Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae)

Discussions aimed at resolution of the Tree of Life are most often focused on the interrelationships of major organismal lineages. In this study, we focus on the resolution of some of the most apical branches in the Tree of Life through exploration of the phylogenetic relationships of darters, a species-rich clade of North American freshwater fishes. With a near-complete taxon sampling of close to 250 species, we aim to investigate strategies for efficient multilocus data sampling and the estimation of divergence times using relaxed-clock methods when a clade lacks a fossil record. Our phylogenetic data set comprises a single mitochondrial DNA (mtDNA) gene and two nuclear genes sampled from 245 of the 248 darter species. This dense sampling allows us to determine if a modest amount of nuclear DNA sequence data can resolve relationships among closely related animal species. Darters lack a fossil record to provide age calibration priors in relaxed-clock analyses. Therefore, we use a near-complete species-sampled phylogeny of the perciform clade Centrarchidae, which has a rich fossil record, to assess two distinct strategies of external calibration in relaxed-clock divergence time estimates of darters: using ages inferred from the fossil record and molecular evolutionary rate estimates. Comparison of Bayesian phylogenies inferred from mtDNA and nuclear genes reveals that heterospecific mtDNA is present in approximately 12.5% of all darter species. We identify three patterns of mtDNA introgression in darters: proximal mtDNA transfer, which involves the transfer of mtDNA among extant and sympatric darter species, indeterminate introgression, which involves the transfer of mtDNA from a lineage that cannot be confidently identified because the introgressed haplotypes are not clearly referable to mtDNA haplotypes in any recognized species, and deep introgression, which is characterized by species diversification within a recipient clade subsequent to the transfer of heterospecific mtDNA. The results of our analyses indicate that DNA sequences sampled from single-copy nuclear genes can provide appreciable phylogenetic resolution for closely related animal species. A well-resolved near-complete species-sampled phylogeny of darters was estimated with Bayesian methods using a concatenated mtDNA and nuclear gene data set with all identified heterospecific mtDNA haplotypes treated as missing data. The relaxed-clock analyses resulted in very similar posterior age estimates across the three sampled genes and methods of calibration and therefore offer a viable strategy for estimating divergence times for clades that lack a fossil record. In addition, an informative rank-free clade-based classification of darters that preserves the rich history of nomenclature in the group and provides formal taxonomic communication of darter clades was constructed using the mtDNA and nuclear gene phylogeny. On the whole, the appeal of mtDNA for phylogeny inference among closely related animal species is diminished by the observations of extensive mtDNA introgression and by finding appreciable phylogenetic signal in a modest sampling of nuclear genes in our phylogenetic analyses of darters.     

Histamine deficiency promotes inflammation-associated carcinogenesis through reduced myeloid maturation and accumulation of CD11b+Ly6G+ immature myeloid cells

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b(+)Ly6G(+) immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b(+)Ly6G(+) cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b(+)Ly6G(+) IMCs in early cancer development.     

Targeting the Ubiquitin E3 Ligase MuRF1 to Inhibit Muscle Atrophy

Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.     

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (～45%) of the 1,101 essential yeast genes, with ～30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.     

Seven new species within western Atlantic Starksia atlantica, S. lepicoelia, and S. sluiteri (Teleostei, Labrisomidae), with comments on congruence of DNA barcodes and species

Specimens of Starksia were collected throughout the western Atlantic, and a 650-bp portion of the mitochondrial gene cytochrome oxidase-c subunit I (COl) was sequenced as part of a re-analysis of species diversity of western Central Atlantic shorefishes. A neighbor-joining tree constructed from the sequence data suggests the existence of several cryptic species. Voucher specimens from each genetically distinct lineage and color photographs of vouchers taken prior to dissection and preservation were examined for diagnostic morphological characters. The results suggest that Starksia atlantica, Starksia lepicoelia, and Starksia sluiteri are species complexes, and each comprises three or more species. Seven new species are described. DNA data usually support morphological features, but some incongruence between genetic and morphological data exists. Genetic lineages are only recognized as species if supported by morphology. Genetic lineages within western Atlantic Starksia generally correspond to geography, such that members of each species complex have a very restricted geographical distribution. Increasing geographical coverage of sampling locations will almost certainly increase the number of Starksia species and species complexes recognized in the western Atlantic. Combining molecular and morphological investigations is bringing clarity to the taxonomy of many genera of morphologically similar fishes and increasing the number of currently recognized species. Future phylogenetic studies should help resolve species relationships and shed light on patterns of speciation in western Atlantic Starksia.     

Inferring mechanisms of compensation from E-MAP and SGA data using local search algorithms for max cut

A new method based on a mathematically natural local search framework for max cut is developed to uncover functionally coherent module and BPM motifs in high-throughput genetic interaction data. Unlike previous methods, which also consider physical protein-protein interaction data, our method utilizes genetic interaction data only; this becomes increasingly important as high-throughput genetic interaction data is becoming available in settings where less is known about physical interaction data. We compare modules and BPMs obtained to previous methods and across different datasets. Despite needing no physical interaction information, the BPMs produced by our method are competitive with previous methods. Biological findings include a suggested global role for the prefoldin complex and a SWR subcomplex in pathway buffering in the budding yeast interactome.     

Production and characterization of Acidothermus cellulolyticus endoglucanase in Pichia pastoris

The endoglucanase (E1) from Acidothermus cellulolyticus has been used extensively in cellulase research. The goal of this work was to produce high levels of this enzyme in a system that facilitates purification. A codon-optimized synthetic gene for A. cellulolyticus E1 with a C-terminal histidine tag was cloned into the genome of Pichia pastoris. Strain KM71H expressed the most enzyme, with a yield of 550mg/L culture supernatant. The temperature optimum (80°C) and pH optimum (5.1) of the purified enzyme agree with previously determined values for the enzyme produced in other systems. Michaelis-Menten kinetic parameters were determined, using a fluorescent substrate (methylumbelliferyl-β-d-cellobioside) at various temperatures. This thermostable enzyme can be used in future cellulosic biofuels-related research.