The role of sexual isolation during rapid ecological divergence: Evidence for a new dimension of isolation in Rhagoletis pomonella.

The pace of divergence and likelihood of speciation often depends on how and when different types of reproductive barriers evolve. Questions remain about how reproductive isolation evolves after initial divergence. We tested for the presence of sexual isolation (reduced mating between populations due to divergent mating preferences and traits) in Rhagoletis pomonella flies, a model system for incipient ecological speciation. We measured the strength of sexual isolation between two very recently diverged (~170 generations) sympatric populations, adapted to different host fruits (hawthorn and apple). We found that flies from both populations were more likely to mate within than between populations. Thus, sexual isolation may play an important role in reducing gene flow allowed by early-acting ecological barriers. We also tested how warmer temperatures predicted under climate change could alter sexual isolation and found that sexual isolation was markedly asymmetric under warmer temperatures - apple males and hawthorn females mated randomly while apple females and hawthorn males mated more within populations than between. Our findings provide a window into the early speciation process and the role of sexual isolation after initial ecological divergence, in addition to examining how environmental conditions could shape the likelihood of further divergence.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1.

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

Engineering of methionine chain elongation part of glucoraphanin pathway in E. coli.

The methionine-derived glucosinolate glucoraphanin is associated with the health-promoting properties of broccoli. This has developed a strong interest in producing this compound in high amounts from a microbial source. Glucoraphanin synthesis starts with a five-gene chain elongation pathway that converts methionine to dihomo-methionine, which is subsequently converted to glucoraphanin by the seven-gene glucosinolate core structure pathway. As dihomo-methionine is the precursor amino acid for glucoraphanin production, a first challenge is to establish an expression system for production of dihomo-methionine. In planta, the methionine chain elongation enzymes are physically separated within the cell with the first enzyme in the cytosol while the rest are located in the chloroplast. A de-compartmentalization approach was applied to produce dihomo-methionine by expression of the respective plant genes in Escherichia coli cytosol. Introduction of two plasmids encoding the methionine chain elongation pathway into E. coli resulted in production of 25mgL(-1) of dihomo-methionine. In addition to chain-elongated methionine products, side-products from chain elongation of leucine were produced. Methionine supplementation enhanced dihomo-methionine production to 57mgL(-1), while keeping a steady level of the chain-elongated leucine products. Engineering of the de-compartmentalized pathway of dihomo-methionine in E. coli cytosol provides an important first step for microbial production of the health-promoting glucoraphanin.

Changing substrate specificity and iteration of amino acid chain elongation in glucosinolate biosynthesis through targeted mutagenesis of Arabidopsis methylthioalkylmalate synthase 1.

Methylthioalkylmalate synthases catalyse the committing step of amino acid chain elongation in glucosinolate biosynthesis. As such, this group of enzymes plays an important role in determining the glucosinolate composition of Brassicaceae species, including Arabidopsis thaliana Based on protein structure modelling of MAM1 from A. thaliana and analysis of 57 MAM sequences from Brassicaceae species, we identified four polymorphic residues likely to interact with the 2-oxo acid substrate. Through site-directed mutagenesis, the natural variation in these residues and the effect on product composition were investigated. Fifteen MAM1 variants as well as the native MAM1 and MAM3 from A. thaliana were characterised by heterologous expression of the glucosinolate chain elongation pathway in Escherichia coli Detected products derived from leucine, methionine or phenylalanine were elongated with up to six methylene groups. Product profile and accumulation were changed in 14 of the variants, demonstrating the relevance of the identified residues. The majority of the single amino acid substitutions decreased the length of methionine-derived products, while approximately half of the substitutions increased the phenylalanine-derived products. Combining two substitutions enabled the MAM1 variant to increase the number of elongation rounds of methionine from three to four. Notably, characterisation of the native MAMs indicated that MAM1 and not MAM3 is responsible for homophenylalanine production. This hypothesis was confirmed by glucosinolate analysis in mam1 and mam3 mutants of A. thaliana.

Optimization of Engineered Production of the Glucoraphanin Precursor Dihomomethionine in Nicotiana benthamiana.

Glucosinolates are natural products characteristic of the Brassicales order, which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Toward our goal of creating a rich source of glucoraphanin for dietary supplements, we have previously reported the feasibility of engineering glucoraphanin in Nicotiana benthamiana through transient expression of glucoraphanin biosynthetic genes from A. thaliana (Mikkelsen et al., 2010). As side-products, we obtained fivefold to eightfold higher levels of chain-elongated leucine-derived glucosinolates, not found in the native plant. Here, we investigated two different strategies to improve engineering of the methionine chain elongation part of the glucoraphanin pathway in N. benthamiana: (1) coexpression of the large subunit (LSU1) of the heterodimeric isopropylmalate isomerase and (2) coexpression of BAT5 transporter for efficient transfer of intermediates across the chloroplast membrane. We succeeded in raising dihomomethionine (DHM) levels to a maximum of 432 nmol g(-1) fresh weight that is equivalent to a ninefold increase compared to the highest production of this intermediate, as previously reported (Mikkelsen et al., 2010). The increased DHM production without increasing leucine-derived side-product levels provides new metabolic engineering strategies for improved glucoraphanin production in a heterologous host.

Intraluminal duodenal diverticula: collective review with report of a laparoscopic excision.

A comprehensive review of intraluminal duodenal diverticulum (IDD) is presented, along with a report of a completely laparoscopic excision of this duodenal abnormality as well as a report of magnetic resonance cholangiopancreatography demonstrating the classic fluoroscopic "wind sock sign" pathognomonic appearance of IDD. IDD may easily be missed unless one specifically considers this entity in patients presenting with symptoms of foregut disease. Patients with IDD typically present in the fourth decade of life with duration of symptoms less than 5 years that typically include pain, nausea and vomiting, pancreatitis, and gastrointestinal bleeding. Diagnosis usually requires imaging studies and upper gastrointestinal endoscopy. Laparoscopic excision is recommended because of superior visualization of significant intestinal anatomic abnormalities, the need for accurate ampullary localization, and the ability to facilitate complete diverticular excision while maintaining biliary and pancreatic ductal integrity. Review of surgical literature suggests that IDD results from congenital duodenal developmental abnormalities matured by long-term duodenal peristalsis.