Identifying deer antler uhrf1 proliferation and s100a10 mineralization genes using comparative RNA-seq.

BACKGROUND: Deer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration. METHODS: To identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (> 60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies. RESULTS: Cell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6-11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10. CONCLUSION: Using a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.

Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands.

BACKGROUND & AIMS: Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology. METHODS: We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. RESULTS: H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. CONCLUSIONS: H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.

The phylogenetic and geographic structure of Y-chromosome haplogroup R1a.

R1a-M420 is one of the most widely spread Y-chromosome haplogroups; however, its substructure within Europe and Asia has remained poorly characterized. Using a panel of 16 244 male subjects from 126 populations sampled across Eurasia, we identified 2923 R1a-M420 Y-chromosomes and analyzed them to a highly granular phylogeographic resolution. Whole Y-chromosome sequence analysis of eight R1a and five R1b individuals suggests a divergence time of ∼25,000 (95% CI: 21,300-29,000) years ago and a coalescence time within R1a-M417 of ∼5800 (95% CI: 4800-6800) years. The spatial frequency distributions of R1a sub-haplogroups conclusively indicate two major groups, one found primarily in Europe and the other confined to Central and South Asia. Beyond the major European versus Asian dichotomy, we describe several younger sub-haplogroups. Based on spatial distributions and diversity patterns within the R1a-M420 clade, particularly rare basal branches detected primarily within Iran and eastern Turkey, we conclude that the initial episodes of haplogroup R1a diversification likely occurred in the vicinity of present-day Iran.

Phylogenetic applications of whole Y-chromosome sequences and the Near Eastern origin of Ashkenazi Levites.

Previous Y-chromosome studies have demonstrated that Ashkenazi Levites, members of a paternally inherited Jewish priestly caste, display a distinctive founder event within R1a, the most prevalent Y-chromosome haplogroup in Eastern Europe. Here we report the analysis of 16 whole R1 sequences and show that a set of 19 unique nucleotide substitutions defines the Ashkenazi R1a lineage. While our survey of one of these, M582, in 2,834 R1a samples reveals its absence in 922 Eastern Europeans, we show it is present in all sampled R1a Ashkenazi Levites, as well as in 33.8% of other R1a Ashkenazi Jewish males and 5.9% of 303 R1a Near Eastern males, where it shows considerably higher diversity. Moreover, the M582 lineage also occurs at low frequencies in non-Ashkenazi Jewish populations. In contrast to the previously suggested Eastern European origin for Ashkenazi Levites, the current data are indicative of a geographic source of the Levite founder lineage in the Near East and its likely presence among pre-Diaspora Hebrews.

Use of whole genome sequencing to determine the microevolution of Mycobacterium tuberculosis during an outbreak.

RATIONALE: Current tools available to study the molecular epidemiology of tuberculosis do not provide information about the directionality and sequence of transmission for tuberculosis cases occurring over a short period of time, such as during an outbreak. Recently, whole genome sequencing has been used to study molecular epidemiology of Mycobacterium tuberculosis over short time periods. OBJECTIVE: To describe the microevolution of M. tuberculosis during an outbreak caused by one drug-susceptible strain. METHOD AND MEASUREMENTS: We included 9 patients with tuberculosis diagnosed during a period of 22 months, from a population-based study of the molecular epidemiology in San Francisco. Whole genome sequencing was performed using Illumina's sequencing by synthesis technology. A custom program written in Python was used to determine single nucleotide polymorphisms which were confirmed by PCR product Sanger sequencing. MAIN RESULTS: We obtained an average of 95.7% (94.1-96.9%) coverage for each isolate and an average fold read depth of 73 (1 to 250). We found 7 single nucleotide polymorphisms among the 9 isolates. The single nucleotide polymorphisms data confirmed all except one known epidemiological link. The outbreak strain resulted in 5 bacterial variants originating from the index case A1 with 0-2 mutations per transmission event that resulted in a secondary case. CONCLUSIONS: Whole genome sequencing analysis from a recent outbreak of tuberculosis enabled us to identify microevolutionary events observable during transmission, to determine 0-2 single nucleotide polymorphisms per transmission event that resulted in a secondary case, and to identify new epidemiologic links in the chain of transmission.

The essential genome of a bacterium.

Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non-coding elements, including 90 intergenic segments of unknown function. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. We identified all essential promoter elements for the cell cycle-regulated genes. The essential elements are preferentially positioned near the origin and terminus of the chromosome. The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.