Micro-Raman study of stress distribution in local isolation structures and correlation with transmission electron microscopy

Stress in local isolation structures is studied by micro‐Raman spectroscopy. The results are correlated with predictions of an analytical model for the stress distribution and with cross‐sectional transmission electron microscopy observations. The measurements are performed on structures on which the Si3N4 oxidation mask is still present. The influence of the pitch of the periodic local isolation pattern, consisting of parallel lines, the thickness of the mask, and the length of the bird"s beak on the stress distribution are studied. It is found that compressive stress is present in the Si substrate under the center of the oxidation mask lines, with a magnitude dependent on the width of the lines. Large tensile stress is concentrated under the bird"s beak and is found to increase with decreasing length of the bird"s beak and with increasing thickness of the Si3N4 film.

Differential distribution of MAP1A isoforms in the adult mouse barrel cortex and comparison with the serotonin 5-HT2A receptor.

Microtubule-associated protein 1A (MAP1A) is essential during the late differentiation phase of neuronal development. Here, we demonstrated the presence of two MAP1A isoforms with a differential spatial distribution in the adult mouse barrel cortex. Antibody A stained MAP1A in pyramidal and stellate cells, including dendrites that crossed layer IV in the septa between barrels. The other antibody, BW6 recognized a MAP1A isoform that was mainly confined to the barrel hollow and identified smaller caliber dendrites. Previously, an interaction of MAP1A and the serotonin 5-hydroxytryptamine 2A (5-HT(2A)) receptor was shown in the rat cortex. Here, we identified, by double-immunofluorescent labeling, MAP1A isoform and serotonin 5-HT(2A) receptor distribution. MAP1A co-localized mainly with 5-HT(2A) receptor in larger apical dendrites situated in septa. This differential staining of MAP1A and a serotonin receptor in defined barrel compartments may be due to changes in the expression or processing of MAP1A during dendritic transport as a consequence of functional differences in processing of whisker-related sensory input.

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

Polymorphous Si thin films from radio frequency plasmas of SiH4 diluted in Ar: A study by transmission electron microscopy and Raman spectroscopy

In this study, we present a detailed structural characterization by means of transmission electron microscopy and Raman spectroscopy of polymorphous silicon (pm-Si:H) thin films deposited using radio-frequency dust-forming plasmas of SiH4 diluted in Ar. Square-wave modulation of the plasma and gas temperature was varied to obtain films with different nanostructures. Transmission electron microscopy and electron diffraction have shown the presence of Si crystallites of around 2 nm in the pm-Si:H films, which are related to the nanoparticles formed in the plasma gas phase coming from their different growth stages, named particle nucleation and coagulation. Raman scattering has proved the role of the film nanostructure in the crystallization process induced ¿in situ¿ by laser heating.

The phagocytosis of gas-filled microbubbles by human and murine antigen-presenting cells.

This study was designed to evaluate the potential of gas-filled microbubbles (MB) to be internalized by antigen-presenting cells (APC). Fluorescently labeled MB were prepared, thus permitting to track binding to, and internalization in, APC. Both human and mouse cells, including monocytes and dendritic cells (DC), prove capable to phagocyte MB in vitro. Observation by confocal laser scanning microscopy showed that interaction between MB and target cells resulted in a rapid internalization in cellular compartments and to a lesser extent in the cytoplasm. Capture of MB by APC resulted in phagolysosomal targeting as verified by double staining with anti-lysosome-associated membrane protein-1 monoclonal antibody and decrease of internalization by phagocytosis inhibitors. Fluorescent MB injected subcutaneously (s.c.) in mice were found to be associated with CD11c(+)DC in lymph nodes draining the injection sites 24 h after administration. Altogether, our study demonstrates that MB can successfully target APC both in vitro and in vivo, and thus may serve as a potent Ag delivery system without requirement for ultrasound-based sonoporation. This adds to the potential of applications of MB already extensively used for diagnostic imaging in humans.

3D reconstruction and comparison of shapes of DNA minicircles observed by cryo-electron microscopy.

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes.

Magnetic microstructures from magnetic force microscopy and Monte Carlo simulation in CoFe-Ag-Cu granular films

CoFe-Ag-Cu granular films, prepared by rf sputtering, displayed magnetic domain microstructures for ferromagnetic concentrations above about 32% at, and below the percolation threshold. All samples have a fcc structure with an (111) texture perpendicular to the film plane. Magnetic force microscopy (MFM) showed a variety of magnetic domain microstructures, extremely sensitive to the magnetic history of the sample, which arise from the balance of the ferromagnetic exchange, the dipolar interactions and perpendicular magnetocrystalline anisotropy, MFM images indicate that in virgin samples, magnetic bubble domains with an out-of-plane component of the magnetization are surrounded by a quasicontinuous background of opposite magnetization domains. The application of a magnetic field in different geometries drastically modifies the microstructure of the system in the remanent state: i) for an in-plane field, the MFM images show that most of the magnetic moments are aligned along the film plane, ii) for an out-of-plane field, the MFM signal increases about one order of magnitude, and out-of-plane striped domains with alternating up and down magnetization are stabilized. Numerical simulations show that a variety of metastable domain structures (similar to those observed experimentally) can be reached, depending on magnetic history, in systems with competing perpendicular anisotropy, exchange and dipolar interactions.

Homeodomain binding motifs modulate the probability of odorant receptor gene choice in transgenic mice.

