A reverse binding motif that contributes to specific protease inhibition by antibodies

The type II transmembrane serine protease family consists of 18 closely related serine proteases that are implicated in multiple functions. To identify selective, inhibitory antibodies against one particular type II transmembrane serine protease, matriptase [MT-SP1 (membrane-type serine protease 1)], a phage display library was created with a natural repertoire of Fabs [fragment antigen binding (Fab)] from human naïve B cells. Fab A11 was identified with a 720 pM inhibition constant and high specificity for matriptase over other trypsin-fold serine proteases. A Trichoderma reesei system expressed A11 with a yield of ～ 200 mg/L. The crystal structure of A11 in complex with matriptase has been determined and compared to the crystal structure of another antibody inhibitor (S4) in complex with matriptase. Previously discovered from a synthetic single-chain variable fragment library, S4 is also a highly selective and potent matriptase inhibitor. The crystal structures of the A11/matriptase and S4/matriptase complexes were solved to 2.1 Å and 1.5 Å, respectively. Although these antibodies, discovered from separate libraries, interact differently with the protease surface loops for their specificity, the structures reveal a similar novel mechanism of protease inhibition. Through the insertion of the H3 variable loop in a reverse orientation at the substrate-binding pocket, these antibodies bury a large surface area for potent inhibition and avoid proteolytic inactivation. This discovery highlights the critical role that the antibody scaffold plays in positioning loops to bind and inhibit protease function in a highly selective manner. Additionally, Fab A11 is a fully human antibody that specifically inhibits matriptase over other closely related proteases, suggesting that this approach could be useful for clinical applications.     

Molecular characterisation and interpretation of genetic diversity within globally distributed germplasm collections of tall fescue (Festuca arundinacea Schreb.) and meadow fescue (F. pratensis Huds.)

Allohexaploid tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum [Schreb.] Darbysh.) is an agriculturally important grass cultivated for pasture and turf world-wide. Genetic improvement of tall fescue could benefit from the use of non-domesticated germplasm to diversify breeding populations through the incorporation of novel and superior allele content. However, such potential germplasm must first be characterised, as three major morphotypes (Continental, Mediterranean and rhizomatous) with varying degrees of hybrid interfertility are commonly described within this species. As hexaploid tall fescue is also a member of a polyploid species complex that contains tetraploid, octoploid and decaploid taxa, it is also possible that germplasm collections may have inadvertently sampled some of these sub-species. In this study, 1,040 accessions from the publicly available United States Department of Agriculture tall fescue and meadow fescue germplasm collections were investigated. Sequence of the chloroplast genome-located matK gene and the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) permitted attribution of accessions to the three previously known morphotypes and also revealed the presence of tall fescue sub-species of varying ploidy levels, as well as other closely related species. The majority of accessions were, however, identified as Continental hexaploid tall fescue. Analysis using 34 simple sequence repeat markers was able to further investigate the level of genetic diversity within each hexaploid tall fescue morphotype group. At least two genetically distinct sub-groups of Continental hexaploid tall fescue were identified which are probably associated with palaeogeographic range expansion of this morphotype. This work has comprehensively characterised a large and complex germplasm collection and has identified genetically diverse accessions which may potentially contribute valuable alleles at agronomic loci for tall fescue cultivar improvement programs.     

Distribution and phenology of Dasineura oxycoccana (Diptera: Cecidomyiidae) in Michigan blueberries

The blueberry gall midge, Dasineura oxycoccana Johnson, is a serious pest of rabbiteye blueberries in Florida, Georgia, and Mississippi, and a potential pest of southern and northern highbush blueberries. Its damage has been observed with increasing frequency in highbush blueberry plantings in the Great Lakes region, including in Wisconsin and in Michigan. Unlike in rabbiteye blueberry plantings, where blueberry gall midge primarily damages flowering buds, it is found to damage only the vegetative shoots of northern highbush blueberry. In this study, farms throughout Michigan were surveyed for the presence of blueberry gall midge and it was found in 43 of 46 sampled farms in 11 counties. From 2009-2011, several monitoring techniques, including yellow sticky traps, emergence traps, observational sampling, and vegetative shoot dissections were used to determine the ecology of this species in blueberry fields in southwest Michigan. Emergence traps were most useful in early detection of blueberry gall midge in April, and observational sampling for damage symptoms and vegetative shoot dissections revealed multiple population peaks throughout July and August. Infestation was detected in vegetative shoot tips in all parts of the bushes, with initial infestation greatest at the base of bushes. Degree day accumulations until first midge detection and peak infestation suggest some potential for predicting key events in the pest's phenology. This information about the distribution and timing of infestation will be useful in developing management strategies for blueberry gall midge infestation.     

Exertional myopathy in pileated woodpeckers (Dryocopus pileatus) subsequent to capture

Out of 33 Pileated Woodpeckers (Dryocopus pileatus) captured and fitted with radio-transmitters, 12 were later found dead. Three carcasses were recovered and submitted for necropsy. One bird had large pale foci in multiple muscles. Microscopically, skeletal muscle in all three had evidence of severe coagulative necrosis, consistent with capture myopathy.     

Another JA/COI1-independent role of OPDA detected in tomato embryo development

Jasmonates (JAs) are ubiquitously occurring signaling compounds in plants formed in response to biotic and abiotic stress as well as in development. (+)-7-iso-jasmonoyl isoleucine, the bioactive JA, is involved in most JA-dependent processes mediated by the F-box protein COI1 in a proteasome-dependent manner. However, there is an increasing number of examples, where the precursor of JA biosynthesis, cis-(+)-12-oxophytodienoic acid (OPDA) is active in a JA/COI1-independent manner. Here, we discuss those OPDA-dependent processes, thereby giving emphasis on tomato embryo development. Recent data on seed coat-generated OPDA and its role in embryo development is discussed based on biochemical and genetic evidences.     

The origin of proteasome-inhibitor resistant HLA class I peptidomes: a study with HLA-A*68:01

Some HLA class I molecules bind a significant fraction of their constitutive peptidomes in the presence of proteasome inhibitors. In this study, A*68:01-bound peptides, and their parental proteins, were characterized through massive mass spectrometry sequencing to refine its binding motif, including the nearly exclusive preference for C-terminal basic residues. Stable isotope tagging was used to distinguish proteasome-inhibitor sensitive and resistant ligands. The latter accounted for less than 20% of the peptidome and, like in HLA-B27, arose predominantly from small and basic proteins. Under the conditions used for proteasome inhibition in vivo, epoxomicin and MG-132 incompletely inhibited the hydrolysis of fluorogenic substrates specific for the tryptic or for both the tryptic and chymotryptic subspecificities, respectively. This incomplete inhibition was also reflected in the cleavage of synthetic peptide precursors of A*68:01 ligands. For these substrates, the inhibition of the proteasome resulted in altered cleavage patterns. However these alterations did not upset the balance between cleavage at peptide bonds resulting in epitope destruction and those leading to their generation. The results indicate that inhibitor-resistant HLA class I ligands are not necessarily produced by non-proteasomal pathways. However, their generation is not simply explained by decreased epitope destruction upon incomplete proteasomal inhibition and may require additional proteolytic steps acting on incompletely processed proteasomal products.     

Proteome of Aedes aegypti in response to infection and coinfection with microsporidian parasites

Hosts are frequently infected with more than one parasite or pathogen at any one time, but little is known as to how they respond to multiple immune challenges compared to those involving single infections. We investigated the proteome of Aedes aegypti larvae following infection with either Edhazardia aedis or Vavraia culicis, and coinfections involving both. They are both obligate intracellular parasites belonging to the phylum microsporidia and infect natural populations of Ae. aegypti. The results found some proteins only showing modified abundance in response to infections involving E. aedis, while others were only differentially abundant when infections involved V. culicis. Some proteins only responded with modified abundance to the coinfection condition, while others were differentially abundant in response to all three types of infection. As time since infection increased, the response to each of the single parasite infections diverged, while the response to the E. aedis and coinfection treatments converged. Some of the proteins differentially abundant in response to infection were identified. They included two vacuolar ATPases, proteins known to have a role in determining the infection success of intracellular parasites. This result suggests microsporidia could influence the infection success of other intracellular pathogens infecting vector species of mosquito, including viruses, Plasmodium and Wolbachia.