The AMSlide for noninvasive time-lapse imaging of arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) symbiosis, the nutritional partnership between AM fungi and most plant species, is globally ubiquitous and of great ecological and agricultural importance. Studying the processes of AM symbiosis is confounded by its highly spatiotemporally dynamic nature. While microscopy methods exist to probe the spatial side of this plant-fungal interaction, the temporal side remains more challenging, as reliable deep-tissue time-lapse imaging requires both symbiotic partners to remain undisturbed over prolonged time periods. Here, we introduce the AMSlide: a noninvasive, high-resolution, live-imaging system optimised for AM symbiosis research. We demonstrate the AMSlide's applications in confocal microscopy of mycorrhizal roots, from whole colonisation zones to subcellular structures, over timeframes from minutes to weeks. The AMSlide's versatility for different microscope set-ups, imaging techniques, and plant and fungal species is also outlined. It is hoped that the AMSlide will be applied in future research to fill in the temporal blanks in our understanding of AM symbiosis, as well as broader root and rhizosphere processes.

Microcomputed tomography visualization and quantitation of the pulmonary arterial microvascular tree in mouse models of chronic lung disease.

Pulmonary vascular dysfunction is characterized by remodeling and loss of microvessels in the lung and is a major manifestation of chronic lung diseases (CLD). In murine models of CLD, the small arterioles and capillaries are the first and most prevalent vessels that are affected by pruning and remodeling. Thus, visualization of the pulmonary arterial vasculature in three dimensions is essential to define pruning and remodeling both temporally and spatially and its role in the pathogenesis of CLD, aging, and tissue repair. To this end, we have developed a novel method to visualize and quantitate the murine pulmonary arterial circulation using microcomputed tomography (µCT) imaging. Using this perfusion technique, we can quantitate microvessels to approximately 6 µM in diameter. We hypothesize that bleomycin-induced injury would have a significant impact on the arterial vascular structure. As proof of principle, we demonstrated that as a result of bleomycin-induced injury at peak fibrosis, significant alterations in arterial vessel structure were visible in the three-dimensional models as well as quantification. Thus, we have successfully developed a perfusion methodology and complementary analysis techniques, which allows for the reconstruction, visualization, and quantitation of the mouse pulmonary arterial microvasculature in three-dimensions. This tool will further support the examination and understanding of angiogenesis during the development of CLD as well as repair following injury.

Gene expression signature of atypical breast hyperplasia and regulation by SFRP1.

BACKGROUND: Atypical breast hyperplasias (AH) have a 10-year risk of progression to invasive cancer estimated at 4-7%, with the overall risk of developing breast cancer increased by ~ 4-fold. AH lesions are estrogen receptor alpha positive (ERα+) and represent risk indicators and/or precursor lesions to low grade ERα+ tumors. Therefore, molecular profiles of AH lesions offer insights into the earliest changes in the breast epithelium, rendering it susceptible to oncogenic transformation. METHODS: In this study, women were selected who were diagnosed with ductal or lobular AH, but no breast cancer prior to or within the 2-year follow-up. Paired AH and histologically normal benign (HNB) tissues from patients were microdissected. RNA was isolated, amplified linearly, labeled, and hybridized to whole transcriptome microarrays to determine gene expression profiles. Genes that were differentially expressed between AH and HNB were identified using a paired analysis. Gene expression signatures distinguishing AH and HNB were defined using AGNES and PAM methods. Regulation of gene networks was investigated using breast epithelial cell lines, explant cultures of normal breast tissue and mouse tissues. RESULTS: A 99-gene signature discriminated the histologically normal and AH tissues in 81% of the cases. Network analysis identified coordinated alterations in signaling through ERα, epidermal growth factor receptors, and androgen receptor which were associated with the development of both lobular and ductal AH. Decreased expression of SFRP1 was also consistently lower in AH. Knockdown of SFRP1 in 76N-Tert cells resulted altered expression of 13 genes similarly to that observed in AH. An SFRP1-regulated network was also observed in tissues from mice lacking Sfrp1. Re-expression of SFRP1 in MCF7 cells provided further support for the SFRP1-regulated network. Treatment of breast explant cultures with rSFRP1 dampened estrogen-induced progesterone receptor levels. CONCLUSIONS: The alterations in gene expression were observed in both ductal and lobular AH suggesting shared underlying mechanisms predisposing to AH. Loss of SFRP1 expression is a significant regulator of AH transcriptional profiles driving previously unidentified changes affecting responses to estrogen and possibly other pathways. The gene signature and pathways provide insights into alterations contributing to AH breast lesions.

Validation and Comparison of a Brief Instrument vs a Single-Item Screen to Predict Entry to Family Medicine at Matriculation to Medical School.

BACKGROUND AND OBJECTIVES: A strong US primary care workforce is necessary to meet health care needs, yet fewer than 9% of allopathic medical students choose family medicine each year. No validated instrument exists to identify students likely to enter family medicine upon medical school matriculation. METHODS: A subset of a larger survey at the University of Washington School of Medicine (UWSOM) was used to create the Family Medicine Interest Survey (FMIS), a 15-item instrument to predict eventual practice in family medicine for a 2003-2007 matriculating cohort. A single-item screen asking about top specialty choice was administered at UWSOM for the same cohort and for a 2006-2012 matriculating cohort of students at Oregon Health & Science University (OHSU). Test performance measures including D (discrimination) and Cronbach α were calculated. Logistic regression determined whether FMIS score or reporting family medicine as the top specialty choice predicted family medicine practice for 601 UWSOM graduates or family medicine residency match for 744 OHSU graduates. RESULTS: The FMIS is reliable (Cronbach α=0.76). Both tests significantly predicted the probability of entering family medicine. Listing family medicine as the preferred specialty choice yielded a 47% predicted probability for UWSOM graduates entering family medicine. OHSU graduates listing family medicine first had an eightfold odds of matching to family medicine residencies. Combining the two instruments for UWSOM graduates showed a dose-response curve for predicted probability of entering family medicine with increasing levels of interest. CONCLUSION: Each screening tool can predict students more likely to enter family medicine upon matriculation.

Observation of decoherence in a carbon nanotube mechanical resonator.

In physical systems, decoherence can arise from both dissipative and dephasing processes. In mechanical resonators, the driven frequency response measures a combination of both, whereas time-domain techniques such as ringdown measurements can separate the two. Here we report the first observation of the mechanical ringdown of a carbon nanotube mechanical resonator. Comparing the mechanical quality factor obtained from frequency- and time-domain measurements, we find a spectral quality factor four times smaller than that measured in ringdown, demonstrating dephasing-induced decoherence of the nanomechanical motion. This decoherence is seen to arise at high driving amplitudes, pointing to a nonlinear dephasing mechanism. Our results highlight the importance of time-domain techniques for understanding dissipation in nanomechanical resonators, and the relevance of decoherence mechanisms in nanotube mechanics.