FISH-Flow to quantify nascent and mature ribosomal RNA in mouse and human cells.

FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis. For complete details on the use and execution of this protocol, please refer to Antony et al. (2022).1.

Control of ribosomal RNA synthesis by hematopoietic transcription factors.

Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2,200 ChIP-seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of C/EBP alpha (CEBPA), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced RNA Pol I occupancy. Our work identifies numerous potential rRNA regulators and provides a template for dissection of TF roles in rRNA transcription.

Technology-enhanced AcceleRation of Germline Evaluation for Therapy (TARGET): A randomized controlled trial of a pretest patient-driven webtool vs. genetic counseling for prostate cancer germline testing.

BACKGROUND: Germline testing has an increasingly important role in prostate cancer care. However, a relative shortage of genetic counselors necessitates alternate strategies for delivery of pre-test education for germline testing. This study, funded by the Prostate Cancer Foundation, seeks to address the need for novel methods of delivery of pre-test germline education beyond traditional germline counseling to facilitate informed patient decision-making for germline testing. METHODS: This is a two-armed randomized controlled trial (RCT) with a target enrollment of 173 participants with prostate cancer per study arm (total anticipated n = 346). Patients who meet criteria for germline testing based on tumor features, family history or Ashkenazi Jewish ancestry are being recruited from 5 US sites including academic, private practice and Veterans healthcare settings. Consenting participants are randomized to the interactive pretest webtool or germline counseling with assessment of key patient-reported outcomes involved in informed decision-making for germline testing. RESULTS: Participants complete surveys at baseline, after pretest education/counseling, and following disclosure of germline results. The primary outcome of the study is decisional conflict for germline testing. Secondary outcomes include genetic knowledge, satisfaction, uptake of germline testing, and understanding of results. CONCLUSION: Our hypothesis is that the web-based genetic education tool is non-inferior to traditional genetic counseling regarding key patient-reported outcomes involved in informed decision-making for germline testing. If proven, the results would support deploying the webtool across various practice settings to facilitate pre-test genetic education for individuals with prostate cancer and developing collaborative care strategies with genetic counseling. CLINICALTRIALS: gov Identifier: NCT04447703.

Thermal stress responses of Sodalis glossinidius, an indigenous bacterial symbiont of hematophagous tsetse flies.

Tsetse flies (Diptera: Glossinidae) house a taxonomically diverse microbiota that includes environmentally acquired bacteria, maternally transmitted symbiotic bacteria, and pathogenic African trypanosomes. Sodalis glossinidius, which is a facultative symbiont that resides intra and extracellularly within multiple tsetse tissues, has been implicated as a mediator of trypanosome infection establishment in the fly's gut. Tsetse's gut-associated population of Sodalis are subjected to marked temperature fluctuations each time their ectothermic fly host imbibes vertebrate blood. The molecular mechanisms that Sodalis employs to deal with this heat stress are unknown. In this study, we examined the thermal tolerance and heat shock response of Sodalis. When grown on BHI agar plates, the bacterium exhibited the most prolific growth at 25oC, and did not grow at temperatures above 30oC. Growth on BHI agar plates at 31°C was dependent on either the addition of blood to the agar or reduction in oxygen levels. Sodalis was viable in liquid cultures for 24 hours at 30oC, but began to die upon further exposure. The rate of death increased with increased temperature. Similarly, Sodalis was able to survive for 48 hours within tsetse flies housed at 30oC, while a higher temperature (37oC) was lethal. Sodalis' genome contains homologues of the heat shock chaperone protein-encoding genes dnaK, dnaJ, and grpE, and their expression was up-regulated in thermally stressed Sodalis, both in vitro and in vivo within tsetse fly midguts. Arrested growth of E. coli dnaK, dnaJ, or grpE mutants under thermal stress was reversed when the cells were transformed with a low copy plasmid that encoded the Sodalis homologues of these genes. The information contained in this study provides insight into how arthropod vector enteric commensals, many of which mediate their host's ability to transmit pathogens, mitigate heat shock associated with the ingestion of a blood meal.