Patterns of change in regulatory modules of chemical reaction systems induced by network modification.

Cellular functions are realized through the dynamics of chemical reaction networks formed by thousands of chemical reactions. Numerical studies have empirically demonstrated that small differences in network structures among species or tissues can cause substantial changes in dynamics. However, a general principle for behavior changes in response to network structure modifications is not known. The chemical reaction system possesses substructures called buffering structures, which are characterized by a certain topological index being zero. It was proven that the steady-state response to modulation of reaction parameters inside a buffering structure is localized in the buffering structure. In this study, we developed a method to systematically identify the loss or creation of buffering structures induced by the addition of a single degradation reaction from network structure alone. This makes it possible to predict the qualitative and macroscopic changes in regulation that will be caused by the network modification. This method was applied to two reaction systems: the central metabolic system and the mitogen-activated protein kinases signal transduction system. Our method enables identification of reactions that are important for biological functions in living systems.

RENGE infers gene regulatory networks using time-series single-cell RNA-seq data with CRISPR perturbations.

Single-cell RNA-seq analysis coupled with CRISPR-based perturbation has enabled the inference of gene regulatory networks with causal relationships. However, a snapshot of single-cell CRISPR data may not lead to an accurate inference, since a gene knockout can influence multi-layered downstream over time. Here, we developed RENGE, a computational method that infers gene regulatory networks using a time-series single-cell CRISPR dataset. RENGE models the propagation process of the effects elicited by a gene knockout on its regulatory network. It can distinguish between direct and indirect regulations, which allows for the inference of regulations by genes that are not knocked out. RENGE therefore outperforms current methods in the accuracy of inferring gene regulatory networks. When used on a dataset we derived from human-induced pluripotent stem cells, RENGE yielded a network consistent with multiple databases and literature. Accurate inference of gene regulatory networks by RENGE would enable the identification of key factors for various biological systems.

Frequent coexistence of early repolarization pattern, J-point elevation, and high Sokolow-Lyon voltage in young men.

Background: Earlier studies have shown male dominance of an early repolarization (ER) pattern and frequent coexistence with high Sokolow-Lyon voltage. Although possible involvement of androgen is speculated, the underlying mechanism has not been clarified yet. Previous studies were conducted in adult populations or only in children, and there has been no study in which the ER pattern was investigated in a series of individuals ranging from children before puberty to adults. Methods: We included 600 individuals comprising six groups according to age: 10-14 years old, 15-19 years old, twenties, thirties, forties, and fifties. Each group had 50 males and 50 females. The distribution of an ER pattern and related ECG parameters were assessed by age and gender. Results: In early teenagers, there was no significant gender difference in the prevalence of an ER pattern (24% in men vs. 28% in women, p = .82). The prevalence of an ER pattern increased after puberty and reached a peak in men in their twenties (42%). With further advance of age, the prevalence of an ER pattern decreased. On the other hand, the prevalence of an ER pattern in women peaked at 28% in teenagers, and it decreased through twenties (20%) to thirties (10%). Similar male dominance after puberty was observed in Sokolow-Lyon voltage and J-point elevation but not in P-wave amplitude. Conclusion: The prevalence of an ER pattern, Sokolow-Lyon voltage, and J-point elevation are all augmented after puberty and decrease with aging, leading to frequent coexistence of these ECG findings in young men.

Controlling complex dynamical systems based on the structure of the networks.

Progress of molecular biology resulted in the accumulation of information on biomolecular interactions, which are complex enough to be termed as networks. Dynamical behavior generated by complex network systems is considered to be the origin of the biological functions. One of the largest missions in modern life science is to obtain logical understanding for the dynamics of complex systems based on experimentally identified networks. However, a network does not provide sufficient information to specify dynamics explicitly, i.e. it lacks information of mathematical formulae of functions or parameter values. One has to develop mathematical models under assumptions of functions and parameter values to know the detail of dynamics of network systems. In this review, on the other hand, we introduce our own mathematical theory to understand the behavior of biological systems from the information of regulatory networks alone. Using the theory, important aspects of dynamical properties can be extracted from networks. Namely, key factors for observing/controlling the whole dynamical system are determined from network structure alone. We also show an application of the theory to a real biological system, a gene regulatory network for cell-fate specification in ascidian. We demonstrate that the system was completely controllable by experimental manipulations of the key factors identified by the theory from the information of network alone. This review article is an extended version of the Japanese article, Controlling Cell-Fate Specification System Based on a Mathematical Theory of Network Dynamics, published in SEIBUTSU BUTSURI Vol. 60, p. 349-351 (2020).

Position of meristems and the angles of the cell division plane regulate the uniqueness of lateral organ shape.

Leaf meristem is a cell proliferative zone present in the lateral organ primordia. In this study, we examined how cell proliferative zones in primordia of planar floral organs and polar auxin transport inhibitor (PATI)-treated leaf organs differ from those of non-treated foliage leaves of Arabidopsis thaliana, with a focus on the accumulation pattern of ANGUSTIFOLIA3 (AN3) protein, a key element for leaf meristem positioning. We found that PATI-induced leaf shape changes were correlated with cell division angle but not with meristem positioning/size or AN3 localisation. In contrast, different shapes between sepals and petals compared with foliage leaves were associated with both altered meristem position, due to altered AN3 expression patterns, and different distributions of cell division angles. A numerical simulation showed that meristem position majorly affected the final shape but biased cell division angles had a minor effect. Taken together, these results suggest that the unique shapes of different lateral organs depend on the position of the meristem in the case of floral organs and cell division angles in the case of leaf organs with different auxin flow.

Enhanced nuclear localization of phosphorylated MLKL predicts adverse events in patients with dilated cardiomyopathy.

AIMS: The role of necroptosis in dilated cardiomyopathy (DCM) remains unclear. Here, we examined whether phosphorylation of mixed lineage kinase domain-like protein (MLKL), an indispensable event for execution of necroptosis, is associated with the progression of DCM. METHODS AND RESULTS: Patients with DCM (n = 56, 56 ± 15 years of age; 68% male) were enrolled for immunohistochemical analyses of biopsies. Adverse events were defined as a composite of death or admission for heart failure or ventricular arrhythmia. Compared with the normal myocardium, increased signals of MLKL phosphorylation were detected in the nuclei, cytoplasm, and intercalated discs of cardiomyocytes in biopsy samples from DCM patients. The phosphorylated MLKL (p-MLKL) signal was increased in enlarged nuclei or nuclei with bizarre shapes in hypertrophied cardiomyocytes. Nuclear p-MLKL level was correlated negatively with septal peak myocardial velocity during early diastole (r = -0.327, P = 0.019) and was correlated positively with tricuspid regurgitation pressure gradient (r = 0.339, P = 0.023), while p-MLKL level in intercalated discs was negatively correlated with mean left ventricular wall thickness (r = -0.360, P = 0.014). During a median follow-up period of 3.5 years, 10 patients (18%) had adverse events. To examine the difference in event rates according to p-MLKL expression levels, patients were divided into two groups by using the median value of nuclear p-MLKL or intercalated disc p-MLKL. A group with high nuclear p-MLKL level (H-nucMLKL group) had a higher adverse event rate than did a group with low nuclear p-MLKL level (L-nucMLKL group) (32% vs. 4%, P = 0.012), and Kaplan-Meier survival curves showed that the adverse event-free survival rate was lower in the H-nucMLKL group than in the L-nucMLKL group (P = 0.019 by the log-rank test). Such differences were not detected between groups divided by a median value of intercalated disc p-MLKL. In δ-sarcoglycan-deficient (Sgcd-/- ) mice, a model of DCM, total p-MLKL and nuclear p-MLKL levels were higher than in wild-type mice. CONCLUSION: The results suggest that increased localization of nuclear p-MLKL in cardiomyocytes is associated with left ventricular diastolic dysfunction and future adverse events in DCM.

A structural approach to understanding enzymatic regulation of chemical reaction networks.

In living cells, chemical reactions are connected by sharing their products and substrates, and form complex systems, i.e. chemical reaction network. One of the largest missions in modern biology is to understand behaviors of such systems logically based on information of network structures. However, there are series of obstacles to study dynamical behaviors of complex network systems in biology. For example, network structure does not provide sufficient information to determine details of the dynamical behaviors. In this review, I will introduce a novel mathematical theory, structural sensitivity analysis, by which the responses of reaction systems upon the changes in enzyme activities/amounts are determined from network structure alone. The patterns of responses exhibit characteristic features, localization and hierarchy, depending on the topology of the network. The theory also shows that ranges of enzymatic regulations are governed by a mathematical law characterized by local topology of substructures. These findings imply that the network topology is one of the origins of biological robustness.

High defibrillation threshold with right-sided ICD implantation was resolved by a dual coil lead via persistent left superior vena cava.

Persistent left superior vena cava (PLSVC) can be problematic when device implantation is scheduled from the left side because of the technical difficulty in delivering leads. Right-sided implantation is an alternative method, but there is a risk of a high defibrillation threshold (DFT). Transvenous implantation of an implantable cardioverter defibrillator (ICD) was scheduled for a 54-year-old man with idiopathic dilated cardiomyopathy and monomorphic non-sustained ventricular tachycardia, but computed tomography revealed the presence of a PLSVC. Right-sided ICD implantation was performed first; however, an ICD shock at 35 J failed to terminate the induced ventricular fibrillation (VF). Re-implantation via the PLSVC by a left subclavian approach with a dual coil lead was performed next. The dual coil right ventricular lead was successfully implanted via the PLSVC, and the induced VF was terminated by a single shock at 25 J. In the present case, the proximal coil was located in the coronary sinus (CS) and it enabled an antero-posterior defibrillation vector across the left ventricle. In addition to the re-location of the ICD generator from the right side to the left side, the new positioning of the proximal coil inside the CS is likely to have contributed to the great improvement of the DFT. .

Early Detection of Cardiac Amyloidosis in Transthyretin Mutation Carriers.

Diagnostic strategies for symptomatic transthyretin (ATTR) cardiac amyloidosis showing typical morphological features such as increased ventricular wall thickness and myocardial injury such as an elevation in serum troponin T level have been established, but those for subclinical cardiac amyloidosis are limited. In the era when effective therapies to suppress/delay progression of ATTR cardiac amyloidosis are available, early detection of cardiac involvement plays a crucial role in appropriate decision-making for treatment in TTR mutation carriers who have a family history of heart failure and death due to ATTR amyloidosis. Findings of three cases with known pathogenic transthyretin (TTR) mutations (p.Ser70Arg, p.Phe53Val, and p.Val50Met) and family histories of death for amyloidosis were presented. Two cases were asymptomatic, and a case carrying p.Phe53Val had gastrointestinal symptoms and autonomic neuropathy. Levels of plasma N-terminal fragment of pro-B-type natriuretic peptide and troponin T were within normal ranges in all cases, but results of cardiac magnetic resonance (CMR) and bone scintigraphy clearly revealed the presence of cardiac involvement in all cases, even in a case without echocardiographic abnormalities including left ventricular hypertrophy and relative apical sparing of longitudinal strain shown by two-dimensional speckle-tracking echocardiography. Electrocardiography revealed modest abnormalities including reduced R wave amplitude in V2 and a trend toward left axis deviation in all cases. In conclusion, CMR, bone scintigraphy, and electrocardiography are useful for early detection of ATTR cardiac amyloidosis in TTR mutation carriers. The role of comprehensive cardiac assessment in the early detection of cardiac amyloidosis in TTR mutation carriers is discussed.

Origin of diverse phosphorylation patterns in the ERBB system.

Intercellular signals induce various cellular responses, including growth, proliferation, and differentiation, via the dynamic processes of signal transduction pathways. For cell fate decisions, ligand-binding induces the phosphorylation of ERBB receptors, which in turn activate downstream molecules. The ERBB family includes four subtypes, which diverged through two gene duplications from a common ancestor. Differences in the expression patterns of the subtypes have been reported between different organs in the human body. However, how these different expression properties influence the diverse phosphorylation levels of ERBB proteins is not well understood. Here we study the origin of the phosphorylation responses by experimental and mathematical analyses. The experimental measurements clarified that the phosphorylation levels heavily depend on the ERBB expression profiles. We developed a mathematical model consisting of the four subtypes as monomers, homodimers, and heterodimers and estimated the rate constants governing the phosphorylation responses from the experimental data. To understand the origin of the diversity, we analyzed the effects of the expression levels and reaction rates of the ERBB subtypes on the diversity. The difference in phosphorylation rates between ERBB subtypes showed a much greater contribution to the diversity than did the dimerization rates. This result implies that divergent evolution in phosphorylation reactions rather than in dimerization reactions after whole genome duplications was essential for increasing the diversity of the phosphorylation responses.

IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay.

Regnase-1 is an endoribonuclease crucial for controlling inflammation by degrading mRNAs encoding cytokines and inflammatory mediators in mammals. However, it is unclear how Regnase-1-mediated mRNA decay is controlled in interleukin (IL)-1ß- or Toll-like receptor (TLR) ligand-stimulated cells. Here, by analyzing the Regnase-1 interactome, we found that IL-1ß or TLR stimulus dynamically induced the formation of Regnase-1-ß-transducin repeat-containing protein (ßTRCP) complex. Importantly, we also uncovered a novel interaction between Regnase-1 and 14-3-3 in both mouse and human cells. In IL-1R/TLR-stimulated cells, the Regnase-1-14-3-3 interaction is mediated by IRAK1 through a previously uncharacterized C-terminal structural domain. Phosphorylation of Regnase-1 at S494 and S513 is critical for Regnase-1-14-3-3 interaction, while a different set of phosphorylation sites of Regnase-1 is known to be required for the recognition by ßTRCP and proteasome-mediated degradation. We found that Regnase-1-14-3-3 and Regnase-1-ßTRCP interactions are not sequential events. Rather, 14-3-3 protects Regnase-1 from ßTRCP-mediated degradation. On the other hand, 14-3-3 abolishes Regnase-1-mediated mRNA decay by inhibiting Regnase-1-mRNA association. In addition, nuclear-cytoplasmic shuttling of Regnase-1 is abrogated by 14-3-3 interaction. Taken together, the results suggest that a novel inflammation-induced interaction of 14-3-3 with Regnase-1 stabilizes inflammatory mRNAs by sequestering Regnase-1 in the cytoplasm to prevent mRNA recognition.

Flux-augmented bifurcation analysis in chemical reaction network systems.

The dynamics of biochemical reaction networks are considered to be responsible for biological functions in living systems. Since real networks are immense and complicated, it is difficult to determine which reactions can cause a significant change of dynamical behaviors, namely, bifurcations. Also to what extent numerical results of network systems depend on the chosen kinetic rate parameters is not known. In this paper, an analytical setting that splits the information of the dynamics into the network structure and reaction kinetics is introduced. This setting possesses a factorization structure for some class of network systems which allows one to determine which subnetworks are responsible for the occurrence of a bifurcation. Subsequently, the bifurcation criteria are reformulated in a manner that allows the efficient determination of relevant reactions for bifurcations.

The gene regulatory system for specifying germ layers in early embryos of the simple chordate.

In animal embryos, gene regulatory networks control the dynamics of gene expression in cells and coordinate such dynamics among cells. In ascidian embryos, gene expression dynamics have been dissected at the single-cell resolution. Here, we revealed mathematical functions that represent the regulatory logics of all regulatory genes expressed at the 32-cell stage when the germ layers are largely specified. These functions collectively explain the entire mechanism by which gene expression dynamics are controlled coordinately in early embryos. We found that regulatory functions for genes expressed in each of the specific lineages contain a common core regulatory mechanism. Last, we showed that the expression of the regulatory genes became reproducible by calculation and controllable by experimental manipulations. Thus, these regulatory functions represent an architectural design for the germ layer specification of this chordate and provide a platform for simulations and experiments to understand the operating principles of gene regulatory networks.

Quantitative analyses reveal extracellular dynamics of Wnt ligands in Xenopus embryos.

The mechanism of intercellular transport of Wnt ligands is still a matter of debate. To better understand this issue, we examined the distribution and dynamics of Wnt8 in Xenopus embryos. While Venus-tagged Wnt8 was found on the surfaces of cells close to Wnt-producing cells, we also detected its dispersal over distances of 15 cell diameters. A combination of fluorescence correlation spectroscopy and quantitative imaging suggested that only a small proportion of Wnt8 ligands diffuses freely, whereas most Wnt8 molecules are bound to cell surfaces. Fluorescence decay after photoconversion showed that Wnt8 ligands bound on cell surfaces decrease exponentially, suggesting a dynamic exchange of bound forms of Wnt ligands. Mathematical modeling based on this exchange recapitulates a graded distribution of bound, but not free, Wnt ligands. Based on these results, we propose that Wnt distribution in tissues is controlled by a dynamic exchange of its abundant bound and rare free populations.

