Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation

Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8⁺ T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.     

Information-Theoretic Analysis of the Dynamics of an Executable Biological Model

To facilitate analysis and understanding of biological systems, large-scale data are often integrated into models using a variety of mathematical and computational approaches. Such models describe the dynamics of the biological system and can be used to study the changes in the state of the system over time. For many model classes, such as discrete or continuous dynamical systems, there exist appropriate frameworks and tools for analyzing system dynamics. However, the heterogeneous information that encodes and bridges molecular and cellular dynamics, inherent to fine-grained molecular simulation models, presents significant challenges to the study of system dynamics. In this paper, we present an algorithmic information theory based approach for the analysis and interpretation of the dynamics of such executable models of biological systems. We apply a normalized compression distance (NCD) analysis to the state representations of a model that simulates the immune decision making and immune cell behavior. We show that this analysis successfully captures the essential information in the dynamics of the system, which results from a variety of events including proliferation, differentiation, or perturbations such as gene knock-outs. We demonstrate that this approach can be used for the analysis of executable models, regardless of the modeling framework, and for making experimentally quantifiable predictions.     

Limiting factors of Striped Hyaena,Hyaena hyaena,distribution and densities across climatic and geographical gradients (Mammalia: Carnivora)

Few previous studies on the factors that affect Striped Hyaena (Hyaena hyaena Linnaeus, 1758) occurrence and densities were done on geographically unrelated populations using different methodologies. In Israel, hyaenas occur throughout the country's steep climatic and geographical gradients, presenting a unique opportunity to study densities and habitat use across adjacent ecosystems using a unified methodology and test previous conceptions regarding the species’ habitat selection. We collected hyaena abundance-absence data using 1440 camera traps placed at 80 sites (2012– 2016). Site location ranged from hyper-arid deserts to dense Mediterranean shrubland. We assessed the effect of climate, habitat structure, elevation, geomorphological attributes (proxy for den availability), and anthropogenic development (proximity to settlements and agriculture) on hyaena densities using N-mixture models. Hyaena densities were negatively affected by anthropogenic development, and were limited by den availability. Hyaena densities did not follow a climatic or geographic gradient. Densities were highest at hyper-arid deserts and Mediterranean coastal shrublands. Despite the former conception that hyaenas prefer semi-arid open habitats and avoid extreme deserts and dense vegetation, we show that hyaenas use and even thrive in these habitats when geomorphological conditions are suitable and resources are available.     

Gene silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant

Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%–80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.