Instant processing of large-scale image data with FACT, a real-time cell segmentation and tracking algorithm.

Quantifying cellular characteristics from a large heterogeneous population is essential to identify rare, disease-driving cells. A recent development in the combination of high-throughput screening microscopy with single-cell profiling provides an unprecedented opportunity to decipher disease-driving phenotypes. Accurately and instantly processing large amounts of image data, however, remains a technical challenge when an analysis output is required minutes after data acquisition. Here, we present fast and accurate real-time cell tracking (FACT). FACT can segment ∼20,000 cells in an average of 2.5 s (1.9-93.5 times faster than the state of the art). It can export quantifiable features minutes after data acquisition (independent of the number of acquired image frames) with an average of 90%-96% precision. We apply FACT to identify directionally migrating glioblastoma cells with 96% precision and irregular cell lineages from a 24 h movie with an average F1 score of 0.91.

Linking the genotypes and phenotypes of cancer cells in heterogenous populations via real-time optical tagging and image analysis.

Linking single-cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, could help unravel molecular mechanisms driving the growth of tumour-cell subpopulations. Here we show that a custom-built optical microscope with an ultrawide field of view, fast automated image analysis and a dye activatable by visible light enables the screening and selective photolabelling of cells of interest in large heterogeneous cell populations on the basis of specific functional cellular dynamics, such as fast migration, morphological variation, small-molecule uptake or cell division. Combining such functional single-cell selection with single-cell RNA sequencing allowed us to (1) functionally annotate the transcriptomic profiles of fast-migrating and spindle-shaped MCF10A cells, of fast-migrating MDA-MB-231 cells and of patient-derived head-and-neck squamous carcinoma cells, and (2) identify critical genes and pathways driving aggressive migration and mesenchymal-like morphology in these cells. Functional single-cell selection upstream of single-cell sequencing does not depend on molecular biomarkers, allows for the enrichment of sparse subpopulations of cells, and can facilitate the identification and understanding of the molecular mechanisms underlying functional phenotypes.

Mucolytic Agents and Statins Use is Associated with a Lower Risk of Acute Exacerbations in Patients with Bronchiectasis-Chronic Obstructive Pulmonary Disease Overlap.

BACKGROUND: Bronchiectasis-chronic obstructive pulmonary disease (COPD) overlap (BCO) is a neglected area of trials, and it is not covered by guidelines for clinical practice. METHODS: Using the National Health Insurance Research Database of Taiwan, COPD patients with or without bronchiectasis from 2000 to 2009 were enrolled as the BCO and COPD alone cohorts, respectively. Patients followed for <28 days, diagnosed with COPD who were not prescribed with COPD medications, and those diagnosed with bronchiectasis who did not receive a chest X-ray or computed tomography were excluded. The primary endpoints were acute exacerbations and mortality. RESULTS: There were 831 patients in the BCO cohort and 3321 patients in the COPD alone cohort, covering 3763.08 and 17,348.95 person-years, respectively, from 2000 to 2011. The BCO cohort had higher risk for exacerbations (adjusted hazard ratio (HR) 2.26, 95% confidence interval (CI) 1.94⁻2.63) and mortality (HR 1.46, 95% CI 1.24⁻1.73) than the COPD alone cohort. In the patients overall, the use of statins, macrolides, and mucolytic agents was associated with significantly lower risks of acute exacerbations (statins, HR 0.37, 95% CI 0.29⁻0.46; macrolides, HR 0.65, 95% CI 0.45⁻0.93; mucolytic agents, HR 0.68, 95% CI 0.59⁻0.78). Statins were associated with a significantly lower risk of mortality (HR 0.32, 95% CI 0.25⁻0.41). In the BCO group, statins and mucolytic agents use was associated with significantly lower risks of acute exacerbations (statins, HR 0.44, 95% CI 0.29⁻0.65; mucolytic agents, HR 0.58, 95% CI 0.45⁻0.75). CONCLUSION: Statins and mucolytic agents use may lower risk of acute exacerbation in patients with BCO.

