Autophagy as a mechanism of antiviral defense at the maternal–fetal interface

Mechanisms to protect against viral infections are crucial during pregnancy as maternal-fetal transmission can have serious pathological outcomes, including fetal infection and its sequelae, such as growth restriction, birth defects, and/or fetal death. The trophoblast forms the interface between the feto-placental unit and the maternal blood, and is therefore a critical physical and immunological barrier to restrict the spread of pathogens into the fetal microenvironment. Recently, we found that primary human placental trophoblast (PHT) cells are highly resistant to infection by diverse viruses. In this study, we also found that conditioned medium from PHT cell cultures transferred viral resistance to nonplacental recipient cells, suggesting that a component secreted by trophoblasts and present within the conditioned medium is responsible for this antiviral effect. We found that specific miRNAs from a unique primate- and placental-specific locus—the C19MC (chromosome 19 miRNA cluster)—are packaged within exosomes produced by PHT cells and confer viral resistance in nonplacental recipient cells. In addition to conveying viral resistance, we found that PHT-derived exosomes and select miRNA members of the C19MC family strongly induce autophagy, which is involved in recipient cell viral resistance. Our findings establish an exciting and novel mechanism by which placental trophoblasts exploit exosome-dependent transfer of placental-specific miRNAs to influence autophagic induction and antiviral immunity at the maternal–fetal interface.     

An RNA Element at the 5′-End of the Poliovirus Genome Functions as a General Promoter for RNA Synthesis

RNA structures present throughout RNA virus genomes serve as scaffolds to organize multiple factors involved in the initiation of RNA synthesis. Several of these RNA elements play multiple roles in the RNA replication pathway. An RNA structure formed around the 5′- end of the poliovirus genomic RNA has been implicated in the initiation of both negative- and positive-strand RNA synthesis. Dissecting the roles of these multifunctional elements is usually hindered by the interdependent nature of the viral replication processes and often pleiotropic effects of mutations. Here, we describe a novel approach to examine RNA elements with multiple roles. Our approach relies on the duplication of the RNA structure so that one copy is dedicated to the initiation of negative-strand RNA synthesis, while the other mediates positive-strand synthesis. This allows us to study the function of the element in promoting positive-strand RNA synthesis, independently of its function in negative-strand initiation. Using this approach, we demonstrate that the entire 5′-end RNA structure that forms on the positive-strand is required for initiation of new positive-strand RNAs. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identify specific nucleotide sequences within “stem a” that are essential for the initiation of positive-strand RNA synthesis. These findings provide direct evidence for a trans-initiation model, in which binding of proteins to internal sequences of a pre-existing positive-strand RNA affects the synthesis of subsequent copies of that RNA, most likely by organizing replication factors around the initiation site.     

The state of the reproductive system of the tench Tinca tinca (1999–2005) from water bodies polluted as a result of the catastrophe at the Chernobyl Nuclear Power Plant

The state of the reproductive system of Tinca tinca—descendants of individuals that were exposed to radiation as a result of the catastrophe at the Chernobyl Nuclear Power Plant in 1986 was studied. Material was collected in the postemergency period in radionuclide-polluted water bodies: in the Ukraine, in Kiev Reservoir, the Teterev River, and Lake Glubokoe (1999–2005) and in Belarus, in Lake Svyatoe (1998–1999). It is demonstrated that the total number and the extent of disturbances in the gonads of T. tinca are positively related to the levels of pollution in water bodies: in the cleanest water body, the Teterev River, the proportion of fish with gonads without considerable deviations was about 93%; in Kiev Reservoir, 79%, and in Lake Svyatoe, only 18%. Among the “postemergency” generations of T. tinca, the maximum number of disturbances in sexual cells and gonads was recorded in F_3–4, which is evidently caused by a phenomenon of the “prolonged mutagenesis”. A relatively high species resistance of the reproductive system of T. tinca to the radiation impact, in comparison with other cyprinids, was recorded.     

High Yield Elution of Proteins from Sodium Dodecyl Sulfate–Polyacrylamide Gels at the Low-Picomole Level. Application to N-Terminal Sequencing of a Scarce Protein and to In-Solution Biological Activity Analysis of On-Gel Renatured Proteins

A simple, reliable procedure for practically quantitative (90–98%) and fast (<30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-m gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 l for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.