Effects of brassinosteroids on microspore embryogenesis in <Emphasis Type="Italic">Brassica</Emphasis> species

Summary Experiments were conducted to determine the effects of brassinosteroids on microspore embryogenesis in Brassica species. Two compounds, 24-epibrassinolide (EBR) and brassinolide (BL), were evaluated. An increase in embryogenesis was observed in all Brassica napus lines evaluated, including Topas 4079 and several recalcitrant cultivars: Garrison, Westar, and Allons. Microspore embryogenesis, calculated as the number of embryos at 21 d of culture, was increased in the recalcitrant cultivars up to 12 times that of the control. An increase in microspore embryogenesis was also observed for B. juncea when EBR or BL was added to the culture medium. In constrast, no significant increases in embryogenesis was observed for several other Brassica species evaluated (i.e. B. carinata, B. nigra, and B. rapa). The addition of brassinosteroids to the induction media did not affect the subsequent conversion of the embryos to plantlets, but did appear to influence chromosome doubling.     

T regulatory cells and allergy

Anergy, tolerance and active suppression may not be independent events, but rather involve similar mechanisms and cell types in immune regulation. Induction of allergen-specific T(Reg) cells seems essential for maintaining a healthy immune response towards allergens. By utilizing multiple secreted cytokines and surface molecules, antigen-specific T(Reg) cells may re-direct an inappropriate immune response against allergens or auto-antigens.     

AtLACS7 interacts with the TPR domains of the PTS1 receptor PEX5

Long-chain acyl-CoA synthetases (LACSs) activate fatty acids for further metabolism and are encoded by a multi-gene family in Arabidopsis. AtLACS6 possesses a type 2 (PTS2) peroxisomal targeting sequence, whilst AtLACS7 has both a type 1 and type 2 peroxisomal targeting sequence. AtLACS7 was used as bait in a yeast two-hybrid screen. Multiple clones of the PTS1 receptor PEX5 were isolated. Quantitative beta-galactosidase assay indicated that full-length PEX5 interacts with AtLACS7 with higher affinity than the TPR domains alone. The interaction between PEX5 and AtLACS7 was confirmed by co-immunoprecipitation and shown to be specific for the PTS1, therefore the AtLACS7 PTS1 is accessible to bind PEX5 in the full-length AtLACS7 protein. The expression profile of AtLACS6, AtLACS7, AtPEX5, and AtPEX7 revealed that AtLACS6 and 7 have distinct patterns of expression and we speculate that the possession of two targeting signals may be advantageous for the import of AtLACS7 when receptors may be limiting.     

Additive effects of herbivory,nectar robbing and seed predation on male and female fitness estimates of the host plant <Emphasis Type="Italic">Ipomopsis aggregata</Emphasis>

Many antagonistic species attack plants and consume specific plant parts. Understanding how these antagonists affect plant fitness individually and in combination is an important research focus in ecology and evolution. We examined the individual and combined effects of herbivory, nectar robbing, and pre-dispersal seed predation on male and female estimates of fitness in the host plant Ipomopsis aggregata. By examining the effects of antagonists on plant traits, we were able to tease apart the direct consumptive effects of antagonists versus the indirect effects mediated through changes in traits important to pollination. In a three-way factorial field experiment, we manipulated herbivory, nectar robbing, and seed predation. Herbivory and seed predation reduced some male and female fitness estimates, whereas plants tolerated the effects of robbing. The effects of herbivory, robbing, and seed predation were primarily additive, and we found little evidence for non-additive effects of multiple antagonists on plant reproduction. Herbivory affected plant reproduction through both direct consumptive effects and indirectly through changes in traits important to pollination (i.e., nectar and phenological traits). Conversely, seed predators primarily had direct consumptive effects on plants. Our results suggest that the effects of multiple antagonists on estimates of plant fitness can be additive, and investigating which traits respond to damage can provide insight into how antagonists shape plant performance.     

Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected

This paper describes a protocol to encapsulate cells in sub-millimeter-sized cylindrical collagen modules and to assemble these modules into a tissue-engineered construct within a continuous loop flow circuit. Modules are fabricated by gelling a solution of collagen, that contains suspended cells, within the lumen of a small-bore polyethylene tube. The tubing is then cut into short lengths using an automated cutter and gently vortexed to remove the cell-containing collagen modules from the tubing lumen. Modules are then randomly assembled into a modular construct by pipetting a suspension of modules into a larger tube that is positioned within a continuous flow circuit. A range of cylinder aspect ratios are achievable; therefore, this method could potentially be used to create short discs or, alternatively, long threads of soft gels, with or without encapsulated cells, for a variety of tissue-engineering applications. Module fabrication requires 1 d and assembly of a modular construct requires 2 h.     

Polarized distribution of Na, K-ATPase α-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na⁺, K⁺-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na⁺, K⁺-ATPase activity 2.6-fold more sensitive to ouabain (I₅₀=1.0±0.1 μM) than the activity of innervated membranes (I₅₀=2.6±0.2 μM). As depicted in [³H]ouabain binding experiments, when the [³H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na⁺, K⁺-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na⁺, K⁺-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Analysis of void volumes in proteins and application to stability of the p53 tumour suppressor protein

We have developed a new method for the analysis of voids in proteins (defined as empty cavities not accessible to solvent). This method combines analysis of individual discrete voids with analysis of packing quality. While these are different aspects of the same effect, they have traditionally been analysed using different approaches. The method has been applied to the calculation of total void volume and maximum void size in a non-redundant set of protein domains and has been used to examine correlations between thermal stability and void size. The tumour-suppressor protein p53 has then been compared with the non-redundant data set to determine whether its low thermal stability results from poor packing. We found that p53 has average packing, but the detrimental effects of some previously unexplained mutations to p53 observed in cancer can be explained by the creation of unusually large voids.     

In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome,a disease with low mutational burden

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34⁺) and normal (CD3⁺) cells isolated from the patients’ blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4⁺ response and 11 (34%) induced a CD8⁺ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.