Showing papers by "Stephen J. O'Brien published in 2016"

Chromosomal-level assembly of the Asian seabass genome using long sequence reads and multi-layered scaffolding

Abstract: We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species’ native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

Genomics in Conservation: Case Studies and Bridging the Gap between Data and Application

Abstract: We agree with Shafer et al. [1] that there is a need for well-documented case studies of the application of genomics in conservation and management as well as increased communication between academics and natural resource managers. However, we challenge Shafer et al.’s [1] relatively pessimistic assertion that ‘conservation genomics is far from seeing regular application’. Here we illustrate by examples that conservation practitioners utilize more genomic research than is often apparent. In addition, we highlight the work of nonacademic laboratories [government and nongovernmental organizations (NGOs)], some of which are not always well represented in peer-reviewed literature.

Comparison of carnivore, omnivore, and herbivore mammalian genomes with a new leopard assembly

Abstract: There are three main dietary groups in mammals: carnivores, omnivores, and herbivores. Currently, there is limited comparative genomics insight into the evolution of dietary specializations in mammals. Due to recent advances in sequencing technologies, we were able to perform in-depth whole genome analyses of representatives of these three dietary groups. We investigated the evolution of carnivory by comparing 18 representative genomes from across Mammalia with carnivorous, omnivorous, and herbivorous dietary specializations, focusing on Felidae (domestic cat, tiger, lion, cheetah, and leopard), Hominidae, and Bovidae genomes. We generated a new high-quality leopard genome assembly, as well as two wild Amur leopard whole genomes. In addition to a clear contraction in gene families for starch and sucrose metabolism, the carnivore genomes showed evidence of shared evolutionary adaptations in genes associated with diet, muscle strength, agility, and other traits responsible for successful hunting and meat consumption. Additionally, an analysis of highly conserved regions at the family level revealed molecular signatures of dietary adaptation in each of Felidae, Hominidae, and Bovidae. However, unlike carnivores, omnivores and herbivores showed fewer shared adaptive signatures, indicating that carnivores are under strong selective pressure related to diet. Finally, felids showed recent reductions in genetic diversity associated with decreased population sizes, which may be due to the inflexible nature of their strict diet, highlighting their vulnerability and critical conservation status. Our study provides a large-scale family level comparative genomic analysis to address genomic changes associated with dietary specialization. Our genomic analyses also provide useful resources for diet-related genetic and health research.

Pangolin genomes and the evolution of mammalian scales and immunity

Abstract: Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.

The Asian arowana (Scleropages formosus) genome provides new insights into the evolution of an early lineage of teleosts.

Abstract: The Asian arowana (Scleropages formosus), one of the world’s most expensive cultivated ornamental fishes, is an endangered species. It represents an ancient lineage of teleosts: the Osteoglossomorpha. Here, we provide a high-quality chromosome-level reference genome of a female golden-variety arowana using a combination of deep shotgun sequencing and high-resolution linkage mapping. In addition, we have also generated two draft genome assemblies for the red and green varieties. Phylogenomic analysis supports a sister group relationship between Osteoglossomorpha (bonytongues) and Elopomorpha (eels and relatives), with the two clades together forming a sister group of Clupeocephala which includes all the remaining teleosts. The arowana genome retains the full complement of eight Hox clusters unlike the African butterfly fish (Pantodon buchholzi), another bonytongue fish, which possess only five Hox clusters. Differential gene expression among three varieties provides insights into the genetic basis of colour variation. A potential heterogametic sex chromosome is identified in the female arowana karyotype, suggesting that the sex is determined by a ZW/ZZ sex chromosomal system. The high-quality reference genome of the golden arowana and the draft assemblies of the red and green varieties are valuable resources for understanding the biology, adaptation and behaviour of Asian arowanas.

Chromosomer: a reference-based genome arrangement tool for producing draft chromosome sequences.

Abstract: As the number of sequenced genomes rapidly increases, chromosome assembly is becoming an even more crucial step of any genome study. Since de novo chromosome assemblies are confounded by repeat-mediated artifacts, reference-assisted assemblies that use comparative inference have become widely used, prompting the development of several reference-assisted assembly programs for prokaryotic and eukaryotic genomes. We developed Chromosomer – a reference-based genome arrangement tool, which rapidly builds chromosomes from genome contigs or scaffolds using their alignments to a reference genome of a closely related species. Chromosomer does not require mate-pair libraries and it offers a number of auxiliary tools that implement common operations accompanying the genome assembly process. Despite implementing a straightforward alignment-based approach, Chromosomer is a useful tool for genomic analysis of species without chromosome maps. Putative chromosome assemblies by Chromosomer can be used in comparative genomic analysis, genomic variation assessment, potential linkage group inference and other kinds of analysis involving contig or scaffold mapping to a high-quality assembly.

Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome-specific DNA sequencing.

Abstract: B chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes. In this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide-primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1.42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition. Our data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements.

A Mutation in LTBP2 Causes Congenital Glaucoma in Domestic Cats (Felis Catus)

Abstract: The glaucomas are a group of diseases characterized by optic nerve damage that together represent a leading cause of blindness in the human population and in domestic animals. Here we report a mutation in LTBP2 that causes primary congenital glaucoma (PCG) in domestic cats. We identified a spontaneous form of PCG in cats and established a breeding colony segregating for PCG consistent with fully penetrant, autosomal recessive inheritance of the trait. Elevated intraocular pressure, globe enlargement and elongated ciliary processes were consistently observed in all affected cats by 8 weeks of age. Varying degrees of optic nerve damage resulted by 6 months of age. Although subtle lens zonular instability was a common feature in this cohort, pronounced ectopia lentis was identified in less than 10% of cats examined. Thus, glaucoma in this pedigree is attributed to histologically confirmed arrest in the early post-natal development of the aqueous humor outflow pathways in the anterior segment of the eyes of affected animals. Using a candidate gene approach, significant linkage was established on cat chromosome B3 (LOD 18.38, θ = 0.00) using tightly linked short tandem repeat (STR) loci to the candidate gene, LTBP2. A 4 base-pair insertion was identified in exon 8 of LTBP2 in affected individuals that generates a frame shift that completely alters the downstream open reading frame and eliminates functional domains. Thus, we describe the first spontaneous and highly penetrant non-rodent model of PCG identifying a valuable animal model for primary glaucoma that closely resembles the human disease, providing valuable insights into mechanisms underlying the disease and a valuable animal model for testing therapies.

A High-Resolution SNP Array-Based Linkage Map Anchors a New Domestic Cat Draft Genome Assembly and Provides Detailed Patterns of Recombination.

Abstract: High-resolution genetic and physical maps are invaluable tools for building accurate genome assemblies, and interpreting results of genome-wide association studies (GWAS). Previous genetic and physical maps anchored good quality draft assemblies of the domestic cat genome, enabling the discovery of numerous genes underlying hereditary disease and phenotypes of interest to the biomedical science and breeding communities. However, these maps lacked sufficient marker density to order thousands of shorter scaffolds in earlier assemblies, which instead relied heavily on comparative mapping with related species. A high-resolution map would aid in validating and ordering chromosome scaffolds from existing and new genome assemblies. Here, we describe a high-resolution genetic linkage map of the domestic cat genome based on genotyping 453 domestic cats from several multi-generational pedigrees on the Illumina 63K SNP array. The final maps include 58,055 SNP markers placed relative to 6637 markers with unique positions, distributed across all autosomes and the X chromosome. Our final sex-averaged maps span a total autosomal length of 4464 cM, the longest described linkage map for any mammal, confirming length estimates from a previous microsatellite-based map. The linkage map was used to order and orient the scaffolds from a substantially more contiguous domestic cat genome assembly (Felis catus v8.0), which incorporated ∼20 × coverage of Illumina fragment reads. The new genome assembly shows substantial improvements in contiguity, with a nearly fourfold increase in N50 scaffold size to 18 Mb. We use this map to report probable structural errors in previous maps and assemblies, and to describe features of the recombination landscape, including a massive (∼50 Mb) recombination desert (of virtually zero recombination) on the X chromosome that parallels a similar desert on the porcine X chromosome in both size and physical location.

Magnetic Resonance Imaging Currently Fails to Fully Evaluate the Biceps-Labrum Complex and Bicipital Tunnel.

