Simplified Approaches for the Production of Monocyte-Derived Dendritic Cells and Study of Antigen Presentation in Bovine.

Dendritic cells are sentinels of the immune system responsible for the initiation of adaptive immune mechanisms. In that respect, the study of these cells is essential for a full understanding of host response to infectious agents and vaccines. In ruminants, the large blood volume facilitates the isolation of abundant monocytes and their derivation to other antigen-presenting cells such as dendritic cells and macrophages. However, the available protocols for the production of bovine monocyte-derived dendritic cells (moDCs) rely mostly on time-consuming and costly techniques such as density gradient centrifugation and magnetic sorting of cells. In this study, we describe a simplified protocol for the production of bovine moDC using conventional and serum-free media. We also employ moDC produced by this approach to carry out a flow cytometry-based antigen presentation assay adapted to blood fresh or frozen cells. The experimental strategies described here might enable the setup of studies involving a large number of individuals, requiring a large number of dendritic cells, or relying on the utilization of cryopreserved blood cells. These simplified protocols might contribute to the elucidation of cell-mediated immune responses in bovine.

Functional Characterization of the GATA-Type Transcription Factor PaNsdD in the Filamentous Fungus Podospora anserina and Its Interplay with the Sterigmatocystin Pathway.

The model ascomycete Podospora anserina, distinguished by its strict sexual development, is a prolific but yet unexploited reservoir of natural products. The GATA-type transcription factor NsdD has been characterized by the role in balancing asexual and sexual reproduction and governing secondary metabolism in filamentous fungi. In the present study, we functionally investigated the NsdD ortholog PaNsdD in P. anserina. Compared to the wild-type strain, vegetative growth, ageing processes, sexual reproduction, stress tolerance, and interspecific confrontations in the mutant were drastically impaired, owing to the loss of function of PaNsdD. In addition, the production of 3-acetyl-4-methylpyrrole, a new metabolite identified in P. anserina in this study, was significantly inhibited in the ΔPaNsdD mutant. We also demonstrated the interplay of PaNsdD with the sterigmatocystin biosynthetic gene pathway, especially as the deletion of PaNsdD triggered the enhanced red-pink pigment biosynthesis that occurs only in the presence of the core polyketide synthase-encoding gene PaStcA of the sterigmatocystin pathway. Taken together, these results contribute to a better understanding of the global regulation mediated by PaNsdD in P. anserina, especially with regard to its unexpected involvement in the fungal ageing process and its interplay with the sterigmatocystin pathway. IMPORTANCE Fungal transcription factors play an essential role in coordinating multiple physiological processes. However, little is known about the functional characterization of transcription factors in the filamentous fungus Podospora anserina. In this study, a GATA-type regulator PaNsdD was investigated in P. anserina. The results showed that PaNsdD was a key factor that can control the fungal ageing process, vegetative growth, pigmentation, stress response, and interspecific confrontations and positively regulate the production of 3-acetyl-4-methylpyrrole. Meanwhile, a molecular interaction was implied between PaNsdD and the sterigmatocystin pathway. Overall, loss of function of PaNsdD seems to be highly disadvantageous for P. anserina, which relies on pure sexual reproduction in a limited life span. Therefore, PaNsdD is clearly indispensable for the survival and propagation of P. anserina in its complex ecological niches.

Determinants of acute mortality of Hippodamia convergens (Coleoptera: Coccinellidae) to ultra-low volume permethrin used for mosquito management.

There are relatively few experimental studies and risk assessments of the effects on non-target insects from ultra-low volume (ULV) insecticides used for management of adult mosquitoes. Therefore, we evaluated factors that may influence the ability of an insect to intercept the insecticide at the time of application by using Hippodamia convergens (Coleoptera: Coccinellidae) in field bioassay experiments in 2011 and 2015. Treatment factors included different distances, two cage heights (ground-level and 1.5 m above ground) to the point of the application, and covered vs. uncovered cage faces (2015 only). Insecticides used included a water-based formulation (Aqua-Reslin®) and an oil-based formulation (Permanone® 30-30) of permethrin. Cage height was highly significant both years, with much less acute (i.e., short-term exposure) mortality at ground-level compared with 1.5 m. In 2011, acute mortality was less at ground-level (mean = 3.2%, median = 0%) compared to 1.5 m (mean = 85.2%, median = 100%). Cage type also was highly significant, with less mortality in covered cages compared to uncovered cages. Mortality by cage height and cage type was as follows: ground level, covered cage (mean = 2.8%, median = 0.1%); ground level, uncovered cage (mean = 41.9%, median = 9.6%); 1.5 m, covered cage (mean = 6.8%, median = 0%); 1.5 m, uncovered cage (mean = 83.7%, median = 100%). Results suggest that acute mortality to non-target insects may vary considerably based on their height and their ability to directly intercept the insecticide as the aerosol passes through the area being sprayed.