Published papers

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 39 portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

The candidate phyla radiation (CPR) is a large bacterial group consisting mainly of uncultured lineages. They have small cells and small genomes, and they often lack ribosomal proteins uL1, bL9, and/or uL30, which are basically ubiquitous in non-CPR bacteria. Here, we comprehensively analyzed the genomic information on CPR bacteria and identified their unique properties. The distribution of protein lengths in CPR bacteria peaks at around 100–150 amino acids, whereas the position of the peak varies in the range of 100–300 amino acids in free-living non-CPR bacteria, and at around 100–200 amino acids in most symbiotic non-CPR bacteria. These results show that the proteins of CPR bacteria are smaller, on average, than those of free-living non-CPR bacteria, like those of symbiotic non-CPR bacteria. We found that ribosomal proteins bL28, uL29, bL32, and bL33 have been lost in CPR bacteria in a taxonomic lineage-specific manner. Moreover, the sequences of approximately half of all ribosomal proteins of CPR differ, in part, from those of non-CPR bacteria, with missing regions or specifically added regions. We also found that several regions in the 16S, 23S, and 5S rRNAs of CPR bacteria are lacking, which presumably caused the total predicted lengths of the three rRNAs of CPR bacteria to be smaller than those of non-CPR bacteria. The regions missing in the CPR ribosomal proteins and rRNAs are located near the surface of the ribosome, and some are close to one another. These observations suggest that ribosomes are smaller in CPR bacteria than those in free-living non-CPR bacteria, with simplified surface structures.

Background: Tardigrades are microscopic animals that are capable of tolerating extreme environments by entering a desiccated state of suspended animation known as anhydrobiosis. While antioxidative stress proteins, antiapoptotic pathways and tardigrade-specific intrinsically disordered proteins have been implicated in the anhydrobiotic machinery, conservation of these mechanisms is not universal within the phylum Tardigrada, suggesting the existence of overlooked components. Results: Here, we show that a novel Mn-dependent peroxidase is an important factor in tardigrade anhydrobiosis. Through time-series transcriptome analysis of Ramazzottius varieornatus specimens exposed to ultraviolet light and comparison with anhydrobiosis entry, we first identified several novel gene families without similarity to existing sequences that are induced rapidly after stress exposure. Among these, a single gene family with multiple orthologs that is highly conserved within the phylum Tardigrada and enhances oxidative stress tolerance when expressed in human cells was identified. Crystallographic study of this protein suggested Zn or Mn binding at the active site, and we further confirmed that this protein has Mn-dependent peroxidase activity in vitro. Conclusions: Our results demonstrated novel mechanisms for coping with oxidative stress that may be a fundamental mechanism of anhydrobiosis in tardigrades. Furthermore, localization of these sets of proteins mainly in the Golgi apparatus suggests an indispensable role of the Golgi stress response in desiccation tolerance.

Many previous studies have reported the various proteins specifically secreted as inducers in the dorsal or ventral regions in vertebrate gastrula. However, little is known about the effect on cell fate of small molecules below 1000 Da. We therefore tried to identify small molecules specifically expressed in the dorsal marginal zone (DMZ) or ventral marginal zone (VMZ) in vertebrate gastrula. Small intracellular and secreted molecules were detected using explants and supernatant samples. Hydrophilic metabolites were analyzed by capillary ion chromatography–mass spectrometry and liquid chromatography–mass spectrometry, and lipids were analyzed by supercritical fluid chromatography–tandem mass spectrometry. In total, 190 hydrophilic metabolites and 396 lipids were identified. The DMZ was found to have high amounts of glycolysis-and glutathione metabolismrelated metabolites in explants, and the VMZ was richer in purine metabolism-related metabolites. We also discovered some hydrophilic metabolites and lipids differentially contained in the DMZ or VMZ. Our research would contribute to a deeper understanding of the cellular physiology that regulates early embryogenesis.

Spider silks are among the toughest known materials and thus provide models for renewable, biodegradable, and sustainable biopolymers. However, the entirety of their diversity still remains elusive, and silks that exceed the performance limits of industrial fibers are constantly being found. We obtained transcriptome assemblies from 1098 species of spiders to comprehensively catalog silk gene sequences and measured the mechanical, thermal, structural, and hydration properties of the dragline silks of 446 species. The combination of these silk protein genotype-phenotype data revealed essential contributions of multicomponent structures with major ampullate spidroin 1 to 3 paralogs in high-performance dragline silks and numerous amino acid motifs contributing to each of the measured properties. We hope that our global sampling, comprehensive testing, integrated analysis, and open data will provide a solid starting point for future biomaterial designs.

