An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios.

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

The Field of Cell Competition Comes of Age: Semantics and Technological Synergy.

Stem cells experience many selective pressures which shape their cellular populations, potentially pushing them to skew towards dominance of a few break-through clones. An evolutionarily conserved answer to curb these aberrant selective pressures is cell competition, the elimination of a subset of cells by their neighbours in a seemingly homogenous population. Cell competition in mammalian systems is a relatively recent discovery that has now been observed across many tissue systems, such as embryonic, haematopoietic, intestinal, and epithelial compartments. With this rapidly growing field, there is a need to revisit and standardize the terminology used, much of which has been co-opted from evolutionary biology. Further, the implications of cell competition across biological scales in organisms have been difficult to capture. In this review, we make three key points. One, we propose new nomenclature to standardize concepts across dispersed studies of different types of competition, each of which currently use the same terminology to describe different phenomena. Second, we highlight the challenges in capturing information flow across biological scales. Third, we challenge the field to incorporate next generation technologies into the cell competition toolkit to bridge these gaps. As the field of cell competition matures, synergy between cutting edge tools will help elucidate the molecular events which shape cellular growth and death dynamics, allowing a deeper examination of this evolutionarily conserved mechanism at the core of multicellularity.

Inhibition of viral RNA-dependent RNA polymerases with clinically relevant nucleotide analogs.

The treatment of viral infections remains challenging, in particular in the face of emerging pathogens. Broad-spectrum antiviral drugs could potentially be used as a first line of defense. The RNA-dependent RNA polymerase (RdRp) of RNA viruses serves as a logical target for drug discovery and development efforts. Herein we discuss compounds that target RdRp of poliovirus, hepatitis C virus, influenza viruses, respiratory syncytial virus, and the growing data on coronaviruses. We focus on nucleotide analogs and mechanisms of action and resistance.

Evening the playing field: microenvironmental control over stem cell competition during fate programming.

Recent advancements in cellular engineering, including reprogramming of somatic cells into pluripotent stem cells, have opened the door to a new era of regenerative medicine. Given that cellular decisions are guided by microenvironmental cues, such as secreted factors and interactions with neighbouring cells, reproducible cell manufacturing requires robust control over cell-cell interactions. Cell competition has recently emerged as a previously unknown interaction that plays a significant role in shaping the growth and death dynamics of multicellular stem cell populations, both in vivo and in vitro. Although recent studies have largely focused on exploring how the differential expression of key genes mediate the competitive elimination of some cells, little is known about the impact of the microenvironment on cell competition, despite its critical role in shaping cell fate outcomes. Here, we explore recent findings that have brought cell competition into the spotlight, while dissecting the role of microenvironmental factors for controlling competition in cell fate programming applications.