Bioenergetic Profiling of Zebrafish Embryonic Development

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility

Crystal Structures, Metal Activation, and DNA-Binding Properties of Two-Domain IdeR from Mycobacterium tuberculosis

The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co^(2+) and Ni^(2+) activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni^(2+) concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co^(2+) required for activation remains the same. We have determined the crystal structures of Ni^(2+)-activated 2D-IdeR at 1.96 Å resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 Å and 2.6 Å, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR−DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His_6. This C-terminal tail promotes crystal contacts by forming a β-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins

Crystal Structures, Metal Activation, and DNA-Binding Properties of Two-Domain IdeR from Mycobacterium tuberculosis

The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co^(2+) and Ni^(2+) activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni^(2+) concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co^(2+) required for activation remains the same. We have determined the crystal structures of Ni^(2+)-activated 2D-IdeR at 1.96 Å resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 Å and 2.6 Å, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR−DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His_6. This C-terminal tail promotes crystal contacts by forming a β-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins

Identification of heparin modifications and polysaccharide inhibitors of Plasmodium falciparum merozoite invasion that have potential for novel drug development

Despite recent successful control efforts, malaria remains a leading global health burden. Alarmingly, resistance to current antimalarials is increasing and the development of new drug families is needed to maintain malaria control. Current antimalarials target the intraerythrocytic developmental stage of the Plasmodium falciparum life cycle. However, the invasive extracellular parasite form, the merozoite, is also an attractive target for drug development. We have previously demonstrated that heparin-like molecules, including those with low molecular weights and low anticoagulant activities, are potent and specific inhibitors of merozoite invasion and blood-stage replication. Here we tested a large panel of heparin-like molecules and sulfated polysaccharides together with various modified chemical forms for their inhibitory activity against P. falciparum merozoite invasion. We identified chemical modifications that improve inhibitory activity and identified several additional sulfated polysaccharides with strong inhibitory activity. These studies have important implications for the further development of heparin-like molecules as antimalarial drugs and for understanding merozoite invasion

The Diploid Genome Sequence of an Individual Human

Presented here is a genome sequence of an individual human. It was produced from ∼32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2–206 bp), 292,102 heterozygous insertion/deletion events (indels)(1–571 bp), 559,473 homozygous indels (1–82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information

Specific Receptor Usage in Plasmodium falciparum Cytoadherence Is Associated with Disease Outcome

Our understanding of the basis of severe disease in malaria is incomplete. It is clear that pathology is in part related to the pro-inflammatory nature of the host response but a number of other factors are also thought to be involved, including the interaction between infected erythrocytes and endothelium. This is a complex system involving several host receptors and a major parasite-derived variant antigen (PfEMP1) expressed on the surface of the infected erythrocyte membrane. Previous studies have suggested a role for ICAM-1 in the pathology of cerebral malaria, although these have been inconclusive. In this study we have examined the cytoadherence patterns of 101 patient isolates from varying clinical syndromes to CD36 and ICAM-1, and have used variant ICAM-1 proteins to further characterise this adhesive phenotype. Our results show that increased binding to CD36 is associated with uncomplicated malaria while ICAM-1 adhesion is raised in parasites from cerebral malaria cases

Light source selection for a solar simulator for thermal applications: A review

Solar simulators are used to test components and systems under controlled and repeatable conditions, often in locations with unsuitable insolation for outdoor testing. The growth in renewable energy generation has led to an increased need to develop, manufacture and test components and subsystems for solar thermal, photovoltaic (PV), and concentrating optics for both thermal and electrical solar applications. At the heart of any solar simulator is the light source itself. This paper reviews the light sources available for both low and high-flux solar simulators used for thermal applications. Criteria considered include a comparison of the lamp wavelength spectrum with the solar spectrum, lamp intensity, cost, stability, durability, and any hazards associated with use. Four main lamp types are discussed in detail, namely argon arc, the metal halide, tungsten halogen lamp, and xenon arc lamps. In addition to describing the characteristics of each lamp type, the popularity of usage of each type over time is also indicated. This is followed by guidelines for selecting a suitable lamp, depending on the requirements of the user and the criteria applied for selection. The appropriate international standards are also addressed and discussed. The review shows that metal halide and xenon arc lamps predominate, since both provide a good spectral match to the solar output. The xenon lamp provides a more intense and stable output, but has the disadvantages of being a high-pressure component, requiring infrared filtering, and the need of a more complex and expensive power supply. As a result, many new solar simulators prefer metal halide lamps