Most cancer cells predominantly produce energy by glycolysis rather than oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, even in the presence of an adequate oxygen supply (Warburg effect). However, little has been reported regarding the direct measurements of global metabolites in clinical tumor tissues. Here, we applied capillary electrophoresis time-of-flight mass spectrometry, which enables comprehensive and quantitative analysis of charged metabolites, to simultaneously measure their levels in tumor and grossly normal tissues obtained from 16 colon and 12 stomach cancer patients. Quantification of 94 metabolites in colon and 95 metabolites in stomach involved in glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acid and nucleotide metabolisms resulted in the identification of several cancer-specific metabolic traits. Extremely low glucose and high lactate and glycolytic intermediate concentrations were found in both colon and stomach tumor tissues, which indicated enhanced glycolysis and thus confirmed the Warburg effect. Significant accumulation of all amino acids except glutamine in the tumors implied autophagic degradation of proteins and active glutamine breakdown for energy production, i.e., glutaminolysis. In addition, significant organ-specific differences were found in the levels of TCA cycle intermediates, which reflected the dependency of each tissue on aerobic respiration according to oxygen availability. The results uncovered unexpectedly poor nutritional conditions in the actual tumor microenvironment and showed that capillary electrophoresis coupled to mass spectrometry-based metabolomics, which is capable of quantifying the levels of energy metabolites in tissues, could be a powerful tool for the development of novel anticancer agents that target cancer-specific metabolism. ©2009 American Association for Cancer Research.

Polyrhachis lamellidens is a temporary social parasitic species. When a newly mated queen encounters a host worker, it opens its jaws and then mounts and rubs the body of the host worker, called rubbing behaviour. This behaviour is different from aggressive behaviour and is considered to be a preparatory action before invasion of the host colony. However, it is unclear what cues trigger rubbing behaviour. Therefore, in this study, we used glass beads that imitated the insect body surfaces and searched for triggers. Although P. lamellidens did not respond to the cuticular compounds only, cuticular compounds and chitin coatings on glass beads elicited responses that were similar to those towards live samples. The rubbing behaviour of P. lamellidens was elicited in response to a cuticle-like surface that mimicked a procuticle by combining the compounds with chitin. These results suggest that host recognition and nest-mate recognition are supported by different mechanisms.

The pulmonary vein contains a myocardial layer that is capable of generating spontaneous or triggered action potentials. Although the electrophysiological and pharmacological characteristics of the pulmonary vein myocardium have been compiled in various studies, a comprehensive understanding of the spontaneous action potentials generated in the myocardial layer has yet to be presented. Here we integrated the electrophysiological properties of the pulmonary vein myocardial layer using a Guinea pig ventricular cell model. Based on the preceding research, which reported that approximately half of the isolated pulmonary vein myocardial layer exhibited spontaneous action potentials and the remaining half was quiescent, we constructed various combinations of the pulmonary vein myocardial models to represent variations of the action potential tracings. As a result, we predicted that the spontaneous action potentials, including burst-like action potentials, are observed with varying currents of the relative densities of Na+ (INa), L-type Ca2+ (ICaL), inwardly rectifying K+ (IK1), rapid component of delayed rectifying K+ (IKr), and ACh-activated K+ (IKACh).

Here, we integrated various characteristics of fetal ventricular cells based on a mathematical model and predicted the contribution of each characteristic to maintenance of intracellular ATP concentration and sarcomere contraction under anoxic conditions. Our simulation results showed that higher glycogen content, higher hexokinase activity, and lower creatine concentration helped to prolong the time that contraction of ventricular cells was maintained under anoxic conditions. The integrated model enabled us to quantitatively assess the contributions of factors related to energy metabolism in ventricular cells.

By the combinations of high-throughput analytical technologies in the fields of transcriptomics, proteomics, and metabolomics, we are now able to gain comprehensive and quantitative snapshots of the intracellular processes. Dynamic intracellular activities and their regulations can be elucidated by systematic observation of these multi-omics data. On the other hand, careful statistical analysis is necessary for such integration, since each of the omics layers as well as the specific analytical methodologies harbor different levels of noise and variations. Moreover, interpretation of such multitude of data requires an intuitive pathway context. Here we describe such statistical methods for the integration and comparison of multiomics data, as well as the computational methods for pathway reconstruction, ID conversion, mapping, and visualization that play key roles for the efficient study of multi-omics information. © Springer Science+Business Media, LLC 2013.

