6-OHDA generated ROS induces DNA damage and p53- and PUMA-dependent cell death

Background

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons in the substantia nigra (SN), resulting in tremor, rigidity, and bradykinesia. Although the etiology is unknown, insight into the disease process comes from the dopamine (DA) derivative, 6-hydroxydopamine (6-OHDA), which produces PD-like symptoms. Studies show that 6-OHDA activates stress pathways, such as the unfolded protein response (UPR), triggers mitochondrial release of cytochrome-c, and activates caspases, such as caspase-3. Because the BH3-only protein, Puma (p53-upregulated mediator of apoptosis), is activated in response to UPR, it is thought to be a link between cell stress and apoptosis.

Results

To test the hypothesis that Puma serves such a role in 6-OHDA-mediated cell death, we compared the response of dopaminergic neurons from wild-type and Puma-null mice to 6-OHDA. Results indicate that Puma is required for 6-OHDA-induced cell death, in primary dissociated midbrain cultures as well as in vivo. In these cultures, 6-OHDA-induced DNA damage and p53 were required for 6-OHDA-induced cell death. In contrast, while 6-OHDA led to upregulation of UPR markers, loss of ATF3 did not protect against 6-OHDA.

Conclusions

Together, our results indicate that 6-OHDA-induced upregulation of Puma and cell death are independent of UPR. Instead, p53 and DNA damage repair pathways mediate 6-OHDA-induced toxicity.     

A mosaic genetic screen for novel mutations affecting Drosophila neuroblast divisions

Background

The asymmetric segregation of determinants during cell division is a fundamental mechanism for generating cell fate diversity during development. In Drosophila, neural precursors (neuroblasts) divide in a stem cell-like manner generating a larger apical neuroblast and a smaller basal ganglion mother cell. The cell fate determinant Prospero and its adapter protein Miranda are asymmetrically localized to the basal cortex of the dividing neuroblast and segregated into the GMC upon cytokinesis. Previous screens to identify components of the asymmetric division machinery have concentrated on embryonic phenotypes. However, such screens are reaching saturation and are limited in that the maternal contribution of many genes can mask the effects of zygotic loss of function, and other approaches will be necessary to identify further genes involved in neuroblast asymmetric division.

Results

We have performed a genetic screen in the third instar larval brain using the basal localization of Miranda as a marker for neuroblast asymmetry. In addition to the examination of pupal lethal mutations, we have employed the MARCM (Mosaic Analysis with a Repressible Cell Marker) system to generate postembryonic clones of mutations with an early lethal phase. We have screened a total of 2,300 mutagenized chromosomes and isolated alleles affecting cell fate, the localization of basal determinants or the orientation of the mitotic spindle. We have also identified a number of complementation groups exhibiting defects in cell cycle progression and cytokinesis, including both novel genes and new alleles of known components of these processes.

Conclusion

We have identified four mutations which affect the process of neuroblast asymmetric division. One of these, mapping to the imaginal discs arrested locus, suggests a novel role for the anaphase promoting complex/cyclosome (APC/C) in the targeting of determinants to the basal cortex. The identification and analysis of the remaining mutations will further advance our understanding of the process of asymmetric cell division. We have also isolated a number of mutations affecting cell division which will complement the functional genomics approaches to this process being employed by other laboratories. Taken together, these results demonstrate the value of mosaic screens in the identification of genes involved in neuroblast division.     

A proof of the DBRF-MEGN method, an algorithm for deducing minimum equivalent gene networks

Background

We previously developed the DBRF-MEGN (difference-based regulation finding-minimum equivalent gene network) method, which deduces the most parsimonious signed directed graphs (SDGs) consistent with expression profiles of single-gene deletion mutants. However, until the present study, we have not presented the details of the method's algorithm or a proof of the algorithm.

Results

We describe in detail the algorithm of the DBRF-MEGN method and prove that the algorithm deduces all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.

Conclusions

The DBRF-MEGN method provides all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.     

The COG database: an updated version includes eukaryotes

Background

The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies.

Results

We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (~1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes.

Conclusion

The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.     

Multiscale Hy3S: Hybrid stochastic simulation for supercomputers

Background

DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell.

Results

We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100, 000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods.

Conclusion

Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.     

Identification and characterization of SHI family genes from Brassica rapa L. ssp. pekinensis

SHI (short internodes) is a negative regulator of gibberellin-induced cell elongation. Extensive searches in the Brassica rapa genome allowed for the prediction of at least six different SHI-related genes on six chromosomes in the genome. Genome structural examination revealed that these genes had one intron each in their corresponding open reading frames. Protein structure comparisons using the CLUSTALW program and based on alignments of all BrSRS (B. r apa SHI-related sequence) proteins revealed broad conservation of the RING finger-like zinc finger and IGGH motifs. According to the phylogenetic relationship based on deduced amino acid sequences, the six BrSRS proteins were most closely related to Arabidopsis SRS (AtSRS) proteins; however, BrSRS proteins were dispersed in the phylogenetic tree. Semi-quantitative RT-PCR analysis indicated that the six BrSRS genes exhibited different expression patterns in various tissues and responded differently to growth phytohormones. The differences among the six BrSRS genes with respect to gene structure and expression pattern suggest that these genes may play diverse physiological roles in the developmental process of B. rapa.     

A novel allele of the P-starvation tolerance gene OsPSTOL1 from African rice (Oryza glaberrima Steud) and its distribution in the genus Oryza

Key message

We have developed allele-specific markers for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs, to improve their tolerance to P-deficient soil.

Abstract

The deficiency of phosphorus (P) in soil is a major problem in Sub-Saharan Africa due to general nutrient depletion and the presence of P-fixing soils. Developing rice cultivars with enhanced P efficiency would, therefore, represent a sustainable strategy to improve the livelihood of resource-poor farmers. Recently the Pup1 locus, a major QTL for tolerance to P deficiency in soil, was successfully narrowed-down to a major gene, the protein kinase OsPSTOL1 (P-starvation tolerance), which was found to be generally absent from modern irrigated rice varieties. Our target is to improve the tolerance of African mega-varieties to P deficiency through marker-assisted introgression of PSTOL1. As a first step, we have determined the Pup1 haplotype and surveyed the presence or absence of PSTOL1 and other genes of the Pup1 locus in African mega-varieties, NERICAs (New Rice for Africa) and their Oryza glaberrima parents. Here, we report the presence of a novel PSTOL1 allele in upland NERICAs that was inherited from the O. glaberrima parent CG14. This allele showed a 35 base-pair substitution when aligned to the Kasalath allele, but maintained a fully conserved kinase domain, and is present in most O. glaberrima accessions evaluated. In-silico and marker analysis indicated that many other genes of the Kasalath Pup1 locus were missing in the O. glaberrima genome, including the dirigent-like gene OsPupK20-2, which was shown to be downstream of PSTOL1. We have developed several allele-specific markers for the use for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs.     

The large (134.9 kb) mitochondrial genome of the glomeromycete <Emphasis Type="Italic">Funneliformis mosseae</Emphasis>

Funneliformis mosseae is among the most ecologically and economically important glomeromycete species and occurs both in natural and disturbed areas in a wide range of habitats and climates. In this study, we report the sequencing of the complete mitochondrial (mt) genome of F. mosseae isolate FL299 using 454 pyrosequencing and Illumina HiSeq technologies. This mt genome is a full-length circular chromosome of 134,925 bp, placing it among the largest mitochondrial DNAs (mtDNAs) in the fungal kingdom. A comparative analysis with publically available arbuscular mycorrhizal fungal mtDNAs revealed that the mtDNA of F. mosseae FL299 contained a very large number of insertions contributing to its expansion. The gene synteny was completely reshuffled compared to previously published glomeromycotan mtDNAs and several genes were oriented in an anti-sense direction. Furthermore, the presence of different types of introns and insertions in rnl (14 introns) made this gene very distinctive in Glomeromycota. The presence of alternative genetic codes in both initiation (GUG) and termination (UGA) codons was another new feature in this mtDNA compared to previously published glomeromycotan mt genomes. The phylogenetic analysis inferred from the analysis of 14 protein mt genes confirmed the position of the Glomeromycota clade as a sister group of Mortierellomycotina. This mt genome is the largest observed so far in Glomeromycota and the first mt genome within the Funneliformis clade, providing new opportunities to better understand their evolution and to develop molecular markers.     

The Smc complexes in DNA damage response

Background

Caenorhabditis elegans sarcomeres have been studied extensively utilizing both forward and reverse genetic techniques to provide insight into muscle development and the mechanisms behind muscle contraction. A previous genetic screen investigating early muscle development produced 13 independent mutant genes exhibiting a Pat (paralyzed and arrested elongation at the two-fold length of embryonic development) muscle phenotype. This study reports the identification and characterization of one of those genes, pat-9.

Results

Positional cloning, reverse genetics, and plasmid rescue experiments were used to identify the predicted C. elegans gene T27B1.2 (recently named ztf-19) as the pat-9 gene. Analysis of pat-9 showed it is expressed early in development and within body wall muscle lineages, consistent with a role in muscle development and producing a Pat phenotype. However, unlike most of the other known Pat gene family members, which encode structural components of muscle attachment sites, PAT-9 is an exclusively nuclear protein. Analysis of the predicted PAT-9 amino acid sequence identified one putative nuclear localization domain and three C2H2 zinc finger domains. Both immunocytochemistry and PAT-9::GFP fusion expression confirm that PAT-9 is primarily a nuclear protein and chromatin immunoprecipitation (ChIP) experiments showed that PAT-9 is present on certain gene promoters.

Conclusions

We have shown that the T27B1.2 gene is pat-9. Considering the Pat-9 mutant phenotype shows severely disrupted muscle attachment sites despite PAT-9 being a nuclear zinc finger protein and not a structural component of muscle attachment sites, we propose that PAT-9 likely functions in the regulation of gene expression for some necessary structural or regulatory component(s) of the muscle attachment sites.     

Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D

Background

Patients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.

Methods

Lung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES (Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.

Results

TNF-α increased the expression of RANTES (307.84 ± 33.56 versus 207.64 ± 31.27 pg/ml of control, p = 0.025) and eotaxin (108.97 ± 10.87 versus 54.00 ± 5.29 pg/ml of control, p = 0.041). Melatonin(10^-10 to 10^-6M) alone didn't change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES (410.88 ± 52.03, 483.60 ± 55.37, 559.92 ± 75.70, 688.42 ± 95.32, 766.39 ± 101.53 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7,10^-6M melatonin, respectively) and eotaxin (151.95 ± 13.88, 238.79 ± 16.81, 361.62 ± 36.91, 393.66 ± 44.89, 494.34 ± 100.95 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7, 10^-6M melatonin, respectively) expression in a dose dependent manner in A549 cells (compared with TNF-α alone, P < 0.05). The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.

Conclusion

Taken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell.     

Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites

Background

High throughput techniques have generated a huge set of biological data, which are deposited in various databases. Efficient exploitation of these databases is often hampered by a lack of appropriate tools, which allow easy and reliable identification of genes that miss functional characterization but are correlated with specific biological conditions (e.g. organotypic expression).

Results

We have developed a simple algorithm (DGSA = Database-dependent Gene Selection and Analysis) to identify genes with unknown functions involved in organ development concentrating on the heart. Using our approach, we identified a large number of yet uncharacterized genes, which are expressed during heart development. An initial functional characterization of genes by loss-of-function analysis employing morpholino injections into zebrafish embryos disclosed severe developmental defects indicating a decisive function of selected genes for developmental processes.

Conclusion

We conclude that DGSA is a versatile tool for database mining allowing efficient selection of uncharacterized genes for functional analysis.     

The Mitochondrial Genome of an Aquatic Plant,Spirodela polyrhiza

Background

Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant.

Methods

Spirodela polyrhiza mt genome was sequenced from total genomic DNA without physical separation of chloroplast and nuclear DNA using the SOLiD platform. Using a genome copy number sensitive assembly algorithm, the mt genome was successfully assembled. Gap closure and accuracy was determined with PCR products sequenced with the dideoxy method.

Conclusions

This is the most compact monocot mitochondrial genome with 228,493 bp. A total of 57 genes encode 35 known proteins, 3 ribosomal RNAs, and 19 tRNAs that recognize 15 amino acids. There are about 600 RNA editing sites predicted and three lineage specific protein-coding-gene losses. The mitochondrial genes, pseudogenes, and other hypothetical genes (ORFs) cover 71,783 bp (31.0%) of the genome. Imported plastid DNA accounts for an additional 9,295 bp (4.1%) of the mitochondrial DNA. Absence of transposable element sequences suggests that very few nuclear sequences have migrated into Spirodela mtDNA. Phylogenetic analysis of conserved protein-coding genes suggests that Spirodela shares the common ancestor with other monocots, but there is no obvious synteny between Spirodela and rice mtDNAs. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly four-fifths of the Spirodela mitochondrial genome is of unknown origin and function. Although it contains a similar chloroplast DNA content and range of RNA editing as other monocots, it is void of nuclear insertions, active gene loss, and comprises large regions of sequences of unknown origin in non-coding regions. Moreover, the lack of synteny with known mitochondrial genomic sequences shed new light on the early evolution of monocot mitochondrial genomes.     

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

Background

Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved.

Results

Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya (S. c. hoya) and Pycnonotus sinensis (P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished.

Conclusion

In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.     

Osteopathic manipulative treatment and its relationship to autonomic nervous system activity as demonstrated by heart rate variability: a repeated measures study

Background

Osteopathic manipulative treatment (OMT) and ultrasound physical therapy (UPT) are commonly used for chronic low back pain. Although there is evidence from a systematic review and meta-analysis that OMT generally reduces low back pain, there are no large clinical trials that specifically assess OMT efficacy in chronic low back pain. Similarly, there is a lack of evidence involving UPT for chronic low back pain.

Methods

The OSTEOPAThic Health outcomes In Chronic low back pain (OSTEOPATHIC) Trial is a Phase III randomized controlled trial that seeks to study 488 subjects between August 2006 and June 2010. It uses a 2 × 2 factorial design to independently assess the efficacy of OMT and UPT for chronic low back pain. The primary outcome is a visual analogue scale score for pain. Secondary outcomes include back-specific functioning, generic health, work disability, and satisfaction with back care.

Conclusion

This randomized controlled trial will potentially be the largest involving OMT. It will provide long awaited data on the efficacy of OMT and UPT for chronic low back pain.

Trial registration

http://www.clinicaltrials.gov, NCT00315120     

The Complete Mitochondrial Genome of Gossypium hirsutum and Evolutionary Analysis of Higher Plant Mitochondrial Genomes

Background

Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes.

Methodology/Principal Findings

We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes.

Conclusion

The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.     

GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists

Background

Although expression microarrays have become a standard tool used by biologists, analysis of data produced by microarray experiments may still present challenges. Comparison of data from different platforms, organisms, and labs may involve complicated data processing, and inferring relationships between genes remains difficult.

Results

S TAR N ET 2 is a new web-based tool that allows post hoc visual analysis of correlations that are derived from expression microarray data. S TAR N ET 2 facilitates user discovery of putative gene regulatory networks in a variety of species (human, rat, mouse, chicken, zebrafish, Drosophila, C. elegans, S. cerevisiae, Arabidopsis and rice) by graphing networks of genes that are closely co-expressed across a large heterogeneous set of preselected microarray experiments. For each of the represented organisms, raw microarray data were retrieved from NCBI's Gene Expression Omnibus for a selected Affymetrix platform. All pairwise Pearson correlation coefficients were computed for expression profiles measured on each platform, respectively. These precompiled results were stored in a MySQL database, and supplemented by additional data retrieved from NCBI. A web-based tool allows user-specified queries of the database, centered at a gene of interest. The result of a query includes graphs of correlation networks, graphs of known interactions involving genes and gene products that are present in the correlation networks, and initial statistical analyses. Two analyses may be performed in parallel to compare networks, which is facilitated by the new H EAT S EEKER module.

Conclusion

S TAR N ET 2 is a useful tool for developing new hypotheses about regulatory relationships between genes and gene products, and has coverage for 10 species. Interpretation of the correlation networks is supported with a database of previously documented interactions, a test for enrichment of Gene Ontology terms, and heat maps of correlation distances that may be used to compare two networks. The list of genes in a S TAR N ET network may be useful in developing a list of candidate genes to use for the inference of causal networks. The tool is freely available at http://vanburenlab.medicine.tamhsc.edu/starnet2.html, and does not require user registration.     

Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives

Background

The apparent scarcity of available sequence data has greatly impeded evolutionary studies in Acari (mites and ticks). This subclass encompasses over 48,000 species and forms the largest group within the Arachnida. Although mitochondrial genomes are widely utilised for phylogenetic and population genetic studies, only 20 mitochondrial genomes of Acari have been determined, of which only one belongs to the diverse order of the Sarcoptiformes. In this study, we describe the mitochondrial genome of the European house dust mite Dermatophagoides pteronyssinus, the most important member of this largely neglected group.

Results

The mitochondrial genome of D. pteronyssinus is a circular DNA molecule of 14,203 bp. It contains the complete set of 37 genes (13 protein coding genes, 2 rRNA genes and 22 tRNA genes), usually present in metazoan mitochondrial genomes. The mitochondrial gene order differs considerably from that of other Acari mitochondrial genomes. Compared to the mitochondrial genome of Limulus polyphemus, considered as the ancestral arthropod pattern, only 11 of the 38 gene boundaries are conserved. The majority strand has a 72.6% AT-content but a GC-skew of 0.194. This skew is the reverse of that normally observed for typical animal mitochondrial genomes. A microsatellite was detected in a large non-coding region (286 bp), which probably functions as the control region. Almost all tRNA genes lack a T-arm, provoking the formation of canonical cloverleaf tRNA-structures, and both rRNA genes are considerably reduced in size. Finally, the genomic sequence was used to perform a phylogenetic study. Both maximum likelihood and Bayesian inference analysis clustered D. pteronyssinus with Steganacarus magnus, forming a sistergroup of the Trombidiformes.

Conclusion

Although the mitochondrial genome of D. pteronyssinus shares different features with previously characterised Acari mitochondrial genomes, it is unique in many ways. Gene order is extremely rearranged and represents a new pattern within the Acari. Both tRNAs and rRNAs are truncated, corroborating the theory of the functional co-evolution of these molecules. Furthermore, the strong and reversed GC- and AT-skews suggest the inversion of the control region as an evolutionary event. Finally, phylogenetic analysis using concatenated mt gene sequences succeeded in recovering Acari relationships concordant with traditional views of phylogeny of Acari.     

Towards a noninvasive anatomical and functional diagnostic work-up of patients with suspected coronary artery disease

Combining multidetector computed tomography and cardiovascular magnetic resonance imaging provides the clinician a strategy to comprehensively evaluate coronary morphology and function noninvasively. In the MARCC trial (Magnetic Resonance and CT in suspected CAD) a new noninvasive diagnostic work-up for patients with suspected coronary artery disease will be developed, involving the sequential use of both imaging techniques. (Neth Heart J 2010;18:270-3.)