Construction and validation of UV-C decontamination cabinets for filtering facepiece respirators.

We present evidence-based design principles for three different UV-C based decontamination systems for N95 filtering facepiece respirators (FFRs) within the context of the SARS-CoV-2 outbreak of 2019-2020. The approaches used here were created with consideration for the needs of low- and middle-income countries (LMICs) and other under-resourced facilities. As such, a particular emphasis is placed on providing cost-effective solutions that can be implemented in short order using generally available components and subsystems. We discuss three optical designs for decontamination chambers, describe experiments verifying design parameters, validate the efficacy of the decontamination for two commonly used N95 FFRs (3M, #1860 and Gerson #1730), and run mechanical and filtration tests that support FFR reuse for at least five decontamination cycles.

Modeling UV-C irradiation chambers for mask decontamination using Zemax OpticStudio.

Ultraviolet decontamination of personal protective equipment, particularly masks, is important in situations where mask reuse is practiced. To assist in the development of UV-C decontamination chambers, we have constructed ray tracing models in Zemax OpticStudio v20.1 for two distinct geometries, namely, a rectangular cabinet and a cylindrical can. These models provide irradiance distributions that can be used for comparison with experiment, as well as to predict local irradiance variation over the surface of a mask. In this paper we describe the model details, including: (1) a mask object in CAD format; (2) our assumptions for modeling surface properties; (3) the use of polygon object detectors for local irradiance analysis; and (4) experimental results that compare favorably to the simulations.

Automated Cell Segmentation for Quantitative Phase Microscopy.

Automated cell segmentation and tracking is essential for dynamic studies of cellular morphology, movement, and interactions as well as other cellular behaviors. However, accurate, automated, and easy-to-use cell segmentation remains a challenge, especially in cases of high cell densities, where discrete boundaries are not easily discernable. Here, we present a fully automated segmentation algorithm that iteratively segments cells based on the observed distribution of optical cell volumes measured by quantitative phase microscopy. By fitting these distributions to known probability density functions, we are able to converge on volumetric thresholds that enable valid segmentation cuts. Since each threshold is determined from the observed data itself, virtually no input is needed from the user. We demonstrate the effectiveness of this approach over time using six cell types that display a range of morphologies, and evaluate these cultures over a range of confluencies. Facile dynamic measures of cell mobility and function revealed unique cellular behaviors that relate to tissue origins, state of differentiation, and real-time signaling. These will improve our understanding of multicellular communication and organization.

Cell sex affects extracellular matrix protein expression and proliferation of smooth muscle progenitor cells derived from human pluripotent stem cells.

BACKGROUND: Smooth muscle progenitor cells (pSMCs) differentiated from human pluripotent stem cells (hPSCs) hold great promise for treating diseases or degenerative conditions involving smooth muscle pathologies. However, the therapeutic potential of pSMCs derived from men and women may be very different. Cell sex can exert a profound impact on the differentiation process of stem cells into somatic cells. In spite of advances in translation of stem cell technologies, the role of cell sex and the effect of sex hormones on the differentiation towards mesenchymal lineage pSMCs remain largely unexplored. METHODS: Using a standard differentiation protocol, two human embryonic stem cell lines (one male line and one female line) and three induced pluripotent stem cell lines (one male line and two female lines) were differentiated into pSMCs. We examined differences in the differentiation of male and female hPSCs into pSMCs, and investigated the effect of 17ß-estradiol (E2) on the extracellular matrix (ECM) metabolisms and cell proliferation rates of the pSMCs. Statistical analyses were performed by using Student's t test or two-way ANOVA, p < 0.05. RESULTS: Male and female hPSCs had similar differentiation efficiencies and generated morphologically comparable pSMCs under a standard differentiation protocol, but the derived pSMCs showed sex differences in expression of ECM proteins, such as MMP-2 and TIMP-1, and cell proliferation rates. E2 treatment induced the expression of myogenic gene markers and suppressed ECM degradation activities through reduction of MMP activity and increased expression of TIMP-1 in female pSMCs, but not in male pSMCs. CONCLUSIONS: hPSC-derived pSMCs from different sexes show differential expression of ECM proteins and proliferation rates. Estrogen appears to promote maturation and ECM protein expression in female pSMCs, but not in male pSMCs. These data suggest that intrinsic cell-sex differences may influence progenitor cell biology.

Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.

Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed µSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the µSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of µSCALE as a new tool for directed evolution.

High-throughput analysis and protein engineering using microcapillary arrays.

We describe a multipurpose technology platform, termed µSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. µSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With µSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the µSCALE platform.

Dynamic and social behaviors of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored.

Lung Cancer Workshop XI: Tobacco-Induced Disease: Advances in Policy, Early Detection and Management.

The Prevent Cancer Foundation Lung Cancer Workshop XI: Tobacco-Induced Disease: Advances in Policy, Early Detection and Management was held in New York, NY on May 16 and 17, 2014. The two goals of the Workshop were to define strategies to drive innovation in precompetitive quantitative research on the use of imaging to assess new therapies for management of early lung cancer and to discuss a process to implement a national program to provide high quality computed tomography imaging for lung cancer and other tobacco-induced disease. With the central importance of computed tomography imaging for both early detection and volumetric lung cancer assessment, strategic issues around the development of imaging and ensuring its quality are critical to ensure continued progress against this most lethal cancer.

Proliferative behavior of vaginal fibroblasts from women with pelvic organ prolapse.

OBJECTIVE: Pelvic organ prolapse (POP) significantly impacts quality of life of women, especially with advancing age. Cell proliferation is a critical parameter in both normal and pathophysiological processes. We sought to examine fibroblast proliferation in premenopausal women with and without POP and menopausal women with POP, and examine whether TGF-ß1, a fibroblast mitogen, could stimulate proliferation in vaginal fibroblasts from these populations. STUDY DESIGN: Vaginal wall biopsies were obtained from asymptomatic women (controls) and women with POP (cases). Fibroblasts were cultured from these tissues. Vaginal fibroblasts were treated with or without TGF-ß1. Cell proliferation rate (mitotic index) was measured with time-lapse dark-field microscopy. Cell mitosis was counted with ImageJ software after analysis of time-lapse images as Quick time movies. RESULTS: There was no significant difference in mitotic index throughout different time points of observation between premenopausal controls and cases of similar ages. However, a significant difference in mitotic index was seen between premenopausal and menopausal cases (p=0.01), with the menopausal group exhibiting significantly lower mitotic indices. When treated with different doses of TGF-ß1, premenopausal control fibroblast proliferation increased with 5ng/ml of TGF-ß1 compared to non-treated fibroblasts (p=0.04). TGF-ß1 stimulation did not affect fibroblasts from either premenopausal or menopausal cases. CONCLUSIONS: Vaginal fibroblast proliferation decreases with age and this association does not appear to be affected by the presence of pelvic organ prolapse. TGF-ß1 stimulation increased cell proliferation of premenopausal control fibroblasts. In contrast, there was no response seen in fibroblasts from premenopausal and menopausal cases.

Application of high-resolution CT imaging data to lung cancer drug development: measuring progress: workshop IX.

BACKGROUND: Lung cancer is the leading cause of cancer death and a major public health challenge across the entire world. Computed tomography (CT) imaging of the lung is a rapidly improving medical imaging technique. Spiral CT has been reported to not only improve the early detection of lung cancer in screening high-risk tobacco-exposed populations but also to assist in the clinical assessment of new agents for therapy in lung cancer. METHODS: The Prevent Cancer Foundation has sponsored a series of workshops to accelerate progress in using quantitative imaging to advance lung cancer research progress, of which this report summarizes the Ninth Workshop. The defining strategy of this forum to support innovation in quantitative research for early lung cancer management was to enable software validations by assembling collections of high-quality images for which long-term clinical follow-up is known. An additional approach was to define a process for high-quality and economical national implementation of lung cancer screening. Representatives from the Quantitative Imaging Biomarker Alliance, the International Association for the Study of Lung Cancer, the Lung Cancer Alliance, and other organizations outlined their efforts in this regard. A major opportunity exists to advance the dialogue on the use of quantitative imaging tools to cross-fertilize and accelerate image-processing research across lung cancer and chronic obstructive pulmonary disease (COPD). CONCLUSION: The use of high-resolution CT imaging provides a window into a much earlier stage of COPD as well as coronary artery disease, both being tobacco-induced diseases. Progress in this area was reviewed and opportunities for enhanced collaborative progress defined. Key sessions reviewed emerging developments with imaging technology and the infrastructure to support the storage and distribution of these high-content modalities. Cooperation among diverse collaborators is essential to enable the rapid organic evolution of this field, so that improved outcomes with lung cancer, artery disease, and COPD can be obtained.

High-speed laser microsurgery of alert fruit flies for fluorescence imaging of neural activity.

Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-µm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12- to 350-µm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca(2+) signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5-20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation.

Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage.

We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.

A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen.

Tamoxifen significantly reduces tumor recurrence in certain patients with early-stage estrogen receptor-positive breast cancer, but markers predictive of treatment failure have not been identified. Here, we generated gene expression profiles of hormone receptor-positive primary breast cancers in a set of 60 patients treated with adjuvant tamoxifen monotherapy. An expression signature predictive of disease-free survival was reduced to a two-gene ratio, HOXB13 versus IL17BR, which outperformed existing biomarkers. Ectopic expression of HOXB13 in MCF10A breast epithelial cells enhances motility and invasion in vitro, and its expression is increased in both preinvasive and invasive primary breast cancer. The HOXB13:IL17BR expression ratio may be useful for identifying patients appropriate for alternative therapeutic regimens in early-stage breast cancer.

Gene expression profiles of human breast cancer progression.

Although distinct pathological stages of breast cancer have been described, the molecular differences among these stages are largely unknown. Here, through the combined use of laser capture microdissection and DNA microarrays, we have generated in situ gene expression profiles of the premalignant, preinvasive, and invasive stages of human breast cancer. Our data reveal extensive similarities at the transcriptome level among the distinct stages of progression and suggest that gene expression alterations conferring the potential for invasive growth are already present in the preinvasive stages. In contrast to tumor stage, different tumor grades are associated with distinct gene expression signatures. Furthermore, a subset of genes associated with high tumor grade is quantitatively correlated with the transition from preinvasive to invasive growth.