RNA viruses include respiratory viruses, such as coronaviruses and influenza viruses, as well as vector-borne viruses, like dengue and West Nile virus. RNA viruses like these encounter various environments when they copy themselves and spread from cell to cell or host to host. Ex vivo differences, such as geographical location and humidity, affect their stability and transmission, while in vivo differences, such as pH and host gene expression, impact viral receptor binding, viral replication, and the host immune response against the viral infection.

Pseudomonas aeruginosais a ubiquitous opportunistic pathogen that forms robust biofilms in the different niches it occupies. Numerous physiological adaptations are required as this organism shifts from soil or aquatic environments to a host-associated lifestyle. While many conditions differ between these niches, temperature shifts are a factor that can contribute to physiological stress during this transition. To understand how temperature impacts biofilm formation in this pathogen, we used proteomic and transcriptomic tools to elucidate physiological responses in environment-relevant vs. host-relevant temperatures. These studies uncovered differential expression of various proteins including a phage protein that is associated with the EPS matrix inP. aeruginosa. This filamentous phage was induced at host temperatures and was required for full biofilm-forming capacity specifically at human body temperature. These data highlight the importance of temperature shift in biofilm formation and suggest bacteriophage proteins could be a possible therapeutic target in biofilm-associated infections.

The association of microorganisms into biofilms produces functionally organized microbial structures that promote community survival in a wide range of environments. Much like when individual cells within a multicellular organism express different genes from the same DNA blueprint, individual microbial cells located within different regions of a biofilm structure can exhibit distinct genetic programs. These spatially defined regions of physiologically differentiated cells are reminiscent of the role of tissues in multicellular organisms, with specific subpopulations in the microbial community serving defined roles to promote the overall health of the biofilm. The functions of these subpopulations are quite diverse and can range from dormant cells that can withstand antibiotic onslaughts to cells actively producing extracellular polymeric substances providing integrity to the entire community. The purpose of this review is to discuss the diverse roles of subpopulations in the stability and function of clonal biofilms, the methods for studying these subpopulations, and the ways these subpopulations can potentially be exploited for therapeutic intervention.

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processesin vivoand development in metazoans remains to be understood. Here, we describe a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in theDrosophilaeye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVGs) and mini viral RNAs (mvRNAs). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid–inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Here, we provide evidence that RNA structures, called template loops (t-loops), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are suggestive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response.