Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

Brd4-bound enhancers drive cell-intrinsic sex differences in glioblastoma.

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited sex-specific responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET (bromodomain and extraterminal) inhibitors than are female cells. Thus, Brd4 activity is revealed to drive sex differences in stem cell and tumorigenic phenotypes, which can be abrogated by sex-specific responses to BET inhibition. This has important implications for the clinical evaluation and use of BET inhibitors.

A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues.

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pretreatment protocols that enabled rapid and sensitive detection of < 102 viral genomes per reaction in contrived saliva controls. Moreover, our saliva pretreatment protocol enabled sensitive viral detection by conventional quantitative reverse transcription polymerase chain reaction (qRT-PCR) without RNA extraction. We validated the high performance of these assays on clinical samples and demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

Role of robotics in managing mesh and suture complications of prior pelvic organ prolapse surgery.

Robotic surgery is proving essential in providing a minimally invasive approach to complex urogynaecological cases. This video highlights the diversity and complexity of cases performed using the robot-assisted approach. The robot-assisted approach was utilised for excellent effect in two complex urogynaecological cases. In the first case the entire left arm of an intravesically placed TVT was removed using a combined vaginal and robotic approach. The second case involved removing four paravaginal sutures, one of which breeched the bladder and was encrusted with calculus. These were placed during a laparoscopic paravaginal repair 2 years previously. She had a concomitant vaginal hysterectomy, Mc Calls culdoplasty and anterior wall repair. The robot-assisted approach allows for excellent access to the pelvis and retropubic space facilitating the surgical management of complex urogynaecology cases.

Increasing rates of operative vaginal delivery across two decades: accompanying outcomes and instrument preferences.

OBJECTIVE: To examine rates and outcomes of operative vaginal delivery over a 20-year study period and the changing preference for various instruments during this period. STUDY DESIGN: This retrospective analysis of prospectively gathered data was carried out at a large tertiary referral center from 1991 to 2010. All cases of operative vaginal delivery during the study period were recorded. The rates of instrumental delivery, as well as neonatal outcomes and instrument preference, were compared for individual 5-year epochs. RESULTS: During the study period there were 156,130 deliveries of which 17,841 were operative vaginal deliveries, an incidence of 11.4/100 deliveries and 13.6/100 vaginal deliveries. There was an increase in the rate of operative vaginal delivery across the 20-year period (P < 0.0001; R(2) = 0.85; Slope = 0.42). When individual 5-year epochs were compared, the incidence of instrumental delivery increased from 7.3% (2340/31,937) in the first five years, 1991-1995, to 13.7% (6179/45,177) in the final five years, 2006-2010 (P < 0.0001; OR 2.34, 95% CI = 2.23-2.47). The perinatal mortality rate in cases of instrumental delivery was decreased when these time periods were compared (7.3/1000 (17/2340) vs. 1.8/1000 (11/6179); P = 0.003, OR 0.24, 95% CI = 0.11-0.52). The choice of instrument also varied, with 68.2% (1596/2340) of instrumental deliveries in 1991-1995 being carried out with forceps compared to 32.9% (2033/6179) in 2006-2010 (P < 0.001). CONCLUSION: Rates of operative vaginal delivery have increased over the 20-year study period. The rate of perinatal mortality in infants who had an assisted vaginal delivery was decreased in the 5-year epoch at the end of the study compared with the period at the beginning. The rate of forceps delivery has fallen significantly, with vacuum delivery now being the choice of the majority of clinicians.