Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs

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Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10⁷ independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near‐infrared fluorescent proteins with enhanced intracellular brightness. The developed near‐infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near‐infrared fluorescent proteins enabled crosstalk‐free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual‐color near‐infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.     

Functional role for MAP kinase signaling in cell lineage and dorsoventral axis specification in the basal gastropod Testudinalia testudinalis (Patellogastropoda, Mollusca)

In spiralians, the specification of cell lines in development is provided by maternal factors. However, recent studies demonstrated the importance of inductive processes whose significant element is cellular signaling. Our data argue the conditional specification of a number of cell lines at the early stages of development of the mollusk Testudinalia testudinalis (Testudinalia tessellata, Patellogastropoda), including the period when the determination of the 3D cell takes place, which is accompanied by a change in the shape and establishing of contacts with animal micromeres by one of the macromeres of the third quartet. Exactly at this moment activation of MAPK was registered in the 3D blastomere-organizer. An analysis of the influence of the U0126 inhibitor of the MAP kinase pathway on the development of Testudinalia revealed that the greatest effect of the inhibitor was observed if the treatment of embryos was performed until the sixth cycle of cleavage. Notably, the degree of defects correlates with the concentration increase. Absence of the functioning retractor, disorganization of the muscle system, and abnormal structure of the shell (to the extent of complete absence of the shell), as well as velum, foot, and mantle fold, were observed in a considerable part of larvae after a lengthy bathing of the embryos in the U0126 solution. At the same time, none of the experiments showed a complete disruption of the specification of the dorsoventral axis, which produces a larva with a tetramerous radial symmetry. Our data indicate the existence of various molecular mechanisms of determination of the secondary body axis among the animals from the group Spiralia.