Teachable, High-Content Analytics for Live-Cell, Phase Contrast Movies

Introduction Chronic wounds represent a major problem in medicine today as their incidence is continuously increasing due to an ageing population and a rise in the incidence of underlying diseases. Cutaneous wound healing is a complex biological process. Chronic wounds are characterised by a prolonged inflammation, persistent infections, formation of drugresistant microbial biofilms and the inability of dermal and/or epidermal cells to respond to regenerative stimuli. As conventional treatment strategies often fail, innovative therapies have been investigated over the last decade, including stem cell-based therapies. After the initial use of embryonic stem cells, the focus has been set on autologous mesenchymal stem cells over the past years. They can be isolated in large amounts from various tissues and hold no ethical concerns. A promising and cost-effective source of autologous mesenchymal stem cells is subcutaneous adipose tissue. Recent in vitro and in vivo studies have shown that adiposederived stem cells have a positive impact on wound healing, as they are attracted to the wound site and influence regeneration processes via paracrine mechanisms. They are pluripotent and secrete a variety of growth factors. The aim of this critical review was to discuss adiposederived stem cells in wound healing. Conclusion Mesenchymal stem cells-derived from adipose tissue are a promising alternative to embryonic or bone marrow-derived stem cells in the therapy of chronic wounds. Because the use of adipose-derived stem cells in wound healing applications is still limited by a lack of clinical data, further studies have to pave the way for their routine clinical application. Introduction Due to demographic changes the number of patients with multimorbidity is continuously increasing in western countries. One of the associated disorders is the chronic wound. This includes a rise in the incidence of impaired wound healing, causing a reduced patients’ quality of life and rising health care costs. Conventional therapies of chronic wounds are increasingly reaching their limits, motivating the search for alternative treatment options, including stem cell-based therapies1. This paper discusses the recent in vitro and in vivo results of adipose-derived stem cells (ASCs) in wound healing. Physiological wound healing Wound healing is a complex biological process consisting of the four different but partially overlying steps coagulation, inflammation, formation of granulation tissue (proliferative phase) and remodelling or scar formation. After injury, initiation of a bloodclotting cascade prevents excessive bleeding and provides temporary protection of the wound area. During this process platelet-derived growth factor and transforming growth factors A1 and 2 (TGF-A1 and TGF-2) are released, causing attraction of inflammatory cells such as leukocytes and macrophages. Within a few days after injury, apoptosis of inflammatory cells occurs. Anti-inflammatory cytokines such as TGF-A1 and Interleukin (IL) 1 as well as bioactive lipids are assumed to be involved in this process (Figure 1). In the following proliferative phase, production of growth factors and activation of dermal and epidermal cells lead to new tissue formation. Endothelial progenitor cells, which are essential for physiological wound healing, are mobilised by nitric oxide, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP9). Formation of extra-cellular matrix (ECM)rich tissue occurs in response to stromal cell-derived factor (SDF)-1 and insulin-like growth factor (IGF). In the last step of wound healing, matrix remodelling and/or scar formation through cellular migration, proliferation and angiogenic induction is initiated by TGF-A, MMPs and tumour necrosis factor (TNF)2. Impaired wound healing A chronic wound is defined as a wound that does not heal in a certain period of time in spite of appropriate therapy. However, the exact-time point is in discussion. Most authors refer to a chronic wound as a wound that exists for more than three months3. Impaired wound healing is associated with senescence, ischaemia and bacterial colonisation. Wound chronification is caused by local factors such as infection, venous insufficiency, mechanical trauma, * Corresponding author Email: Carolin.Fromm-Dornieden@uni-wh.de 1 Institute for Research in Operative Medicine, University of Witten/Herdecke, Ostmerheimer Str. 200, D-51109 Cologne, Germany 2 Department of Trauma and Orthopaedic Surgery, Cologne-Merheim Medical Center, University of Witten/Herdecke, Cologne, Germany

