Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.     

Adenocarcinoma of the gallbladder in an owl monkey (<Emphasis Type="Italic">Aotus trivirgatus</Emphasis>)

This paper describes an adenocarcinoma of the gallbladder in a 12-year-old male owl monkey. Whereas the histological appearance in the gallbladder itself was relatively benign, well-differentiated cystic structures were found in the pancreatic lymph node. In addition, paradox metastazing behavior revealed a few cystic structures in the lymph nodes of the gut.     

The German wildlife information system: population densities and development of European Hare (<Emphasis Type="Italic">Lepus europaeus</Emphasis> PALLAS) during 2002–2005 in Germany

The German Wildlife Information System, founded in 2001, is a long-term monitoring program documenting occurrence, number, and development of game populations throughout Germany. Population numbers are recorded by standardized counting methods in so-called reference areas. The population densities of the European hare are calculated by spotlight strip censuses in the reference areas each spring and autumn all across Germany. From 2002 to 2005, the censuses were carried out by local hunters in 510 to 676 reference areas each year. During these years, the calculated spring densities increased significantly from 11.0 (2002) to 14.5 hares/km² (2005) nationwide. The overall increase in spring densities was primarily caused by the population rise from spring 2003 to 2004, which correlates with the high net growth rate in 2003. In 2005, the number of counted hares varied between less than 1 and more than 107 hares/km² in spring and between 0 and more than 170 hares/km² in autumn. Because of differing landscapes in Germany, three regions were differentiated. In spring 2005, the average population densities (median) in East Germany (5.4 hares/km²) and Southwest Germany (14.6 hares/km²) were significantly lower than in Northwest Germany (23.9 hares/km²). These regional differences had been similarly distinct in former years.     

Primary sex ratio in fertilized chicken eggs (Gallus gallus domesticus) depends on reproductive age and selection

Recent studies of several avian species have shown that the primary sex ratio can change as a result of prevailing conditions, especially in the female bird's first reproductive season. In this study, we sought to determine the primary sex ratio of the first 15 eggs produced in chickens. The study compared chickens which had been commercially selected over many generations for egg-laying performance (Leghorns) with "fancy-bred" chickens selected for feather coloration. These fancy-bred chickens are known to reach reproductive maturity 4 weeks later than Leghorns. A group of precociously matured Leghorn chickens was produced by modification of diet and day length to investigate the effect of age at reproductive maturity on sex ratio. Sex diagnosis was performed on embryos which had died on or before embryonic day 10 by polymerase chain reaction (PCR). Living embryos were allowed to hatch before sex diagnosis. The group of precociously matured White Leghorns reached egg-laying age 3 weeks earlier than normal. In this group, the sex ratio of hatched chicks was in tendency skewed to females. In the White Leghorns maintained under normal conditions for commercial layers, sex ratio was balanced with a tendency to more males only in the first five eggs. In the group of fancy-bred chickens, the primary sex ratio was significantly biased toward more males and dependent on the laying sequence. Our data suggested a sex ratio bias toward males in the very first eggs at onset of reproduction in chickens depending on genetic background.     

Antibiotic innovation may contribute to slowing the dissemination of multiresistant Streptococcus pneumoniae: the example of ketolides

Background

Despite increasingly frequent bacterial resistance to antibiotics, antibacterial innovation is rare. Ketolides constitute one of the very few new antibiotic classes active against Streptococcus pneumoniae developed during the last 25 years. Their mechanism of action resembles that of macrolides, but they are unaffected by common resistance mechanisms. However, cross-resistance to ketolides has been observed in some macrolide-resistant strains. We examined how new antibiotic exposure may affect overall pneumococcal resistance patterns in the population. The aims of this study were to assess the potential dissemination of newly emerged resistances and to control the selection of strains already multiresistant to existing antimicrobials.

Methodology/Principal Findings

We developed an age-structured population model for S. pneumoniae transmission in a human community exposed to heptavalent vaccine, and β-lactams, macrolides and ketolides. The dynamics of intra-individual selection of resistant strains under antibiotic exposure and interindividual transmission were simulated, with antibiotic-specific resistance mechanisms defining the path to co-resistances and cross-resistances, and parameters concerning the French situation. Results of this simulation study suggest that new antibiotic consumption could markedly slow the diffusion of multiresistant strains. Wider use was associated with slower progression of multiresistance. When ketolides were prescribed to all ages, resistance to them reached 10% after >15 years, while it took >40 years when they were prescribed only to adults. In the scenario according to which new antibiotics totally replaced former antimicrobials, the β-lactam resistance rate was limited at 70%.

Conclusions

In a context of widespread vaccination and rational use of antibiotics, innovative antibiotic, prescribed to all age groups, may have an added impact on multiresistant-strain dissemination in the population.     

Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4⁺ T cells but not in cynomolgus T lymphocytes. The sustained Ca⁺⁺-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.