Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Multilineage differentiation potential of bone and cartilage cells derived from explant culture

To date, mesenchymal stem cells (MSCs) from various tissues have been reported, but the yield and differentiation potential of different tissue-derived MSCs is still not clear. This study was undertaken in an attempt to investigate the multilineage stem cell potential of bone and cartilage explant cultures in comparison with bone marrow derived mesenchymal stem cells (BMSCs). The results showed that the surface antigen expression of tissue-derived cells was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing markers related to adhesion (CD29, CD166) and stem cells (CD90, CD105). The tissue-derived cells were able to differentiate into osteoblast, chondrocyte and adipocyte lineage pathways when stimulated in the appropriate differentiating conditions. However, compared with BMSCs, tissue-derived cells showed less capacity for multilineage differentiation when the level of differentiation was assessed in monolayer culture by analysing the expression of tissue-specific genes by reverse transcription polymerase chain reaction (RT-PCR) and histology. In high density pellet cultures, tissue-derived cells were able to differentiate into chondrocytes, expressing chondrocyte markers such as proteoglycans, type II collagen and aggrecan. Taken together, these results indicate that cells derived from tissue explant cultures reserved certain degree of differentiation properties of MSCs in vitro.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Amniotic fluid stem cells produce robust mineral deposits on biodegradable scaffolds

Insufficient availability of osteogenic cells limits bone regeneration through cell-based therapies. This study investigated the potential of amniotic fluid–derived stem (AFS) cells to synthesize mineralized extracellular matrix within porous medical-grade poly-e-caprolactone (mPCL) scaffolds. The AFS cells were initially differentiated in two-dimensional (2D) culture to determine appropriate osteogenic culture conditions and verify physiologic mineral production by the AFS cells. The AFS cells were then cultured on 3D mPCL scaffolds (6-mm diameter9-mm height) and analyzed for their ability to differentiate to osteoblastic cells in this environment. The amount and distribution of mineralized matrix production was quantified throughout the mPCL scaffold using nondestructive micro computed tomography (microCT) analysis and confirmed through biochemical assays. Sterile microCT scanning provided longitudinal analysis of long-term cultured mPCL constructs to determine the rate and distribution of mineral matrix within the scaffolds. The AFS cells deposited mineralized matrix throughout the mPCL scaffolds and remained viable after 15 weeks of 3D culture. The effect of predifferentiation of the AFS cells on the subsequent bone formation in vivo was determined in a rat subcutaneous model. Cells that were pre-differentiated for 28 days in vitro produced seven times more mineralized matrix when implanted subcutaneously in vivo. This study demonstrated the potential of AFS cells to produce 3D mineralized bioengineered constructs in vitro and in vivo and suggests that AFS cells may be an effective cell source for functional repair of large bone defects

Cellular senescence and longevity of osteophyte-derived mesenchymal stem cells compared to patient-matched bone marrow stromal cells

This study aimed to determine the cellular aging of osteophyte-derived mesenchymal cells (oMSCs) in comparison to patient-matched bone marrow stromal cells (bMSCs). Extensive expansion of the cell cultures was performed and early and late passage cells (passages 4 and 9, respectively) were used to study signs of cellular aging, telomere length, telomerase activity, and cell-cycle-related gene expression. Our results showed that cellular aging was more prominent in bMSCs than in oMSCs, and that oMSCs had longer telomere length in late passages compared with bMSCs, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSCs and not in bMSCs. In osteophyte tissues telomerase-positive cells were found to be located perivascularly and were Stro-1 positive. Fifteen cell-cycle regulator genes were investigated and only three genes (APC, CCND2, and BMP2) were differentially expressed between bMSC and oMSC. Our results indicate that oMSCs retain a level of telomerase activity in vitro, which may account for the relatively greater longevity of these cells, compared with bMSCs, by preventing replicative senescence. J. Cell. Biochem. 108: 839-850, 2009. (c) 2009 Wiley-Liss, Inc.

Cryoreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.

Vitreous cryopreservation of nanofibrous tissue-engineered constructs generated using mesenchymal stromal cells

Development of an effective preservation strategy to fulfill off-the-shelf availability of tissue-engineered constructs (TECs) is demanded for realizing their clinical potential. In this study, the feasibility of vitrification, ice-free cryopreservation, for precultured ready-to-use TECs was evaluated. To prepare the TECs, bone marrow-derived porcine mesenchymal stromal cells (MSCs) were seeded in polycaprolactone-gelatin nanofibrous scaffolds and cultured for 3 weeks before vitrification treatment. The vitrification strategy developed, which involved exposure of the TECs to low concentrations of cryoprotectants followed by a vitrification solution and sterile packaging in a pouch with its subsequent immersion directly into liquid nitrogen, was accomplished within 11min. Stepwise removal of cryoprotectants, after warming in a 38 degrees C water bath, enabled rapid restoration of the TECs. Vitrification did not impair microstructure of the scaffold or cell viability. No significant differences were found between the vitrified and control TECs in cellular metabolic activity and proliferation on matched days and in the trends during 5 weeks of continuous culture postvitrification. Osteogenic differentiation ability in vitrified and control groups was similar. In conclusion, we have developed a time- and cost-efficient cryopreservation method that maintains integrity of the TECs while preserving MSCs viability and metabolic activity, and their ability to differentiate.