Abstract: Purpose To determine the diagnostic accuracy of magnetic resonance imaging (MRI) for biceps-labrum complex (BLC) lesions, including the extra-articular bicipital tunnel. Methods A retrospective review of 277 shoulders with chronic refractory BLC symptoms that underwent arthroscopic subdeltoid transfer of the long head of the biceps tendon (LHBT) to the conjoint tendon was conducted. Intraoperative lesions were categorized as "inside" (labral tears and dynamic LHBT incarceration), "junctional" (LHBT partial tears, LHBT subluxation, and biceps chondromalacia), or "bicipital tunnel" (extra-articular bicipital tunnel scar/stenosis, loose bodies, LHBT instability, and LHBT partial tears) based on anatomic location. Attending radiologist–generated MRI reports were graded dichotomously as positive or negative for biceps and labral damage and then compared with intraoperative findings. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for MRI with respect to intraoperative findings. Results With regard to inside lesions, MRI had an overall sensitivity, specificity, PPV, and NPV for labrum lesions of 77.3%, 68.2%, 57.3%, and 84.5% respectively. The sensitivity, specificity, PPV, and NPV of MRI for junctional lesions were 43.3%, 55.6%, 73.1%, and 26.0%, respectively. For the bicipital tunnel, MRI had a sensitivity, specificity, PPV, and NPV of 50.4%, 61.4%, 48.7%, and 63.0%, respectively. Conclusions MRI was unreliable for ruling out BLC lesions among chronically symptomatic patients, including when the bicipital tunnel was affected. Level of Evidence Level IV, diagnostic case-control study.

The Population Origins and Expansion of Feral Cats in Australia

Abstract: The historical literature suggests that in Australia, the domestic cat (Felis catus) had a European origin [~200 years before present (ybp)], but it is unclear if cats arrived from across the Asian land bridge contemporaneously with the dingo (4000 ybp), or perhaps immigrated ~40000 ybp in association with Aboriginal settlement from Asia. The origin of cats in Australia is important because the continent has a complex and ancient faunal assemblage that is dominated by endemic rodents and marsupials and lacks the large placental carnivores found on other large continents. Cats are now ubiquitous across the entire Australian continent and have been implicit in the range contraction or extinction of its small to medium sized (<3.5kg) mammals. We analyzed the population structure of 830 cats using 15 short tandem repeat (STR) genomic markers. Their origin appears to come exclusively from European founders. Feral cats in continental Australia exhibit high genetic diversity in comparison with the low diversity found in populations of feral cats living on islands. The genetic structure is consistent with a rapid westerly expansion from eastern Australia and a limited expansion in coastal Western Australia. Australian cats show modest if any population structure and a close genetic alignment with European feral cats as compared to cats from Asia, the Christmas and Cocos (Keeling) Islands (Indian Ocean), and European wildcats (F. silvestris silvestris).

Bone-associated gene evolution and the origin of flight in birds.

Abstract: Bones have been subjected to considerable selective pressure throughout vertebrate evolution, such as occurred during the adaptations associated with the development of powered flight. Powered flight evolved independently in two extant clades of vertebrates, birds and bats. While this trait provided advantages such as in aerial foraging habits, escape from predators or long-distance travels, it also imposed great challenges, namely in the bone structure. We performed comparative genomic analyses of 89 bone-associated genes from 47 avian genomes (including 45 new), 39 mammalian, and 20 reptilian genomes, and demonstrate that birds, after correcting for multiple testing, have an almost two-fold increase in the number of bone-associated genes with evidence of positive selection (~52.8 %) compared with mammals (~30.3 %). Most of the positive-selected genes in birds are linked with bone regulation and remodeling and thirteen have been linked with functional pathways relevant to powered flight, including bone metabolism, bone fusion, muscle development and hyperglycemia levels. Genes encoding proteins involved in bone resorption, such as TPP1, had a high number of sites under Darwinian selection in birds. Patterns of positive selection observed in bird ossification genes suggest that there was a period of intense selective pressure to improve flight efficiency that was closely linked with constraints on body size.

