Killer whale depredation and associated costs to Alaskan sablefish, Pacific halibut and Greenland turbot longliners.

Killer whale (Orcinus orca) depredation (whales stealing or damaging fish caught on fishing gear) adversely impacts demersal longline fisheries for sablefish (Anoplopoma fimbria), Pacific halibut (Hippoglossus stenolepis) and Greenland turbot (Reinhardtius hippoglossoides) in the Bering Sea, Aleutian Islands and Western Gulf of Alaska. These interactions increase direct costs and opportunity costs associated with catching fish and reduce the profitability of longline fishing in western Alaska. This study synthesizes National Marine Fisheries Service observer data, National Marine Fisheries Service sablefish longline survey and fishermen-collected depredation data to: 1) estimate the frequency of killer whale depredation on longline fisheries in Alaska; 2) estimate depredation-related catch per unit effort reductions; and 3) assess direct costs and opportunity costs incurred by longliners in western Alaska as a result of killer whale interactions. The percentage of commercial fishery sets affected by killer whales was highest in the Bering Sea fisheries for: sablefish (21.4%), Greenland turbot (9.9%), and Pacific halibut (6.9%). Average catch per unit effort reductions on depredated sets ranged from 35.1-69.3% for the observed longline fleet in all three management areas from 1998-2012 (p<0.001). To compensate for depredation, fishermen set additional gear to catch the same amount of fish, and this increased fuel costs by an additional 82% per depredated set (average $433 additional fuel per depredated set). In a separate analysis with six longline vessels in 2011 and 2012, killer whale depredation avoidance measures resulted in an average additional cost of $494 per depredated vessel-day for fuel and crew food. Opportunity costs of time lost by fishermen averaged $522 per additional vessel-day on the grounds. This assessment of killer whale depredation costs represents the most extensive economic evaluation of this issue in Alaska to date and will help longline fishermen and managers consider the costs and benefits of depredation avoidance and alternative policy solutions.

A nondiscriminating glutamyl-tRNA synthetase in the plasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.

The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA(Gln) biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA(Glu) and tRNA(Gln), determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA(Gln) biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.

Combining different design strategies for rational affinity maturation of the MICA-NKG2D interface.

We redesigned residues on the surface of MICA, a protein that binds the homodimeric immunoreceptor NKG2D, to increase binding affinity with a series of rational, incremental changes. A fixed-backbone RosettaDesign protocol scored a set of initial mutations, which we tested by surface plasmon resonance for thermodynamics and kinetics of NKG2D binding, both singly and in combination. We combined the best four mutations at the surface with three affinity-enhancing mutations below the binding interface found with a previous design strategy. After curating design scores with three cross-validated tests, we found a linear relationship between free energy of binding and design score, and to a lesser extent, enthalpy and design score. Multiple mutants bound with substantial subadditivity, but in at least one case full additivity was observed when combining distant mutations. Altogether, combining the best mutations from the two strategies into a septuple mutant enhanced affinity by 50-fold, to 50 nM, demonstrating a simple, effective protocol for affinity enhancement.

Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence.

We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5-12 new techniques learned, which are transferrable to graduate research in academia and industry.

Out of time and out of pocket: experiences of women seeking state-subsidized insurance for abortion care in Massachusetts.

BACKGROUND: Massachusetts has implemented reforms aimed at providing universal health care coverage and covers abortion through subsidized state insurance programs. Three Massachusetts abortion funds evaluated their referral processes for low-income women from April to October 2010 to learn about women's experiences applying for subsidized insurance and to identify barriers to obtaining insurance or its use for abortion services. METHODS: Follow-up interviews were conducted with 39 low-income women thought eligible for subsidized insurance at least 1 month after their initial contact with the funds. RESULTS: Health insurance literacy was low, and participants reported confusion distinguishing between levels of subsidized insurance. The process of applying for subsidized insurance delayed a substantial proportion of procedures. More than two thirds of the women who applied for state coverage had become insured or expected to become insured shortly, but only one third of respondents who applied were able to secure insurance in time for their abortion care. Two women were unable to obtain abortions as a result of delays. Delays also limited low-income women's ability to obtain medication abortion. CONCLUSION: This analysis suggests that the process for enrolling in subsidized insurance does not currently meet the goal of providing women with coverage for abortion care (and other health needs) in a timely way. Systemic improvements are needed to ensure that enrollments are processed quickly and disruptions in coverage are minimized. Information resources should be developed to help women and their families understand health insurance and coverage of services.