A microsatellite linkage map of Drosophila mojavensis

Background

Drosophila mojavensishas been a model system for genetic studies of ecological adaptation and speciation. However, despite its use for over half a century, no linkage map has been produced for this species or its close relatives.

Results

We have developed and mapped 90 microsatellites in D. mojavensis, and we present a detailed recombinational linkage map of 34 of these microsatellites. A slight excess of repetitive sequence was observed on the X-chromosome relative to the autosomes, and the linkage groups have a greater recombinational length than the homologous D. melanogaster chromosome arms. We also confirmed the conservation of Muller's elements in 23 sequences between D. melanogaster and D. mojavensis.

Conclusions

The microsatellite primer sequences and localizations are presented here and made available to the public. This map will facilitate future quantitative trait locus mapping studies of phenotypes involved in adaptation or reproductive isolation using this species.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.     

Breast cancer tissue slices as a model for evaluation of response to rapamycin

Rapamycin is a selective inhibitor of the mammalian target of rapamycin (mTOR), a regulator kinase that integrates growth factors signaling via the phosphoinositide-3-kinase pathway and that has emerged as a novel therapeutic modality in breast cancer (BC). We propose a pre-clinical “ex-vivo” personalized organotypic culture of BC that preserves the microenvironment to evaluate rapamycin-mediated gene expression changes. Freshly excised ductal invasive BC slices, 400 μm thick (n=30), were cultured in the presence or absence (control) of rapamycin (20 nM) for 24 h. Some slices were formalin-fixed for immunohistochemical determinations and some were processed for microarray analysis. Control slices in culture retained their tissue morphology and tissue viability (detected by BrdU uptake). The percentage of proliferating cells (assessed by Ki67) did not change up to 24 h of treatment. Immunohistochemical evaluation of p-AKT, p-mTOR, p-4EBP1 and p-S6K1 indicated that AKT/mTOR pathway activation was maintained during cultivation. For microarray analysis, slices were divided into two groups, according to the presence/absence of epidermal growth factor receptor-type 2 and analyzed separately. Limited overlap was seen among differentially expressed genes after treatment (P<0.01) in both groups suggesting different responses to rapamycin between these BC subtypes. Ontology analysis indicated that genes involved in biosynthetic processes were commonly reduced by rapamycin. Our network analysis suggested that concerted expression of these genes might distinguish controls from treated slices. Thus, breast carcinoma slices constitute a suitable physiological tool to evaluate the short-term effects of rapamycin on the gene profile of individual BC samples.     

Biomechanical spinal growth modulation and progressive adolescent scoliosis – a test of the 'vicious cycle' pathogenetic hypothesis: Summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The text for this debate was written by Dr Ian A Stokes. It evaluates the hypothesis that in progressive scoliosis vertebral body wedging during adolescent growth results from asymmetric muscular loading in a "vicious cycle" (vicious cycle hypothesis of pathogenesis) by affecting vertebral body growth plates (endplate physes). A frontal plane mathematical simulation tested whether the calculated loading asymmetry created by muscles in a scoliotic spine could explain the observed rate of scoliosis increase by measuring the vertebral growth modulation by altered compression. The model deals only with vertebral (not disc) wedging. It assumes that a pre-existing scoliosis curve initiates the mechanically-modulated alteration of vertebral body growth that in turn causes worsening of the scoliosis, while everything else is anatomically and physiologically 'normal' The results provide quantitative data consistent with the vicious cycle hypothesis. Dr Stokes' biomechanical research engenders controversy. A new speculative concept is proposed of vertebral symphyseal dysplasia with implications for Dr Stokes' research and the etiology of AIS. What is not controversial is the need to test this hypothesis using additional factors in his current model and in three-dimensional quantitative models that incorporate intervertebral discs and simulate thoracic as well as lumbar scoliosis. The growth modulation process in the vertebral body can be viewed as one type of the biologic phenomenon of mechanotransduction. In certain connective tissues this involves the effects of mechanical strain on chondrocytic metabolism a possible target for novel therapeutic intervention.     

New developments in developmental biology

A report on the 15th International Society of Developmental Biologists Congress, Sydney, Australia, 3-7 September 2005.     

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species

The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.