Using de novo transcriptomes to decipher the relationships in cutthroat trout subspecies ().

For almost 200 years, the taxonomy of cutthroat trout (), a salmonid native to Western North America, has been in flux as ichthyologists and fisheries biologists have tried to describe the diversity within these fishes. Starting in the 1950s, Robert Behnke reexamined the cutthroat trout and identified 14 subspecies based on morphological traits, Pleistocene events, and modern geographic ranges. His designations became instrumental in recognizing and preserving the remaining diversity of cutthroat trout.

Heterochronic shift in gene expression leads to ontogenetic morphological divergence between two closely related polyploid species.

Heterochrony-alteration to the rate or timing of development-is an important mechanism of trait differentiation associated with speciation. Heterochrony may explain the morphological divergence between two polyploid species, June sucker () and Utah sucker (). The larvae of both species have terminal mouths; however, as adults, June sucker and Utah sucker develop subterminal and ventral mouths, respectively.

Phylogenetic relationships of three rockfish: , and (Scorpaeniformes, Scorpaenidae) based on complete mitochondrial genome sequences.

We characterise the complete mitochondrial genomes (mitogenomes) of Black rockfish ( Girard, 1856; n = 1), Dark rockfish ( Tilesius, 1813; n = 2) and Dusky rockfish ( Pallas, 1814; n = 2). The lengths of the mitogenomes are 16,405 bp for , 16,400 bp for both and 16,400 and 16,401 bp for . We examine these species' phylogenetic relationships using 35 previously published rockfish mitogenomes, representing 27 species.

Structure and Dynamics of Three NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.

NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S.

Oxygen-insensitive nitroreductase E. coli NfsA, but not NfsB, is inhibited by fumarate.

Escherichia coli NfsA and NfsB are founding members of two flavoprotein families that catalyze the oxygen-insensitive reduction of nitroaromatics and quinones by NAD(P)H. This reduction is required for the activity of nitrofuran antibiotics and the enzymes have also been proposed for use with nitroaromatic prodrugs in cancer gene therapy and biocatalysis, but the roles of the proteins in vivo in bacteria are not known. NfsA is NADPH-specific whereas NfsB can also use NADH.

Complete mitochondrial genomes of two rockfish: and (Scorpaenidae, Scorpaeniformes).

We report the complete mitochondrial genomes of two rockfish: and . The mitogenomes consist of 13 protein-coding regions, 22 tRNAs, two rRNAs, and one control region. mitogenome control regions are highly variable due to the presence of repeat sequences.

The 3D-structure, kinetics and dynamics of the E. coli nitroreductase NfsA with NADP provide glimpses of its catalytic mechanism.

Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP shows that a mobile loop forms a phosphate-binding pocket.

Complete mitochondrial genomes of June sucker and Utah sucker ( and ).

The relationship between June sucker (, Jordan, 1878) and Utah sucker (, Jordan & Gilbert, 1881) has been a matter of controversy since the mid 1900s. is endemic to Utah Lake, UT and has a subterminal mouth adapted for pelagic feeding. is widely distributed throughout the Bonneville Basin and Upper Snake River Basin and has a ventral mouth adapted for benthic feeding.

Species boundaries in the messy middle-A genome-scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi.

Species delimitation among closely related species is challenging because traditional phenotype-based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high-throughput sequencing, it has become increasingly cost-effective to acquire genome-scale data which can resolve previously ambiguous species boundaries. As the availability of genome-scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries.

The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN.

NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands.

Ontogenetic shape trajectory of Trichomycterus areolatus varies in response to water velocity environment.

Body and head shape among fishes both vary between environments influenced by water velocity and across ontogeny. Although the shape changes associated with variation in average water velocity and ontogeny are well documented, few studies have tested for the interaction between these two variables (i.e.

An Automated Tube Labeler for High-Throughput Purification Laboratories.

Centralized high-throughput purification laboratories routinely produce large numbers of test tubes with fractions containing the purified compounds of interest interspersed with test tubes containing fractions collected from undesired peaks. Because the next step after purification entails the removal of the solvent in a centrifugal evaporator with multiple sample positions per rotor, select test tubes must be labeled prior to dry-down to track the identity of each compound. The diversity of test tube sizes and tray configurations from different chromatography system vendors complicates this labeling task.

