Genetic basis of tetrodotoxin resistance in pufferfishes

Tetrodotoxin (TTX) is a highly potent neurotoxin that selectively binds to the outer vestibule of voltage-gated sodium channels. Pufferfishes accumulate extremely high concentrations of TTX without any adverse effect. A nonaromatic amino acid (Asn) residue present in domain I of the pufferfish, Takifugu pardalis, Na v1.4 channel has been implicated in the TTX resistance of pufferfishes . However, the effect of this residue on TTX sensitivity has not been investigated, and it is not known if this residue is conserved in all pufferfishes. We have investigated the genetic basis of TTX resistance in pufferfishes by comparing the sodium channels from two pufferfishes (Takifugu rubripes [fugu] and Tetraodon nigroviridis) and the TTX-sensitive zebrafish. Although all three fishes contain duplicate copies of Na v1.4 channels (Na v1.4a and Na v1.4b), several substitutions were found in the TTX binding outer vestibule of the two pufferfish channels. Electrophysiological studies showed that the nonaromatic residue (Asn in fugu and Cys in Tetraodon) in domain I of Na v1.4a channels confers TTX resistance. The Glu-to-Asp mutation in domain II of Tetraodon channel Na v1.4b is similar to that in the saxitoxin- and TTX-resistant Na+ channels of softshell clams . Besides helping to deter predators, TTX resistance enables pufferfishes to selectively feed on TTX-bearing organisms.     

Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare)

The least ambiguous genetic markers are those based on completely characterized DNA sequence polymorphisms. Unfortunately, assaying allele states by allele sequencing is slow and cumbersome. The most desirable type of genetic marker would be unambiguous, inexpensive to assay and would be assayable singly or in parallel with hundreds of other markers (multiplexable). In this report we sequenced alleles at 54 barley (Hordeum vulgare ssp. vulgare) loci, 38 of which contained single-nucleotide polymorphisms (SNPs). Many of these 38 loci contained multiple polymorphisms, and a total of 112 polymorphisms were scored in five barley genotypes. The polymorphism data set was analyzed both by using the individual mutations as cladistic characters and by reducing data for each locus to haplotypes. We compared the informativeness of these two approaches by consensus tree construction and bootstrap analysis. Both approaches provided similar results. Since some of the loci sequenced contained insertion/deletion events and multiple point mutations, we thought that these multiple-mutated loci might represent old alleles that predated the divergence of barley from H. spontaneum. We evaluated sequences from a sample of H. spontaneum accessions from the Eastern Mediterranean, and observed similar alleles present in both cultivated barley and H. spontaneum, suggesting either multiple domestication events or multiple transfers of genes between barley and its wild ancestor.     

Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture

Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.     

Estimating the global distribution of field size using crowdsourcing

There is an increasing evidence that smallholder farms contribute substantially to food production globally, yet spatially explicit data on agricultural field sizes are currently lacking. Automated field size delineation using remote sensing or the estimation of average farm size at subnational level using census data are two approaches that have been used. However, both have limitations, for example, automatic field size delineation using remote sensing has not yet been implemented at a global scale while the spatial resolution is very coarse when using census data. This paper demonstrates a unique approach to quantifying and mapping agricultural field size globally using crowdsourcing. A campaign was run in June 2017, where participants were asked to visually interpret very high resolution satellite imagery from Google Maps and Bing using the Geo‐Wiki application. During the campaign, participants collected field size data for 130 K unique locations around the globe. Using this sample, we have produced the most accurate global field size map to date and estimated the percentage of different field sizes, ranging from very small to very large, in agricultural areas at global, continental, and national levels. The results show that smallholder farms occupy up to 40% of agricultural areas globally, which means that, potentially, there are many more smallholder farms in comparison with the two different current global estimates of 12% and 24%. The global field size map and the crowdsourced data set are openly available and can be used for integrated assessment modeling, comparative studies of agricultural dynamics across different contexts, for training and validation of remote sensing field size delineation, and potential contributions to the Sustainable Development Goal of Ending hunger, achieve food security and improved nutrition and promote sustainable agriculture.     

A genome‐wide association study for gestation length in swine

Selection for increased litter size in swine has potentially resulted in a correlated increase in preweaning mortality. Additional selection criteria should be considered when selecting for increased litter size to account for associated decreases in piglet quality, specifically piglet survival, initial weight and growth. Traits such as gestation length (GL), which have been associated with piglet performance, could be utilized to improve piglet development and survivability. The objective of this study was to conduct a genome‐wide association study to identify genomic regions associated with GL in differing parities in swine (n = 831) from the University of Nebraska–Lincoln reproductive longevity project. Gestation length was calculated as the number of days between last insemination administered and farrowing. Sows were genotyped with the Illumina SNP60 BeadArray, and the data were analyzed using Bayesian mixture models for GL at parity 1, 2, 3 and 4 (GL1, GL2, GL3 and GL4 respectively). Means (SD) for GL1–GL4 were 113 (1.4), 114 (1.2), 114 (1.3) and 115 (1.2) respectively. Posterior mean heritability estimates (PSD) for GL1, GL2, GL3 and GL4 were 0.33 (0.06), 0.34 (0.07), 0.32 (0.08) and 0.20 (0.08) respectively. Rank correlations between genomic estimated breeding values between GL1 and GL2, GL3 and GL4 respectively were moderate: 0.67, 0.65 and 0.60. The top SNP (ASGA0017859, SSC4, 7.8 Mb), located in the top common genomic region associated with GL1, GL2 and GL3, was associated with a difference of 1.1 days in GL1 between homozygote genotypes (P < 0.0001). The results of this study suggest that GL is a largely polygenic trait with relatively minor contributions from multiple genomic regions.     

Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker

We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression.     

Taxonomic study of Asian species of poroid Auriculariales

A taxonomic study of poroid Asian species in the Auriculariales was conducted using phylogenetic and morphological methods. Phylogenetic analyses based on the LSU and ITS regions reveal that Elmerina was a polyphyletic genus. Moreover, E. hexagonoides collected from Malaysia and “E. hexagonoides” from Japan formed two distinct clades within a well-supported larger clade with Protomerulius caryae. These taxa comprise a different lineage from the type species of both Protomerulius and Protodaedalea, and we conclude that they are members of the genus Aporpium. The phylogenetic position of E. cladophora (type species of Elmerina) is still unclear, and we retain Aporpium and Protodaedalea as genera distinct from Elmerina. Aporpium strigosum sp. nov. is described to represent Japanese specimens misidentified as E. hexagonoides. Aporpium is characterized by resupinate to sessile basidiocarps, tough-fleshy to leathery context, poroid hymenophore, dimitic hyphal systems with unbranched skeletal hyphae, clavate to pyriform probasidia, and ellipsoid to cylindrical or allantoid basidiospores. Aporpium strigosum is similar to A. hexagonoides, but can be differentiated by the ochraceous to straw-colored basidiocarps, strigose pileus surface with long and stiff hairs, and the larger pores, basidia, and basidiospores.