Using linkage logic theory to control dynamics of a gene regulatory network of a chordate embryo.

Linkage logic theory provides a mathematical criterion to control network dynamics by manipulating activities of a subset of network nodes, which are collectively called a feedback vertex set (FVS). Because many biological functions emerge from dynamics of biological networks, this theory provides a promising tool for controlling biological functions. By manipulating the activity of FVS molecules identified in a gene regulatory network (GRN) for fate specification of seven tissues in ascidian embryos, we previously succeeded in reproducing six of the seven cell types. Simultaneously, we discovered that the experimentally reconstituted GRN lacked information sufficient to reproduce muscle cells. Here, we utilized linkage logic theory as a tool to find missing edges in the GRN. Then, we identified a FVS from an updated version of the GRN and confirmed that manipulating the activity of this FVS was sufficient to induce all seven cell types, even in a multi-cellular environment. Thus, linkage logic theory provides tools to find missing edges in experimentally reconstituted networks, to determine whether reconstituted networks contain sufficient information to fulfil expected functions, and to reprogram cell fate.

Expression of a gene for an MLX56 defense protein derived from mulberry latex confers strong resistance against a broad range of insect pests on transgenic tomato lines.

Insect pests cause serious damage in crop production, and various attempts have been made to produce insect-resistant crops, including the expression of genes for proteins with anti-herbivory activity, such as Bt (Bacillus thuringiensis) toxins. However, the number of available genes with sufficient anti-herbivory activity is limited. MLX56 is an anti-herbivory protein isolated from the latex of mulberry plants, and has been shown to have strong growth-suppressing activity against the larvae of a variety of lepidopteran species. As a model of herbivore-resistant plants, we produced transgenic tomato lines expressing the gene for MLX56. The transgenic tomato lines showed strong anti-herbivory activities against the larvae of the common cutworm, Spodoptera litura. Surprisingly, the transgenic tomato lines also exhibited strong activity against the attack of western flower thrips, Frankliniera occidentalis. Further, growth of the hadda beetle, Henosepilachna vigintioctopunctata, fed on leaves of transgenic tomato was significantly retarded. The levels of damage caused by both western flower thrips and hadda beetles were negligible in the high-MLX56-expressing tomato line. These results indicate that introduction of the gene for MLX56 into crops can enhance crop resistance against a wide range of pest insects, and that MLX56 can be utilized in developing genetically modified (GM) pest-resistant crops.

A novel scoring system for stroke risk stratification in Japanese patients with low CHADS2 scores: Study using a transesophageal-echocardiogram endpoint.

BACKGROUND: Catheter ablation is an effective treatment for atrial fibrillation (AF), but it carries risk of perioperative thromboembolism even in cases with low CHADS2 scores. Here, we examined whether a combination of clinical variables can predict stroke risk factors that are assessed by transesophageal echocardiography (TEE). METHODS: The study population consisted of 209 consecutive AF patients with a CHADS2 score of 0 or 1 (58.7 ± 10.6 years old; persistent AF, 33.0%). All patients underwent TEE, and TEE-determined stroke risk (TEE risk) was defined as cardiac thrombus/sludge, dense spontaneous echo contrast (SEC), and/or peak left atrial appendage (LAA) flow velocity <0.25 m/s. RESULTS: Transesophageal echocardiography risk was observed in 10.5% of the patients. In multivariate logistic analysis, persistent AF [odds ratio (OR): 11.5, CI: 3.14-42.1, P = .0002], left atrial diameter (LAD) (OR: 1.10, CI: 1.01-1.20, P = .0293), contrast medium defect (CMD) in the LAA detected by computed tomography (OR: 20.2, CI: 6.3-65.0, P < .0001), and serum brain natriuretic peptide (BNP) level (OR: 1.00, CI: 1.00-1.01, P = .0056) were independent predictors of TEE risk. A new scoring system comprising LAD > 41 mm (1 point), BNP > 47 pg/mL (1 point), CMD (2 points), and persistent AF (2 points) was constructed and defined as TEE-risk score. The area under the curve (AUC) for prediction of TEE risk was 0.631 in modified CHADS2 score and it was 0.852 in TEE-risk score. CONCLUSION: Transesophageal echocardiography risk is predictable by TEE-risk score, and its combination with CHADS2 score may improve the stroke risk stratification in AF patients with a low CHADS2 score.