Integrated Platform for Expedited Synthesis-Purification-Testing of Small Molecule Libraries.

The productivity of medicinal chemistry programs can be significantly increased through the introduction of automation, leading to shortened discovery cycle times. Herein, we describe a platform that consolidates synthesis, purification, quantitation, dissolution, and testing of small molecule libraries. The system was validated through the synthesis and testing of two libraries of binders of polycomb protein EED, and excellent correlation of obtained data with results generated through conventional approaches was observed.

An Automated Microwave-Assisted Synthesis Purification System for Rapid Generation of Compound Libraries.

A novel methodology for the synthesis and purification of drug-like compound libraries has been developed through the use of a microwave reactor with an integrated high-performance liquid chromatography-mass spectrometry (HPLC-MS) system. The strategy uses a fully automated synthesizer with a microwave as energy source and robotic components for weighing and dispensing of solid reagents, handling liquid reagents, capper/crimper of microwave reaction tube assemblies, and transportation. Crude reaction products were filtered through solid-phase extraction cartridges and injected directly onto a reverse-phase chromatography column via an injection valve.

An automated synthesis-purification-sample-management platform for the accelerated generation of pharmaceutical candidates.

A flexible and integrated flow-chemistry-synthesis-purification compound-generation and sample-management platform has been developed to accelerate the production of small-molecule organic-compound drug candidates in pharmaceutical research. Central to the integrated system is a Mitsubishi robot, which hands off samples throughout the process to the next station, including synthesis and purification, sample dispensing for purity and quantification analysis, dry-down, and aliquot generation.

Long-term proliferation of functional human NK cells, with conversion of CD56(dim) NK cells to a CD56 (bright) phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12.

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL + IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21 day) NK cell proliferation.

Synthesis and structure-activity relationships of nitrobenzyl phosphoramide mustards as nitroreductase-activated prodrugs.

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E.

Minimal RB-responsive E1A promoter modification to attain potency, selectivity, and transgene-arming capacity in oncolytic adenoviruses.

Oncolytic adenoviruses are promising anticancer agents due to their ability to self-amplify at the tumor mass. However, tumor stroma imposes barriers difficult to overcome by these agents. Transgene expression is a valuable strategy to counteract these limitations and to enhance antitumor activity.

CD40 ligand induced cytotoxicity in carcinoma cells is enhanced by inhibition of metalloproteinase cleavage and delivery via a conditionally-replicating adenovirus.

Background: CD40 and its ligand (CD40L) play a critical role in co-ordinating immune responses. CD40 is also expressed in lymphoid malignancies and a number of carcinomas. In carcinoma cells the physiological outcome of CD40 ligation depends on the level of receptor engagement with low levels promoting cell survival and high levels inducing cell death.

Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: effects of combining beneficial single mutations.

Prodrug activation gene therapy for cancer involves expressing prodrug-activating enzymes in tumour cells, so they can be selectively killed by systemically administered prodrug. For example, Escherichia colinfsB nitroreductase (E.C.

Purification of drugs from biological fluids by counter-current chromatography.

Experiments were performed to demonstrate the potential of counter-current chromatography (CCC) for the isolation of drugs and their metabolites from biological matrices relevant to the metabolism studies of pharmaceutical research. Examples of typical drugs are spiked into biological media ex vivo to provide test samples for analysis. A mass spectrometer hyphenated to a CCC allows for the detection of small molecule drugs within the matrix through selected ion monitoring, and fraction collection can provide material for further structural elucidation by NMR.

Steady-state and stopped-flow kinetic studies of three Escherichia coli NfsB mutants with enhanced activity for the prodrug CB1954.

The enzyme nitroreductase, NfsB, from Escherichia coli has entered clinical trials for cancer gene therapy with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, CB1954 is a poor substrate for the enzyme. Previously we made several NfsB mutants that show better activity with CB1954 in a cell-killing assay in E.

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent.