[Early Experience of Robot-assisted Mitral Valve Surgeries in Our Local Hospital].

We report the early experience of robot-assisted mitral valve repair in our local hospital. It took about two years from the application for the robot-assisted cardiac surgery until the first case of robot-assisted mitral repair. Since July 2020 to June 2022, we have performed 23 cases of robot-assisted mitral valve repair with da Vinci Xi system. There was no hospital death. The mean cross-clamp and total operation time were 118±22 and 295±41 min, respectively. Pre-discharge echocardiograms showed none-to-mild residual mitral regurgitation (MR) in all patients. The mean post-operative hospital stay was 7.6±5 days. Robot-assisted mitral valve repair could safely be started in our hospital. Early results were acceptable. Further experiences will be needed to confirm the efficacy of robotic mitral valve repair.

StemPanTox: A fast and wide-target drug assessment system for tailor-made safety evaluations using personalized iPS cells.

An alternative model that reliably predicts human-specific toxicity is necessary because the translatability of effects on animal models for human disease is limited to context. Previously, we developed a method that accurately predicts developmental toxicity based on the gene networks of undifferentiated human embryonic stem (ES) cells. Here, we advanced this method to predict adult toxicities of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, two types of nephrotoxins, and non-genotoxic carcinogens) and achieved high predictability (AUC = 0.90-1.00) in all categories. Moreover, we screened for an induced pluripotent stem (iPS) cell line to predict the toxicities based on the gene networks of iPS cells using transfer learning of the gene networks of ES cells, and predicted toxicities in four categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) with high performance (AUC = 0.82-0.99). This method holds promise for tailor-made safety evaluations using personalized iPS cells.

Trans-omic Analysis Reveals ROS-Dependent Pentose Phosphate Pathway Activation after High-Frequency Electrical Stimulation in C2C12 Myotubes.

Skeletal muscle adaptation is mediated by cooperative regulation of metabolism, signal transduction, and gene expression. However, the global regulatory mechanism remains unclear. To address this issue, we performed electrical pulse stimulation (EPS) in differentiated C2C12 myotubes at low and high frequency, carried out metabolome and transcriptome analyses, and investigated phosphorylation status of signaling molecules. EPS triggered extensive and specific changes in metabolites, signaling phosphorylation, and gene expression during and after EPS in a frequency-dependent manner. We constructed trans-omic network by integrating these data and found selective activation of the pentose phosphate pathway including metabolites, upstream signaling molecules, and gene expression of metabolic enzymes after high-frequency EPS. We experimentally validated that activation of these molecules after high-frequency EPS was dependent on reactive oxygen species (ROS). Thus, the trans-omic analysis revealed ROS-dependent activation in signal transduction, metabolome, and transcriptome after high-frequency EPS in C2C12 myotubes, shedding light on possible mechanisms of muscle adaptation.

Single-Cell Information Analysis Reveals That Skeletal Muscles Incorporate Cell-to-Cell Variability as Information Not Noise.

Cell-to-cell variability in signal transduction in biological systems is often considered noise. However, intercellular variation (i.e., cell-to-cell variability) has the potential to enable individual cells to encode different information. Here, we show that intercellular variation increases information transmission of skeletal muscle. We analyze the responses of multiple cultured myotubes or isolated skeletal muscle fibers as a multiple-cell channel composed of single-cell channels. We find that the multiple-cell channel, which incorporates intercellular variation as information, not noise, transmitted more information in the presence of intercellular variation than in the absence according to the "response diversity effect," increasing in the gradualness of dose response by summing the cell-to-cell variable dose responses. We quantify the information transmission of human facial muscle contraction during intraoperative neurophysiological monitoring and find that information transmission of muscle contraction is comparable to that of a multiple-cell channel. Thus, our data indicate that intercellular variation can increase the information capacity of tissues.

Automatic Quantitative Segmentation of Myotubes Reveals Single-cell Dynamics of S6 Kinase Activation.

