Collagen in the scarless fetal skin wound: Detection with Picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a response to burn injury.

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus

White spot syndrome virus ( WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultrathin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal stuctures upon hydration

The aim of the present study was to prepare solid Quil A-cholesterol-phospholid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal stuctures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-temary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems. (c) 2004 Elsevier B.V. All rights reserved.

Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles

Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1 null (Cav1 -/-) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1 -/- MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveoloe and to identify noncaveolar endocytic vehicles. In WT MEFs a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2 % per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.

Biocompatible properties of surgical mesh using an animal model

To study the biocompatibility of surgical meshes for use in pelvic reconstructive surgery using an animal model. Eight different types of mesh: Atrium, Dexon, Gynemesh, IVS tape, Prolene, SPARC tape, TVT tape and Vypro II, were implanted into the abdominal walls of rats for 3 months' duration. Explanted meshes were assessed, using light microscopy, for parameters of rejection and incorporation. Type 1 (Atrium, Gynemesh, Prolene, SPARC and TVT) and type 3 (Vypro II, Dexon and IVS) meshes demonstrated different biocompatible properties. Inflammatory cellular response and fibrosis at the interface of mesh and host tissue was most marked with Vypro II and IVS. All type 1 meshes displayed similar cellular responses despite markedly different mesh architecture. The inflammatory response and fibrous reaction in the non-absorbable type 3 meshes tested (IVS and Vypro II) was more marked than the type 1 meshes. The increased inflammatory and fibrotic response may be because of the multifilamentous polypropylene components of these meshes. Material and filament composition of mesh is the main factor in determining cellular response.

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: A role for caveolar endocytosis

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.

Diversity of pigmentation in cultured human melanocytes is due to differences in the type as well as quantity of melanin

Cultured human melanocytes differ tremendously in visual pigmentation, and recapitulate the pigmentary phenotype of the donor's skin. This diversity arises from variation in type as well as quantity of melanin produced. Here, we measured contents of eumelanin (EM) and pheomelanin (PM) in 60 primary human melanocyte cultures (51 neonatal and nine adults), and correlated some of these values with the respective activity and protein levels of tyrosinase, and the melanocortin-1 receptor (MC1R) genotype. Melanocytes were classified into four phenotypes (L, L+, D, D+) as depicted by visual pigmentation using light microscopy, and by the pigmentary phenotype of the donor's skin. There were large differences in total melanin (TM) and EM, which increased progressively for L, L+, D and D+ melanocytes. TM content, the sum of EM and PM, showed a good correlation with TM measured spectrophotometrically, and with the activity and protein levels of tyrosinase. Log EM/PM ratio did not correlate with MC1R genotype. We conclude that: (i) EM consistently correlates with the visual phenotype; (ii) lighter melanocytes tend to be more pheomelanic in composition than darker melanocytes; (iii) in adult melanocyte cultures, EM correlates with the ethnic background of the donors (African-American > Indian > Caucasian); and (iv) MC1R loss-of-function mutations do not necessarily alter the phenotype of cultured melanocytes.

Postharvest infection of Freesia hybrida flowers by Botrytis cinerea

'Specking' on harvested freesia (Freesia hybrida) flowers is a problem worldwide. The disease is caused by the fungal pathogen Botrytis cinerea. This disease symptom detracts from appearance and reduces marketability of the flowers. Unlike other important cut flower crops (e.g. gerbera), the mode of infection and epidemiology of postharvest freesia flower specking caused by B. cinerea has not been reported. Epidemiological studies were carried out under simulated conditions typical of those occurring during postharvest handling of freesia flowers. Infection of freesia flowers by B. cinerea occurred when a conidium germinated, formed a germ tube(s) and penetrated epidermal cells. Fungal hyphae then colonised adjacent cells, resulting in visible lesions. Different host reactions were observed on freesia 'Cote d'Azur' petals at 20 degrees C compared to 5 degrees C. The infection process was relatively rapid at 20 degrees C, with visible lesions produced within 7 h of incubation. However, lesion expansion ceased after 24 h of incubation. Infection was slower at 5 degrees C, with visible lesions produced after 48 h of incubation. However, lesion development at 5 degrees C was continuous, with lesions expanding over 4 days. Light microscopy observations revealed increased host defence reactions during infection. These reactions involved production of phenolic compounds, probably lignin and/or callose, around infection sites. Such substances may play a role in restricting petal colonisation and lesion expansion. Disease severity and lesion numbers on freesia flowers incubated at 12 degrees C were higher, but not significantly higher (P > 0.05), than on those incubated at 20 degrees C. Disease severity and progression were differentially mediated by temperature and relative humidity (R. H.). Infection of freesia flowers was severe at 100% R. H. for all three incubation temperatures of 5, 12 and 20 degrees C. In contrast, no lesions were produced at 80 to 90% R. H. at either 5 or 20 degrees C.