The impact of natural disasters on residential property markets

Over the past ten years various residential property markets throughout Australia in general and NSW in particular have been subject to substantial natural disasters. These occurrences have included floods, bushfires and hailstorms. In extreme cases the actual rectification costs have been up to AUD$1.5 billion, which occurred with the severe hailstorm in Sydney in April 1999 and cyclone Tracey in Darwin in 1974. Natural disasters such as severe storms and hailstorms have tended to be very indiscriminate in relation to frequency and the actual location of damage, whereas the nature of bushfire and flooding tends to be more defined. Although these extreme natural disasters tend to be infrequent, occurrences of floods and bushfires in residential property areas are more frequent, particularly as urban sprawl encroaches closer to national Parks, State recreation Parks and State forests. Considerable work has been carried out on flood effects on property markets by Bell (1999), Donnelly (1988), McClusky and Rausser (2001), Skrantz and Strickland (1987) in the US, and Chou and Shih (2001) in Taiwan. Fibbens (1994), Lambley and Cordery (1991) and Eves (1999, 2001, 2002) have carried out studies in relation to the effect of flooding on residential property values in the Sydney region, including the tracking of flood prone property values over time. However, no similar rigorous research has been carried out in relation to the impact of bushfires on residential property markets in the Sydney region.

Electron paramagnetic resonance spectral studies on natural sapphire

EPR study of both blue and green sapphire samples confirms the presence of Cr(III) in four different octahedral sites. The g (1.98) value is the same but D values differ for the two the samples. The EPR spectra suggest that the blue sapphire contains more chromium than the green sapphire. No Fe(III) impurity was noted in the EPR spectrum.

Weather variability, sunspots, and the blooms of cyanobacteria

The roles of weather variability and sunspots in the occurrence of cyanobacteria blooms, were investigated using cyanobacteria cell data collected from the Fred Haigh Dam, Queensland, Australia. Time series generalized linear model and classification and regression (CART) model were used in the analysis. Data on notified cell numbers of cyanobacteria and weather variables over the periods 2001 and 2005 were provided by the Australian Department of Natural Resources and Water, and Australian Bureau of Meteorology, respectively. The results indicate that monthly minimum temperature (relative risk [RR]: 1.13, 95% confidence interval [CI]: 1.02-1.25) and rainfall (RR: 1.11; 95% CI: 1.03-1.20) had a positive association, but relative humidity (RR: 0.94; 95% CI: 0.91-0.98) and wind speed (RR:0.90; 95% CI: 0.82-0.98) were negatively associated with the cyanobacterial numbers, after adjustment for seasonality and auto-correlation. The CART model showed that the cyanobacteria numbers were best described by an interaction between minimum temperature, relative humidity, and sunspot numbers. When minimum temperature exceeded 18%C and relative humidity was under 66%, the number of cyanobacterial cells rose by 2.15-fold. We conclude that the weather variability and sunspot activity may affect cyanobacterial blooms in dams.

Natural enemies of Paropsis atomaria Olivier (Coleoptera: Chrysomelidae) in south-eastern Queensland eucalypt plantations

Abstract A field survey for natural enemies of Paropsis atomaria was conducted at two south-eastern Queensland Eucalyptus cloeziana plantation sites during 2004–2005. Primary egg and larval parasitoids and associated hyperparasitoids were identified to genus or species, and parasitism rates were determined throughout the season. Predators were identified to family level but their impact was not quantified. P. atomaria adults were also examined as potential hosts for parasitic mites and nematodes. An undescribed species of Neopolycystus (Pteromalidae) was the major primary egg parasitoid species reared from egg batches, parasitising half of all egg batches collected. Three hyperparasitoid species (Baeoanusia albifunicle (Encyrtidae), Neblatticida sp. (Encyrtidae) and Aphaneromella sp. (Platygasteridae) were present, representing around one-quarter to one-third of all emergent wasps; this is the first host association record for Neopolycystus–B. albifunicle. In contrast to populations of P. atomaria from the Australian Capital Territory, primary larval parasitism was very low, around 1%, and attributable only to the tachinid flies Anagonia sp. and Paropsivora sp. However, the presence of the sit-and-wait larval hyperparasitoid, Perilampus sp. (Perilampidae) was high, emerging from around 17% of tachinid pupae, with planidia infesting a further 40% of unparasitised hosts. Three species of podapolipid mites parasitised sexually mature P. atomaria adults, while no nematodes were found in this study. Spiders were the most common predators and their abundance was positively correlated with P. atomaria adult and egg numbers. Although natural enemy species composition was identical between our two study sites, significant differences in abundance and frequency were found between sites