Automatic cell segmentation is a powerful method for quantifying signaling dynamics at single-cell resolution in live cell fluorescence imaging. Segmentation methods for mononuclear and round shape cells have been developed extensively. However, a segmentation method for elongated polynuclear cells, such as differentiated C2C12 myotubes, has yet to be developed. In addition, myotubes are surrounded by undifferentiated reserve cells, making it difficult to identify background regions and subsequent quantification. Here we developed an automatic quantitative segmentation method for myotubes using watershed segmentation of summed binary images and a two-component Gaussian mixture model. We used time-lapse fluorescence images of differentiated C2C12 cells stably expressing Eevee-S6K, a fluorescence resonance energy transfer (FRET) biosensor of S6 kinase (S6K). Summation of binary images enhanced the contrast between myotubes and reserve cells, permitting detection of a myotube and a myotube center. Using a myotube center instead of a nucleus, individual myotubes could be detected automatically by watershed segmentation. In addition, a background correction using the two-component Gaussian mixture model permitted automatic signal intensity quantification in individual myotubes. Thus, we provide an automatic quantitative segmentation method by combining automatic myotube detection and background correction. Furthermore, this method allowed us to quantify S6K activity in individual myotubes, demonstrating that some of the temporal properties of S6K activity such as peak time and half-life of adaptation show different dose-dependent changes of insulin between cell population and individuals.Key words: time lapse images, cell segmentation, fluorescence resonance energy transfer, C2C12, myotube.

Laguerre Filter Analysis with Partial Least Square Regression Reveals a Priming Effect of ERK and CREB on c-FOS Induction.

Signaling networks are made up of limited numbers of molecules and yet can code information that controls different cellular states through temporal patterns and a combination of signaling molecules. In this study, we used a data-driven modeling approach, the Laguerre filter with partial least square regression, to describe how temporal and combinatorial patterns of signaling molecules are decoded by their downstream targets. The Laguerre filter is a time series model used to represent a nonlinear system based on Volterra series expansion. Furthermore, with this approach, each component of the Volterra series expansion is expanded by Laguerre basis functions. We combined two approaches, application of a Laguerre filter and partial least squares (PLS) regression, and applied the combined approach to analysis of a signal transduction network. We applied the Laguerre filter with PLS regression to identify input and output (IO) relationships between MAP kinases and the products of immediate early genes (IEGs). We found that Laguerre filter with PLS regression performs better than Laguerre filter with ordinary regression for the reproduction of a time series of IEGs. Analysis of the nonlinear characteristics extracted using the Laguerre filter revealed a priming effect of ERK and CREB on c-FOS induction. Specifically, we found that the effects of a first pulse of ERK enhance the subsequent effects on c-FOS induction of treatment with a second pulse of ERK, a finding consistent with prior molecular biological knowledge. The variable importance of projections and output loadings in PLS regression predicted the upstream dependency of each IEG. Thus, a Laguerre filter with partial least square regression approach appears to be a powerful method to find the processing mechanism of temporal patterns and combination of signaling molecules by their downstream gene expression.

Robustness of MEK-ERK Dynamics and Origins of Cell-to-Cell Variability in MAPK Signaling.

Cellular signaling processes can exhibit pronounced cell-to-cell variability in genetically identical cells. This affects how individual cells respond differentially to the same environmental stimulus. However, the origins of cell-to-cell variability in cellular signaling systems remain poorly understood. Here, we measure the dynamics of phosphorylated MEK and ERK across cell populations and quantify the levels of population heterogeneity over time using high-throughput image cytometry. We use a statistical modeling framework to show that extrinsic noise, particularly that from upstream MEK, is the dominant factor causing cell-to-cell variability in ERK phosphorylation, rather than stochasticity in the phosphorylation/dephosphorylation of ERK. We furthermore show that without extrinsic noise in the core module, variable (including noisy) signals would be faithfully reproduced downstream, but the within-module extrinsic variability distorts these signals and leads to a drastic reduction in the mutual information between incoming signal and ERK activity.

