Co‐expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor‐β1 into a biologically active protein

Transforming growth factor beta (TGF‐β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF‐β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF‐β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF‐β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF‐β1 in the absence of the latency‐associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP‐TGF‐β1, we were able to show that processing of the latent complex by a furin‐like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP‐TGF‐β1, and co‐expression of human furin enabled the proteolytic processing of latent TGF‐β1. Engineering the plant post‐translational machinery by co‐expressing human furin also enhanced the accumulation of biologically active TGF‐β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.     

Timing of simulated aboveground herbivory influences population dynamics of root-feeding nematodes

Aims

A soil-plant-atmosphere continuum (SPAC) model for simulating tree transpiration (Ep) with variable water stress and water distribution in the soil is presented. The model couples a sun/shade approach for the canopy with a discrete representation of the soil in different layers and compartments.

Methods

To test its performance, the outputs from the simulations are compared to those from an experiment using trees of olive ‘Picual’ and almond ‘Marinada’ with the root system split into two. Trees are subjected to different irrigation phases in which one side of the root system is dried out while the other is kept wet.

Results

The model is able to accurately predict Ep (R² and the efficiency factor (EF) around 0.9) in the two species studied. The use of a function that modulates the uptake capacity of a root according to the soil water content was necessary to track the fluxes observed from each split part. It was also appropriate to account for root clumping to match the measured and modelled leaf water potential.

Conclusions

Coupling the sun/shade approach with the soil multi-compartment solution provides a useful tool to explore tree Ep for different degrees of water availability and distribution.

     

Development of a wingless morph in the ladybird beetle, Adalia bipunctata

SUMMARY Many taxa of winged insects have independently lost the ability to fly and often possess reduced wings. Species exhibiting natural variation in wing morphology provide opportunities to investigate the genetics and developmental processes underlying the evolution of alternative wing morphs. Although many wing dimorphic species of beetles are known, the underlying mechanisms of variation are not well understood in this insect order. Here, we examine wing development of wild type and natural wingless morphs of the two-spot ladybird beetle, Adalia bipunctata . We show that both pairs of wings are distally truncated in the wingless adults. A laboratory population of the wingless morph displays heritable variation in the degree of wing truncation, reflecting reduced growth of the larval wing discs. The coexistence of variable wingless morphs supports the idea that typical monomorphic wingless insects may be the result of a gradual evolution of wing loss. Gene expression patterns in wing discs suggest that the conserved gene network controlling wing development in wild-type Adalia is disrupted in the dorsoventral patterning pathway in the wingless morphs. Previous research on several species of ant has revealed that the anteroposterior wing patterning pathway is disrupted in wingless workers. Future investigations should confirm whether interruptions in both taxa are limited to the patterning pathways found thus far, or whether there are also shared interruption points. Nevertheless, our results highlight that diverse mechanisms of development are likely to underlie the evolution of wingless insects.     

Short Term Depression Unmasks the Ghost Frequency

Short Term Plasticity (STP) has been shown to exist extensively in synapses throughout the brain. Its function is more or less clear in the sense that it alters the probability of synaptic transmission at short time scales. However, it is still unclear what effect STP has on the dynamics of neural networks. We show, using a novel dynamic STP model, that Short Term Depression (STD) can affect the phase of frequency coded input such that small networks can perform temporal signal summation and determination with high accuracy. We show that this property of STD can readily solve the problem of the ghost frequency, the perceived pitch of a harmonic complex in absence of the base frequency. Additionally, we demonstrate that this property can explain dynamics in larger networks. By means of two models, one of chopper neurons in the Ventral Cochlear Nucleus and one of a cortical microcircuit with inhibitory Martinotti neurons, it is shown that the dynamics in these microcircuits can reliably be reproduced using STP. Our model of STP gives important insights into the potential roles of STP in self-regulation of cortical activity and long-range afferent input in neuronal microcircuits.     

Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco

Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant physiology and plant development on the yield and quality of the heterologous proteins produced in plants. To investigate this, tobacco (Nicotiana tabacum cv Samsun NN) was transformed with a single construct that contained behind constitutive promotors the light- and heavy-chain genes of a mouse antibody. The in planta stability of the antibody was analyzed in transgenic plants that were grown under high and low irradiation at 15 degrees C and 25 degrees C. High-light conditions favored the production of biomass, of total soluble protein, and of antibody. The plants grown at 25 degrees C developed faster and contained less antibody per amount of leaf tissue than the plants grown at 15 degrees C. Both endogenous protein and antibody content showed a strong decline during leaf development. The heavy chains of the antibody underwent in planta degradation via relatively stable fragments. In vitro incubations of purified plantibody with leaf extracts of wild-type tobacco indicated the involvement of acidic proteases. It is interesting that the same antibody produced by mouse hybridoma cells exhibited higher stability in this in vitro assay. This may be explained by the assumption that the plant type of N-glycosylation contributes less to the stability of the antibody than the mouse-type of N-glycosylation. The results of this study indicate that proteolytic degradation during plant development can be an important factor affecting yield and homogeneity of heterologous protein produced by transgenic plants.     

Chemical fingerprinting for the evaluation of unintended secondary metabolic changes in transgenic food crops

A common element in designed guidelines for assessment of the food safety of transgenic crops is centred on a comparative analytical analysis with conventionally bred crop plants, assuming that these products have a long history of safe use (i.e. OECD-principle of substantial equivalence). In this study we examine the utility of an off-line combination of 400 MHz proton (1H)-NMR spectroscopy and liquid chromatography (LC) for the multi-component comparison of low-molecular weight compounds (i.e. chemical fingerprinting) in complex plant matrices. The developed NMR-methodology can contribute to the demonstration of substantial equivalence by its ability to compare possible compositional alterations in a novel food crop with respect to related non-transgenic reference lines. In this respect a hierarchical approach is proposed by comparing the chemical fingerprints of the transgenic crop plant to those of: (1) isogenic parental or closely related lines bred at identical and multiple sites; (2) extended ranges of commercial varieties of that plant; and (3) downstream processing effects. This is of importance to assess the likelihood that some of the statistical differences in a transgenic crop plant may be false positives due to chance alone or arose from natural genetic and/or physiologic variations.     

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.     

Fate of Carbohydrates and Lignin during Composting and Mycelium Growth of Agaricus bisporus on Wheat Straw Based Compost

In wheat straw based composting, enabling growth of Agaricus bisporus mushrooms, it is unknown to which extent the carbohydrate-lignin matrix changes and how much is metabolized. In this paper we report yields and remaining structures of the major components. During the Phase II of composting 50% of both xylan and cellulose were metabolized by microbial activity, while lignin structures were unaltered. During A. bisporus’ mycelium growth (Phase III) carbohydrates were only slightly consumed and xylan was found to be partially degraded. At the same time, lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was found to be modified by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 during mycelium growth, while fewer decorations on the phenolic skeleton of both S and G units remained.