Vitellogenin levels in male and female rainbow trout (Salmo gairdneri Richardson) at various stages of the reproductive cycle

The immunoassayable vitellogenin (VTG) in plasma from male rainbow trout had the same molecular weight as authentic VTG from female fish. The VTG level in male trout was low (usually nanograms, occasionally up to a few micrograms, per ml) and did not correlate with the stage of sexual maturity. The plasma VTG level of female trout that were two years from first spawning was 200-fold higher than males of the same strain and age. The plasma VTG level of female rainbow trout rose approximately a million-fold during the two or three years required to attain sexual maturity.     

The ribosomal binding domain of the Escherichia coli release factors. Modification of tyrosine in the N-terminal domain of ribosomal protein L11 affects release factors 1 and 2 differentially

Ribosomal protein L11 is one of only two ribosomal proteins significantly iodinated when Escherichia coli 50 S subunits are modified by immobilized lactoperoxidase, and the major target has been shown previously to be tyrosine at position 7 in the N-terminal domain. This modification reduces in vitro termination activity with release factor (RF)-1 by 70-90%, but RF-2 activity is less affected (30-50%). The loss of activity parallels incorporation of iodine into the subunit. The 50 S subunits from L11-lacking strains of bacteria have highly elevated activity with RF-2 and low activity with RF-1. The iodination does not affect RF-2 activity but reduces the RF-1 activity further. Ribosomal proteins, L2, L6, and L25, are significantly labeled in L11-lacking ribosomes in contrast to the control 50 S subunits. L11 has been modified in isolation and incorporated back efficiently into L11-lacking ribosomes. This L11, iodinated also predominantly at Tyr 7, is unable to restore RF-1 activity to L11-lacking ribosomes in contrast to mock-iodinated protein. These results suggest the involvement of the N terminus of L11 in the binding domain of the bacterial release factors and indicate that there are subtle differences in how the two factors interact with the ribosome.     

Symmetry Restoring Bifurcation in Collective Decision-Making

How social groups and organisms decide between alternative feeding sites or shelters has been extensively studied both experimentally and theoretically. One key result is the existence of a symmetry-breaking bifurcation at a critical system size, where there is a switch from evenly distributed exploitation of all options to a focussed exploitation of just one. Here we present a decision-making model in which symmetry-breaking is followed by a symmetry restoring bifurcation, whereby very large systems return to an even distribution of exploitation amongst options. The model assumes local positive feedback, coupled with a negative feedback regulating the flow toward the feeding sites. We show that the model is consistent with three different strains of the slime mold Physarum polycephalum, choosing between two feeding sites. We argue that this combination of feedbacks could allow collective foraging organisms to react flexibly in a dynamic environment.     

Quantitative Cross-Species Extrapolation between Humans and Fish: The Case of the Anti-Depressant Fluoxetine

Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 µg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (H_TPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the H_TPC range, whereas no effects were observed at plasma concentrations below the H_TPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation.     

Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization

Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.     

Quorum decision-making in foraging fish shoals

Quorum responses provide a means for group-living animals to integrate and filter disparate social information to produce accurate and coherent group decisions. A quorum response may be defined as a steep increase in the probability of group members performing a given behaviour once a threshold minimum number of their group mates already performing that behaviour is exceeded. In a previous study we reported the use of a quorum response in group decision-making of threespine sticklebacks (Gasterosteus aculeatus) under a simulated predation threat. Here we examine the use of quorum responses by shoals of sticklebacks in first locating and then leaving a foraging patch. We show that a quorum rule explains movement decisions by threespine sticklebacks toward and then away from a food patch. Following both to and from a food patch occurred when a threshold number of initiators was exceeded, with the threshold being determined by the group size.     

Ultrasonic pregnancy diagnosis in the camel

Application of ultrasound for pregnancy diagnosis has been tested and evaluated in 15 Iranian camels (Camelus dromedarius), all of which ultimately calved. Transabdominal examinations were unsuccessful, while intrapelvic application resulted in the reception of sounds characteristic for foetal life, similar to those found in other domestic animals. Signals of foetal heart, pulse of umbilical vessels and uterine artery as well as foetal movement could be recognized as distinct sounds and have been recorded for further studies. An attempt was made to verify the findings of the ultrasonic diagnosis through rectal palpation. The ultrasonic technique resulted in 12 correct and three incorrect diagnoses.     

Molecular evolution of the mammalian ribosomal protein gene, RPS14

Ribosomal protein S14 genes (RPS14) in eukaryotic species from protozoa to primates exhibit dramatically different intron-exon structures yet share homologous polypeptide-coding sequences. To recognize common features of RPS14 gene architectures in closely related mammalian species and to evaluate similarities in their noncoding DNA sequences, we isolated the intron-containing S14 locus from Chinese hamster ovary (CHO) cell DNA by using a PCR strategy and compared it with human RPS14. We found that rodent and primate S14 genes are composed of identical protein-coding exons interrupted by introns at four conserved DNA sites. However, the structures of corresponding CHO and human RPS14 introns differ significantly. Nonetheless, individual intron splice donor, splice acceptor, and upstream flanking motifs have been conserved within mammalian S14 homologues as well as within RPS14 gene fragments PCR amplified from other vertebrate genera (birds and bony fish). Our data indicate that noncoding, intronic DNA sequences within highly conserved, single-copy ribosomal protein genes are useful molecular landmarks for phylogenetic analysis of closely related vertebrate species.      

Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley Fever vaccine in mice

Background

Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens.

Methods

Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice.

Results

A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8⁺ T cell response.

Conclusions

Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.