Anterior lamella actinic changes as a factor in involutional eyelid malposition

Purpose: We conducted a noncomparative, retrospective chart review of 45 patients and 51 eyelids with the diagnosis of involutional entropion or ectropion that underwent full-thickness lower eyelid shortening between June 2001 and February 2004, in whom the severity of actinic damage was analyzed in relation to the eyelid position. Patients with any different surgical approach or other primary causes of abnormal eyelid position, such as paralytic, congenital, or mechanical factors, were excluded. Methods: After excision, all eyelid specimens were examined by a single anatomic pathologist, who was masked to the type of eyelid malposition. The extent of dermal actinic change was evaluated under light microscopy, according to a previously validated grading system. Results: Fifty-one eyelids from 26 male and 19 female patients were analyzed. The mean age at the surgery was 76 +/- 10 years (range, 52 to 92 years), affecting one side in 39 cases and both sides in 6 cases. The most frequent eyelid malposition was ectropion, which affected two thirds of the cases (35 eyelids). Half of the patients presented with mild actinic skin changes; however, the severity of the histologic skin actinic changes was significantly worse in patients with ectropion in comparison to those with entropion (p < 0.0001). Conclusions: Actinic damage affecting the anterior lamella of the lower eyelid contributes as an additional factor in final eyelid position in patients with involutional eyelid changes. More severe and extensive actinic changes were present in eyelids with ectropion.

Textural and microstructural studies of zinc sulfide and associated phases in certain base metal deposits

A textural and microstructural study of a variety of zinc sulfide-containing ores has been undertaken, and the possible depositional and deformational controls of textural and microstructural development considered. Samples for the study were taken from both deformed and undeformed zinc ores of the Central U.S. Appalachians, and deformed zinc ores of the English Pennines. A variety of mineralogical techniques were employed, including transmitted and reflected light microscopy of etched and unetched material, transmission electron microscopy and electron microprobe analysis. For the Pennine zinc sulfides, spectroscopic, x-ray diffraction and fluid inclusion studies were also undertaken. Optical and electron optical examination of the Appalachian material confirmed the suitability of zinc sulfide for detailed study with such techniques. Growth and deformation-related microstructures could be distinguished from specimen-preparation induced artifacts. A deformationally-mduced lamelliform optical anisotropy is seen to be developed in areas hosting a dense planar microstructure of {111} twin- and slip-planes. The Pennine zinc sulfide texturally records a changing depositional environment. Thus, for example, delicately growth- zoned crystals are truncated and cross-cut by solution disconformities. Fluid inclusion studies indicate a highly saline (20-25 wt. % equiv. NaCl), low temperature (100-150°C.) fluid. Texturally, two varieties of zinc sulfide can be recognised; a widely developed, iron- banded variety, and a paragenetically early variety, banded due to horizons rich in crystal defects and microscopic inclusions. The zinc sulfide takes the form of a disordered 3C-polytype, with much of the disorder being deformational in origin. Twin- and slip-plane fabrics are developed . A deformation-related optical anisotropy is seen to overprint growth-related anisotropy, along with cuprian alteration of certain {111} deformation planes.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)

Introduction: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). Methods and materials: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells’ location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells’ VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium GreenTM-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. Results: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K+. This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. Conclusion: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

The Application of Fluorescent Nanocrystals to Study the Distribution of Neuropeptides in the Aedes Aegypti Mosquito

