Optimizing Multi-Photon Fluorescence Microscopy Light Collection from Living Tissue by Non-Contact Total Emission Detection (TEDII)

Farrando Sicilia, Jordi; Fuente Fuente, Carlo

FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

Abstract Background: Antigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved. Methods: In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by F\uf6rster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100?). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor. Results: We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. Conclusions: The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates

The N2K Consortium. III. Short-Period Planets Orbiting HD 149143 and HD 109749

We report the detection of two short-period planets discovered at Keck Observatory. HD 149143 is a metal-rich G0 IV star with a planet of M sin i = 1.33M_J and an orbital radius of 0.053 AU. The best-fit Keplerian model has an orbital period, P = 4.072 days, semivelocity amplitude, K = 149.6 m s^(-1), and eccentricity, e = 0.016 ± 0.01. The host star is chromospherically inactive and metal-rich, with [Fe/H] = 0.26. Based on the T_(eff) and stellar luminosity, we derive a stellar radius of 1.49 R_☉. Photometric observations of HD 149143 were carried out using the automated photometric telescopes at Fairborn Observatory. HD 149143 is photometrically constant over the radial velocity period to 0.0003 ± 0.0002 mag, supporting the existence of the planetary companion. No transits were detected down to a photometric limit of approximately 0.02%, eliminating transiting planets with a variety of compositions and constraining the orbital inclination to less than 83°. A short-period planet was also detected around HD 109749, a G3 IV star. HD 109749 is chromospherically inactive, with [Fe/H] = 0.25 and a stellar radius of 1.24. The radial velocities for HD 109749 are modeled by a Keplerian with P = 5.24 days and K = 28.7 m s^(-1). The inferred planet mass is M sin i = 0.28M_J and the semimajor axis of this orbit is 0.0635 AU. Photometry of HD 109749 was obtained with the SMARTS consortium telescope, the PROMPT telescope, and by transitsearch.org observers in Adelaide and Pretoria. These observations did not detect a decrement in the brightness of the host star at the predicted ephemeris time, and they constrain the orbital inclination to less than 85° for gas giant planets with radii down to 0.7R_J

Monitoring and Understanding Trends in Extreme Storms: State of Knowledge

Review of the climate science for severe convective storms, extreme precipitation, hurricanes and typhoons, and severe snowstorms and ice storms in the US shows that the ability to detect and attribute trends varies, depending on the phenomenon. A specific subset of extreme weather and climate types affecting the country is discussed to examine these extreme weather conditions. The categories of storms described were selected as they caused property damage and loss of life. The identification of an extreme occurrence was based on meteorological properties in place of the destructiveness. The primary purpose was to examine the scientific evidence for the prevailing capability to detect trends and understand their causes for certain weather types, including severe convective storms and hurricanes and typhoons

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts