A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells

BACKGROUND: Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation. RESULTS: We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR. CONCLUSIONS: Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses

Cell Invasion Dynamics into a Three Dimensional Extracellular Matrix Fibre Network

The dynamics of filopodia interacting with the surrounding extracellular matrix (ECM) play a key role in various cell-ECM interactions, but their mechanisms of interaction with the ECM in 3D environment remain poorly understood. Based on first principles, here we construct an individual-based, force-based computational model integrating four modules of 1) filopodia penetration dynamics; 2) intracellular mechanics of cellular and nuclear membranes, contractile actin stress fibers, and focal adhesion dynamics; 3) structural mechanics of ECM fiber networks; and 4) reaction-diffusion mass transfers of seven biochemical concentrations in related with chemotaxis, proteolysis, haptotaxis, and degradation in ECM to predict dynamic behaviors of filopodia that penetrate into a 3D ECM fiber network. The tip of each filopodium crawls along ECM fibers, tugs the surrounding fibers, and contracts or retracts depending on the strength of the binding and the ECM stiffness and pore size. This filopodium-ECM interaction is modeled as a stochastic process based on binding kinetics between integrins along the filopodial shaft and the ligands on the surrounding ECM fibers. This filopodia stochastic model is integrated into migratory dynamics of a whole cell in order to predict the cell invasion into 3D ECM in response to chemotaxis, haptotaxis, and durotaxis cues. Predicted average filopodia speed and that of the cell membrane advance agreed with experiments of 3D HUVEC migration at r[superscript 2] > 0.95 for diverse ECMs with different pore sizes and stiffness.Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology)National Science Foundation (U.S.). Science and Technology Center and Emergent Behaviors of Integrated Cellular Systems (Grant EFRI-0735997)National Science Foundation (U.S.). Science and Technology Center and Emergent Behaviors of Integrated Cellular Systems (Grant STC-0902396)National Science Foundation (U.S.). Science and Technology Center and Emergent Behaviors of Integrated Cellular Systems (Grant CBET-0939511

Adipose saturation reduces lipotoxic systemic inflammation and explains the obesity paradox

Obesity sometimes seems protective in disease. This obesity paradox is predominantly described in reports from the Western Hemisphere during acute illnesses. Since adipose triglyceride composition corresponds to long-term dietary patterns, we performed a meta-analysis modeling the effect of obesity on severity of acute pancreatitis, in the context of dietary patterns of the countries from which the studies originated. Increased severity was noted in leaner populations with a higher proportion of unsaturated fat intake. In mice, greater hydrolysis of unsaturated visceral triglyceride caused worse organ failure during pancreatitis, even when the mice were leaner than those having saturated triglyceride. Saturation interfered with triglyceride\u27s interaction and lipolysis by pancreatic triglyceride lipase, which mediates organ failure. Unsaturation increased fatty acid monomers in vivo and aqueous media, resulting in greater lipotoxic cellular responses and organ failure. Therefore, visceral triglyceride saturation reduces the ensuing lipotoxicity despite higher adiposity, thus explaining the obesity paradox

Photometric and Spectroscopic Properties of Type Ia Supernova 2018oh with Early Excess Emission from the Kepler 2 Observations

Supernova (SN) 2018oh (ASASSN-18bt) is the first spectroscopically confirmed Type Ia supernova (SN Ia) observed in the Kepler field. The Kepler data revealed an excess emission in its early light curve, allowing us to place interesting constraints on its progenitor system. Here we present extensive optical, ultraviolet, and near-infrared photometry, as well as dense sampling of optical spectra, for this object. SN 2018oh is relatively normal in its photometric evolution, with a rise time of 18.3 ± 0.3 days and Δm 15(B) = 0.96 ± 0.03 mag, but it seems to have bluer B − V colors. We construct the "UVOIR" bolometric light curve having a peak luminosity of 1.49 × 1043 erg s−1, from which we derive a nickel mass as 0.55 ± 0.04 M ⊙ by fitting radiation diffusion models powered by centrally located 56Ni. Note that the moment when nickel-powered luminosity starts to emerge is +3.85 days after the first light in the Kepler data, suggesting other origins of the early-time emission, e.g., mixing of 56Ni to outer layers of the ejecta or interaction between the ejecta and nearby circumstellar material or a nondegenerate companion star. The spectral evolution of SN 2018oh is similar to that of a normal SN Ia but is characterized by prominent and persistent carbon absorption features. The C ii features can be detected from the early phases to about 3 weeks after the maximum light, representing the latest detection of carbon ever recorded in an SN Ia. This indicates that a considerable amount of unburned carbon exists in the ejecta of SN 2018oh and may mix into deeper layers

Temporal evaluation of efficacy and quality of tissue repair upon laser‐activated sealing

Abstract Injuries caused by surgical incisions or traumatic lacerations compromise the structural and functional integrity of skin. Immediate approximation and robust repair of skin are critical to minimize occurrences of dehiscence and infection that can lead to impaired healing and further complication. Light‐activated skin sealing has emerged as an alternative to sutures, staples, and superficial adhesives, which do not integrate with tissues and are prone to scarring and infection. Here, we evaluate both shorter‐ and longer‐term efficacy of tissue repair response following laser‐activated sealing of full‐thickness skin incisions in immunocompetent mice and compare them to the efficacy seen with sutures. Laser‐activated sealants (LASEs) in which, indocyanine green was embedded within silk fibroin films, were used to form viscous pastes and applied over wound edges. A hand‐held, near‐infrared laser was applied over the incision, and conversion of the light energy to heat by the LASE facilitated rapid photothermal sealing of the wound in approximately 1 min. Tissue repair with LASEs was evaluated using functional recovery (transepidermal water loss), biomechanical recovery (tensile strength), tissue visualization (ultrasound [US] and photoacoustic imaging [PAI]), and histology, and compared with that seen in sutures. Our studies indicate that LASEs promoted earlier recovery of barrier and mechanical function of healed skin compared to suture‐closed incisions. Visualization of sealed skin using US and PAI indicated integration of the LASE with the tissue. Histological analyses of LASE‐sealed skin sections showed reduced neutrophil and increased proresolution macrophages on Days 2 and 7 postclosure of incisions, without an increase in scarring or fibrosis. Together, our studies show that simple fabrication and application methods combined with rapid sealing of wound edges with improved histological outcomes make LASE a promising alternative for management of incisional wounds and lacerations

A Virus-Derived Immune Modulating Serpin Accelerates Wound Closure with Improved Collagen Remodeling

Numerous treatments have been developed to promote wound healing based on current understandings of the healing process. Hemorrhaging, clotting, and associated inflammation regulate early wound healing. We investigated treatment with a virus-derived immune modulating serine protease inhibitor (SERPIN), Serp-1, which inhibits thrombolytic proteases and inflammation, in a mouse excisional wound model. Saline or recombinant Serp-1 were applied directly to wounds as single doses of 1 μg or 2 µg or as two 1 µg boluses. A chitosan-collagen hydrogel was also tested for Serp-1 delivery. Wound size was measured daily for 15 days and scarring assessed by Masson’s trichrome, Herovici’s staining, and immune cell dynamics and angiogenesis by immunohistochemistry. Serp-1 treatment significantly accelerated wound healing, but was blocked by urokinase-type plasminogen activator (uPAR) antibody. Repeated dosing at a lower concentration was more effective than single high-dose serpin. A single application of Serp-1-loaded chitosan-collagen hydrogel was as effective as repeated aqueous Serp-1 dosing. Serp-1 treatment of wounds increased arginase-1-expressing M2-polarized macrophage counts and periwound angiogenesis in the wound bed. Collagen staining also demonstrated that Serp-1 improves collagen maturation and organization at the wound site. Serp-1 has potential as a safe and effective immune modulating treatment that targets thrombolytic proteases, accelerating healing and reducing scar in deep cutaneous wounds

Four modules for the cell invasion dynamics.

<p><b>A</b>) Filopodia dynamics showing outgrowing, tugging and contractile phases (see ‘a’ in <b>E</b>). <b>B</b>) cellular membrane mechanics showing the membrane is not only connected by actin stress fibers, but also anchored to elastic ECM fibers by forming focal adhesions (FAs) (See ‘b’ in <b>e</b>), and viscoelastic behaviors in cellular membrane is modeled using Kelvin-Voigt model. <b>C</b>) Schematic diagram of MMP-2 activation [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004535#pcbi.1004535.ref014" target="_blank">14</a>] and selected simulation results of VEGF, MMP-2 and TIMP-2 concentration distributions using simplified model for MMP-2 activation. <b>D</b>) a magnified schematic of ‘c’ in <b>E)</b> showing the elastic ECM fiber networks is organized with collagen fibers, crosslinking molecules and free (or non-crosslinked) molecules. <b>E</b>) An integrated schematic representation of MT1-MMP and TIMP-2 secretions at the membrane near the roots of filopodia; directions of outgrowing filopodia are guided when tips of filopodia sense local gradients of VEGF toward a chemotactic cue.</p