Elastin-like polypeptides (ELPs) are modular, stimuli-responsive materials that self-assemble into protein-rich microdomains in response to heating. By cloning ELPs to effector proteins, expressed intracellular fusions can even modulate cellular pathways. A critical step in engineering these fusions is to determine and control their intracellular phase transition temperature (Tt). To do so, this Method paper describes a simple live-cell imaging technique to estimate the Tt of non-fluorescent ELP fusion proteins by co-transfection with a fluorescent ELP marker. Intracellular microdomain formation can then be visualized in live cells through the co-assembly of the non-fluorescent and fluorescent ELP fusion proteins. If the two ELP fusions have different Tt, the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower Tt. In addition, co-assembled ELP microdomains often exhibit pronounced differences in size or number, compared to single transfected treatments. These features enable live-cell imaging experiments and image analysis to determine the intracellular Tt of a library of related ELP fusions. As a case study, we employ the recently reported Caveolin1-ELP library (CAV1-ELPs). In addition to providing a detailed protocol, we also report the development of a useful FIJI plugin named SIAL (Simple Image Analysis Library), which contains programs for image randomization and blinding, phenotype scoring, and ROI selection. These tasks are important parts of the protocol detailed here and are also commonly employed in other image analysis workflows.

37] Cell motility assay and inhibition by Rho-GDP dissociation inhibitor

Stem cells have recently been shown to play important roles in wound healing. The aim of this study was to investigate the role of dermal CD271+ cells in wound healing. Full-thickness wounds were produced on the backs of 5-year-old and 24-week-old mice, and time-course of wound closure, CD271+ cell counts, and gene expression levels were compared. Delayed wound healing was observed in 24-week-old mice. The peak of CD271+ cell increase was delayed in 24-week-old mice, and gene expression levels of growth factors in wounded tissue were significantly increased in 5-year-old mice. Dermal CD271+ cells purified by fluorescence-activated cell sorting (FACS) expressed higher growth factors than CD271- cells, suggesting that CD271+ cells play important roles by producing growth factors. This study also investigated dermal CD271+ cells in patients with chronic skin ulcers. Dermal CD271+ cells in patients were significantly reduced compared with in healthy controls. Thus, dermal CD271+ cells are closely associated with wound healing.

We previously reported that relative to normal cervical mucus, microRNA 126-3p (miR-126-3p) is present in significantly greater amounts in the cervical mucus of patients with overt cervical cancer or precursor lesions. Here, we investigated the effects of enforced miR-126-3p expression in the cervical cancer cell line, HeLa, on proliferation, migration, invasion, apoptosis and protein expression. We transfected HeLa cells with miR-126-3p miRNA and found that proliferation, migration and invasion by cell counting, wound healing, cell migration and invasion assay were significantly reduced in these cells relative to those transfected with a negative control mimic. The levels of phosphoinositide 3 kinase (PI3K), phosphorylated 3-phosphoinositide-dependent protein kinase-1 (p-PDK1) and p-AKT proteins were lower in the miR-126-3p-transfected cells. Phosphorylated 70S6K (p-p70S6K), phosphorylated glycogen synthase kinase 3β (p-GSK3β), phosphorylated S6K (p-S6K), cyclin D1, phosphorylated p21-activated kinase 1 (p-PAK1), Rho associated coiled-coil containing protein kinase 1 (ROCK1), myotonic dystrophy-related CDC42-binding kinases α (MRCKα) and phospholipase C γ1 (p-PLCγ1) were also downregulated. This suggests that downstream effectors of the PI3K/PDK1/AKT pathway are targets for inhibition by miR-126-3p. In contrast, apoptotic-related proteins including the BCL-2-associated agonist of cell death (Bad), B-cell lymphoma-extra-large (Bcl-xL) and BCL-2-associated X (Bax), were all upregulated by miR-126-3p, resulting in increased caspase 3/7 activity and apoptosis. Thus, enforced expression of miR-126-3p inhibited cell migration and invasion and also induced apoptosis by regulating the PI3K/PDK1/AKT pathway in HeLa cells. Hence, high levels of miR-126-3p may inhibit cervical carcinogenesis, and targeting the PI3K/PDK1/AKT pathway via miR-126-3p could represent a new approach for treating patients with cervical cancer.

IntroductionAsbestos-induced formation of mesothelioma has been attributed to phenotypic and morphological changes in cells caused by polyploidization and aneuploidization, and multiwalled carbon nanotubes (MWCNTs) are suspected to have similar adverse effects due to the similarity in their physical form. MWCNTs and crocidolite, a kind of asbestos, show similar genotoxicity characteristics in vitro, including induction of binucleated cells. We here focused on the mechanisms underlying polyploidization during cell division on exposure to MWCNTs and conducted confocal live-cell imaging analysis using MDA-435 human breast cancer cells in which chromosomes and centromeres were visualized using fluorescent proteins.FindingsDuring anaphase, relatively short MWCNT fibers (approximately 5 μm) migrated rapidly to either of the daughter cells, whereas some long MWCNT fibers (approximately 20 μm) remained inside the contractile ring and induced the formation of binucleated cells through impairment of cytokinesis. This toxicity mechanism has also been observed with crocidolite.ConclusionsOur findings indicate that the mechanism of polyploidization by MWCNTs is very similar to that observed with crocidolite.Electronic supplementary materialThe online version of this article (doi:10.1186/s41021-015-0003-y) contains supplementary material, which is available to authorized users.

Wound healing phases comprise of highly synchronized process that begins due to a damage and restores the integrity of the injured tissues. Wound healing reduces the damage in tissue and supply sufficient oxygen and tissue perfusion, provide proper nourishment and humid wound healing atmosphere to re-establish the essential status of exaggerated parts. The untreated wound becomes susceptible for pus development, bacterial infection and complications like sepsis. Traditional and modern approaches are in practice to treat acute, open and chronic injuries, however, present wound care management has met with challenges and minimal positive effects. Stem cells have possible wound healing capability to overwhelm restrictions of the current wound care practices as it produces faster tissue regeneration in wound repair. Stem cells are unspecialized cells derived from adult body tissues and embryos that differentiate into any cell of an organism and capable of self-regeneration. The understanding on molecular mechanisms of stem cells has become the central and promising field in scientific study. This review focuses on the pre-existing traditional and modern treatments for wound healing, and types and roles of stem cells in wound care management. This review also focuses on the fundamental molecular characterization and factors influencing the molecular mechanisms of stem cells in wound healing.

Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine {bf{text{synthetase}}}{boldsymbol{/}}{{bf{text{tRNA}}}}_{{bf{text{CUA}}}}^{{bf{text{Pyl}}}} pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

Balance deficits are present in a variety of clinical populations and can negatively impact quality of life. The integration of wearable sensors and machine learning technology (ML) provides unique opportunities to quantify biomechanical characteristics related to balance outside of a laboratory setting. This article provides a general overview of recent developments in using wearable sensors and ML to estimate or predict biomechanical characteristics such as center of pressure (CoP) and center of mass (CoM) motion. This systematic review was conducted according to PRISMA guidelines. Databases including Scopus, PubMed, CINHAL, Trip PRO, Cochrane, and Otseeker databases were searched for publications on the use of wearable sensors combined with ML to predict biomechanical characteristics. Fourteen publications met the inclusion criteria and were included in this review. From each publication, information on study characteristics, testing conditions, ML models applied, estimated biomechanical characteristics, and sensor positions were extracted. Additionally, the study type, level of evidence, and Downs and Black scale score were reported to evaluate methodological quality and bias. Most studies tested subjects during walking and utilized some type of neural network (NN) ML model to estimate biomechanical characteristics. Many of the studies focused on minimizing the necessary number of sensors and placed them on areas near or below the waist. Nearly all studies reporting RMSE and correlation coefficients had values <15% and >0.85, respectively, indicating strong ML model estimation accuracy. Overall, this review can help guide the future development of ML algorithms and wearable sensor technologies to estimate postural mechanics.

Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing

Wound healing is a dynamic and well-ordered biological process, in which re-epithelialization plays an important role. Re-epithelialization of skin wounds depends upon epidermal cells migration from the wound edge. Hypoxia is a microenvironmental stress that occurs immediately after injury, likely due to the vascular disruption and increased oxygen consumption by cells surrounding the wound edge, which has been shown to promote epidermal cells migration and wound healing. Endogenous electric field (EF) is generated instantaneously after injury due to the collapse of the trans-epithelial potentials, which plays an overriding guidance role in directing epidermal cells migration towards the wound center in wound healing. In addition, nitric monoxide (NO) as an inflammatory factor produced after wounds occur has been found to promote epidermal cells migration and wound healing. In conclusion, hypoxia, EF and NO accelerate the healing process though promoting epidermal cells migration towards the wound center. Key words: Cell movement; Wounds and injuries; Wound healing; Nitric oxide; Hypoxia; Electric field

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

BackgroundDamaged intestinal epithelial barrier is characteristic of inflammatory bowel diseases (IBD) and mucosal healing is the primary goal for IBD treatment. We previously showed the direct beneficial effects of rhamnogalacturonan (RGal), a polysaccharide isolated from the plant Acmella oleracea, on intestinal epithelial barrier function with participation of TLR4 and PKC activation. We also observed that RGal accelerates wound healing in human colonic epithelial Caco-2 cells. RNAseq data and pathway analysis have indicated the involvement of the canonical nuclear factor kB (NF-kB) signaling pathway.AimsWe hypothesize that RGal increases intestinal epithelial wound healing through NF-kB signaling pathway.MethodsCaco-2 cells monolayers were scratched and treated with vehicle (media or 0.5% DMSO in media) or RGal (1000 mg/ml) for 48 h. Wound healing was assessed using the IncuCyte live cell imaging system. Proliferation and apoptosis of cells were evaluated using EdU and TUNEL assays, respectively. Inhibitors were added at the same time (transcription inhibitor Actinomycin D, 5 mg/ml) or 1 h (NF-kB inhibitors Bay 11–7082 and JSH-23, 10 and 20 mM, respectively, or COX-2 inhibitor NS-398, 20 mM) before RGal treatment. Unwounded Caco-2 monolayers treated with RGal (1000 mg/ml) were collected for Western blotting for COX-2 protein.ResultsIn the wound healing assay, RGal at a concentration of 1000 µg/ml enhanced wound healing by 12.5% at 48 h compared to control group, under 10% serum conditions. Neither proliferation nor apoptosis were involved in the RGal effect on wound healing, suggesting the response was due solely to cell migration. Actinomycin D (5 mg/ml), Bay 11–7082 (10 mM) or JSH-23 (20 mM) treatment significantly reversed the effect of RGal on wound healing, showing that the response was transcriptionally dependent and involved NF-kB signaling. Treatment of cells with NS-398 (20 mM) also reversed the effect of RGal on wound healing. COX-2 protein expression was significantly increased at 6 and 12 h after RGal addition to Caco-2 monolayers.ConclusionsThese data suggest that the plant-based polysaccharide RGal increases intestinal epithelial cell wound healing by increasing cell migration. The RGal effect is dependent on the activation of the transcription factor NF-kB and downstream COX-2 protein expression and activity. Our findings show a novel mechanism of action of RGal in wound healing that could help in the resolution of intestinal inflammation and mucosal healing.Funding AgenciesNSERC

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

The growth factor signaling network, including the Akt, ERK, mTOR and other pathways, is deregulated in many diseases and is a prominent target for therapeutic inhibitors. The effectiveness of these inhibitors is limited by the existence of homeostatic feedback loops that act to restore signaling and attenuate the effects of inhibition. To obtain the single-cell data necessary to fully characterize these rapid and heterogeneous responses, we developed a library of mammary epithelial cells stably expressing over 90 fluorescent protein-based reporters. This library comprises reporters for: cellular processes including cell death, cell cycle, protein translation, and metabolism; kinases including ERK, Akt and AMPK; and microRNAs involved in the regulation of cellular differentiation and stress response. Using high-throughput live-cell microscopy and computational image analysis, we monitored the responses of these pathways to inhibitors of EGFR, ErbB2, PI3K, mTOR, MEK, Raf, and Bcl-2. This analysis revealed unexpectedly dynamic fluctuations in key pathways including ERK, Akt, and glycolysis. In one example, inhibition of EGFR transformed constitutive ERK activity into intermittent bursts of activity ranging in duration from 20-90 minutes with high cell-to-cell variability. Characterization of ERK activity pulses revealed that their frequency depended on the level of EGF receptor activity and that their duration was essential for controlling the commitment to cell proliferation. In sharp contrast, inhibition of MEK altered the amplitude, rather than the frequency, of ERK activity. Using these live-cell measurements in combination with high content immunofluorescence, we constructed a computational model of feedback loops controlling ERK activity pulses. Quantitative mapping of ERK output to proliferation rate revealed a distinct and exquisitely sensitive threshold of ERK output required for proliferation. Potential strategies for reducing ERK output below this threshold and effectively blocking proliferation include dual inhibition of upstream regulators or intermittent high-dose inhibition. Single-cell quantitative analysis of inhibitor responses is now being extended to other pathways to identify signaling dynamics and information transfer properties that enable effective therapeutic targeting of the network. This proffered talk is also presented as Poster A19. Citation Format: John Albeck, Yin P. Hung, Gregory Leya, Gary Yellen, Gordon Mills, Joan Brugge. Visualizing the dynamic and heterogeneous responses of single cells to inhibitors of the growth factor signaling network [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr PR7.