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We consolidate and present data for the sexual stages of five North American species of Orphella, fungal members of trichomycetes previously classified within Harpellales. Three species emendations accommodate the newly recognized characters, including not only the coiled zygospores and accompanying cells but also other morphological traits not provided in the original descriptions for O. avalonensis, O. haysii, and O. hiemalis. We describe three new species, Orphella cataloochensis from both the Smoky Mountains in USA and two provinces in Canada as well as O. pseudoavalonensis and O. pseudohiemalis, both from the Cascade Range, in Oregon, USA. Key morphological features for all known species are summarized and reviewed, with illustrations of some of the North American taxa to update and supplement the literature. The entire suite of morphological characters is discussed, with emphasis on species relationships and hypotheses on possible vicariant origins. We also present a molecular phylogeny based on nuc rDNA 18S and 28S, which supports Orphella as a lineage distinct from Harpellales, and we establish a new order, Orphellales, for it. With the combination of sexual features, now known for 12 of the 14 species of Orphella, and new molecular data, the group is now better characterized, facilitating and hopefully also promoting future studies toward a better understanding of their relationships, origins, and evolutionary history as stonefly gut-dwelling fungi. Fil: White, Merlin M.. Boise State University; Estados Unidos Fil: Guàrdia Valle, Laia. Universitat Autònoma de Barcelona; España Fil: Lichtwardt, Robert W.. Kansas State University; Estados Unidos Fil: Siri, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; Argentina Fil: Strongman, Douglas. Saint Mary's University; Canadá Fil: William, Robert T.. Saint Mary's University; Canadá Fil: Gause, W. Justin. Boise State University; Estados Unidos Fil: Tretter, Eric D.. Boise State University; Estados Unidos

Objective Intraoperative tumor shedding may facilitate tumor dissemination. In earlier studies, shed tumor cells were defined primarily by cytomorphological examination, and normal epithelial cells could not always be distinguished from tumor cells. We sought to accurately identify tumor cells using single-cell sequencing and determine whether these cells were mobilized into the circulation during pulmonary lobectomy. Methods Forty-two blood samples collected from the tumor-draining pulmonary vein at the end of lobectomy procedures were analyzed. Arrays of nanowells were used to enumerate and retrieve single EpCAM[superscript +] cells. Targeted sequencing of 10 to 15 cells and nested polymerase chain reaction of single cells detected somatic mutations in shed epithelial cells consistent with patient-matched tumor but not normal tissue. Results The mean number of EpCAM[superscript +] cells in video-assisted thoracoscopy (VATS) lobectomy (no wedge) specimens (n = 16) was 165 (median, 115; range, 0-509) but sampling cells from 3 patients indicated that only 0% to 38% of the EpCAM[superscript +] cells were tumor cells. The mean number of EpCAM[superscript +] cells in VATS lobectomy (wedge) specimens (n = 12) was 1128 (median, 197; range, 47-9406) and all of the EpCAM[superscript +] cells were normal epithelial cells in 2 patients sampled. The mean number of EpCAM[superscript +] cells in thoracotomy specimens (n = 14) was 238 (median, 22; range, 9-2920) and 0% to 50% of total EpCAM[superscript +] cells were tumor cells based on 4 patients sampled. Conclusions Surgery mobilizes tumor cells into the pulmonary vein, along with many normal epithelial cells. EpCAM alone cannot differentiate between normal and tumor cells. On the other hand, single-cell genetic approaches with patient-matched normal and tumor tissues can accurately quantify the number of shed tumor cells., National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), Singapore. Agency for Science, Technology and Research, National Science Foundation (U.S.). Graduate Research Fellowship, Janssen Pharmaceutical Ltd.