Restriction of protein synthesis abolishes senescence features at cellular and organismal levels.

Cellular senescence or its equivalence is induced by treatment of cells with an appropriate inducer of senescence in various cell types. Mild restriction of cytoplasmic protein synthesis prevented induction of all aspects of cellular senescence in normal and tumor-derived human cells. It allowed the cells to continuously grow with no sign of senescent features in the presence of various inducers. It also delayed replicative senescence in normal human fibroblasts. Moreover, it allowed for growth of the cells that had entered a senescent state. When adult worms of the nematode C. elegans were grown under protein-restricted conditions, their average and maximal lifespans were significantly extended. These results suggest that accumulation of cytoplasmic proteins due to imbalance in macromolecule synthesis is a fundamental cause of cellular senescence.

Transcriptional profile of tomato roots exhibiting Bacillus thuringiensis-induced resistance to Ralstonia solanacearum.

KEY MESSAGE: Activation of SA-dependent signaling pathway and suppression of JA-dependent signaling pathway seem to play key roles inB. thuringiensis-induced resistance toR. solanacearumin tomato plants. Bacillus thuringiensis, a well-known and effective bio-insecticide, has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. Treatment of tomato roots with a filter-sterilized cell-free filtrate (CF) of B. thuringiensis systemically suppresses bacterial wilt caused by Ralstonia solanacearum through systemic activation of the plant defense system. Comparative analysis of the expression of the Pathogenesis-Related 1(P6) gene, a marker for induced resistance to pathogens, in various tissues of tomato plants treated with CF on their roots suggested that the B. thuringiensis-induced defense system was activated in the leaf, stem, and main root tissues, but not in the lateral root tissue. At the same time, the growth of R. solanacearum was significantly suppressed in the CF-treated main roots but not in the CF-treated lateral roots. This distinct activation of the defense reaction and suppression of R. solanacearum were reflected by the differences in the transcriptional profiles of the main and lateral tissues in response to the CF. In CF-treated main roots, but not CF-treated lateral roots, the expression of several salicylic acid (SA)-responsive defense-related genes was specifically induced, whereas jasmonic acid (JA)-related gene expression was either down-regulated or not induced in response to the CF. On the other hand, genes encoding ethylene (ET)-related proteins were induced equally in both the main and lateral root tissues. Taken together, the co-activation of SA-dependent signaling pathway with ET-dependent signaling pathway and suppression of JA-dependent signaling pathway may play key roles in B. thuringiensis-induced resistance to R. solanacearum in tomato.

Substrate-induced conformational changes of the truncated catalytic domain of Geobacillus stearothermophilus lysyl-tRNA synthetase as examined by fluorescence.

The substrate-induced conformational change of the truncated C-terminal catalytic domain (CAT) of Geobacillus stearothermophilus lysyl-tRNA synthetase was examined by measuring tryptophan fluorescence of the truncated CAT domain in the presence or absence of the truncated N-terminal tRNA anticodon-binding domain (TAB). The fluorescence spectrum of CAT was not changed by the addition of l-lysine or ATP, whereas the intensity increased by adding a lysyl-adenylate analogue, suggesting that the CAT fluorescence increases when lysyl-adenylate is formed in the active site of CAT in l-lysine activation. In the presence of TAB, the addition of l-lysine to CAT decreased the fluorescence, and the subsequent addition of ATP recovered partially the decreased intensity, as is similar to the case of the intact enzyme. The static parameters of the CAT-TAB complex were similar to those of the intact enzyme, suggesting that a somewhat impaired structure of CAT is repaired on the formation of the complex with TAB. The mutational analysis of the fluorescence showed that Trp314 but not Trp332 is responsible for the observed fluorescence changes. The role of the TAB domain in the intact enzyme is considered to enhance the binding efficiency of lysyl-adenylate to the CAT domain.

Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability.

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures.