Ian L. Ross

HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA.

The mammalian innate immune system is activated by foreign nucleic acids. Detection of double-stranded DNA (dsDNA) in the cytoplasm triggers characteristic antiviral responses and macrophage cell death. Cytoplasmic dsDNA rapidly activated caspase 3 and caspase 1 in bone marrow–derived macrophages. We identified the HIN-200 family member and candidate lupus susceptibility factor, p202, as a dsDNA binding protein that bound stably and rapidly to transfected DNA. Knockdown studies showed p202 to be an inhibitor of DNA-induced caspase activation. Conversely, the related pyrin domain–containing HIN-200 factor, AIM2 (p210), was required for caspase activation by cytoplasmic dsDNA. This work indicates that HIN-200 proteins can act as pattern recognition receptors mediating responses to cytoplasmic dsDNA.

The mononuclear phagocyte system revisited

The mononuclear phagocyte system (MPS) was defined as a family of cells comprising bone marrow progenitors, blood monocytes, and tissue macrophages. In this review, we briefly consider markers for cells of this lineage in the mouse, especially the F4/80 surface antigen and the receptor for macrophage colony‐stimulating factor. The concept of the MPS is challenged by evidence that there is a separate embryonic phagocyte lineage, the blurring of the boundaries between macrophages and other cells types arising from phenotypic plasticity and transdifferentiation, and evidence of local renewal of tissue macrophage populations as opposed to monocyte recruitment. Nevertheless, there is a unity to cells of the MPS suggested by their location, morphology, and shared markers. We discuss the origins of macrophage heterogeneity and argue that macrophages and antigen‐representing dendritic cells are closely related and part of the MPS.

An economic and technical evaluation of microalgal biofuels

In her News Feature “Biotech’s green gold”, Emily Waltz details the ‘hype’ being propagated around emerging microalgal biofuel technologies, which often exceeds the physical and thermodynamic constraints that ultimately define their economic viability. Our calculations counter such excessive claims and demonstrate that 22 MJ m−2 d−1 solar radiation supports practical yield maxima of ∼60 to 100 kl oil ha−1 y−1 (∼6,600 to 10,800 gal ac−1 y−1) and an absolute theoretical ceiling of ∼94 to 155 kl oil ha−1 y−1, assuming a maximum photosynthetic conversion efficiency of 10%. To evaluate claims and provide an accurate analysis of the potential of microalgal biofuel systems, we have conducted industrial feasibility studies and sensitivity analyses based on peer-reviewed data and industrial expertise. Given that microalgal biofuel research is still young and its development still in flux, we anticipate that the stringent assessment of the technologys economic potential presented below will assist R&D investment and policy development in the area going forward.

Future prospects of microalgal biofuel production systems.

Climate change mitigation, economic growth and stability, and the ongoing depletion of oil reserves are all major drivers for the development of economically rational, renewable energy technology platforms. Microalgae have re-emerged as a popular feedstock for the production of biofuels and other more valuable products. Even though integrated microalgal production systems have some clear advantages and present a promising alternative to highly controversial first generation biofuel systems, the associated hype has often exceeded the boundaries of reality. With a growing number of recent analyses demonstrating that despite the hype, these systems are conceptually sound and potentially sustainable given the available inputs, we review the research areas that are key to attaining economic reality and the future development of the industry.

Selection, breeding and engineering of microalgae for bioenergy and biofuel production.

Microalgal production technologies are seen as increasingly attractive for bioenergy production to improve fuel security and reduce CO(2) emissions. Photosynthetically derived fuels are a renewable, potentially carbon-neutral and scalable alternative reserve. Microalgae have particular promise because they can be produced on non-arable land and utilize saline and wastewater streams. Furthermore, emerging microalgal technologies can be used to produce a range of products such as biofuels, protein-rich animal feeds, chemical feedstocks (e.g. bioplastic precursors) and higher-value products. This review focuses on the selection, breeding and engineering of microalgae for improved biomass and biofuel conversion efficiencies.

Transcription of individual genes in eukaryotic cells occurs randomly and infrequently

Experimental evidence is presented indicating that the expression of a lacZ reporter gene driven by the HIV‐1 long terminal repeat in a series of stably transfected. cloned macrophage cell lines occurs in a very small proportion of cells. The proportion of cells expressing lacZ, rather than the level of expression in each cell, is regulated by external stimuli such as LPS and phorbol ester. Based upon these and published data we propose that transcription in eukaryotic cells occurs in short pulses interspersed by long periods of inactivity of indeterminate duration. Transcriptional regulation is envisaged as involving changes in the probability rather than the rate of transcription. A probabilistic model of transcription may explain many biological phenomena, such as stem cell division and clonogenic activity, heterogeneous gene expression among clonal cell populations, retroviral latency and cell cycle progression, which appear to involve stochastic decisions.