Proton pump inhibitors use is associated with a lower risk of acute exacerbation and mortality in patients with coexistent COPD and GERD.

OBJECTIVE: The effect of antacid therapy for patients with COPD and gastroesophageal reflux disease (GERD) remains unclear. PATIENTS AND METHODS: This nationwide population-based study was conducted using data from Taiwan's National Health Insurance Research Database, and enrolled COPD patients with or without GERD. Patients with COPD who were not prescribed COPD medications were excluded. Patients with GERD who underwent upper gastrointestinal endoscopy or 24-hour pH monitoring and received at least 1 antacid were enrolled as symptomatic GERD group. The primary endpoint was acute exacerbation and mortality. RESULTS: This study included 3,485 patients with COPD and symptomatic GERD, and 13,938 patients with COPD alone and covered 12,806.57 and 56,809.78 person-years, respectively, from 2000 to 2011. After multivariate adjustment, symptomatic GERD was associated with acute exacerbation (adjusted hazard ratio [HR]: 1.35, 95% CI: 1.23-1.48, p<0.0001) and mortality (HR: 1.42, 95% CI: 1.25-1.61, p<0.0001). In the COPD with symptomatic GERD group, use of proton pump inhibitors was associated with a lower risk of acute exacerbation and mortality (acute exacerbation, HR 0.31, 95% CI: 0.20-0.50, p<0.0001; mortality, HR 0.36, 95% CI: 0.20-0.65, p=0.0007), whereas no significant benefit was observed for histamine2-receptor antagonists. CONCLUSION: Use of proton pump inhibitors was associated with a lower risk of acute exacerbation and mortality in the patients with COPD and symptomatic GERD.

Toward reliable retrieval of functional information of papillary dermis using spatially resolved diffuse reflectance spectroscopy.

Spatially resolved diffuse reflectance spectroscopy (SRDRS) has been employed to quantify tissue optical properties and its interrogation volume is majorly controlled by the source-to-detector separations (SDSs). To noninvasively quantify properties of dermis, a SRDRS setup that includes SDS shorter than 1 mm is required. It will be demonstrated in this study that Monte Carlo simulations employing the Henyey-Greenstein phase function cannot always precisely predict experimentally measured diffuse reflectance at such short SDSs, and we speculated this could be caused by the non-negligible backward light scattering at short SDSs that cannot be properly modeled by the Henyey-Greenstein phase function. To accurately recover the optical properties and functional information of dermis using SRDRS, we proposed the use of the modified two-layer (MTL) geometry. Monte Carlo simulations and phantom experiment results revealed that the MTL probing geometry was capable of faithfully recovering the optical properties of upper dermis. The capability of the MTL geometry in probing the upper dermis properties was further verified through a swine study, and it was found that the measurement results were reasonably linked to histological findings. Finally, the MTL probe was utilized to study psoriatic lesions. Our results showed that the MTL probe was sensitive to the physiological condition of tissue volumes within the papillary dermis and could be used in studying the physiology of psoriasis.

Broadband absorption and reduced scattering spectra of in-vivo skin can be noninvasively determined using δ-P1 approximation based spectral analysis.

Previously, we revealed that a linear gradient line source illumination (LGLSI) geometry could work with advanced diffusion models to recover the sample optical properties at wavelengths where sample absorption and reduced scattering were comparable. In this study, we employed the LGLSI geometry with a broadband light source and utilized the spectral analysis to determine the broadband absorption and scattering spectra of turbid samples in the wavelength range from 650 to 1350 nm. The performance of the LGLSI δ-P1 diffusion model based spectral analysis was evaluated using liquid phantoms, and it was found that the sample optical properties could be properly recovered even at wavelengths above 1000 nm where µs' to µa ratios were in the range between 1 to 20. Finally, we will demonstrate the use of our system for recovering the 650 to 1350 nm absorption and scattering spectra of in-vivo human skin. We expect this system can be applied to study deep vessel dilation induced hemoglobin concentration variation and determine the water and lipid concentrations of in-vivo skin in clinical settings in the future.

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane.

BACKGROUND: Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. RESULTS: The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. CONCLUSIONS: We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.