The Origin Recognition Complex Interacts with a Subset of Metabolic Genes Tightly Linked to Origins of Replication

The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP–on–chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1–resistant ORC–interacting chromosomal sites (ORF–ORC sites) that did not function as replication origins or silencers. Instead, ORF–ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC–silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF–ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism

Contribution of voltage-dependent ion channels to subthreshold resonance

Subthreshold resonance has been observed in many excitatory/'inhibitory neurons in the brain and it is suggested that such resonance phenomena play an important role in behavioral or perceptual functions in animals. Various voltage-dependent channels are thought to be involved in the generation of these resonance oscillations. For a compartmental neuron model with Ca2+-dependent K+ channel and low-threshold Ca2+ channel, conductance-based channel dynamics are linearized around equilibrium states and a neuron model can be treated as an equivalent RLC electric circuit, which indicates that the subthreshold resonance may be attributable to inductive properties of voltage-dependent channels. By computer simulation, we examine how parameters of these voltage-dependent channels, such as an equilibrium potential and the amplitude, effect to generate a subthreshold resonance

A Novel In Vitro Simulator to Investigate Promotion of Reconstruction of Damaged Neuronal Cell Colony Differentiated from iPS Cells with the Aid of Micro Dynamic Stimulation

Neuronal cells are equipped with the function of a sensor that senses stimulation and elongates neurites to connect nearby neuronal cells in forming a neuronal network, as they are generally said to be hard to recover from physical damage, such as in the case of a spinal cord injury. Therefore, in this study, a novel in vitro simulator in which micro dynamic stimulations are applied to a damaged neuronal cell colony artificially is proposed to investigate the possibility of promoting the reconstruction of damaged neuronal cells on a colony basis. A neuronal cell colony differentiated from iPS cells is physically damaged by cutting off treatment, and micro dynamic stimulations are applied to the colony by utilizing a developed mini-vibration table system. NeuroFluor NeuO is used to establish a method for fluorescent staining of the living neuronal cells, and morphologies of the reconstructing neurons are analysed, revealing a relationship between the stimulation and the reconstructing process of the damaged neurons. It is found that significant differences are observed in the reconstructing efficiency between the statically cultured damaged neuronal cell colony and the dynamically stimulated one. The results suggest that applying appropriate micro dynamic stimulations is a promising approach to promote the reconstruction of a damaged neuronal cell colony

Liquid-in-liquid printing of 3D and mechanically tunable conductive hydrogels

Abstract Conductive hydrogels require tunable mechanical properties, high conductivity and complicated 3D structures for advanced functionality in (bio)applications. Here, we report a straightforward strategy to construct 3D conductive hydrogels by programable printing of aqueous inks rich in poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) inside of oil. In this liquid-in-liquid printing method, assemblies of PEDOT:PSS colloidal particles originating from the aqueous phase and polydimethylsiloxane surfactants from the other form an elastic film at the liquid-liquid interface, allowing trapping of the hydrogel precursor inks in the designed 3D nonequilibrium shapes for subsequent gelation and/or chemical cross-linking. Conductivities up to 301 S m−1 are achieved for a low PEDOT:PSS content of 9 mg mL−1 in two interpenetrating hydrogel networks. The effortless printability enables us to tune the hydrogels’ components and mechanical properties, thus facilitating the use of these conductive hydrogels as electromicrofluidic devices and to customize near-field communication (NFC) implantable biochips in the future

Modulation of the mechanical properties of ventricular extracellular matrix hydrogels with a carbodiimide crosslinker and investigation of their cellular compatibility

Hydrogels made from the cardiac extracellular matrix (ECM) as two-dimensional (2D) or 3D cell-culture substrates have beneficial biochemical effects on the differentiation of stem cells into cardiomyocytes. The mechanical properties of the substrates that match those of the host tissues have been identified as critical biophysical cues for coaxing the tissue-specific differentiation of stem cells. The objectives of the present study are (1) to fabricate hydrogels comprising pure ventricular ECM (vECM), (2) to make the gels possess mechanical properties similar to those of the decellularized ventricular tissue, and (3) to evaluate the cellular compatibility of the hydrogels. In order to achieve these aims, (1) a simplified protocol was developed to produce vECM solution easily and rapidly, (2) N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC) was chosen to crosslink the hydrogels made from the vECM solution to enhance their mechanical properties and stabilize the microstructure of the gels, (3) rat embryonic fibroblasts or cardiomyocytes were cultured on these gels to determine the cellular compatibility of the gels. In particular, the nonlinearity and viscoelasticity of the gels were characterized quantitatively using a newly proposed nonlinear Kelvin model. The results showed that EDAC treatment allowed modulation of the mechanical properties of the gels to the same level as those of decellularized ventricular tissue in terms of the equilibrium elasticity and relaxation coefficient. Cell culture confirmed the cellular compatibility of the gels. Furthermore, an empirical relationship between the equilibrium elastic modulus of the gels and the vECM and EDAC concentrations was derived, which is important to tailor the mechanical properties of the gels. Finally, the influence of the mechanical properties of the gels on the behavior of cultured fibroblasts and cardiomyocytes was discussed