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The rise of "-omics" related technologies makes it essential for students to have experience working with large bioinformatics data sets. Although”-omic” datasets are complex and abstract, effective instruction can be improved when students see the direct connections between the data on a computer screen and the results of "wet lab" experimentation. Here we describe the use of protein mass spectrometry as a means for students to gain experience in connecting bioinformatic data with work done at the lab bench. Course-based Research Experiences (CREs) based on these techniques are accessible to institutions of all types as a result of rapidly declining costs for whole genome and proteome analysis. Our implementation is within a CRE based on viral infection of a bacterial host; however, this basic paradigm may be applied to other experimental systems of interest.

Four bacteriophages infecting Mycobacterium smegmatis mc 2 155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages.

Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T(4) transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17beta-glucuronide (E(2)17betaG). Intriguingly, published data suggest that E(2)17betaG inhibition of Oatp1c1-mediated T(4) transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T(4) and E(2)17betaG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T(4) and E(2)17betaG concentrations (100 pm to 300 nm and 27 nm to 200 mum, respectively). Eadie-Hofstee plots derived from these detailed T(4) and E(2)17betaG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T(4) and E(2)17betaG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T(4) and E(2)17betaG. T(4) and E(2)17betaG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E(2)17betaG transport more strongly than T(4) transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.

Organic anion transporting polypeptide (Oatp) 1c1 is a high-affinity T4 transporter with narrow substrate specificity expressed at the blood-brain barrier. A transport model using cells overexpressing Oatp1c1 was created to identify novel Oatp1c1 substrates and inhibitors. Rat Oatp1c1 was cloned and stably expressed in human embryonic kidney 293 cells. Oatp1c1-transfected human embryonic kidney 293 cells transported 125I-labeled T4 in a time-dependent manner that was completely abolished in the presence of excess unlabeled T4. Next, various compounds, including inhibitors of thyroid hormone uptake, were screened for inhibitory effects on Oatp1c1-mediated T4 uptake. Phenytoin (64%), indocyanine green (17%), fenamic acid (68%), diclofenac (51%), and meclofenamic acid (33%) all reduced T4 uptake by Oatp1c1 when assayed at concentrations of 10 μM. Dose-response assays for the fenamic acids, iopanoic acid, indocyanine green, and phenytoin revealed IC50 values for Oatp1c1 T4 uptake below or near the blood plasma levels after therapeutic doses. Further kinetic assays and reciprocal plot analyses demonstrated that the fenamic acid diclofenac inhibited in a competitive manner. Finally, microvessels were isolated from adult rat brain and assessed for T4 uptake. Ten micromolar of fenamate concentrations inhibited T4 microvessel uptake with a similar hierarchical inhibition profile [fenamic acid (43%), diclofenac (78%), and meclofenamic acid (85%)], as observed for Oatp1c1 transfected cells. Oatp1c1 is expressed luminally and abluminally in the blood-brain barrier endothelial cell, and exhibits bidirectional transport capabilities. Together, these data suggest that Oatp1c1 transports fenamates into, and perhaps across, brain barrier cells. The fenamate class of nonsteroidal anti-inflammatory drugs is transported by organic anion transporting polypeptides and competitively inhibits thyroxine transport in brain microvessels.

Mycobacteriophages – viruses of mycobacterial hosts – are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages – Corndog, Catdawg, Dylan, Firecracker, and YungJamal – designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8–9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

Organic anion transporting polypeptide 1c1 (Oatp1c1) is a high-affinity T4 transporter expressed in brain barrier cells. To identify Oatp1c1 amino acid residues critical for T4 transport, consensus membrane topology was predicted and a three-dimensional Oatp1c1 structure was generated using the known structures of major facilitator superfamily (MFS) transporters, glycerol 3-phosphate transporter, lactose permease, and the multidrug transporter Escherichia coli multidrug resistance protein D as templates. A total of nine amino acid mutations were generated based on amino acid conservation, localization to putative transmembrane domains, and side chain functionality. Mutant constructs were transiently transfected into human embryonic kidney 293 cells and assessed for plasma membrane localization and the capacity to transport substrate 125I-T4. Wild-type Oatp1c1, R601S, P609A, W277A/W278A, W277F/W278F, G399A/G409A, and G399L/G409L were all expressed at the plasma membrane. Wild-type Oatp1c1 and W277F/W278F displayed biphasic T4 transport kinetics, albeit the mutant did so with an approximately 10-fold increase in high-affinity Michaelis constant. The W277A/W278A mutation abolished Oatp1c1 T4 transport. G399A/G409A and G399V/G409V mutants displayed near wild-type activity in an uptake screen but exhibited diminished T4 transport activity at high-substrate concentrations, suggesting a substrate binding site collapse or inability to convert between input and output states. Finally, transmembrane domain 11 mutants R601S and P609A displayed partial T4 transport activity with significantly reduced maximum velocities and higher Michaelis constant. Arg601 is functionally strongly conserved with members of the MFS whose structures and function have been extensively studied. These data provide the experimental foundation for mapping Oatp1c1 substrate binding sites and reveal evolutionary conservation with bacterial MFS transporter members.

Organic anion-transporting polypeptides (Oatps) are solute carrier family members that exhibit marked evolutionary conservation. Mammalian Oatps exhibit wide tissue expression with an emphasis on expression in barrier cells. In the brain, Oatps are expressed in the blood-brain barrier endothelial cells and blood-cerebrospinal fluid barrier epithelial cells. This expression profile serves to illustrate a central role for Oatps in transporting endo- and xenobiotics across brain barrier cells. This chapter will detail the expression patterns and substrate specificities of Oatps expressed in the brain, and will place special emphases on the role of Oatps in prostaglandin synthesis and in the transport of conjugated endobiotics.

Organic anion‐transporting polypeptides (Oatps) are solute carrier family members that exhibit marked evolutionary conservation. Mammalian Oatps exhibit wide tissue expression with an emphasis on expression in barrier cells. In the brain, Oatps are expressed in the blood–brain barrier endothelial cells and blood–cerebrospinal fluid barrier epithelial cells. This expression profile serves to illustrate a central role for Oatps in transporting endo‐ and xenobiotics across brain barrier cells. This chapter will detail the expression patterns and substrate specificities of Oatps expressed in the brain, and will place special emphases on the role of Oatps in prostaglandin synthesis and in the transport of conjugated endobiotics.