Whole-Genome Identification, Phylogeny, and Evolution of the Cytochrome P450 Family 2 (CYP2) Subfamilies in Birds

Abstract: The cytochrome P450 (CYP) superfamily defends organisms from endogenous and noxious environmental compounds, and thus is crucial for survival. However, beyond mammals the molecular evolution of CYP2 subfamilies is poorly understood. Here, we characterized the CYP2 family across 48 avian whole genomes representing all major extant bird clades. Overall, 12 CYP2 subfamilies were identified, including the first description of the CYP2F, CYP2G, and several CYP2AF genes in avian genomes. Some of the CYP2 genes previously described as being lineage-specific, such as CYP2K and CYP2W, are ubiquitous to all avian groups. Furthermore, we identified a large number of CYP2J copies, which have been associated previously with water reabsorption. We detected positive selection in the avian CYP2C, CYP2D, CYP2H, CYP2J, CYP2K, and CYP2AC subfamilies. Moreover, we identified new substrate recognition sites (SRS0, SRS2_SRS3, and SRS3.1) and heme binding areas that influence CYP2 structure and function of functional importance as under significant positive selection. Some of the positively selected sites in avian CYP2D are located within the same SRS1 region that was previously linked with the metabolism of plant toxins. Additionally, we find that selective constraint variations in some avian CYP2 subfamilies are consistently associated with different feeding habits (CYP2H and CYP2J), habitats (CYP2D, CYP2H, CYP2J, and CYP2K), and migratory behaviors (CYP2D, CYP2H, and CYP2J). Overall, our findings indicate that there has been active enzyme site selection on CYP2 subfamilies and differential selection associated with different life history traits among birds.

The Clinical Impact of Bicipital Tunnel Decompression During Long Head of the Biceps Tendon Surgery: A Systematic Review and Meta-analysis.

Abstract: Purpose (1) To identify existing outcomes studies and (2) to use meta-analysis techniques to summarize pooled clinical outcomes for surgical techniques that decompress the bicipital tunnel and those that do not, to identify important areas for future clinical investigation. Methods A systematic review of the PubMed database was performed in accordance with PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines. Patients were categorized into 2 groups for analysis. Group 1, the "tunnel decompressed" group, included open subpectoral tenodesis, long head of the biceps tendon transfer procedures, and proximal tenodesis techniques that explicitly released the bicipital sheath. Group 2, the "tunnel not decompressed" group, included proximal tenodesis techniques and tenotomy. Validated clinical outcome measures (Constant; University of California, Los Angeles; Simple Shoulder Test; visual analog scale for pain; and American Shoulder and Elbow Surgeons) and revision rates were summarized using inverse-variance weighting in a random-effects model. Because the constituent studies were largely single-cohort observational studies, direct between-group statistical comparisons could not be made. Results Thirty studies (comprising 1,881 patients) met the inclusion and exclusion criteria. The Constant score was the most commonly reported outcome measure (16 cohorts, 961 patients) and was seemingly higher in group 1 (88.3 v 81.7). Revision rates; University of California, Los Angeles scores; Simple Shoulder Test scores; visual analog scale scores for pain; and American Shoulder and Elbow Surgeons scores appeared to be similar between groups. The mean patient age was 50.7 ± 5.7 years for group 1 and 58.9 ± 6.3 years for group 2. The Egger intercept method showed an intercept of −13.29 ( P Conclusions Bicipital tunnel–decompressing techniques showed apparently higher Constant scores compared with non-decompressing techniques but may have been affected by differences in mean patient age between groups. Existing literature consists of largely single-cohort retrospective observational Level IV studies, which are likely influenced by significant publication bias. Level of Evidence Level IV, systematic review of Level II through IV studies.

The Genome-Wide Analysis of Carcinoembryonic Antigen Signaling by Colorectal Cancer Cells Using RNA Sequencing

Abstract: Сarcinoembryonic antigen (CEA, CEACAM5, CD66) is a promoter of metastasis in epithelial cancers that is widely used as a prognostic clinical marker of metastasis. The aim of this study is to identify the network of genes that are associated with CEA-induced colorectal cancer liver metastasis. We compared the genome-wide transcriptomic profiles of CEA positive (MIP101 clone 8) and CEA negative (MIP 101) colorectal cancer cell lines with different metastatic potential in vivo. The CEA-producing cells displayed quantitative changes in the level of expression for 100 genes (over-expressed or down-regulated). They were confirmed by quantitative RT-PCR. The KEGG pathway analysis identified 4 significantly enriched pathways: cytokine-cytokine receptor interaction, MAPK signaling pathway, TGF-beta signaling pathway and pyrimidine metabolism. Our results suggest that CEA production by colorectal cancer cells triggers colorectal cancer progression by inducing the epithelial- mesenchymal transition, increasing tumor cell invasiveness into the surrounding tissues and suppressing stress and apoptotic signaling. The novel gene expression distinctions establish the relationships between the existing cancer markers and implicate new potential biomarkers for colorectal cancer hepatic metastasis.

De novo sequencing, assembly and analysis of eight different transcriptomes from the Malayan pangolin.

Abstract: Pangolins are scale-covered mammals, containing eight endangered species. Maintaining pangolins in captivity is a significant challenge, in part because little is known about their genetics. Here we provide the first large-scale sequencing of the critically endangered Manis javanica transcriptomes from eight different organs using Illumina HiSeq technology, yielding ~75 Giga bases and 89,754 unigenes. We found some unigenes involved in the insect hormone biosynthesis pathway and also 747 lipids metabolism-related unigenes that may be insightful to understand the lipid metabolism system in pangolins. Comparative analysis between M. javanica and other mammals revealed many pangolin-specific genes significantly over-represented in stress-related processes, cell proliferation and external stimulus, probably reflecting the traits and adaptations of the analyzed pregnant female M. javanica. Our study provides an invaluable resource for future functional works that may be highly relevant for the conservation of pangolins.

The Quality of Open-Access Video-Based Orthopaedic Instructional Content for the Shoulder Physical Exam is Inconsistent:

Abstract: BackgroundThe internet has an increasing role in both patient and physician education. While several recent studies critically appraised the quality and accuracy of web-based written information av...

DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history (class I genes/restriction fragment length polymorphism/felids)

Abstract: The major histocompatibility complex (MHC) is a multigene complex of tightly linked homologous genes that encode cell surface antigens that play a key role in immune regulation and response to foreign antigens. In most species, MHC gene products display extreme antigenic poly- morphism, and their variability has been interpreted to reflect an adaptive strategy for accommodating rapidly evolving in- fectious agents that periodically afflict natural populations. Determination of the extent of MHC variation has been limited to populations in which skin grafting is feasible or for which serological reagents have been developed. We present here a quantitative analysis of restriction fragment fength polymor- phism of MHC class I genes in several mammalian species (cats, rodents, humans) known to have very different levels of genetic diversity based on functional MHC assays and on allozyme surveys. When homologous class I probes were employed, a notable concordance was observed between the extent of MHC restriction fragment variation and functional MHC variation detected by skin grafts or genome-wide diversity estimated by allozyme screens. These results confirm the genetically depau- r~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Tenotomy, Tenodesis, Transfer: A Review of Treatment Options for Biceps-Labrum Complex Disease.

Abstract: Biceps-labrum complex (BLC) disease is a well recognized source of shoulder pain. The BLC can be divided into 3 anatomical zones: inside, junction, and bicipital tunnel. Despite our evolving understanding, diagnosis of BLC pathology through physical examination, standard imaging techniques, and standard diagnostic glenohumeral arthroscopy can be challenging. Selection of the most appropriate surgical technique in refractory cases should take into account decompression of the extra-articular bicipital tunnel. In this article we review the recent literature regarding diagnosis and treatment options for BLC disease.

Positive Selection Linked with Generation of Novel Mammalian Dentition Patterns.

Abstract: A diverse group of genes are involved in the tooth development of mammals. Several studies, focused mainly on mice and rats, have provided a detailed depiction of the processes coordinating tooth formation and shape. Here we surveyed 236 tooth-associated genes in 39 mammalian genomes and tested for signatures of selection to assess patterns of molecular adaptation in genes regulating mammalian dentition. Of the 236 genes, 31 (∼13.1%) showed strong signatures of positive selection that may be responsible for the phenotypic diversity observed in mammalian dentition. Mammalian-specific tooth-associated genes had accelerated mutation rates compared with older genes found across all vertebrates. More recently evolved genes had fewer interactions (either genetic or physical), were associated with fewer Gene Ontology terms and had faster evolutionary rates compared with older genes. The introns of these positively selected genes also exhibited accelerated evolutionary rates, which may reflect additional adaptive pressure in the intronic regions that are associated with regulatory processes that influence tooth-gene networks. The positively selected genes were mainly involved in processes like mineralization and structural organization of tooth specific tissues such as enamel and dentin. Of the 236 analyzed genes, 12 mammalian-specific genes (younger genes) provided insights on diversification of mammalian teeth as they have higher evolutionary rates and exhibit different expression profiles compared with older genes. Our results suggest that the evolution and development of mammalian dentition occurred in part through positive selection acting on genes that previously had other functions.

Primary cultures of human colon cancer as a model to study cancer stem cells.

Abstract: The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

Koalas (Phascolarctos cinereus) From Queensland Are Genetically Distinct From 2 Populations in Victoria

Abstract: The koala (Phascolarctos cinereus) suffered population declines and local extirpation due to hunting in the early 20th century, especially in southern Australia. Koalas were subsequently reintroduced to the Brisbane Ranges (BR) and Stony Rises (SR) by translocating individuals from a population on French Island descended from a small number of founders. To examine genetic diversity and north-south differentiation, we genotyped 13 microsatellite markers in 46 wild koalas from the BR and SR, and 27 Queensland koalas kept at the US zoos. The Queensland koalas displayed much higher heterozygosity (H O = 0.73) than the 2 southern Australian koala populations examined: H O = 0.49 in the BR, whereas H O = 0.41 in the SR. This is consistent with the historical accounts of bottlenecks and founder events affecting the southern populations and contrasts with reports of high genetic diversity in some southern populations. The 2 southern Australian koala populations were genetically similar (F ST = 0.018, P = 0.052). By contrast, northern and southern Australian koalas were highly differentiated (F ST = 0.27, P < 0.001), thereby suggesting that geographic structuring should be considered in the conservation management of koalas. Sequencing of 648bp of the mtDNA control region in Queensland koalas found 8 distinct haplotypes, one of which had not been previously detected among koalas. Queensland koalas displayed high mitochondrial haplotype diversity (H = 0.753) and nucleotide diversity (π = 0.0072), indicating along with the microsatellite data that North American zoos have maintained high levels of genetic diversity among their Queensland koalas.

PGD: a pangolin genome hub for the research community

Abstract: Pangolins (order Pholidota) are the only mammals covered by scales. We have recently sequenced and analyzed the genomes of two critically endangered Asian pangolin species, namely the Malayan pangolin (Manis javanica) and the Chinese pangolin (Manis pentadactyla). These complete genome sequences will serve as reference sequences for future research to address issues of species conservation and to advance knowledge in mammalian biology and evolution. To further facilitate the global research effort in pangolin biology, we developed the Pangolin Genome Database (PGD), as a future hub for hosting pangolin genomic and transcriptomic data and annotations, and with useful analysis tools for the research community. Currently, the PGD provides the reference pangolin genome and transcriptome data, gene sequences and functional information, expressed transcripts, pseudogenes, genomic variations, organ-specific expression data and other useful annotations. We anticipate that the PGD will be an invaluable platform for researchers who are interested in pangolin and mammalian research. We will continue updating this hub by including more data, annotation and analysis tools particularly from our research consortium.Database URL: http://pangolin-genome.um.edu.my.

Correction: A Mutation in LTBP2 Causes Congenital Glaucoma in Domestic Cats (Felis catus)

Abstract: In Fig 1, the numbers (n) of subjects are missing from the bars of the bar graph. Please see corrected Fig 1 here. Fig 1 Intraocular pressure in glaucomatous and normal cats.

Genetic Evidence for Contrasting Wetland and Savannah Habitat Specializations in Different Populations of Lions (Panthera leo).

Abstract: South-central Africa is characterized by an archipelago of wetlands, which has evolved in time and space since at least the Miocene, providing refugia for animal species during Pleistocene arid episodes. Their importance for biodiversity in the region is reflected in the evolution of a variety of specialist mammal and bird species, adapted to exploit these wetland habitats. Populations of lions (Panthera leo) across south-central and east Africa have contrasting signatures of mitochondrial DNA haplotypes and biparental nuclear DNA in wetland and savannah habitats, respectively, pointing to the evolution of distinct habitat preferences. This explains the absence of genetic admixture of populations from the Kalahari savannah of southwest Botswana and the Okavango wetland of northern Botswana, despite separation by only 500 km. We postulate that ancestral lions were wetland specialists and that the savannah lions evolved from populations that were isolated during arid Pleistocene episodes. Expansion of grasslands and the resultant increase in herbivore populations during mesic Pleistocene climatic episodes provided the stimulus for the rapid population expansion and diversification of the highly successful savannah lion specialists. Our model has important implications for lion conservation.