In cancer, apoptosis or programmed cell death has been demonstrated through the tumor necrosis factor related apoptosis-inducing ligand (TRAIL) signal transduction. As a result, TRAIL-based therapies have been widely investigated to fight cancers. However, several malignant cancer types still remain resistant to TRAIL. Recently, we developed a dynamic computational model to investigate the resistance mechanisms in TRAIL-stimulated human fibrosarcoma (HT1080) cells. The macroscopic average-cell response model, based on the law of mass action and signaling flux conservation, successfully simulates the semi-quantitative temporal profiles of cell survival (I?B, JNK, p38) and apoptotic (caspase-8 and -3) molecules in wildtype and several mutants (FADD, RIP1 and TRAF2 knockdowns or KD). However, cancer populations are known to be highly heterogeneous, and various studies have demonstrated the importance of stochasticity and variability for phenotypic diversity between identical cells. Here, we extend our original model to investigate the effect of such fluctuations on TRAIL signaling response by adopting probabilities of signaling reactions through the Gillespie algorithm. Notably, when we stimulated the model 1000 times to indicate the variability of 1000 single cell responses in all 4 experimental conditions with different levels of stochasticity, we notice that TRAF2 KD produced the most variable signaling response. This variance subsequently affected the level of cellular apoptosis analysed through the cell-survival metric (CSM). Our work highlights the necessity to understand variable responses of cell signaling reactions to different levels of stochasticity. Thus, prior to the actual development of potential drug targets for killing cancer cells, the effect of stochastic variance could be investigated through dynamic models. © 2012 IEEE.

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Large numbers of miRNAs have been identified in a wide range of species, and most of them are known to downregulate translation of messenger RNAs (mRNAs) via imperfect binding of the miRNA to a specific site or sites in the 3′ untranslated region (UTR) of the mRNA. Identification of genes targeted by miRNAs is widely believed to be an important step toward understanding the role of miRNAs in gene regulatory networks. As part of the effort to understand interactions between miRNAs and their targets, computational algorithms have been developed based on observed rules for features such as the degree of hybridization between the two RNA molecules. These in silico approaches provide important tools for miRNA target detection, and together with experimental validation, help to reveal regulated targets of miRNAs. Here, we summarize the knowledge that has been accumulated about the principles of target recognition by miRNAs and the currently available computational methodologies for prediction of miRNA target genes. © 2007 Elsevier Inc. All rights reserved.

Brain-inspired Computing Architecture (Bri CA) is a generic software platform for modular composition of machine learning algorithms. It can combine and schedule an arbitrary number of machine learning components in a brain-inspired fashion to construct higher level structures such as cognitive architectures. We would like to report and discuss the core concepts of Bri CA version 1 and prospects toward future development.

During rodent ventricular cell development, the sarcoplasmic reticulum (SR) is scarce and poorly organized. A Ca2+ transient in embryonic ventricular cells depends mostly on Ca2+ influx through L-type Ca2+ channels. In rat ventricular cells, transverse tubules (t-tubules) begin to form postnatally. As such, Ca2+-induced Ca2+ release (CICR), in which ryanodine receptor channels on the SR are activated via Ca2+ influx through L-type Ca2+ channels on the t-tubules, is not established in embryonic ventricular cells. Here, we modeled developmental changes in Guinea pig ventricular cells and identified the factors that enhance contraction of the cells with an increase in CICR.

During development, ventricular cells utilize different isoforms of both myosin heavy chain (MHC) and troponin I. The differences in these isoforms affect Ca2+ sensitivity, ATPase activity, and velocity of contraction. In order to consider the differences in isoforms, we integrated a new contraction model with the Kyoto model. Briefly, the new model considered tropomyosin, which inhibits formation of a cross-bridge between actin and myosin filaments. We varied the level of Ca2+ sensitivity in order to obtain similar traces for contractile force between the original Kyoto model and the modified model. We also modified the new contraction model to consider ATP consumption by myosin-ATPase in order to simulate the changes in ATPase activity caused by the difference in MHC isoforms. The modified model enabled us to compare the contribution of developmental changes in ATP consumption via contraction to excitation-contraction coupling, which is regulated differently in fetal and adult Guinea pigs.