Electroporation and DNA-dependent cell death in murine macrophages

The difficulty of transfecting primary macrophages and macrophage ceil lines has meant that relatively few studies on regulation of gene expression have been performed in these cells. This study has optimized an electroporation procedure for the macrophage cell line RAW 264, but shows that introduction of DNA into the cytoplasm of primary macrophages by electroporation is toxic to the cells. It is proposed that this cell death may have a physiological role in defence against certain viral infections which result in accumulation of cytoplasmic DNA. RAW 264 cells were efficiently transfected by electroporation, but electroporated bone marrow derived macrophages (BMM) showed large scale cell death over a period of 12 h. Electroporation without DNA was not toxic and DNase treatment of samples before transfection prevented cell death. The toxicity of DNA was concentration‐dependent and sequence independent. Synthetic, genomic and plasmid DNA all caused cell death. This sensitivity to DNA seems to be distinct from the antiviral state induced by double‐stranded RNA and may be part of an uncharacterized viral defence system.

RNAi Knock-Down of LHCBM1, 2 and 3 Increases Photosynthetic H2 Production Efficiency of the Green Alga Chlamydomonas reinhardtii

Single cell green algae (microalgae) are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA) is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels). Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3) in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%), LHCBM2 (81.2% ±0.037%) and LHCBM3 (41.4% ±0.05%) compared to 100% control levels, and improved light to H2 (180%) and biomass (165%) conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m−2 s−1) and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface; 2) improved light distribution in the reactor; 3) reduced photoinhibition; 4) early onset of HYDA expression and 5) reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems.

PU.1 and ICSBP control constitutive and IFN‐γ‐regulated Tlr9 gene expression in mouse macrophages

Macrophages are activated by unmethylated CpG‐containing DNA (CpG DNA) via TLR9. IFN‐γ and LPS can synergize with CpG DNA to enhance proinflammatory responses in murine macrophages. Here, we show that LPS and IFN‐γ up‐regulated Tlr9 mRNA in murine bone marrow‐derived macrophages (BMM). The ability of LPS and IFN‐γ to induce Tlr9 mRNA expression in BMM was dependent on the presence of the growth factor, CSF‐1, which is constitutively present in vivo. However, there were clear differences in mechanisms of Tlr9 mRNA induction. LPS stimulation rapidly removed the CSF‐1 receptor (CSF‐1R) from the cell surface, thereby blocking CSF‐1‐mediated transcriptional repression and indirectly inducing Tlr9 mRNA expression. By contrast, IFN‐γ activated the Tlr9 promoter directly and only marginally affected cell surface CSF‐1R expression. An ∼100‐bp proximal promoter of the murine Tlr9 gene was sufficient to confer basal and IFN‐γ‐inducible expression in RAW264.7 cells. A composite IFN regulatory factor (IRF)/PU.1 site upon the major transcription start site was identified. Mutation of the binding sites for PU.1 or IRF impaired basal promoter activity, but only the IRF‐binding site was required for IFN‐γ induction. The mRNA expression of the IRF family member IFN consensus‐binding protein [(ICSBP)/IRF8] was coregulated with Tlr9 in macrophages, and constitutive and IFN‐γ‐inducible Tlr9 mRNA expression was reduced in ICSBP‐deficient BMM. This study therefore characterizes the regulation of mouse Tlr9 expression and defines a molecular mechanism by which IFN‐γ amplifies mouse macrophage responses to CpG DNA.

Isolation of 3,4-dihydroxyphenylalanine-containing proteins using boronate affinity chromatography

A rapid procedure for the isolation of 3,4-dihydroxyphenylalanine-containing proteins has been developed in which the protein is selectively bound to a m-phenylboronate agarose column, and eluted with 1.0 M ammonium acetate, pH 3.0. The method is based on the affinity of boronates for diols including catechol. The chromatography is carried out in the absence of oxygen to prevent oxidation of the catechol. Other proteins are eluted beforehand with 0.25 M ammonium acetate, pH 8.5, or for glycoproteins with a Tris buffer containing 0.2 M sorbitol, pH 8.5.