PhotoMorphs™: A novel light‐activated reagent for controlling gene expression in zebrafish

Manipulating gene expression in zebrafish is critical for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly used antisense technology for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel light‐ activatable knockdown reagent called PhotoMorph?. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand containing a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradiation of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein, No tail, and E‐cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E‐cadherin knockdown. A splice‐blocking PhotoMorph directed to the rheb gene showed light‐dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of laboratory reagents suitable for the spatiotemporal control of gene expression in the zebrafish. genesis 47:736–743, 2009. © 2009 Wiley‐Liss, Inc.     

BIODIVERSITY RESEARCH: Conserving macroinvertebrate diversity in headwater streams: the importance of knowing the relative contributions of α and β diversity

Aim We investigated partitioning of aquatic macroinvertebrate diversity in eight headwater streams to determine the relative contributions of α and β diversity to γ diversity, and the scale dependence of α and β components. Location Great Dividing Range, Victoria, Australia. Methods We used the method of Jost (Ecology, 2007, 88, 2427–2439) to partition γ diversity into its α and β components. We undertook the analyses at both reach and catchment scales to explore whether inferences depended on scale of observation. Results We hypothesized that β diversity would make a large contribution to the γ diversity of macroinvertebrates in our dendritic riverine landscape, particularly at the larger spatial scale (among catchments) because of limited dispersal among sites and especially among catchments. However, reaches each had relatively high taxon richness and high α diversity, while β diversity made only a small contribution to γ diversity at both the reach and catchment scales. Main conclusions Dendritic riverine landscapes have been thought to generate high β diversity as a consequence of limited dispersal and high heterogeneity among individual streams, but this may not hold for all headwater stream systems. Here, α diversity was high and β diversity low, with individual headwater stream reaches each containing a large portion of γ diversity. Thus, each stream could be considered to have low irreplaceability since losing the option to use one of these sites in a representative reserve network does not greatly diminish the options available for completing the reserve network. Where limited information on individual taxonomic distributions is available, or time and money for modelling approaches are limited, diversity partitioning may provide a useful ‘first‐cut’ for obtaining information about the irreplaceability of individual streams or subcatchments when establishing representative freshwater reserves.     

Solubility engineering and crystallization of human apolipoprotein D

Human apolipoprotein D (ApoD) is a physiologically important member of the lipocalin protein family that was discovered as a peripheral subunit of the high-density lipoprotein (HDL) but is also abundant in other body fluids and organs, including neuronal tissue. Although it has been possible to produce functional ApoD in the periplasm of Escherichia coli and to demonstrate its ligand-binding activity for progesterone and arachidonic acid, the recombinant protein suffers from a pronounced tendency to aggregate and to adsorb to vessel surfaces as well as chromatography matrices, thus hampering further structural investigation. Here, we describe a systematic mutagenesis study directed at presumably exposed hydrophobic side chains of the unglycosylated recombinant protein. As a result, one ApoD mutant with just three new amino acid substitutions--W99H, I118S, and L120S--was identified, which exhibits the following features: (1) improved yield upon periplasmic biosynthesis in E. coli, (2) elution as a monomeric protein from a gel permeation chromatography column, and (3) unchanged binding activity for its physiological ligands. In addition, the engineered ApoD was successfully crystallized (space group I4 with unit cell parameters a = 75.1 A, b = 75.1 A, c = 166.0 A, alpha = beta = gamma = 90 degrees), thus demonstrating its conformationally homogeneous behavior and providing a basis for the future X-ray structural analysis of this functionally still puzzling protein.     

Erythropoietin protects against 6-hydroxydopamine-induced dopaminergic cell death

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopaminergic neurons. In the present study, erythropoietin, a trophic factor that has both hematopoietic and neural protective characteristics, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. Using both the dopaminergic cell line, MN9D, and primary dopamine neurons, we show that erythropoietin (1-3 U/mL) is neuroprotective against the dopaminergic neurotoxin, 6-hydroxydopamine. Protection was mediated by the erythropoietin receptor, as neutralizing anti-erythropoietin receptor antibody abrogated the protection. Activation of Akt/protein kinase B (PKB), via the phosphoinositide 3-kinase pathway, is a critical mechanism in erythropoietin-induced protection, while activation of extracellular signal-regulated kinase (ERK)1/2 contributes only moderately. Indeed, transfection of constitutively active Akt/PKB into dopaminergic cells was sufficient to protect against cell death. Furthermore, erythropoietin diminished markers of apoptosis in MN9D cells, including caspase 9 and caspase 3 activation and internucleosomal DNA fragmentation, suggesting that erythropoietin interferes with the apoptosis-execution process. When erythropoietin was administered to mice unilaterally lesioned with 6-hydroxydopamine, it prevented the loss of nigral dopaminergic neurons and maintained striatal catecholamine levels for at least 8 weeks. Erythropoietin-treated mice also had significantly reduced behavioral asymmetries. These studies suggest that erythropoietin can be an effective neuroprotective agent for dopaminergic neurons, and may be useful in reversing behavioral deficits associated with Parkinson's disease.     

A cytochrome b561 with ferric reductase activity from the parasitic blood fluke, Schistosoma japonicum

Background

Iron has an integral role in numerous cellular reactions and is required by virtually all organisms. In physiological conditions, iron is abundant in a largely insoluble ferric state. Ferric reductases are an essential component of iron uptake by cells, reducing iron to the soluble ferrous form. Cytochromes b561 (cyts-b561) are a family of ascorbate reducing transmembrane proteins found in most eukaryotic cells. The identification of the ferric reductase duodenal cytochrome b (dcytb) and recent observations that other cyts-b561 may be involved in iron metabolism have opened novel perspectives for elucidating their physiological function.

Methodology/Principal Findings

Here we have identified a new member of the cytochrome b561 (Sjcytb561) family in the pathogenic blood fluke Schistosoma japonicum that localises to the outer surface of this parasitic trematode. Heterologous expression of recombinant Sjcyt-b561 in a Saccharomyces cerevisiae mutant strain that lacks plasma membrane ferrireductase activity demonstrated that the molecule could rescue ferric reductase activity in the yeast.

Significance/Conclusions

This finding of a new member of the cytochrome b561 family further supports the notion that a ferric reductase function is likely for other members of this protein family. Additionally, the localisation of Sjcytb561 in the surface epithelium of these blood-dwelling schistosomes contributes further to our knowledge concerning nutrient acquisition in these parasites and may provide novel targets for therapeutic intervention.     

Prenatal screening of fetal ventriculoarterial connections: benefits of 4D technique in fetal heart imaging

Background

Identification of prenatal ventriculoarterial connections in fetuses with conotruncal anomalies (CTA) remains one of the greatest challenges for sonographers performing screening examinations. Herein, we propose a novel protocol of 4D volume analysis that identifies ventriculoarterial connections and evaluate its clinical utility in routine screenings.

Methods

Twenty-nine cases of transposition of the great arteries (TGA), 22 cases of double-outlet right ventricle (DORV), 36 cases of tetralogy of Fallot (TOF), 14 cases of truncus arteriosus (TCA), and randomly selected 70 normal fetuses were reviewed in this study. All cases were evaluated using 2D data alone (2D method), post-processing volumes with no exact algorithm (4D-1 method), or with the proposed algorithm (4D-2 method), or using the 2D and 4D data together (combined method). Comparisons were made to evaluate the detection rate of ventriculoarterial connections for these different methods.

Results

During 18–28 gestational weeks, the detection rate of 4D-2 modality was satisfactory. The detection rate of the combined method was significantly higher than 2D method in the identification of TGA, TOF, and TCA. The detection rate of 4D-1 method was significantly lower than 4D ?2 modality for CTA fetuses. During late pregnancy, the detection rate for both 4D modalities was very low due to the poor quality of the 4D volumes.

Conclusions

We proposed a detailed protocol, which allowed the examiner to identify fetal ventriculoarterial connections by 4D volumes. Inclusion of blood information into the volumes improved diagnosis. Our findings suggest that the incorporation of 4D STIC into routine screenings could improve the detection for TGA, TOF, and TCA.

     

Mapping interactions between germinants and Clostridium difficile spores

Germination of Clostridium difficile spores is the first required step in establishing C. difficile-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of C. difficile spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit C. difficile spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The C. difficile germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However, L-phenylalanine and L-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6- and 7-hydroxyl groups are required for inhibition of C. difficile spore germination. Similarly, C. difficile spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an m-aminobenzenesulfonic acid side chain is a strong inhibitor of C. difficile spore germination. In conclusion, C. difficile spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.