Phosphorus limitation and heat stress decrease calcification in Emiliania huxleyi

Calcifying haptophytes (coccolithophores) sequester carbon in the form of organic and inorganic cellular components (coccoliths). We examined the effect of phosphorus (P) limitation and heat stress on particulate organic and inorganic carbon (calcite) production in the coccolithophore Emiliania huxleyi. Both environmental stressors are related to rising CO2 levels and affect carbon production in marine microalgae, which in turn impacts biogeochemical cycling. Using semi-continuous cultures, we show that P limitation and heat stress decrease the calcification rate in E. huxleyi. However, using batch cultures, we show that different culturing approaches (batch versus semi-continuous) induce different physiologies. This affects the ratio of particulate inorganic (PIC) to organic carbon (POC) and complicates general predictions on the effect of P limitation on the PIC = POC ratio. We found heat stress to increase P requirements in E. huxleyi, possibly leading to lower standing stocks in a warmer ocean, especially if this is linked to lower nutrient input. In summary, the predicted rise in global temperature and resulting decrease in nutrient availability may decrease CO2 sequestration by E. huxleyi through lower overall carbon production. Additionally, the export of carbon may be diminished by a decrease in calcification and a weaker coccolith ballasting effect

High temperature decreases the PIC / POC ratio and increases phosphorus requirements in <i>Coccolithus pelagicus</i> (Haptophyta)

Rising ocean temperatures will likely increase stratification of the water column and reduce nutrient input into the photic zone. This will increase the likelihood of nutrient limitation in marine microalgae, leading to changes in the abundance and composition of phytoplankton communities, which in turn will affect global biogeochemical cycles. Calcifying algae, such as coccolithophores, influence the carbon cycle by fixing CO₂ into particulate organic carbon through photosynthesis (POC production) and into particulate inorganic carbon through calcification (PIC production). As calcification produces a net release of CO₂, the ratio of PIC to POC production determines whether coccolithophores act as a source (high PIC / POC) or a sink (low PIC / POC) of atmospheric CO₂. We studied the effect of phosphorus (P-) limitation and high temperature on the physiology and the PIC / POC ratio of two subspecies of Coccolithus pelagicus. This large and heavily calcified species is a major contributor to calcite export from the photic zone into deep-sea reservoirs. Phosphorus limitation did not influence exponential growth rates in either subspecies, but P-limited cells had significantly lower cellular P-content. One of the subspecies was subjected to a 5 °C temperature increase from 10 °C to 15 °C, which did not affect exponential growth rates either, but nearly doubled cellular P-content under both high and low phosphate availability. This temperature increase reduced the PIC / POC ratio by 40–60%, whereas the PIC / POC ratio did not differ between P-limited and nutrient-replete cultures when the subspecies were grown near their respective isolation temperature. Both P-limitation and elevated temperature significantly increased coccolith malformations. Our results suggest that a temperature increase may intensify P-limitation due to a higher P-requirement to maintain growth and POC production rates, possibly reducing abundances in a warmer ocean. Under such a scenario <i>C. pelagicus</i> may decrease its calcification rate relative to photosynthesis, thus favouring CO₂ sequestration over release. It seems unlikely that P-limitation by itself causes changes in the PIC / POC ratio in this species

The Expanded Central Dogma:Genome Resynthesis, Orthogonal Biosystems, Synthetic Genetics

Synthetic biology seeks to probe fundamental aspects of biological form and function by construction [i.e., (re)synthesis] rather than deconstruction (analysis). In this sense, biological sciences now follow the lead given by the chemical sciences. Synthesis can complement analytic studies but also allows novel approaches to answering fundamental biological questions and opens up vast opportunities for the exploitation of biological processes to provide solutions for global problems. In this review, we explore aspects of this synthesis paradigm as applied to the chemistry and function of nucleic acids in biological systems and beyond, specifically, in genome resynthesis, synthetic genetics (i.e., the expansion of the genetic alphabet, of the genetic code, and of the chemical make-up of genetic systems), and the elaboration of orthogonal biosystems and components.</p

Human Embryonic Stem Cell Technology: Large Scale Cell Amplification and Differentiation

Embryonic stem cells (ESC) hold the promise of overcoming many diseases as potential sources of, for example, dopaminergic neural cells for Parkinson’s Disease to pancreatic islets to relieve diabetic patients of their daily insulin injections. While an embryo has the innate capacity to develop fully functional differentiated tissues; biologists are finding that it is much more complex to derive singular, pure populations of primary cells from the highly versatile ESC from this embryonic parent. Thus, a substantial investment in developing the technologies to expand and differentiate these cells is required in the next decade to move this promise into reality. In this review we document the current standard assays for characterising human ESC (hESC), the status of ‘defined’ feeder-free culture conditions for undifferentiated hESC growth, examine the quality controls that will be required to be established for monitoring their growth, review current methods for expansion and differentiation, and speculate on the possible routes of scaling up the differentiation of hESC to therapeutic quantities

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al

Physical Passaging of Embryoid Bodies Generated from Human Pluripotent Stem Cells

Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1∶4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells

Loss of viability during freeze-thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to apoptosis rather than cellular necrosis

10.1007/s11373-005-9051-9Journal of Biomedical Science133433-44

Combinatorial Development of Biomaterials for Clonal Growth of Human Pluripotent Stem Cells

July 3, 2012Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however, present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure–function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content, have a moderate wettability and employ integrin α[subscript v]β[subscript 3] and α[subscript v]β[subscript 5] engagement with adsorbed vitronectin to promote colony formation. The structure–function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture.National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022)National Institutes of Health (U.S.) (Grant DE016516)Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (Contract W911NF-07-D-0004

Molecular Evidence of the Toxic Effects of Diatom Diets on Gene Expression Patterns in Copepods

Diatoms are dominant photosynthetic organisms in the world's oceans and are considered essential in the transfer of energy through marine food chains. However, these unicellular plants at times produce secondary metabolites such as polyunsaturated aldehydes and other products deriving from the oxidation of fatty acids that are collectively termed oxylipins. These cytotoxic compounds are responsible for growth inhibition and teratogenic activity, potentially sabotaging future generations of grazers by inducing poor recruitment in marine organisms such as crustacean copepods.Here we show that two days of feeding on a strong oxylipin-producing diatom (Skeletonema marinoi) is sufficient to inhibit a series of genes involved in aldehyde detoxification, apoptosis, cytoskeleton structure and stress response in the copepod Calanus helgolandicus. Of the 18 transcripts analyzed by RT-qPCR at least 50% were strongly down-regulated (aldehyde dehydrogenase 9, 8 and 6, cellular apoptosis susceptibility and inhibitor of apoptosis IAP proteins, heat shock protein 40, alpha- and beta-tubulins) compared to animals fed on a weak oxylipin-producing diet (Chaetoceros socialis) which showed no changes in gene expression profiles.Our results provide molecular evidence of the toxic effects of strong oxylipin-producing diatoms on grazers, showing that primary defense systems that should be activated to protect copepods against toxic algae can be inhibited. On the other hand other classical detoxification genes (glutathione S-transferase, superoxide dismutase, catalase, cytochrome P450) were not affected possibly due to short exposure times. Given the importance of diatom blooms in nutrient-rich aquatic environments these results offer a plausible explanation for the inefficient use of a potentially valuable food resource, the spring diatom bloom, by some copepod species