Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, γ-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera^® (γ-Zein ER-accumulating domain) is the N-terminal proline-rich domain of γ-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera^® domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera^® domain. We demonstrated that Zera^®-F1-V protein accumulation was at least 3× higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera^® technology to induce protein body formation in non-seed tissues. Zera^® expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera^® technology to substantially increase the accumulation of value-added proteins in plants.     

Asn112 in Plasmodium falciparum glutathione S-transferase is essential for induced reversible tetramerization by phosphate or pyrophosphate

The glutathione S-transferase from Plasmodium falciparum presents distinct features which are absent from mammalian GST isoenzyme counterparts. Most apparent among these are the ability to tetramerize and the presence of a flexible loop. The loop, situated between the 113–119 residues, has been reported necessary for the tetramerization process. In this article, we report that a residue outside of this loop, Asn112, is a key to the process — to the point where the single Asn112Leu mutation prevents tetramerization altogether. We propose that a structural pattern involving the interaction of the Asn112 and Lys117 residues from two neighboring subunits plays a role in keeping the tetramer structure stable. We also report that, for the tetramerization of the wild-type PfGST to occur, phosphate or pyrophosphate anions must be present. In other words, tetramerization is a phosphate- or pyrophosphate-induced process. Furthermore, the presence of magnesium reinforces this induction. We present experimental evidence for these claims as well as a preliminary calorimetric and kinetic study of the dimeric Asn112Leu PfGST mutant. We also propose a putative binding site for phosphate or pyrophosphate anions through a comparative structural analysis of PfGST and pyrophosphatases from several organisms. Our results highlight the differences between PfGST and the human isoenzymes, which make the parasite enzyme a suitable antimalarial target.     

Colony foraging allocation is finely tuned to food distance and sweetness even close to a bee colony

Social bee colonies can allocate their foraging resources over a large spatial scale, but how they allocate foraging on a small scale near the colony is unclear and can have implications for understanding colony decision‐making and the pollination services provided. Using a mass‐foraging stingless bee, Scaptotrigona pectoralis (Dalla Torre) (Hymenoptera: Apidae: Meliponini), we show that colonies will forage near their nests and allocate their foraging labor on a very fine spatial scale at an array of food sources placed close to the colony. We counted the foragers that a colony allocated to each of nine feeders containing 1.0, 1.5, or 2.0 M sucrose solution [31, 43, and 55% sucrose (wt/wt), respectively] at distances of 10, 15, and 20 m from the nest. A significantly greater number of foragers (2.6–5.3 fold greater) visited feeders placed 10 vs. 20 m away from the colony. Foraging allocation also corresponded to food quality. At the 10‐m feeders, 4.9‐fold more foragers visited 2.0 M as compared to 1.0 M sucrose feeders. Colony forager allocation thus responded to both differences in food distance and quality even when the travel cost was negligible compared to normal colony foraging distances (10 m vs. an estimated 800–1 710 m). For a nearby floral patch, this could result in unequal floral visitation and pollination.     

A molecular phylogeny of bivalve mollusks: ancient radiations and divergences as revealed by mitochondrial genes

Background

Bivalves are very ancient and successful conchiferan mollusks (both in terms of species number and geographical distribution). Despite their importance in marine biota, their deep phylogenetic relationships were scarcely investigated from a molecular perspective, whereas much valuable work has been done on taxonomy, as well as phylogeny, of lower taxa.

Methodology/Principal Findings

Here we present a class-level bivalve phylogeny with a broad sample of 122 ingroup taxa, using four mitochondrial markers (MT-RNR1, MT-RNR2, MT-CO1, MT-CYB). Rigorous techniques have been exploited to set up the dataset, analyze phylogenetic signal, and infer a single final tree. In this study, we show the basal position of Opponobranchia to all Autobranchia, as well as of Palaeoheterodonta to the remaining Autobranchia, which we here propose to call Amarsipobranchia. Anomalodesmata were retrieved as monophyletic and basal to (Heterodonta + Pteriomorphia).

Conclusions/Significance

Bivalve morphological characters were traced onto the phylogenetic trees obtained from the molecular analysis; our analysis suggests that eulamellibranch gills and heterodont hinge are ancestral characters for all Autobranchia. This conclusion would entail a re-evaluation of bivalve symplesiomorphies.     