All-Arthroscopic Modified Rotator Interval Slide for Massive Anterosuperior Cuff Tears Using the Subdeltoid Space: Surgical Technique and Early Results:

Abstract: BackgroundTraditional intra-articular arthroscopic repair techniques for massive anterosuperior rotator cuff tears are technically demanding and necessitate sacrifice of the rotator interval to ena...

Response to Comment by Faurby, Werdelin and Svenning

Abstract: Faurby, Werdelin, and Svenning raise some interesting and valid points around the interpretation of the genomics and fossil data on the history of the African cheetah, a species that has fascinated humankind for millennia. They emphasize a controversy to which we alluded in our article [1] and they offer an alternative explanation to the scenario we had suggested for the time and place of demographic events that preceded modern African cheetah populations. We fully agree that there are important areas of uncertainty in this conundrum and welcome the opportunity to reconsider and to offer our view into the natural history of Acinonyx jubatus. We do believe that the integration of fossil and molecular data seems a good way to get toward the truth, so we shall try to emphasize two considerations in our comments: first, the most parsimonious interpretation of data; and second, the robustness of available and relevant fossil and molecular data. Some aspects to consider: The extreme genetic depletion of modern cheetahs seems true and validated by Dobrynin et al., as has been suggested with multiple metrics across several decades [1–3]. Two possible historic events that could account for the genetic reduction are extended continental migration events and/or population bottlenecks (a.k.a. founder effect). We offered possible (but not proven) explanations based upon dating the latest founder effect at 10,000–12,000 during the late Pleistocene by multiple molecular methods, coincident with abrupt mammal extinction events in North America and Eurasia at that time. The genome-wide Dadi result suggested an earlier more ancient demographic contraction ~100,000 years ago. Something caused the massive diversity loss in their history. We suggested two scenarios: the earliest—a migration of North American cheetahs out of North America >100,000 years BP, and a second—when cheetahs went extinct from North America 10,000–12,000 years ago. It may be relevant that North American pumas show a near identical quantity of genomic depletion as African cheetahs, suggesting they were also extirpated form North America during the quaternary mammal extinction and repopulated from South America subsequently [3–5]. Was this an irrelevant coincidence or was a single North American or global event responsible? The answer is uncertain. Please refer to [6, 7] which detail the latest relatively robust molecular phylogenies for the Felidae and a derivative study [8] that postulates parsimonious historic migration scenarios based upon phylogeny, the fossil record, present and past geographical occurrence, and geological sea levels. That phylogenetic study reported in [6] examined 18,853 bp of nuclear sequence (autosome, X and Y chromosome), which yielded 3440 parsimony informative sites, and mtDNA sequence, from all 37 Felidae species calibrated with 16 fossil dates [6]. That molecular phylogeny (combined with paleontological, geological and present range) posited the arrival of the ancestors of three Felidae lineages (Ocelot preceding Lynx, and then preceding Puma lineage) into North America, around 8–8.5 million years ago (MYA). All three lineages have roots in North America with ocelot ancestors appearing first 8.0 MYA, Lynx ancestors 7.2 MYA, and Puma ancestors 6.7 MYA. Cheetah-like genera (Acinonyx lineage) appear first ~4.9 MYA. Support for this scenario comes from molecular evolutionary analyses that were supported by 13 cladistic synapomorphies (large insertion/deletions) [6]. Sometime thereafter, the Acinonyx lineage ancestors, derived from the North American puma lineage precursors, would make their way back to Asia from North America. The fossil record puts the oldest cheetah fossils in Asia 3 million years before present (MYBP). Fossils of Acinonyx and cheetah-looking cats were in both in Asia and North America from 3 MYA to the late Pleistocene. So the comment in their title “There were never any true cheetahs in North America” depends on what we mean by “true cheetahs.” Faurby, Werdelin, and Svenning argue cogently that North American cheetah fossils are all from the genus Miracinonyx, which is not closely related to modern Acinonyx cheetahs, which occur in Africa today and in Eurasia throughout the Pliocene and Pleistocene fossil deposits. Thus, the trivial name of North American “cheetah” is misleading (they imply we fell into that trap, although these authors themselves use the trivial name “cheetah” ten times in their note). The data supporting the distinctions of North American species in Miracinonyx from Eurasian Acinonyx are the bases of the controversy. A seminal and elegant report by Van Valkenburgh et al. [9] concluded that North American cheetah