Hemodynamic Collapse After Influenza Vaccination: A Vaccine-Induced Fulminant Myocarditis?

A 14-year-old girl with cardiopulmonary arrest was referred to our hospital. She had received an injection of inactivated influenza vaccine 7 days before the referral. Her cardiac rhythm was pulseless wide QRS tachycardia, and mechanical circulatory support was immediately begun. Results of endomyocardial biopsy showed that there was massive infiltration of CD3- and CD68-positive cells and various degrees of cardiomyocyte necrosis in all of 3 endomyocardial specimens, whereas infiltration of eosinophils or giant cells was not observed. A histologic diagnosis of lymphocytic myocarditis was made. Acute myocarditis is a rare but potentially fatal complication of the influenza vaccination.

Is Incomplete Left Atrial Posterior Wall Isolation Associated With Recurrence of Atrial Fibrillation After Radiofrequency Catheter Ablation?

Background: Complete left atrial posterior wall isolation (LAPI) is not always achieved. We examined whether incomplete LAPI has an effect on outcomes after catheter ablation (CA). Methods and Results: This study enrolled 75 consecutive patients (mean [±SD] age 62.6±8.9 years, 74.7% male) who underwent LAPI by radiofrequency CA for persistent atrial fibrillation (AF). The median follow-up period was 541 days (interquartile range 338-840 days). Incomplete LAPI was defined as the presence of a successfully created roof or floor linear lesion. The rate of complete LAPI was 41.3% (31/75). Either a roof or floor linear lesion was created in 38 patients, whereas neither was created in 6. Multivariate Cox proportional hazards regression analysis revealed that female sex (hazard ratio [HR] 5.29; 95% confidence interval [CI] 1.81-16.8; P=0.002) and complete or incomplete LAPI (HR 0.17; 95% CI 0.03-0.79; P=0.027) were independent predictors of AF recurrence. Kaplan-Meier curves indicated that better outcome was associated with at least one rather than no successful linear lesion (86.5% vs. 50.0% at 1 year; P=0.043). There were no significant differences in outcomes between the complete LAPI and incomplete LAPI groups. Conclusions: Complete LAPI is unachievable in a significant percentage of patients with persistent AF. However, incomplete LAPI, as a result of aiming for complete LAPI, may have a benefit comparable to that of complete LAPI.

Revaluation of the Significance of Demonstrable Exit Block After Radiofrequency Pulmonary Vein Isolation.

Background: Demonstration of exit block from the pulmonary vein (PV) to the left atrium after PV isolation (PVI) is not always possible after demonstration of entrance block. We examined factors associated with demonstrable exit block and the relationship between demonstrable exit block and subsequent PV reconnection. Methods and Results: The subjects consisted of 227 patients (908 PV; mean patient age, 59.2±10.8 years; 72.2% male) who underwent radiofrequency PVI, 49 of whom proceeded to the second session after a mean duration of 563.4±456.3 days after the first session. In the first session, exit block was demonstrated in 73.1% of PV, and the predictors were superior PV, longitudinal diameter of the PV, and spontaneous activity in the PV. In the second session (n=49), exit block was demonstrated in 51.0% (33.1% in PV without reconnection vs. 79.7% in PV with reconnection, P<0.0001). Spontaneous activity (OR, 2.74; 95% CI: 1.12-7.03, P=0.0272) and use of a contact force-sensing catheter (OR, 0.42, 95% CI: 0.20-0.85, P=0.0151) were independent predictors of PV reconnection, but demonstrable exit block was not (OR, 1.58; 95% CI: 0.74-3.46, P=0.2377). Conclusions: Inability to demonstrate exit block was not associated with increased risk of future PV reconnection.