Stability Analysis for Large-Scale Multi-Agent Molecular Communication Systems.

Molecular communication (MC) is recently featured as a novel communication tool to connect individual biological nanorobots. It is expected that a large number of nanorobots can form large multi-agent MC systems through MC to accomplish complex and large-scale tasks that cannot be achieved by a single nanorobot. However, most previous models for MC systems assume a unidirectional diffusion communication channel and cannot capture the feedback between each nanorobot, which is important for multi-agent MC systems. In this paper, we introduce a system theoretic model for large-scale multi-agent MC systems using transfer functions, and then propose a method to analyze the stability for multi-agent MC systems. The proposed method decomposes the multi-agent MC system into multiple single-input and single-output (SISO) systems, which facilitates the application of simple analysis technique for SISO systems to the large-scale multi-agent MC system. Finally, we demonstrate the proposed method by analyzing the stability of a specific large-scale multi-agent MC system and clarify a parameter region to synchronize the states of nanorobots, which is important to make cooperative behaviors at a population level.

Multimolecular Competition Effect as a Modulator of Protein Localization and Biochemical Networks in Cell-Size Space.

Cells are small, closed spaces filled with various types of macromolecules. Although it is shown that the characteristics of biochemical reactions in vitro are quite different from those in living cells, the role of the co-existence of various macromolecules in cell-size space remains still elusive. Here, using a constructive approach, it is demonstrated that the co-existence of various macromolecules themselves has the ability to tune protein localization for spatiotemporal regulation and a biochemical reaction system in a cell-size space. Both experimental and theoretical analyses reveal that enhancement of interfacial effects by a large surface-area-to-volume ratio facilitates membrane localization of molecules in the cell-size space, and the interfacial effects are alleviated by competitive binding to lipid membranes among multiple proteins even if their membrane affinities are weak. These results indicate that competition for membrane binding among various macromolecules in the cell-size space plays a role in regulating the spatiotemporal molecular organization and biochemical reaction networks. These findings shed light on the importance of surrounding molecules for biochemical reactions using purified elements in small spaces.

L-2L ladder digital-to-analogue converter for dynamics generation of chemical concentrations.

Cellular response to dynamic chemical stimulation encodes rich information about the underlying reaction pathways and their kinetics. Microfluidic chemical stimulators play a key role in generating dynamic concentration waveforms by mixing several aqueous solutions. In this article, we propose a multi-layer microfluidic chemical stimulator capable of modulating chemical concentrations by a simple binary logic based on the electronic-hydraulic analogy of electronic R-2R ladder circuits. The proposed device, which we call L-2L ladder digital-to-analogue converter (DAC), allows us to systematically modulate 2 n levels of concentrations from single sources of solution and solvent by a single operation of 2n membrane valves, which contrasts with existing devices that require complex channel geometry with multiple input sources and valve operations. We fabricated the L-2L ladder DAC with n = 3 bit resolution and verified the concept by comparing the generated waveforms with computational simulations. The response time of the proposed DAC was within the order of seconds because of its simple operation logic of membrane valves. Furthermore, detailed analysis of the waveforms revealed that the transient concentration can be systematically predicted by a simple addition of the transient waveforms of 2n = 6 base patterns, enabling facile optimization of the channel geometry to fine-tune the output waveforms.

A parametric logistic equation with light flux and medium concentration for cultivation planning of microalgae.

Microalgae are considered to be promising producers of bioactive chemicals, feeds and fuels from carbon dioxide by photosynthesis. Thus, the prediction of microalgal growth profiles is important for the planning of cost-effective and sustainable cultivation-harvest cycles. This paper proposes a mathematical model capable of predicting the effect of light flux into culture and medium concentration on the growth profiles of microalgae by incorporating these growth-limiting factors into a logistic equation. The specific form of the equation is derived based on the experimentally measured growth profiles of Monoraphidium sp., a microalgal strain isolated by the authors, under 16 conditions consisting of combinations of incident light fluxes into culture and initial medium concentrations. Using a cross-validation method, it is shown that the proposed model has the ability to predict necessary incident light flux into culture and initial medium concentration for harvesting target biomass at a target time. Finally, model-guided cultivation planning is performed and is evaluated by comparing the result with experimental data.

Microfiber-Shaped Programmable Materials with Stimuli-Responsive Hydrogel.

Programmable materials have artificially designed physical shapes responding to external stimuli, as well as high design capability and high flexibility. Here, we propose a microfiber-shaped programmable material with an axial pattern of stimuli-responsive (SR) and nonresponsive hydrogels. The SR pre-gel solution was mixed to sodium alginate pre-gel solution for instantaneous gelation with ionic crosslinking and solidified on a nonresponsive hydrogel microfiber with a valve-controlled microfluidic system. A design of microfiber-shaped programmable material (patterned position of SR regions) could be flexibly altered by changing a coded sequence program. We confirmed that the three-dimensional (3D) coil-like structures were self-folded at the patterned SR regions responding to the thermal stimulus and that the chirality of the self-folded 3D coil-like structures depends on the condition of the stimulus to the microfiber. Finally, interaction with objects using the programmable microfiber as a soft actuator was demonstrated. Our microfiber-shaped programmable materials expand possibilities of fiber-based materials in biomimetics and soft robotics fields.