Odorant receptor (OR) genes constitute with 1200 members the largest gene family in the mouse genome. A mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and from one allele. The cell bodies of OSNs that express a given OR gene display a mosaic pattern within a particular region of the main olfactory epithelium. The mechanisms and cis-acting DNA elements that regulate the expression of one OR gene per OSN - OR gene choice - remain poorly understood. Here, we describe a reporter assay to identify minimal promoters for OR genes in transgenic mice, which are produced by the conventional method of pronuclear injection of DNA. The promoter transgenes are devoid of an OR coding sequence, and instead drive expression of the axonal marker tau-β-galactosidase. For four mouse OR genes (M71, M72, MOR23, and P3) and one human OR gene (hM72), a mosaic, OSN-specific pattern of reporter expression can be obtained in transgenic mice with contiguous DNA segments of only ~300 bp that are centered around the transcription start site (TSS). The ~150bp region upstream of the TSS contains three conserved sequence motifs, including homeodomain (HD) binding sites. Such HD binding sites are also present in the H and P elements, DNA sequences that are known to strongly influence OR gene expression. When a 19mer encompassing a HD binding site from the P element is multimerized nine times and added upstream of a MOR23 minigene that contains the MOR23 coding region, we observe a dramatic increase in the number of transgene-expressing founders and lines and in the number of labeled OSNs. By contrast, a nine times multimerized 19mer with a mutant HD binding site does not have these effects. We hypothesize that HD binding sites in the H and P elements and in OR promoters modulate the probability of OR gene choice.

Spatial analysis of erythrocyte membrane fluctuations by digital holographic microscopy.

Red blood cell (RBC) membrane fluctuations provide important insights into cell states. We present a spatial analysis of red blood cell membrane fluctuations by using digital holographic microscopy (DHM). This interferometric and dye-free technique, possessing nanometric axial and microsecond temporal sensitivities enables to measure cell membrane fluctuations (CMF) on the whole cell surface. DHM acquisition is combined with a model which allows extracting the membrane fluctuation amplitude, while taking into account cell membrane topology. Uneven distribution of CMF amplitudes over the RBC surface is observed, showing maximal values in a ring corresponding to the highest points on the RBC torus as well as in some scattered areas in the inner region of the RBC. CMF amplitudes of 35.9+/-8.9 nm and 4.7+/-0.5 nm (averaged over the cell surface) were determined for normal and ethanol-fixed RBCs, respectively.

A novel PIKFYVE mutation in fleck corneal dystrophy.

Purpose: To report the findings of the clinical and molecular evaluation in a Greek family with fleck corneal dystrophy (CFD).Methods: A 58-year-old woman was seen on routine ophthalmic examination and diagnosed as having CFD. All available family members were examined to evaluate the clinical findings and inheritance of the disease. Twenty members of the family in five generations underwent slit-lamp examination. Eleven were females and nine males, aged from two years to 85 years old. Blood samples were available from four patients with CFD and seven unaffected relatives, and the DNAs were subjected to molecular screening of the phosphoinositide kinase, five finger-containing (PIKFYVE) gene by direct sequencing or denaturing high performance liquid chromatography (DHPLC).Results: The clinical evaluation revealed six family members (five females and one male) with CFD. In two CFD patients early cataract formation was noticed. All patients affected with the corneal dystrophy were asymptomatic. The molecular analyses demonstrated the existence of a novel c. 3060-3063delCCTT (p.P968Vfs23) mutation in PIKFYVE in all CFD patients tested but in none of the six unaffected family members. No molecular screening was performed in the seventh unaffected member as the causative mutation was clearly transmitted from his affected wife to his affected son.Conclusions: We report on the clinical and molecular findings of a five generation Greek family with CFD and we conclude that the novel c. 3060-3063delCCTT (p. P968Vfs23) mutation in PIKFYVE, which segregated with the disease, was the causative mutation in this family.

On the role of kerato-epithelin in the pathogenesis of 5q31-linked corneal dystrophies.

PURPOSE: Recently, the authors identified a gene, BIGH3, in which different mutations cause a group of hereditary corneal dystrophies: lattice type I and IIIA (CDLI and CDLIIIA), granular Groenouw type I (CDGGI), Avellino (CDA), and Reis-Bücklers' (CDRB). All these disorders are characterized by the progressive accumulation of corneal deposits with different structural organization. Experiments were conducted to determine the role of kerato-epithelin (KE), the product of BIGH3, in the pathogenesis of the diseases. METHODS: KE-15 and KE-2, two rabbit antisera raised against peptides from the 69-364 and 426 - 682 amino acid regions of KE respectively, were used for immunohistology of the corneas obtained after keratoplasty in six CDLI patients, three CDGGI patients, and one CDA patient. RESULTS: The nonamyloid deposits observed in CDGGI stained intensively with KE-15 and KE-2, whereas the amyloid deposits in all analyzed CDLI corneas reacted to KE-2 but not to KE-15. In the CDA cornea, where amyloid and nonamyloid inclusions were present, positive staining with both antisera was observed. CONCLUSIONS: Pathologic amyloid and nonamyloid deposits observed in CDLI, CDGGI-, and CDA-affected corneas are caused by KE accumulation. Different staining patterns of amyloid and nonamyloid deposits observed with antibodies against the amino and carboxyl termini of KE suggest that two mechanisms of KE misfolding are implicated in the pathogenesis of 5q31-linked corneal dystrophies.