In order to evaluate developmental changes in embryonic ventricular cells at early embryonic (EE) and late embryonic (LE) stage, we aimed to predict potentially unstable action potentials (APs) that could be lethal to developing ventricular cells. Two models of the Kyoto and the Luo-Rudy model were used for simulation of 512 representative combinations by switching the relative activities of 9 ionic components whose activities vary between the EE and LE stages. Out of these 512 combinations in Kyoto model, 144 combinations were predicted potentially unstable resulting from combinations of funny current (If), inward rectifier current (IK1), sustained inward current (Ist), L-type Ca2+ current (ICaL), and Na+ current (INa). Other 208 and 160 combinations were predicted quiescent membrane potentials and regular spontaneous APs. Based on these results, we suggest that sequential switches of the relative activities of INa, If, and IK1 enable cells to avoid unstable patterns. © 2012 CCAL.

The Na+-Ca2+ exchange assumes considerable importance in neonatal cells with a morphologically sparse sarcoplasmic reticulum. Our simulation shows that the neonatal ventricular cell consumed larger amount of ATP than the adult ventricular cell, owing to a long relaxation time. The amount of ATP consumed per beat was reduced to the amount equivalent to the adult ventricular cells, thus increasing the relative current density of Na +-Ca2+ exchange by 4-fold, which is consistent with the observed current density in the neonatal rabbit ventricular cells. © 2012 CCAL.

As the workhorse of early studies on metabolism, the metabolic pathways of E. coli are arguably the best characterized. The richness of information available about its pathways is broader than for any other model. However, in spite of decades of descriptive work, only recently can a significant number of E. coli metabolic network constituents be analyzed simultaneously. The advent of metabolomic methods that allow to capture qualitative as well as quantitative information about the intracellular and extracellular metabolite profiles is starting to shed light on the remaining complexity of this simpler model. Here we describe important findings about the physiology of E. coli resulting from emerging metabolomic studies. While a vast number of intracellular metabolites in E. coli still remain to be characterized, the information obtained from those studies can provide an unprecedented amount of information about metabolic pathways including their functional elucidation, enzyme activity, metabolic fluxes, network robustness, or even the discovery of completely novel reactions or pathways. These results are also being used to populate rich databases and to develop computational models of E. coli metabolism that have already proven effective to predict cellular states and will shed light on complex and until now still elusive regulatory principles. © 2007 Springer-Verlag Berlin Heidelberg.

The present paper expanded an integrated mathematical model, which successfully simulated developmental changes in electrophysiological activities of rodent ventricular cells, to contrive models that reflect one of the hypothesis on the role of Na+-Ca"+ exchange in neonatal ventricular cell: the Na+-Ca+ exchange assumes a relatively greater role in neonatal cells with a morphologically sparse SR. Our simulation with the expanded model showed that neonatal ventricular cell consumed larger amount of adenosine triphosphate (ATP) than adult ventricular cell did, owing to long relaxation time. The amount of ATP consumed per beat was reduced to the amount equivalent to adult ventricular cell, as increasing the relative current density of Na+-Ca2+ exchange (F NCx) by 4-fold, which is consistent with the observed current density in neonatal rabbit ventricular cell. To further validate the hypothesis, the relative functional activity of SR (FSR) was incrementally increased from neonatal to adult levels and subsequently increased FNCX, and found that increasing FNcx had negative impact on sarcomere relaxation velocity in ventricular cell model with large FSR. All the results thus theoretically supported the hypothesis that the Na+-Ca2+ exchange assumes a relatively greater role in neonatal cells with a morphologically sparse SR.

Complex living systems have shown remarkably well-orchestrated, self-organized, robust, and stable behavior under a wide range of perturbations. However, despite the recent generation of high-throughput experimental datasets, basic cellular processes such as division, differentiation, and apoptosis still remain elusive. One of the key reasons is the lack of understanding of the governing principles of complex living systems. Here, we have reviewed the success of perturbation-response approaches, where without the requirement of detailed in vivo physiological parameters, the analysis of temporal concentration or activation response unravels biological network features such as causal relationships of reactant species, regulatory motifs, etc. Our review shows that simple linear rules govern the response behavior of biological networks in an ensemble of cells. It is daunting to know why such simplicity could hold in a complex heterogeneous environment. Provided physical reasons can be explained for these phenomena, major advancement in the understanding of basic cellular processes could be achieved. © 2009 Imperial College Press.