Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway

The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared with adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression, respectively. This links BRN2 as an activator, and conversely, MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense-ablated cell lines decreased the melanoma sphere-forming capability, cell adhesion during 3D-spheroid formation and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere-culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than the growth as adherent melanoma cells.

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge : A proof-of-concept study

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A deletion and point mutation study of the human papillomavirus type 16 major capsid gene

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A → T mutation at residue 266 (A266T), and of a C → G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428-483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2-9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55 nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428-465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines. © 2006 Elsevier B.V. All rights reserved.

Micromarrows—Three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells

Hematopoietic stem cell (HSC) transplant is a well established curative therapy for some hematological malignancies. However, achieving adequate supply of HSC from some donor tissues can limit both its application and ultimate efficacy. The theory that this limitation could be overcome by expanding the HSC population before transplantation has motivated numerous laboratories to develop ex vivo expansion processes. Pioneering work in this field utilized stromal cells as support cells in cocultures with HSC to mimic the HSC niche. We hypothesized that through translation of this classic coculture system to a three-dimensional (3D) structure we could better replicate the niche environment and in turn enhance HSC expansion. Herein we describe a novel high-throughput 3D coculture system where murine-derived HSC can be cocultured with mesenchymal stem/stromal cells (MSC) in 3D microaggregates—which we term “micromarrows.” Micromarrows were formed using surface modified microwells and their ability to support HSC expansion was compared to classic two-dimensional (2D) cocultures. While both 2D and 3D systems provide only a modest total cell expansion in the minimally supplemented medium, the micromarrow system supported the expansion of approximately twice as many HSC candidates as the 2D controls. Histology revealed that at day 7, the majority of bound hematopoietic cells reside in the outer layers of the aggregate. Quantitative polymerase chain reaction demonstrates that MSC maintained in 3D aggregates express significantly higher levels of key hematopoietic niche factors relative to their 2D equivalents. Thus, we propose that the micromarrow platform represents a promising first step toward a high-throughput HSC 3D coculture system that may enable in vitro HSC niche recapitulation and subsequent extensive in vitro HSC self-renewal.

The El Chichon stratospheric cloud : solid particulates and settling rates

Sampling of the El Chichón stratospheric cloud in early May and in late July, 1982, showed that a significant proportion of the cloud consisted of solid particles between 2 μm and 40 μm size. In addition, many particles may have been part of larger aggregates or clusters that ranged in size from < 10 μm to > 50 μm. The majority of individual grains were angular aluminosilicate glass shards with various amounts of smaller, adhering particles. Surface features on individual grains include sulfuric acid droplets and larger (0.5 μm to 1 μm) sulfate gel droplets with various amounts of Na, Mg, Ca and Fe. The sulfate gels probably formed by the interaction of sulfur-rich gases and solid particles within the cloud soon after eruption. Ca-sulfate laths may have formed by condensation within the plume during eruption, or alternatively, at a later stage by the reaction of sulfuric acid aerosols with ash fragments within the stratospheric cloud. A Wilson-Huang formulation for the settling rate of individual particles qualitatively agrees with the observed particle-size distribution for a period at least four months after injection of material into the stratosphere. This result emphasizes the importance of particle shape in controlling the settling rate of volcanic ash from the stratosphere.

Bismuth in interplanetary dust

Samples of a large (~60 µm) chondritic porous (CP) aggregate collected from the stratosphere have been analysed in detail by analytical electron microscopy (AEM). Previous studies of CP aggregates have shown that they are extraterrestrial in origin1–3 and may be related to cometary debris4. CP aggregates are dissimilar to C1 and C2 carbonaceous chondrite matrices and many have not been significantly altered by thermal or radiation processes since their assembly5. We report here a high concentration of Bi2O3 grains within the large CP aggregate designated W7029* A (~60 µm) and suggest they formed by rapid heating (~300 °C) of elemental Bi grains within the aggregate during atmospheric entry. We examine the possibilities for terrestrial Bi contamination of CP aggregate W7029* A but judge them unlikely. Enrichment of elemental Bi within components of extraterrestrial materials is consistent with a nebula condensation model6 and implies that Bi within CP aggregate W7029* A may have formed at a late stage of the condensation process.