Ontogenetic integration between force production and force reception: a case study in Ctenomys (Rodentia: Caviomorpha)

During ontogeny, complex adaptations undergo changes that sometimes entail different functional capabilities. This fact constrains the behaviour of organisms at each developmental stage. Rodents have ever‐growing incisors for gnawing, and a powerful jaw musculature. The incisors are long enough, relative to their diameter, to be affected by bending stresses. This is particularly true in the subterranean Ctenomys that uses its incisors for digging. We measured bite force (BF) in individuals of different ages using a force transducer. We estimated incisor section modulus Z, a geometrical parameter proportional to bending strength. A relative strength indicator was calculated as S = Z/BF incisor length. We found that ontogenetic BF scales to body mass with positive allometry. However, an anova showed non‐significant differences in S, neither between sexes nor among age classes. This result implies that during growth, incisors might have a rather similar ability to withstand bending stresses from increasing masticatory forces, what may be considered evidence of ontogenetic integration of force production (by muscles) and force reception (by the incisors). This fact well correlates with the observation that pups and juveniles of C. talarum incorporate solid foods shortly after birth, and they are able to dig burrows early in life.     

Transgenic mice recapitulate the phenotypic heterogeneity of genetic prion diseases without developing prion infectivity: Role of intracellular PrP retention in neurotoxicity

Genetic prion diseases are degenerative brain disorders caused by mutations in the gene encoding the prion protein (PrP). Different PrP mutations cause different diseases, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome and fatal familial insomnia (FFI). The reason for this variability is not known. It has been suggested that prion strains with unique self-replicating and neurotoxic properties emerge spontaneously in individuals carrying PrP mutations, dictating the phenotypic expression of disease. We generated transgenic mice expressing the FFI mutation, and found that they developed a fatal neurological illness highly reminiscent of FFI, and different from those of similarly generated mice modeling genetic CJD and GSS. Thus transgenic mice recapitulate the phenotypic differences seen in humans. The mutant PrPs expressed in these mice are misfolded but unable to self-replicate. They accumulate in different compartments of the neuronal secretory pathway, impairing the membrane delivery of ion channels essential for neuronal function. Our results indicate that conversion of mutant PrP into an infectious isoform is not required for pathogenesis, and suggest that the phenotypic variability may be due to different effects of mutant PrP on intracellular transport.     

Spirohydantoin derivatives of thiopyrano[2,3-b]pyridin-4(4H)-one as potent in vitro and in vivo aldose reductase inhibitors

The 2,3-dihydrospiro[4H-thiopyrano[2,3-b]pyridin-4,4'-imidazolidine]-2',5'-dione 3 and its 7-methyl analogue 4 were synthesized and tested for their ability to inhibit aldose reductase (ALR2). To expand the structure-activity relationships, the sulfone 5 and the acetic acid derivative 7 were also prepared and tested. Compounds 3 and 4 proved to be potent ALR2 inhibitors, with IC50 values in the submicromolar range (0.96 and 0.94 microM, respectively) similar to that of sorbinil (0.65 microM). Moreover, compound 3 was found to be highly potent in preventing cataract development in severely galactosemic rats, like tolrestat, when administered as an eyedrop solution. Docking simulations of both R- and S-isomers of 3 into the ALR2 crystal structure were carried out to guide, prospectively, the design of new analogues.     

Pulsatile Flow Inside Moderately Elastic Arteries,Its Modelling and Effects of Elasticity

Pulsatile flow inside a moderately elastic circular conduit with a smooth expansion is studied as a model to understand the influence of wall elasticity in artery flow. The solution of the simultaneous fluid-wall evolution is evaluated by a perturbative method, where the zeroth order solution is represented by the flow in a rigid vessel; the first order correction gives the wall motion and induced flow modification without the need to solve the difficult coupled problem. Such an approach essentially assumes a locally infinite celerity, therefore it represent a good approximation for the fluid-wall interaction in sites of limited extent (branches, stenosis, aneurism, etc.), which include typical situations associated with vascular diseases. The problem is solved numerically in the axisymmetric approximation; the influence of wall elasticity on the flow and on the unsteady wall shear stress is studied in correspondence of parameters taken from realistic artery flow. Attention is posed to the role of phase difference between the incoming pressure and flow pulses.     

Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone

We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.