Activation of DNA Damage Checkpoint Pathways During Skeletal Myoblast Differentiation and Apoptosis

A subset of skeletal myoblasts undergo apoptosis rather than differentiation when cultured in differentiation media (DM: absence of growth factors). While the muscle regulatory transcription factor MyoD is known to control the process of differentiation, our lab has recently discovered that MyoD is also controlling the apoptotic process in response to culture in DM by direct up-regulation of the pro-apoptotic Bcl2 family member PUMA. We similarly discovered that MyoD plays a role in the increased expression of PUMA and apoptosis in response to the DNA damaging agent, etoposide. This led to the hypothesis that culture in DM may lead to stalled replication forks during DNA synthesis that are “recognized” as DNA damage. We are testing our hypothesis by determining if culture in DM results in the activation of pathways known to respond to DNA damage. We have determined that p38, p53, and c-abl are all up-regulated in response to culture in DM. Next, we will determine the significance of MyoD to the increased expression of these molecules.https://engagedscholarship.csuohio.edu/u_poster_2014/1027/thumbnail.jp

Creating and Maintaining Chemical Artificial Life by Robotic Symbiosis

We present a robotic platform based on the open source RepRap 3D printer that can print and maintain chemical artificial life in the form of a dynamic, chemical droplet. The robot uses computer vision, a self-organizing map, and a learning program to automatically categorize the behavior of the droplet that it creates. The robot can then use this categorization to autonomously detect the current state of the droplet and respond. The robot is programmed to visually track the droplet and either inject more chemical fuel to sustain a motile state or introduce a new chemical component that results in a state change (e.g., division). Coupling inexpensive open source hardware with sensing and feedback allows for replicable real-time manipulation and monitoring of nonequilibrium systems that would be otherwise tedious, expensive, and error-prone. This system is a first step towards the practical confluence of chemical, artificial intelligence, and robotic approaches to artificial life

Role of geometrical cues in bone marrow-derived mesenchymal stem cell survival, growth and osteogenic differentiation

Mesenchymal stem cells play a vital role in bone formation process by differentiating into osteoblasts, in a tissue that offers not a flat but a discontinuous three-dimensional (3D) topography in vivo. In order to understand how geometry may be affecting mesenchymal stem cells, this study explored the influence of 3D geometry on mesenchymal stem cell-fate by comparing cell growth, viability and osteogenic potential using monolayer (two-dimensional, 2D) with microsphere (3D) culture systems normalised to surface area. The results suggested lower cell viability and reduced cell growth in 3D. Alkaline phosphatase activity was higher in 3D; however, both collagen and mineral deposition appeared significantly lower in 3D, even after osteogenic supplementation. Also, there were signs of patchy mineralisation in 3D with or without osteogenic supplementation as early as day 7. These results suggest that the convex surfaces on microspheres and inter-particulate porosity may have led to variable cell morphology and fate within the 3D culture. This study provides deeper insights into geometrical regulation of mesenchymal stem cell responses applicable for bone tissue engineering

Direct three-dimensional printing of polymeric scaffolds with nanofibrous topography

Three-dimensional (3D) printing is a powerful manufacturing tool for making 3D structures with well-defined architectures for a wide range of applications. The field of tissue engineering has also adopted this technology to fabricate scaffolds for tissue regeneration. The ability to control architecture of scaffolds, e.g. matching anatomical shapes and having defined pore size, has since been improved significantly. However, the material surface of these scaffolds is smooth and does not resemble that found in natural extracellular matrix (ECM), in particular, the nanofibrous morphology of collagen. This natural nanoscale morphology plays a critical role in cell behaviour. Here, we have developed a new approach to directly fabricate polymeric scaffolds with an ECM-like nanofibrous topography and defined architectures using extrusion-based 3D printing. 3D printed tall scaffolds with interconnected pores were created with disparate features spanning from nanometres to centimetres. Our approach removes the need for a sacrificial mould and subsequent mould removal compared to previous methods. Moreover, the nanofibrous topography of the 3D printed scaffolds significantly enhanced protein absorption, cell adhesion and differentiation of human mesenchymal stem cells when compared to those with smooth material surfaces. These 3D printed scaffolds with both defined architectures and nanoscale ECM-mimicking morphologies have potential applications in cartilage and bone regeneration

Comprehensive Mutation Analysis in Colorectal Flat Adenomas

Background: Flat adenomas are a subgroup of colorectal adenomas that have been associated with a distinct biology and a more aggressive clinical behavior compared to their polypoid counterparts. In the present study, we aimed to compare the mutation spectrum of 14 cancer genes, between these two phenotypes. Methods: A consecutive series of 106 flat and 93 polypoid adenomas was analyzed retrospectively for frequently occurring mutations in “hot spot” regions of KRAS, BRAF, PIK3CA and NRAS, as well as selected mutations in CTNNB1 (β-catenin), EGFR, FBXW7 (CDC4), PTEN, STK11, MAP2K4, SMAD4, PIK3R1 and PDGFRA using a high-throughput genotyping technique. Additionally, APC was analyzed using direct sequencing. Results: APC mutations were more frequent in polypoid adenomas compared to flat adenomas (48.5% versus 30.3%, respectively, p = 0.02). Mutations in KRAS, BRAF, NRAS, FBXW7 and CTNNB1 showed similar frequencies in both phenotypes. Between the different subtypes of flat adenomas (0-IIa, LST-F and LST-G) no differences were observed for any of the investigated genes. Conclusion: The lower APC mutation rate in flat adenomas compared to polypoid adenomas suggests that disruption of the Wnt-pathway may occur via different mechanisms in these two phenotypes. Furthermore, in contrast to previous observations our results in this large well-defined sample set indicate that there is no significant association between the different morphological phenotypes and mutations in key genes of the RAS-RAF-MAPK pathway

Appropriate referral and selection of patients with chronic pain for spinal cord stimulation: European consensus recommendations and e-health tool

Background: Spinal cord stimulation (SCS) is an established treatment for chronic neuropathic, neuropathic-like and ischaemic pain. However, the heterogeneity of patients in daily clinical practice makes it often challenging to determine which patients are eligible for this treatment, resulting in undesirable practice variations. This study aimed to establish patient-specific recommendations for referral and selection of SCS in chronic pain. Methods: A multidisciplinary European panel used the RAND/UCLA Appropriateness Method (RUAM) to assess the appropriateness of (referral for) SCS for 386 clinical scenarios in four pain areas: chronic low back pain and/or leg pain, complex regional pain syndrome, neuropathic pain syndromes and ischaemic pain syndromes. In addition, the panel identified a set of psychosocial factors that are relevant to the decision for SCS treatment. Results: Appropriateness of SCS was strongly determined by the neuropathic or neuropathic-like pain component, location and spread of pain, anatomic abnormalities and previous response to therapies targeting pain processing (e.g. nerve block). Psychosocial factors considered relevant for SCS selection were as follows: lack of engagement, dysfunctional coping, unrealistic expectations, inadequate daily activity level, problematic social support, secondary gain, psychological distress and unwillingness to reduce high-dose opioids. An educational e-health tool was developed that combines clinical and psychosocial factors into an advice on referral/selection for SCS. Conclusions: The RUAM was useful to establish a consensus on patient-specific criteria for referral/selection for SCS in chronic pain. The e-health tool may help physicians learn to apply an integrated approach of clinical and psychosocial factors. Significance: Determining the eligibility of SCS in patients with chronic pain requires careful consideration of a variety of clinical and psychosocial factors. Using a systematic approach to combine evidence from clinical studies and expert opinion, a multidisciplinary European expert panel developed detailed recommendations to support appropriate referral and selection for SCS in chronic pain. These recommendations are available as an educational e-health tool (https://www.scstool.org/)

Culturing Aerobic and Anaerobic Bacteria and Mammalian Cells with a Microfluidic Differential Oxygenator

In this manuscript, we report on the culture of anaerobic and aerobic species within a disposable multilayer polydimethylsiloxane (PDMS) microfluidic device with an integrated differential oxygenator. A gas-filled microchannel network functioning as an oxygen−nitrogen mixer generates differential oxygen concentration. By controlling the relative flow rate of the oxygen and nitrogen input gases, the dissolved oxygen (DO) concentration in proximal microchannels filled with culture media are precisely regulated by molecular diffusion. Sensors consisting of an oxygen-sensitive dye embedded in the fluid channels permit dynamic fluorescence-based monitoring of the DO concentration using low-cost light-emitting diodes. To demonstrate the general utility of the platform for both aerobic and anaerobic culture, three bacteria with differential oxygen requirements (E. coli, A. viscosus, and F. nucleatum), as well as a model mammalian cell line (murine embryonic fibroblast cells (3T3)), were cultured. Growth characteristics of the selected species were analyzed as a function of eight discrete DO concentrations, ranging from 0 ppm (anaerobic) to 42 ppm (fully saturated)

Expression of a Dominant Negative CELF Protein In Vivo Leads to Altered Muscle Organization, Fiber Size, and Subtype

CUG-BP and ETR-3-like factor (CELF) proteins regulate tissue- and developmental stage-specific alternative splicing in striated muscle. We previously demonstrated that heart muscle-specific expression of a nuclear dominant negative CELF protein in transgenic mice (MHC-CELFΔ) effectively disrupts endogenous CELF activity in the heart in vivo, resulting in impaired cardiac function. In this study, transgenic mice that express the dominant negative protein under a skeletal muscle-specific promoter (Myo-CELFΔ) were generated to investigate the role of CELF-mediated alternative splicing programs in normal skeletal muscle.Myo-CELFΔ mice exhibit modest changes in CELF-mediated alternative splicing in skeletal muscle, accompanied by a reduction of endomysial and perimysial spaces, an increase in fiber size variability, and an increase in slow twitch muscle fibers. Weight gain and mean body weight, total number of muscle fibers, and overall muscle strength were not affected.Although these findings demonstrate that CELF activity contributes to the normal alternative splicing of a subset of muscle transcripts in vivo, the mildness of the effects in Myo-CELFΔ muscles compared to those in MHC-CELFΔ hearts suggests CELF activity may be less determinative for alternative splicing in skeletal muscle than in heart muscle. Nonetheless, even these small changes in CELF-mediated splicing regulation were sufficient to alter muscle organization and muscle fiber properties affected in myotonic dystrophy. This lends further evidence to the hypothesis that dysregulation of CELF-mediated alternative splicing programs may be responsible for the disruption of these properties during muscle pathogenesis