fossils (there were six specimens, one complete and five fragmented) were cladistically similar based upon ten synapomorphic morphological features that were distinct from Eurasian Acinonyx fossils. This study proposed the distinction of North America (Miracinonyx) and Eurasian (Acinonyx) genera in 1990. Faurby, Werdelin, and Svenning affirm this conclusion by stating “… No fossils of Acinonyx are known from North America, despite the extensive fossil record from the continent, while no fossils of Miracinonyx are known outside North America.” This declarative statement discounts a longstanding controversy that Van Valkenburgh et al. [9] acknowledge. First, there are several important adaptive morphometric characters that are shared between North America and Eurasian cheetah species including long slender limbs, small aerodynamic skulls, and reduced canines which Van Valkenburgh et al. [9] attribute to homoplasy or parallelisms. But at least three respected authors groups have disagreed [10–12] and opined that the adaptive similarities are evidence for homology of North American and African cheetahs supporting the modern cheetah origins in North America. Adams’ study titled “The cheetah-native American” [10], classifies the North American cheetah specimens as Acinonyx (precursors of modern Acinonyx) and suggests that pronouncing these important cursorial characters as convergence or parallelism “pushes the concept of parallel evolution to an unprecedented extreme”. Van Valkenburgh et al. [9] actually place the Eurasian Acinonyx and the North American Miracinonyx genera as sister taxa, emphasizing the difficulty in sorting this controversy cleanly. Further they state “… The common ancestor (of Acinonyx and Miracinonyx) … could have been either Old or New World.” Mark Springer, a noted expert of interpreting molecular and paleontological evidence across vertebrate taxa [13], states that “… morphological studies of eutherian … relationships have failed to separate homology and homoplasy and have consistently been misled by the latter … one of the greatest challenges ahead for mammalian systematists is to tease apart homology and homoplasy in morphological characters.” Springer’s study described explicitly how morphological inference erred in over 50 % of ordinal assignments within the mammalian radiations due to this issue [13]. Faurby, Werdelin, and Svenning refer to Barnet et al. [14] who presented a molecular phylogeny that uses ancient DNA to place the North American Miracinonyx as a sister to Puma concolor (and not modern Acinonyx), but they acknowledge the limited power of a 1302 bp mtDNA dataset. There are other details of the Barnett study that may be relevant. First, the actual nucleotide alignment is not presented which limits our ability to evaluate their conclusions or assess the extent of homoplasy. Further, when Barnet et al. performed a jack-knife analyses, excluding H. jaguarundi from the phylogenic analysis (H. jaguarundi is a sister taxon to Puma concolor [6–8]), statistical support for the sister relationship of Puma-Miracinonyx actually increased, an indication that molecular homoplasy was operative in their results [14]. Their conclusion that American cheetah Miracinonyx is not within the African-Asian Acinonyx group is tentative at best and has not been confirmed convincingly. Faurby, Werdelin, and Svenning raise an important caveat concerning the confidence interval of the Dadi and PSMC and question the average generation of modern and ancient cheetahs. We do agree these aspects are not as firm as we would prefer. They hypothesize that “… the simplest explanation of the patterns may be a gradual decline in Acinonyx as inferred by the PSMC algorithm, potentially with sharper declines in some periods as suggested by the DaDi analysis.” Their points are true and we acknowledge the tentative aspects of our derived conclusions. In sum, we welcome the engagement of Faurby, Werdelin, and Svenning in the interpretation of the new and older data around the natural history for cheetah origins. They may be correct in their interpretation that Eurasian Cheetahs are the most recent ancestors of modern African cheetahs but their North American presence at one or several stages also seems plausible from the available data. We were not there at the time, so we cannot be sure of what the timing or influence of any possible North American cheetah migration to Asia might be, but both fossil and molecular evidence would be consistent with cheetah-like cats in both Asia and North America for some 3 MY. A correct explanation for African cheetah origins would seem to be one of three possibilities: (1) Eurasian origin exclusively; (2) North American origins exclusively and a migration across Asia to Africa; or (3) a combination of continental evolution on both continents, combined with periodic migration (and perhaps admixture) allowing both continental populations to contribute to modern African cheetah genetic disposition and locations. The resolution of these alternatives represents a fascinating challenge for the genome archeologists reading this exchange.