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bioRxiv2021

Functional characterization of 5' UTR cis-acting sequence elements that modulate translational efficiency in P. falciparum and humans

BackgroundThe eukaryotic parasite Plasmodium falciparum causes millions of malarial infections annually while drug resistance to common antimalarials is further confounding eradication efforts. Translation is an attractive therapeutic target that will benefit from a deeper mechanistic understanding. As the rate limiting step of translation, initiation is a primary driver of translational efficiency. It is a complex process regulated by both cis and trans acting factors, providing numerous potential targets. Relative to model organisms and humans, P. falciparum mRNAs feature unusual 5 untranslated regions suggesting cis-acting sequence complexity in this parasite may act to tune levels of protein synthesis through their effects on translational efficiency. MethodsHere, we deployed in vitro translation to compare the role of cis-acting regulatory sequences in P. falciparum and humans. Using parasite mRNAs with high or low translational efficiency, the presence, position, and termination status of upstream "AUG"s, in addition to the base composition of the 5 untranslated regions, were characterized. ResultsThe density of upstream "AUG"s differed significantly among the most and least efficiently translated genes in P. falciparum, as did the average "GC" content of the 5 untranslated regions. Using exemplars from highly translated and poorly translated mRNAs, multiple putative upstream elements were interrogated for impact on translational efficiency. Upstream "AUG"s were found to repress translation to varying degrees, depending on their position and context, while combinations of upstream "AUG"s had nonadditive effects. The base composition of the 5 untranslated regions also impacted translation, but to a lesser degree. Surprisingly, the effects of cis-acting sequences were remarkably conserved between P. falciparum and humans. ConclusionWhile translational regulation is inherently complex, this work contributes toward a more comprehensive understanding of parasite and human translational regulation by examining the impact of discrete cis-acting features, acting alone or in context.

molecular biology↗

bioRxiv2020

Label-free imaging and classification of live P. falciparum

Manual microscopic inspection of fixed and stained blood smears has remained the gold standard for Plasmodium parasitemia analysis for over a century. Unfortunately, smear preparation consumes time and reagents, while manual microscopy is skill-dependent and labor-intensive. Here, we demonstrate that label-free microscopy combined with deep learning enables both life stage classification and accurate parasitemia quantification. Using a custom-built microscope, we find that deep-ultraviolet light enhances image contrast and resolution, achieving four-category classification of Plasmodium falciparum blood stages at an overall accuracy greater than 99%. To increase accessibility, we extended our method to a commercial brightfield microscope using near-ultraviolet and visible light. Both systems were tested extrinsically by parasitemia titration, revealing superior performance over manually-scored Giemsa-stained smears, and a limit of detection below 0.1%. Our results suggest that label-free microscopy combined with deep learning could eliminate the need for conventional blood smear analysis.

biophysics↗