GTSF-2: A new, versatile cell culture medium for diverse normal and transformed mammalian cells

Summary The aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.     

Seasonal rainfall and runoff promote coral disease on an inshore reef

Background

Declining water quality coupled with the effects of climate change are rapidly increasing coral diseases on reefs worldwide, although links between coral diseases and environmental parameters remain poorly understood. This is the first study to document a correlation between coral disease and water quality on an inshore reef.

Methodology/Principal Findings

The temporal dynamics of the coral disease atramentous necrosis (AN) was investigated over two years within inshore populations of Montipora aequituberculata in the central Great Barrier Reef, in relation to rainfall, salinity, temperature, water column chlorophyll a, suspended solids, sedimentation, dissolved organic carbon, and particulate nitrogen, phosphorus and organic carbon. Overall, mean AN prevalence was 10-fold greater during summer wet seasons than winter dry seasons. A 2.5-fold greater mean disease abundance was detected during the summer of 2009 (44 ± SE 6.7 diseased colonies per 25 m²), when rainfall was 1.6-fold greater than in the summer of 2008. Two water quality parameters explained 67% of the variance in monthly disease prevalence in a Partial Least Squares regression analysis; disease abundance was negatively correlated with salinity (R2 = −0.6) but positively correlated with water column particulate organic carbon concentration (R2 = 0.32). Seasonal temperature patterns were also positively correlated with disease abundance, but explained only a small portion of the variance.

Conclusions/Significance

The results suggest that rainfall and associated runoff may facilitate seasonal disease outbreaks, potentially by reducing host fitness or by increasing pathogen virulence due to higher availability of nutrients and organic matter. In the future, rainfall and seawater temperatures are likely to increase due to climate change which may lead to decreased health of inshore reefs.     

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The “static bioreactor” provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.     

Solar radiation and tidal exposure as environmental drivers of Enhalus acoroides dominated seagrass meadows

There is strong evidence of a global long-term decline in seagrass meadows that is widely attributed to anthropogenic activity. Yet in many regions, attributing these changes to actual activities is difficult, as there exists limited understanding of the natural processes that can influence these valuable ecosystem service providers. Being able to separate natural from anthropogenic causes of seagrass change is important for developing strategies that effectively mitigate and manage anthropogenic impacts on seagrass, and promote coastal ecosystems resilient to future environmental change. The present study investigated the influence of environmental and climate related factors on seagrass biomass in a large ≈250 ha meadow in tropical north east Australia. Annual monitoring of the intertidal Enhalus acoroides (L.f.) Royle seagrass meadow over eleven years revealed a declining trend in above-ground biomass (54% significant overall reduction from 2000 to 2010). Partial Least Squares Regression found this reduction to be significantly and negatively correlated with tidal exposure, and significantly and negatively correlated with the amount of solar radiation. This study documents how natural long-term tidal variability can influence long-term seagrass dynamics. Exposure to desiccation, high UV, and daytime temperature regimes are discussed as the likely mechanisms for the action of these factors in causing this decline. The results emphasise the importance of understanding and assessing natural environmentally-driven change when interpreting the results of seagrass monitoring programs.     

The hydrogenosomes of Psalteriomonas lanterna

Background  

Hydrogenosomes are organelles that produce molecular hydrogen and ATP. The broad phylogenetic distribution of their hosts suggests that the hydrogenosomes of these organisms evolved several times independently from the mitochondria of aerobic progenitors. Morphology and 18S rRNA phylogeny suggest that the microaerophilic amoeboflagellate Psalteriomonas lanterna, which possesses hydrogenosomes and elusive "modified mitochondria", belongs to the Heterolobosea, a taxon that consists predominantly of aerobic, mitochondriate organisms. This taxon is rather unrelated to taxa with hitherto studied hydrogenosomes.     

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.Download video file.^{(453M, mp4)}     

Chemical synthesis and characterization of chemokine RANTES and its analogues

RANTES, a polypeptide of 68 amino acid residues, is a member of the C-C chemokine subfamily including other monocyte chemoattractants such as MIP-1alpha, MIP-1beta, MCP-1, MCP-2 and MCP-3. To provide a chemically defined RANTES in quantity suitable for structure-function studies, RANTES and its analogues were synthesized, using a modified solid-phase chemistry approach. The fully protected RANTES and RANTES(3-68) were assembled by automated solid-phase methodology using Fmoc chemistry. Deprotection and cleavage of the resin bound peptides yielded crude peptides, which were then folded and further purified by reverse-phase HPLC. The chemically synthesized RANTES with its identity and purity established, was found to be immunochemically and functionally indistinguishable from the recombinant human RANTES. RANTES and its analog, RANTES(3-68), have recently been used as the substrate in the study of dipeptidyl peptidase IV (CD26)-mediated processing of RANTES and its effect on receptor specificity.     

Measuring and modelling seasonal variation of carbon dioxide and water vapour exchange of a Pinus ponderosa forest subject to soil water deficit

We conducted ecosystem carbon and water vapour exchange studies in an old‐growth Pinus ponderosa forest in the Pacific North‐west region of the United States. The canopy is heterogeneous, with tall multiaged trees and an open, clumped canopy with low leaf area. Carbon assimilation can occur throughout relatively mild winters, although night frosts can temporarily halt the process and physiological factors limit its efficiency. In contrast, carbon assimilation is often limited in the ‘growing season’ by stomatal closure associated with high evaporative demand (D) and soil water deficits. All of these factors present a challenge to effectively modelling ecosystem processes. Our objective was to generate an understanding of the controls on ecosystem processes across seasonal and annual cycles from a combination of fine‐scale process modelling, ecophysiological measurements, and carbon and water vapour fluxes measured by the eddy covariance method. Flux measurements showed that 50% and 70% of the annual carbon uptake occurred outside the ‘growing season’ (defined as bud break to senescence, ～ days 125–275) in 1996 and 1997. On a daily basis in summer, net ecosystem productivity (NEP) was low when D and soil water deficits were large. Whole ecosystem water vapour fluxes (LE) increased from spring to summer (1.0–1.9 mm d?1) as conducting leaf area increased by 30% and as evaporative demand increased, while evaporation from the soil surface became a smaller portion of total LE as soil water deficits increased. The models underestimated soil evaporation, particularly following rain. In the SPA model, varying the temperature optimum for photosynthesis seasonally resulted in overestimation of carbon uptake in winter and spring, showing that in coniferous forests, assumptions about temperature optima are clearly important. Daily estimates of soil surface CO₂ flux from measurements and site meteorological data demonstrated that modelling of soil CO₂ flux based on an Arrhenius‐type equation in CANPOND overestimated CO₂ respired from the soil during drought and when temperatures were low.