Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library

Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation

Acetylcholine (ACh) has been shown to modulate neuronal differentiation during early development. Both muscarinic and nicotinic acetylcholine receptors (AChRs) regulate a wide variety of physiological responses, including apoptosis, cellular proliferation and neuronal differentiation. However, the intracellular mechanisms underlying these effects of AChR signaling are not fully understood. It is known that activation of AChRs increase cellular proliferation and neurogenesis and that regulation of intracellular calcium through AChRs may underlie the many functions of ACh. Intriguingly, activation of diverse signaling molecules such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src is modulated by AChRs. Here we discuss the roles of ACh in neuronal differentiation, cell proliferation and apoptosis. We also discuss the pathways involved in these processes, as well as the effects of novel endogenous AChRs agonists and strategies to enhance neuronal-differentiation of stem and neural progenitor cells. Further understanding of the intracellular mechanisms underlying AChR signaling may provide insights for novel therapeutic strategies, as abnormal AChR activity is present in many diseases

On the design of a MEMS piezoelectric accelerometer coupled to the middle ear as an implantable sensor for hearing devices

Abstract The presence of external elements is a major limitation of current hearing aids and cochlear implants, as they lead to discomfort and inconvenience. Totally implantable hearing devices have been proposed as a solution to mitigate these constraints, which has led to challenges in designing implantable sensors. This work presents a feasibility analysis of a MEMS piezoelectric accelerometer coupled to the ossicular chain as an alternative sensor. The main requirements of the sensor include small size, low internal noise, low power consumption, and large bandwidth. Different designs of MEMS piezoelectric accelerometers were modeled using Finite Element (FE) method, as well as optimized for high net charge sensitivity. The best design, a 2 × 2 mm2 annular configuration with a 500 nm thick Aluminum Nitride (AlN) layer was selected for fabrication. The prototype was characterized, and its charge sensitivity and spectral acceleration noise were found to be with good agreement to the FE model predictions. Weak coupling between a middle ear FE model and the prototype was considered, resulting in equivalent input noise (EIN) lower than 60 dB sound pressure level between 600 Hz and 10 kHz. These results are an encouraging proof of concept for the development of MEMS piezoelectric accelerometers as implantable sensors for hearing devices

IDyOT: A Computational Theory of Creativity as Everyday Reasoning from Learned Information

We present progress towards a computational cognitive architecture, IDyOT (Information Dynamics of Thinking) that is intended to account for certain aspects of human creativity and other forms of cognitive processing in terms of a pre-conscious predictive loop. The theory is motivated in terms of the evolutionary pressure to be efficient. It makes several predictions that may be tested by building computational implementations and studying their behaviour