Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer''s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.     

ESS++: a C++ objected-oriented algorithm for Bayesian stochastic search model exploration

SUMMARY: ESS++ is a C++ implementation of a fully Bayesian variable selection approach for single and multiple response linear regression. ESS++ works well both when the number of observations is larger than the number of predictors and in the 'large p, small n' case. In the current version, ESS++ can handle several hundred observations, thousands of predictors and a few responses simultaneously. The core engine of ESS++ for the selection of relevant predictors is based on Evolutionary Monte Carlo. Our implementation is open source, allowing community-based alterations and improvements. AVAILABILITY: C++ source code and documentation including compilation instructions are available under GNU licence at http://bgx.org.uk/software/ESS.html.     

In the right place at the right time: Parnassia resolves the herkogamy dilemma by accurate repositioning of stamens and stigmas

Background and Aims

Spatial (herkogamy) and temporal (dichogamy) separation of pollen presentation and stigma receptivity have been interpreted as reducing interference between male and female functions in hermaphroditic flowers. However, spatial separation leads to a potential conflict: reduced pollination accuracy, where pollen may be placed in a location on the pollinator different from the point of stigma contact.

Methods

To understand better how herkogamous flowers resolve this conflict, a study was made of a subalpine herb, Parnassia epunctulata, the nectariferous flowers of which exhibit sequential anther dehiscence (staggered pollen presentation) and stamen movements; usually one newly dehisced anther is positioned each day over the central gynoecium, while the older stamens bend away from the central position.

Key Results

The open flowers were visited by a variety of pollinators, most of which were flies. Seed set was pollinator-dependent (bagged flowers set almost no seeds) and pollen-limited (manual pollination increased seed set over open pollination). Analyses of adaptive accuracy showed that coordinated stamen movements and style elongation (movement herkogamy) dramatically increased pollination accuracy. Specifically, dehiscing anthers and receptive stigmas were positioned accurately in the vertical and horizontal planes in relation to the opposite sexual structure and pollinator position. By contrast, the spatial correspondence between anthers and stigma was dramatically lower before the anthers dehisced and after stamens bent outwards, as well as before and after the period of stigmatic receptivity.

Conclusions

It is shown for the first time that a combination of movement herkogamy and dichogamy can maintain high pollination accuracy in flowers with generalized pollination. Staggered pollen and stigma presentation with spatial correspondence can both reduce sexual interference and improve pollination accuracy.     

Activation of a Cysteine Protease in MCF-7 and T47D Breast Cancer Cells during β-Lapachone-Mediated Apoptosis

β-Lapachone (β-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, β-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of β-lap, a novel non-caspase-mediated “atypical” cleavage of PARP (i.e., an 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a β-lap-mediated activation of calpain. β-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that β-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.     

Sequence analysis and editing for bisulphite genomic sequencing projects

Bisulphite genomic sequencing is a widely used technique for detailed analysis of the methylation status of a region of DNA. It relies upon the selective deamination of unmethylated cytosine to uracil after treatment with sodium bisulphite, usually followed by PCR amplification of the chosen target region. Since this two-step procedure replaces all unmethylated cytosine bases with thymine, PCR products derived from unmethylated templates contain only three types of nucleotide, in unequal proportions. This can create a number of technical difficulties (e.g. for some base-calling methods) and impedes manual analysis of sequencing results (since the long runs of T or A residues are difficult to align visually with the parent sequence). To facilitate the detailed analysis of bisulphite PCR products (particularly using multiple cloned templates), we have developed a visually intuitive program that identifies the methylation status of CpG dinucleotides by analysis of raw sequence data files produced by MegaBace or ABI sequencers as well as Staden SCF trace files and plain text files. The program then also collates and presents data derived from independent templates (e.g. separate clones). This results in a considerable reduction in the time required for completion of a detailed genomic methylation project.     

Epstein-Barr virus-encoded BILF1 is a constitutively active G protein-coupled receptor

Both beta- and gammaherpesviruses encode G protein-coupled receptors (GPCRs) with unique pharmacological phenotypes and important biological functions. An example is ORF74, the gamma2-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded GPCR, which is highly constitutively active and considered the key oncogene in Kaposi's sarcoma pathogenesis. In contrast, the current annotation of the Epstein-Barr virus (EBV) genome does not reveal any GPCR homolog encoded by this human oncogenic gamma1-herpesvirus. However, by employing bioinformatics, we recognized that the previously established EBV open reading frame BILF1 indeed encodes a GPCR. Additionally, BILF1 is a member of a new family of related GPCRs exclusively encoded by gamma1-herpesviruses. Expression of hemagglutinin-tagged BILF1 in the HEK293 epithelial cell line revealed that BILF1 is expressed as an approximately 50-kDa glycosylated protein. Immunocytochemistry and confocal microscopy revealed that BILF1 localizes predominantly to the plasma membrane, similar to the localization of KSHV ORF74. Using chimeric G proteins, we found that human and rhesus EBV-encoded BILF1 are highly potent constitutively active receptors, activating Galphai. Furthermore, BILF1 is able to inhibit forskolin-triggered CREB activation via stimulation of endogenous G proteins in a pertussis toxin-sensitive manner, verifying that BILF1 signals constitutively through Galphai. We suggest that EBV may use BILF1 to regulate Galphai-activated pathways during viral lytic replication, thereby affecting disease progression.     

Structure of the bundle-forming pilus from enteropathogenic Escherichia coli

Bundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization.