Multiple independent origins of mitochondrial control region duplications in the order Psittaciformes

Mitochondrial genomes are generally thought to be under selection for compactness, due to their small size, consistent gene content, and a lack of introns or intergenic spacers. As more animal mitochondrial genomes are fully sequenced, rearrangements and partial duplications are being identified with increasing frequency, particularly in birds (Class Aves). In this study, we investigate the evolutionary history of mitochondrial control region states within the avian order Psittaciformes (parrots and cockatoos). To this aim, we reconstructed a comprehensive multi-locus phylogeny of parrots, used PCR of three diagnostic fragments to classify the mitochondrial control region state as single or duplicated, and mapped these states onto the phylogeny. We further sequenced 44 selected species to validate these inferences of control region state. Ancestral state reconstruction using a range of weighting schemes identified six independent origins of mitochondrial control region duplications within Psittaciformes. Analysis of sequence data showed that varying levels of mitochondrial gene and tRNA homology and degradation were present within a given clade exhibiting duplications. Levels of divergence between control regions within an individual varied from 0-10.9% with the differences occurring mainly between 51 and 225 nucleotides 3' of the goose hairpin in domain I. Further investigations into the fates of duplicated mitochondrial genes, the potential costs and benefits of having a second control region, and the complex relationship between evolutionary rates, selection, and time since duplication are needed to fully explain these patterns in the mitochondrial genome.     

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

Introduction  

Recent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage.     

Production of Tiger Puffer <Emphasis Type="Italic">Takifugu rubripes</Emphasis> Offspring from Triploid Grass Puffer <Emphasis Type="Italic">Takifugu niphobles</Emphasis> Parents

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2–5 kg) much larger than average market size (0.6–1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.     

DNA barcode detects high genetic structure within neotropical bird species

Background

Towards lower latitudes the number of recognized species is not only higher, but also phylogeographic subdivision within species is more pronounced. Moreover, new genetically isolated populations are often described in recent phylogenies of Neotropical birds suggesting that the number of species in the region is underestimated. Previous COI barcoding of Argentinean bird species showed more complex patterns of regional divergence in the Neotropical than in the North American avifauna.

Methods and Findings

Here we analyzed 1,431 samples from 561 different species to extend the Neotropical bird barcode survey to lower latitudes, and detected even higher geographic structure within species than reported previously. About 93% (520) of the species were identified correctly from their DNA barcodes. The remaining 41 species were not monophyletic in their COI sequences because they shared barcode sequences with closely related species (N = 21) or contained very divergent clusters suggestive of putative new species embedded within the gene tree (N = 20). Deep intraspecific divergences overlapping with among-species differences were detected in 48 species, often with samples from large geographic areas and several including multiple subspecies. This strong population genetic structure often coincided with breaks between different ecoregions or areas of endemism.

Conclusions

The taxonomic uncertainty associated with the high incidence of non-monophyletic species and discovery of putative species obscures studies of historical patterns of species diversification in the Neotropical region. We showed that COI barcodes are a valuable tool to indicate which taxa would benefit from more extensive taxonomic revisions with multilocus approaches. Moreover, our results support hypotheses that the megadiversity of birds in the region is associated with multiple geographic processes starting well before the Quaternary and extending to more recent geological periods.     

Laboratory diagnosis of melioidosis: Past,present and future

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized “in house” assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis. Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.     

Phylogeny and comparative phylogeography of Sclerurus (Aves: Furnariidae) reveal constant and cryptic diversification in an old radiation of rain forest understorey specialists

Aim To evaluate the role of historical processes in the evolution of Sclerurus leaftossers by integrating phylogenetic and phylogeographical approaches. Location Humid forests of the Neotropical region. Methods We reconstructed the evolutionary history of Sclerurus based on DNA sequences representing all species and 20 of the 26 recognized subspecies using one autosomal nuclear locus and three protein‐coding mitochondrial gene sequences. Phylogenetic relationships were inferred using Bayesian and maximum‐likelihood methods. We used Bayesian coalescent‐based approaches to evaluate demographic changes through time, and to estimate the timing of diversification events. Based on these results, we examined the temporal accumulation of divergence events using lineage‐through‐time plots. Results The monophyly of all Sclerurus species was strongly supported except for Sclerurus mexicanus, which was paraphyletic in relation to Sclerurus rufigularis, and for the sister pair Sclerurus scansor–Sclerurus albigularis, which were not reciprocally monophyletic in the nuclear tree. We found remarkably deep phylogeographical structure within all Sclerurus species, and overall this structure was congruent with currently recognized subspecies and Neotropical areas of endemism. Diversification within Sclerurus has occurred at a relatively constant rate since the Middle Miocene. Main conclusions Our results strongly support the relevance of physiographical (e.g. Nicaragua Depression, Isthmus of Panama, Andean Cordillera, great rivers of Amazonia) and ecological barriers (open vegetation corridor) and ecological gradients (elevational zonation) to the diversification of Neotropical forest‐dwelling organisms. Despite the high congruence among the spatial patterns identified, the variance in divergence times suggests multiple speciation events occurring independently across the same barrier, and a role for dispersal. The phylogenetic patterns and cryptic diversity uncovered in this study demonstrate that the current taxonomy of Sclerurus underestimates the number of species.     

Mechanism of increased susceptibility to 4-nitroquinoline-1-oxide in cultured skin fibroblasts from patients with familial polyposis coli

About 50% of the strains of cultured fibroblasts from patients with familial polyposis coli (FPC) exhibited increased susceptibility to cytotoxicity of 4-nitroquinoline-1-oxide (4NQO) compared with cells from normal individuals. The FPC cells that showed hyper-sensitivity to 4NQO were also hyper-sensitive to mitomycin C (MMC), but susceptibilities of these cells to UV radiation, methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were within the normal range. The extent of single-strand scission of DNA in the 4NQO-sensitive FPC cells was greater than in normal cells, and the amount of [14C]4NQO bound to DNA in the FPC cells was twice as high as in normal cells. The rate of release of [14C]4NQO from DNA by the post-culture was the same as in both FPC and normal cells. The 4NQO-sensitive FPC cells exhibited increased 4NQO-reductase activity; the level of this activity was consistent with the extent of the decrease in colony formation by 4NQO. These results suggest that the enhanced ability to activate 4NQO might be an important factor in the mechanism of susceptibility of FPC cells to 4NQO rather than the reduced ability to repair DNA.     

Loss of normal allele of the APC gene in an adrenocortical carcinoma from a patient with familial adenomatous polyposis

Summary Salla disease is a lysosomal storage disorder due to impaired transport of free sialic acid across the lysosomal membrane. The clinical presentation of this autosomal recessive trait is severe psychomotor retardation from early infancy on. In order to determine the gene locus for the disease we have initiated a genetic linkage study using polymorphic gene markers in rep-resentative family material comprising about 60% of all families known to be affected with Salla disease. Here we present an exclusion map based on combined linkage data from 64 informative loci on 19 autosomes. Theoretically, at least 55% of the genome has been excluded as a locus for the disease gene, while some chromosome areas, particularly the long arm of chromosome 2, are highlighted as possible sites for the gene locus.     

Novel germline hMSH2 genomic deletion and somatic hMSH2 mutations in a hereditary nonpolyposis colorectal cancer family

Germline and somatic mutations of the hMSH2 gene were determined in a Japanese hereditary nonpolyposis colorectal cancer (HNPCC) family fulfilling the Amsterdam criteria. PCR-SSCP-sequencing of genomic DNA detected a somatic hMSH2 mutation of an A deletion at codon 227-229 in a duodenal carcinoma and a somatic hMSH2 mutation of an A insertion at codon 21 in a gastric carcinoma from affected family members, both carcinomas exhibiting high microsatellite instability. However, no germline hMSH2 mutation was detected by the PCR-SSCP-sequencing method. Genomic DNA was then analyzed by Southern blot hybridization using three hMSH2 cDNA probes (probe A involving exons 1-5, probe B involving exons 4-11 and probe C involving exons 9-16) after digestion by restriction enzymes, EcoRI, HindIII and NsiI. The NsiI digest of DNA from normal tissues of affected members exhibited an aberrant 8.6 kb restriction fragment, in addition to the normal 10.6 kb fragment, when hybridized to probes A and B. This suggested the presence of a heterozygous 2kb genomic deletion encompassing exon 4, 5 or 6. RT-PCR-sequencing revealed that the deleted region encompassed exon 5. This novel genomic deletion of the hMSH2 gene was confirmed to be pathogenic, and the Southern hybridization pattern was applied to the pre-symptomatic diagnosis.