Tanaka, C. | bioRxiv Science

bioRxiv2025

Adapting a commercial anti-Aspergillus IgG ELISA kit for penguin sera: A novel approach using anti-chicken IgY antibody for the detection of anti-Aspergillus IgY titer

Penguins are susceptible to aspergillosis and are affected by the causative fungal pathogens Aspergillus spp., under immunocompromised conditions. Currently, there are limited serological tests available for diagnosing Aspergillus infections in penguins, highlighting the need for new diagnostic methods. While an anti-Aspergillus IgG detection kit using ELISA is commercially available and widely used in human medicine, it is not applicable for penguins because it incorporates anti-human antibodies as the secondary antibody for detection. To address this issue, an anti-chicken IgY antibody was incorporated into a commercial anti-Aspergillus IgG detection ELISA kit. First, anti-chicken IgY antibody was examined for cross-reactivity to penguin IgY, and the antibody recognized IgY from King, Gentoo, and African penguins in sera. Subsequently, serum samples from healthy penguins and penguins with aspergillosis were examined using anti-chicken IgY antibody incorporated into the anti-Aspergillus IgG ELISA kit. The results suggest that the combination method is applicable for the detection of anti-Aspergillus penguin IgY antibodies. However, because background titers can vary among individuals, we propose that routine monitoring could aid in the early detection of aspergillosis, even before the onset of symptoms.

microbiology↗

bioRxiv2024

Slow diffusion and signal amplification on membranes regulated by phospholipase D

Control of molecular diffusion is pivotal for highly fluidic membranes to serve as substrates for biochemical reactions and the self-assembly of molecular machinery driving membrane protrusions. Lateral diffusion in membranes depends on lipid composition, which is highly diverse and homeostatically controlled in living cells. Due to the complexity of the underlying processes, its impact on molecular diffusion remain largely unclear. In this study, we show that lipid diffusion in model membranes is markedly decreased in cytosolic extracts. The reduction in lipid diffusivity could be pharmacologically inhibited by targeting phospholipase D (PLD), and addition of PLD to membranes mimicked the reduction in diffusion. Phosphatidic acid, a direct product of PLD, diffused slowly in model membranes and reduced the diffusivity of surrounding lipids. Furthermore, we demonstrated that PLD specifically controls the lateral diffusion of a myristoylated protein in cells, possibly due to auxiliary electrostatic interactions between cationic residues located near the lipidated tail and anionic phospholipids. PLD controlled the size and lifetime of localized patches of phosphatidylinositol (3,4,5) triphosphates that specify regions of membrane protrusions. Overall, the results of this study suggest that PLD controls the lateral diffusion of certain membrane proteins, which play key roles in phosphoinositide signaling. Significance StatementIn living cells, many biochemical reactions occur in confined regions on the membranes, facilitating the local occurrence of specific events, such as membrane protrusion. This is puzzling from a physical perspective because the membrane is a two-dimensional fluidic structure that should allow molecules to spread freely. Herein, we found that the fluidity of artificial membranes was markedly reduced by adding extracts from the cell cytoplasm. A lipid-modifying protein phospholipase D (PLD) was found to be responsible for this and it regulates the diffusion of membrane proteins in cells. This study suggests the novel role of PLD as a regulator of molecular diffusion and its impact on phosphoinositide production that serves as an important signal for cell deformation.

biophysics↗