Persistence and Significance of Chlamydia trachomatis in the Housefly, Musca domestica L.

Trachoma is the leading cause of infectious blindness worldwide. Ocular infection by the obligate intracellular pathogen, Chlamydia trachomatis, causes the eyelashes to turn in and scratch the cornea, leading to blindness if left untreated. The disease is most prevalent in poor, rural communities that lack the infrastructure for basic hygiene, clean water, and proper sanitation. Infection is often spread through infected clothes, contaminated hands, and face seeking flies. The goal of this research was to understand the biological role of Musca domestica flies in the transmission of C. trachomatis. PCR, tissue culture, and immunofluorescence microscopy were used to determine the presence, viability, and the anatomical location of C. trachomatis within the digestive tract of M. domestica. Flies were fed with C. trachomatis and then harvested at various time intervals after feeding. The data confirmed the presence of C. trachomatis DNA and viable elementary bodies (EBs) in fly crops, up to 24 h postfeeding. C. trachomatis DNA was also isolated from the upper portions of the alimentary tract of flies up to 48 h postfeeding. In addition, DNA was isolated from the regurgitation material from fly crops up to 12 h postfeeding. The viability of isolated C. trachomatis EBs was repeatedly confirmed between 12 and 48 h and up to 7 days in ex vivo crops stored at room temperature. Our data suggest that eye-seeking flies such as M. domestica can ingest C. trachomatis during regular feeding. Because M. sorbens does not occur in continental United States, we did not use it in any of our studies. These data also confirm, for the first time, that ingested chlamydia remains viable inside the flies for 24-48 h postfeeding. We further show that these flies can regurgitate and transmit the trachoma agent at their next feeding. We believe that these findings reveal an opportunity for efficient intervention strategies through fly vector control, especially as we near new target date for global elimination of trachoma.

Systematic tissue collection during clinical breast biopsy is feasible, safe and enables high-content translational analyses.

Systematic collection of fresh tissues for research at the time of diagnostic image-guided breast biopsy has the potential to fuel a wide variety of innovative studies. Here we report the initial experience, including safety, feasibility, and laboratory proof-of-principle, with the collection and analysis of research specimens obtained via breast core needle biopsy immediately following routine clinical biopsy at a single institution over a 14-month period. Patients underwent one or two additional core biopsies following collection of all necessary clinical specimens. In total, 395 patients were approached and 270 consented to the research study, yielding a 68.4% consent rate. Among consenting patients, 238 lesions were biopsied for research, resulting in 446 research specimens collected. No immediate complications were observed. Representative research core specimens showed high diagnostic concordance with clinical core biopsies. Flow cytometry demonstrated consistent recovery of hundreds to thousands of viable cells per research core. Among a group of HER2 + tumor research specimens, HER2 assessment by flow cytometry correlated highly with immunohistochemistry (IHC) staining, and in addition revealed extensive inter- and intra-tumoral variation in HER2 levels of potential clinical relevance. Suitability for single-cell transcriptomic analysis was demonstrated for a triple-negative tumor core biopsy, revealing substantial cellular diversity in the tumor immune microenvironment, including a prognostically relevant T cell subpopulation. Thus, collection of fresh tissues for research purposes at the time of diagnostic breast biopsy is safe, feasible and efficient, and may provide a high-yield mechanism to generate a rich tissue repository for a wide variety of cross-disciplinary research.

RASAL2 Confers Collateral MEK/EGFR Dependency in Chemoresistant Triple-Negative Breast Cancer.

PURPOSE: While chemotherapy remains the standard treatment for triple-negative breast cancer (TNBC), identifying and managing chemoresistant tumors has proven elusive. We sought to discover hallmarks and therapeutically actionable features of refractory TNBC through molecular analysis of primary chemoresistant TNBC specimens. EXPERIMENTAL DESIGN: We performed transcriptional profiling of tumors from a phase II clinical trial of platinum chemotherapy for advanced TNBC (TBCRC-009), revealing a gene expression signature that identified de novo chemorefractory tumors. We then employed pharmacogenomic data mining, proteomic and other molecular studies to define the therapeutic vulnerabilities of these tumors. RESULTS: We reveal the RAS-GTPase-activating protein (RAS-GAP) RASAL2 as an upregulated factor that mediates chemotherapy resistance but also an exquisite collateral sensitivity to combination MAP kinase kinase (MEK1/2) and EGFR inhibitors in TNBC. Mechanistically, RASAL2 GAP activity is required to confer kinase inhibitor sensitivity, as RASAL2-high TNBCs sustain basal RAS activity through suppression of negative feedback regulators SPRY1/2, together with EGFR upregulation. Consequently, RASAL2 expression results in failed feedback compensation upon co-inhibition of MEK1/2 and EGFR that induces synergistic apoptosis in vitro and in vivo. In patients with TNBC, high RASAL2 levels predict clinical chemotherapy response and long-term outcomes, and are associated via direct transcriptional regulation with activated oncogenic Yes-Associated Protein (YAP). Accordingly, chemorefractory patient-derived TNBC models exhibit YAP activation, high RASAL2 expression, and tumor regression in response to MEK/EGFR inhibitor combinations despite well-tolerated intermittent dosing. CONCLUSIONS: These findings identify RASAL2 as a mediator of TNBC chemoresistance that rewires MAPK feedback and cross-talk to confer profound collateral sensitivity to combination MEK1/2 and EGFR inhibitors.