Semiconductor nanocrystals, also known as quantum dots (QDs), have been used in studies involving mice and human tissues, but never before in research on insects. We used QDs to study the distribution of two neuropeptides in the Aedes aegypti mosquito, the vector of both dengue and yellow fever. These neuropeptides play a significant role in the production of juvenile hormone, a hormone that controls biting behavior, metamorphosis, and reproduction throughout the life of the mosquito. The two neuropeptides allatostatin-C (AS-C) and allatotropin (AT) function as inhibitory (AS-C) and stimulatory (AT) regulators of juvenile hormone synthesis in the corpus allatum gland. In other insects, they also affect heart rate, gut movement, and nutrient uptake. Conjugating these neuropeptides to quantum dots via a streptavidinlbiotin link, we were able to expose the mosquito corpus allatum and abdomen to allatostatin-C and allatotropin and then to visualize their distribution under UV light using confocal and compound light microscopy. Histological sections of the whole mosquito, incubations of tissues with conjugates (in vitro), and microinjections of conjugates into the mosquito (in vivo) were performed. The results showed that quantum dots can be used to detect neuropeptide distribution in the mosquito. The more we understand about these neuropeptides and juvenile hormone, the more we can contribute to stopping the spread of infectious diseases, such as dengue and yellow fever.

Event layer chronology from Lake Suigetsu (Japan, SG06 Project) for the last 40 kyr

Event layers in lake sediments are indicators of past extreme events, mostly the results of floods or earthquakes. Detailed characterisation of the layers allows the discrimination of the sedimentation processes involved, such as surface runoff, landslides or subaqueous slope failures. These processes can then be interpreted in terms of their triggering mechanisms. Here we present a 40 kyr event layer chronology from Lake Suigetsu, Japan. The event layers were characterised using a multi-proxy approach, employing light microscopy and µXRF for microfacies analysis. The vast majority of event layers in Lake Suigetsu was produced by flood events (362 out of 369), allowing the construction of the first long-term, quantitative (with respect to recurrence) and well dated flood chronology from the region. The flood layer frequency shows a high variability over the last 40 kyr, and it appears that extreme precipitation events were decoupled from the average long-term precipitation. For instance, the flood layer frequency is highest in the Glacial at around 25 kyr BP, at which time Japan was experiencing a generally cold and dry climate. Other cold episodes, such as Heinrich Event 1 or the Late Glacial stadial, show a low flood layer frequency. Both observations together exclude a simple, straightforward relationship with average precipitation and temperature. We argue that, especially during Glacial times, changes in typhoon genesis/typhoon tracks are the most likely control on the flood layer frequency, rather than changes in the monsoon front or snow melts. Spectral analysis of the flood chronology revealed periodic variations on centennial and millennial time scales, with 220 yr, 450 yr and a 2000 yr cyclicity most pronounced. However, the flood layer frequency appears to have not only been influenced by climate changes, but also by changes in erosion rates due to, for instance, earthquakes.

(Table 2) Relative contents of clay minerals in aeolian pelagic clays from the Pacific Ocean

For determining importance, composition, and history of aerosol material accumulation in formation of pelagic clays a study with use of light microscopy and scanning electron microscopy, X-ray diffractometry, and chemical methods has been carried out.

Global distributions of diazotrophs abundance, biomass and nitrogen fixation rates - Collection of source datasets - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

The MAREDAT atlas covers 11 types of plankton, ranging in size from bacteria to jellyfish. Together, these plankton groups determine the health and productivity of the global ocean and play a vital role in the global carbon cycle. Working within a uniform and consistent spatial and depth grid (map) of the global ocean, the researchers compiled thousands and tens of thousands of data points to identify regions of plankton abundance and scarcity as well as areas of data abundance and scarcity. At many of the grid points, the MAREDAT team accomplished the difficult conversion from abundance (numbers of organisms) to biomass (carbon mass of organisms). The MAREDAT atlas provides an unprecedented global data set for ecological and biochemical analysis and modeling as well as a clear mandate for compiling additional existing data and for focusing future data gathering efforts on key groups in key areas of the ocean. The present collection presents the original data sets used to compile Global distributions of diazotrophs abundance, biomass and nitrogen fixation rates