Biology subjects

Ochoa, M.

Publications and source records attributed to Ochoa, M..

3 recordsLinked to original sources

bioRxiv2019

Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. To date, non-invasive quantitative imaging modalities that can comprehensively assess simultaneous cellular drug delivery efficacy and therapeutic response are lacking. In this regard, Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Forster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in vivo and subsequent intracellular internalization, which is critical to assess the delivery efficacy of targeted therapeutics. However, implementation of multiplexing optical imaging with FRET in vivo is challenging to achieve due to spectral crowding and cross-contamination. Herein, we report on a strategy that relies on a dark quencher that enables simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700) using in vitro NIR FLI microscopy and in vivo wide-field MFLI imaging. Second, we report on simultaneous in vivo imaging of the metabolic probe IRDye 800CW 2-deoxyglucose (2-DG) and MFLI-FRET imaging of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery. Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

bioengineering↗

bioRxiv2019

Spectral and lifetime fluorescence unmixing via deep learning

Hyperspectral Fluorescence Lifetime Imaging allows for the simultaneous acquisition of spectrally resolved temporal fluorescence emission decays. In turn, the acquired rich multidimensional data set enables simultaneous imaging of multiple fluorescent species for a comprehensive molecular assessment of biotissues. However, to enable quantitative imaging, inherent spectral overlap between the considered fluorescent probes and potential bleed-through must be taken into account. Such task is performed via either spectral or lifetime unmixing, typically independently. Herein, we present UNMIX-ME (unmix multiple emissions), a deep learning-based fluorescence unmixing routine, capable of quantitative fluorophore unmixing by simultaneously using both spectral and temporal signatures. UNMIX-ME was trained and validated using an in silico framework replicating the data acquisition process of a compressive hyperspectral fluorescent lifetime imaging platform (HMFLI). It was benchmarked against a conventional LSQ method for both tri and quadri-exponential simulated samples. Last, UNMIX-MEs potential was assessed for NIR FRET in vitro and in vivo for small animal experimental data.

bioengineering↗

bioRxiv2018

High Throughput Mechanobiological Screens Enable Mechanical Priming of Pluripotency in Mouse Fibroblasts

Transgenic methods for direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) are effective in cell culture systems but ultimately limit the utility of iPSCs due to concerns of mutagenesis and tumor formation. Recent studies have suggested that some transgenes can be eliminated by using small molecules as an alternative to transgenic methods of iPSC generation. We developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. Using this system, we screened for optimized conditions to stimulate the activation of Oct-4 and other transcription factors to prime the development of pluripotency in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the priming of pluripotency of mouse fibroblasts in combination with mechanical loading. Taken together, our findings demonstrate the ability of mechanical forces to induce reprograming factors and support that biophysical conditioning can act cooperatively with small molecules to priming the induction pluripotency in somatic cells.

bioengineering↗