Assessing transfer entropy from biochemical data.

We address the problem of evaluating the transfer entropy (TE) produced by biochemical reactions from experimentally measured data. Although these reactions are generally nonlinear and nonstationary processes making it challenging to achieve accurate modeling, Gaussian approximation can facilitate the TE assessment only by estimating covariance matrices using multiple data obtained from simultaneously measured time series representing the activation levels of biomolecules such as proteins. Nevertheless, the nonstationary nature of biochemical signals makes it difficult to theoretically assess the sampling distributions of TE, which are necessary for evaluating the statistical confidence and significance of the data-driven estimates. We resolve this difficulty by computationally assessing the sampling distributions using techniques from computational statistics. The computational methods are tested by using them in analyzing data generated from a theoretically tractable time-varying signal model, which leads to the development of a method to screen only statistically significant estimates. The usefulness of the developed method is examined by applying it to real biological data experimentally measured from the ERBB-RAS-MAPK system that superintends diverse cell fate decisions. A comparison between cells containing wild-type and mutant proteins exhibits a distinct difference in the time evolution of TE while any apparent difference is hardly found in average profiles of the raw signals. Such a comparison may help in unveiling important pathways of biochemical reactions.

p52Shc regulates the sustainability of ERK activation in a RAF-independent manner.

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant but that SHC plays the critical role in the temporal regulation of ERK activation.

Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation.

RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS.

Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral.

RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras-Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS-Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP-Ras/RalGDS clusters, to induce the full activation of Ral.

Tegotae-based decentralised control scheme for autonomous gait transition of snake-like robots.

Snakes change their locomotion patterns in response to the environment. This ability is a motivation for developing snake-like robots with highly adaptive functionality. In this study, a decentralised control scheme of snake-like robots that exhibited autonomous gait transition (i.e. the transition between concertina locomotion in narrow aisles and scaffold-based locomotion on unstructured terrains) was developed. Additionally, the control scheme was validated via simulations. A key insight revealed is that these locomotion patterns were not preprogrammed but emerged by exploiting Tegotae, a concept that describes the extent to which a perceived reaction matches a generated action. Unlike local reflexive mechanisms proposed previously, the Tegotae-based feedback mechanism enabled the robot to 'selectively' exploit environments beneficial for propulsion, and generated reasonable locomotion patterns. It is expected that the results of this study can form the basis to design robots that can work under unpredictable and unstructured environments.

Training with an Electric Exercise Bike versus a Conventional Exercise Bike during Hemodialysis for Patients with End-stage Renal Disease: A Randomized Clinical Trial.

OBJECTIVE: Hemodialysis (HD) patients have lower fitness levels than healthy subjects because of various structural, metabolic, and functional abnormalities secondary to uremic changes in skeletal muscles. Aerobic and resistance exercises are beneficial in improving not only physical function, including maximal oxygen uptake and muscle strength, but also anthropometrics, nutritional status, and hematologic indices. The use of electric ergometers that place light loads on patients has been implemented at many dialysis facilities in Japan. However, reports comparing the effects on body function of electric and variable-load ergometers are few. This study aimed to compare electric ergometers and variable-load ergometers in terms of exercise outcomes in HD patients. METHODS: A total of 15 ambulatory HD patients were randomly divided into two groups: the variable-load ergometer group (n=8) and the electric ergometer group (n=7). HD patients exercised at a level based on their physical function three times a week for 12 weeks. RESULTS: After the 12-week intervention period, only the variable-load ergometer group experienced significant increases in lower extremity muscle strength and exercise tolerance. CONCLUSION: This study confirmed that conventional aerobic training and electric bike exercise during HD were efficacious and safe without causing sudden hypotension or any other side effects. However, exercise using a variable-load ergometer may be more effective than exercise using an electric bike in improving the physical function of HD patients. Exercise using a variable-load ergometer elicited specific whole-body and local effects.