Mineralogy of chondritic interplanetary dust particles

From a mineralogical survey of approximately 30 chondritic micrometeorites collected from the lower stratosphere and studied in detail using current electron microscopy techniques, it is concluded that these particles represent a unique group of extraterrestrial materials. These micrometeorites differ significantly in form and texture from components of carbonaceous chondrites and contain some mineral assemblages which do not occur in any meteorite class. Electron microscope investigations of chondritic micrometeorites have established that these materials (1) are extraterrestrial in origin, (2) existed in space as small objects, (3) endured minimal alteration by planetary processes since formation, and (4) can suffer minimal pulse heating (<600°C) on entering earth's atmosphere. The probable sources for chondritic interplanetary dust particles (IDPs) are cometary and asteroidal debris and, perhaps to a lesser extent, interstellar regions. These sources have not been conclusively linked to any specific mineralogical subset of IDP, although the chondritic porous (CP) aggregate is considered of likely cometary origin. Chondritic IDPs occur in two predominant mineral assemblages: (1) carbonaceous phases and phyllosilicates and (2) carbonaceous phases and nesosilicates or inosilicates, although particles with both types of silicate assemblages are observed. Olivines, pyroxenes, layer silicates, and carbon-rich phases are the most commonly occurring minerals in many chondritic IDPs. Other phases often observed in variable proportions include sulphides, spinels, metals, metal carbides, carbonates, and minor amounts of sulphates and phosphates. Individual mineral grain sizes range from micrometers (primarily pyroxenes and olivines) to nanometers, with the predominant size for all phases less than 100 nm. Specific mineral characteristics for particular chondritic IDPs provide an indication of processes which may have occurred prior to collection in the earth's stratosphere. For example, pyroxene mineralogy in some chondritic aggregates is consistent with condensation from a vapor phase and, we consider, with condensation in a turbulent solar nebula at relatively low temperatures (<1000°C). Carbonaceous phases present in other CP aggregates have been used to imply low-temperature formation processes such as Fischer-Tropsch synthesis (∼530°C) or carbonization and graphitization (∼315°C). Alteration processes have been implicated in the formation of some layer silicates in CP aggregates and may have involved hydrocryogenic alteration at <0°C. In general, interpretations of transformation processes on submicrometer-size minerals in chondritic IDPs are consistent with formation at a radius equivalent to the asteroid belt or greater during the later stages of solar nebula evolution using currently available models.

Poorly graphitized carbon as a new cosmothermometer for primitive extraterrestrial materials

The presence of carbon in primitive extraterrestrial materials has long been considered a useful indicator of prevailing geochemical conditions early in the formation of the Solar System. A recent addition to the suite of primitive materials available for study by cosmochemists includes particles collected from the stratosphere called chondritic porous (CP) aggregates1. Carbon-rich CP aggregates are less abundant in stratospheric collections and contain many low-temperature phases (such as layer silicates) as minor components2,3. We describe here the nature of the most abundant carbon phase in a carbon-rich CP aggregate (sample no. W7029* A) collected from the stratosphere as part of the Johnson Space Center (JSC) Cosmic Dust Program4. By comparison with experimental and terrestrial studies of poorly graphitized carbon (PGC), we show that the graphitization temperature, or the degree of ordering in the PGC, may provide a useful cosmothermometer for primitive extraterrestrial materials.

A multi-stage history for carbonaceous material in extraterrestrial chondritic porous aggregate W7029*A and a new cosmothermometer

A new set of primitive extraterrestrial materials collected in the Earth's stratosphere include Chondritic Porous Aggregates (CPA's) [1]. CPAs have a complex and variable mineralogy [1-3] that include 'organic compounds' [4,5] and poorly graphitised carbon (PGC)[6]. This study presents a continuation of our detailed Analytical Electron Microscope study on carbon-rich CPA W7029*A from the JSC Cosmic Dust Collection. This CPA is an uncontaminated sample that survived atmospheric entry without appreciable alteration [7] and which contains ~44% carbonaceous material. The carbonaceous composition of selected particles was confirmed by Electron Energy Loss Spectroscopy and Selected Area Electron Diffraction (SAED). Possible carbonaceous contaminants introduced by specimen preparation techniques are easily recognised from indigenous CPA carbon particles [8] and do not bias our interpretations.

Metastable carbon in two chondritic porous interplanetary dust particles

An understanding of carbonaceous matter in primitive extraterrestrial materials is an essential component of studies on dust evolution in the interstellar medium and the early history of the Solar System. We have suggested previously that a record of graphitization is preserved in chondritic porous (CP) aggregates and carbonaceous chondrites1,2 and that the detailed mineralogy of CP aggregates can place boundary conditions on the nature of both physical and chemical processes which occurred at the time of their formation2,3. Here, we report further analytical electron microscope (AEM) studies on carbonaceous material in two CP aggregates which suggest that a record of hydrocarbon carbonization may also be preserved in these materials. This suggestion is, based upon the presence of well-ordered carbon-2H (lonsdaleite) in CP aggregates W7029*A and W7010*A2. This carbon is a metastable phase resulting from hydrous pyrolysis below 300-350°C and may be a precursor to poorly graphitized carbons (PGCs) in primitive extraterrestrial materials2. © 1987 Nature Publishing Group.

Layer silicates in a chondritic porous interplanetary dust particle

Analytical electron microscopy on individual grains from a portion of a chondritic porous interplanetary dust particle (aggregate W7029C1 from the NASA Johnson Space Center Cosmic Dust Collection) shows that layer silicates compose 50 percent of the silicate fraction examined. These layer silicates can be classified into two distinct crystallochemical groups: (1) fine-grained, polycrystalline smectite minerals; and (2) well-ordered, single crystals of kaolinite and Mg-poor talc. The layer silicates in this portion of sample W7029(asterisk)A are dissimilar to those described in other chondritic porous aggregates. The predominant layer silicate assemblage in W7029(asterisk)A indicates that heating of the aggregate during atmospheric entry was brief and probably to a temperature less than 300C. Comparison with terrestrial phyllosilicate occurrences suggests that some layer silicates in aggregate W7029(asterisk)A may have been formed by alteratiton from preexisting silicate minerals at low temperatures (less than 25C) after aggregate formation.

Classification of the Johnson Space Center stratospheric dust collection

A review of 291 catalogued particles on the bases of particle size, shape, bulk chemistry, and texture is used to establish a reliable taxonomy. Extraterrestrial materials occur in three defined categories: spheres, aggregates and fragments. Approximately 76% of aggregates are of probable extraterrestrial origin, whereas spheres contain the smallest amount of extraterrestrial material (approx 43%). -B.M.

Microbeam analyses of stratospheric particles

Collections of solid particles from the Earth's stratosphere by high-flying aircraft have been reported since 1965, with the initial primary objective of understanding the nature of the aerosol layer that occurs in the lower stratosphere. With the advent of efficient collection procedures and sophisticated electron- and ion-beam techniques, the primary aim of current stratospheric collections has been to study specific particle types that are extraterrestrial in origin and have survived atmospheric entry processes. The collection program provided by NASA at Johnson Space Center (JSC) has conducted many flights over the past 4 years and retrieved a total of 99 collection surfaces (flags) suitable for detailed study. Most of these collections are part of dedicated flights and have occurred during volcanically quiescent periods, although solid particles from the El Chichon eruptions have also been collected. Over 800 individual particles (or representative samples from larger aggregates) have been picked from these flags, examined in a preliminary fashion by SEM and EDS, and cataloged in a manner suitable for selection and study by the wider scientific community.

Paclitaxel resistance and multicellular spheroid formation are induced by kallikrein-related peptidase 4 in serous ovarian cancer cells in an ascites mimicking microenvironment

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.

Transmission X-ray microscopy : a new tool in clay mineral floccules characterization

Effective flocculation and dewatering of mineral processing streams containing clays are microstructure dependent in clay-water systems. Initial clay flocculation is crucial in the design and for the development of a new methodology of gas exploitation. Microstructural engineering of clay aggregates using covalent cations and Keggin macromolecules have been monitored using the new state of the art Transmission X-ray Microscope (TXM) with 60 nm tomography resolution installed in a Taiwanese synchrotron. The 3-D reconstructions from TXM images show complex aggregation structures in montmorillonite aqueous suspensions after treatment with Na+, Ca2+ and Al13 Keggin macromolecules. Na-montmorillonite displays elongated, parallel, well-orientated and closed-void cellular networks, 0.5–3 μm in diameter. After treatment by covalent cations, the coagulated structure displays much smaller, randomly orientated and openly connected cells, 300–600 nm in diameter. The average distances measured between montmorillonite sheets was around 450 nm, which is less than half of the cell dimension measured in Na-montmorillonite. The most dramatic structural changes were observed after treatment by Al13 Keggin; aggregates then became arranged in compacted domains of a 300 nm average diameter composed of thick face-to-face oriented sheets, which forms porous aggregates with larger intra-aggregate open and connected voids.