analyzed by semiquantitative polymerase chain reaction in serial blood samples obtained from 11 untreated and 11 ART-treated patients. Repertoire variations were evaluated longitudinally. Stabilization of the TCRBV repertoire was more consistently observed in treated as compared with untreated patients. Furthermore, the extent and the rapidity of stabilization were significantly different in treated versus untreated patients. TCRBV repertoire stabilization was positively correlated with the slope of HIV viremia in the treated group, suggesting an association between repertoire stabilization and virologic response to treatment. To test whether stabilization was associated with variations in the clonal complexity of T-cell populations, T-cell receptor (TCR) heteroduplex mobility shift assays (HMAs) were performed on sequential samples from 4 HAART-treated subjects. Densitometric analysis of HMA profiles showed a reduction in the number of TCR clonotypes in most TCRBV families and a significant decrease in the total number of clonotypes following 7 months of HAART. Furthermore, a biphasic decline in HIV-specific but not heterologous CTL clones was observed. This indicates that ART leads to a global reduction of CD8(+) T-cell oligoclonality and significantly modulates the mobilization of HIV-specific CTL during primary infection. (Blood. 2000;95:1743-1751)

Molecular Probe Data Base: a database on synthetic oligonucleotides.

The Molecular Probe Data Base (MPDB) was designed to collect and make information on synthetic oligonucleotides available on-line. This paper briefly describes its purpose, contents and structure, forms and mode of data distribution. Particular emphasis is given to recent data extension and system enhancements that have been carried out in order to simplify access to MPDB for unskilled users.

[Study of cardiac arrhythmias in the healthy newborn infant]. / Studio delle aritmie cardiache nel neonato sano.

Of 426 infants born at the end of a regular pregnancy and delivery, which are apparently in good health, thirteen, showed cardiac arrhythmias (atrial, supraventricular, ventricular premature beats, and parasistole) on a standard E.C.G. and on a unique lead (DII carried out in the first 24 hours of life. This study points out the rise of disorders of the rhythm in healthy newborn infants and suggest further prospectives of research in the natural history of arrhythmias. Furthermore it underlines as the neonatal electrocardiography, simple and non invasive procedure, could be a good help in comparison with the screenings of the newborn. In all cases we have noticed the spontaneous resolution of arrhythmias the second week of life.