Stereocontrolled Total Synthesis of (+)-Isolaurenidificin and (-)-Bromlaurenidificin.

We report the first total syntheses of (+)-isolaurenidificin (1) and (-)-bromlaurenidificin (2), the latest acetogenins of the 2,6-dioxabicyclo[3.3.0]octane class. The synthesis features a completely stereoselective one-pot epimerization-ring contraction to establish the cis configuration with respect to C10-H and C12-H of the tetrahydrofuran ring. Six stereogenic centers and an olefin geometry were constructed in a highly stereoselective manner. Absolute configurations of the natural products were deduced by the comparison of NMR data and specific rotations.

Analysing diffusion and flow-driven instability using semidefinite programming.

Diffusion and flow-driven instability, or transport-driven instability, is one of the central mechanisms to generate inhomogeneous gradient of concentrations in spatially distributed chemical systems. However, verifying the transport-driven instability of reaction-diffusion-advection systems requires checking the Jacobian eigenvalues of infinitely many Fourier modes, which is computationally intractable. To overcome this limitation, this paper proposes mathematical optimization algorithms that determine the stability/instability of reaction-diffusion-advection systems by finite steps of algebraic calculations. Specifically, the stability/instability analysis of Fourier modes is formulated as a sum-of-squares optimization program, which is a class of convex optimization whose solvers are widely available as software packages. The optimization program is further extended for facile computation of the destabilizing spatial modes. This extension allows for predicting and designing the shape of the concentration gradient without simulating the governing equations. The streamlined analysis process of self-organized pattern formation is demonstrated with a simple illustrative reaction model with diffusion and advection.

Optimization-based synthesis of stochastic biocircuits with statistical specifications.

Model-guided design has become a standard approach to engineering biomolecular circuits in synthetic biology. However, the stochastic nature of biomolecular reactions is often overlooked in the design process. As a result, cell-cell heterogeneity causes unexpected deviation of biocircuit behaviours from model predictions and requires additional iterations of design-build-test cycles. To enhance the design process of stochastic biocircuits, this paper presents a computational framework to systematically specify the level of intrinsic noise using well-defined metrics of statistics and design highly heterogeneous biocircuits based on the specifications. Specifically, we use descriptive statistics of population distributions as an intuitive specification language of stochastic biocircuits and develop an optimization-based computational tool that explores parameter configurations satisfying design requirements. Sensitivity analysis methods are also performed to ensure the robustness of a biocircuit design against extrinsic perturbations. These design tools are formulated with convex optimization programs to enable rigorous and efficient quantification of the statistics. We demonstrate these features by designing a stochastic negative feedback biocircuit that satisfies multiple statistical constraints and perform an in-depth study of noise propagation and regulation in negative feedback pathways.

Estimation of tibialis anterior muscle stiffness during the swing phase of walking with various footwear.

The current study examined stiffness in the tibialis anterior muscle during the swing phase of walking while wearing various footwear. Seven healthy young men participated in this study. Participants were instructed to walk on a treadmill at 3 km/h while wearing sports shoes, slippers, or slippers with belts. The common peroneal nerve was electrically stimulated every two steps at toe-off during walking. Mechanomyograms (MMGs), electromyograms, and ankle angle were measured. Evoked MMG was extracted using a Kalman filter and subtraction of walking acceleration. The transfer function from the electrical stimulation to the evoked MMG was identified using a singular value decomposition method, and the natural frequency of the transfer function was calculated as an index of muscle stiffness. The natural frequency did not show a clear relationship with footwear type. Four participants showed the lowest natural frequency when they wore slippers with belts. The remaining subjects showed the lowest natural frequency when they wore slippers or shoes. These contrasting findings may have been caused by different degrees of adaptation of participants to the footwear.

Cell-free extract based optimization of biomolecular circuits with droplet microfluidics.

Engineering an efficient biomolecular circuit often requires time-consuming iterations of optimization. Cell-free protein expression systems allow rapid testing of biocircuits in vitro, speeding the design-build-test cycle of synthetic biology. In this paper, we combine this with droplet microfluidics to densely scan a transcription-translation biocircuit space. Our system assays millions of parameter combinations per hour, providing a detailed map of function. The ability to comprehensively map biocircuit parameter spaces allows accurate modeling to predict circuit function and identify optimal circuits and conditions.

A population-based temporal logic gate for timing and recording chemical events.

Engineered bacterial sensors have potential applications in human health monitoring, environmental chemical detection, and materials biosynthesis. While such bacterial devices have long been engineered to differentiate between combinations of inputs, their potential to process signal timing and duration has been overlooked. In this work, we present a two-input temporal logic gate that can sense and record the order of the inputs, the timing between inputs, and the duration of input pulses. Our temporal logic gate design relies on unidirectional DNA recombination mediated by bacteriophage integrases to detect and encode sequences of input events. For an E. coli strain engineered to contain our temporal logic gate, we compare predictions of Markov model simulations with laboratory measurements of final population distributions for both step and pulse inputs. Although single cells were engineered to have digital outputs, stochastic noise created heterogeneous single-cell responses that translated into analog population responses. Furthermore, when single-cell genetic states were aggregated into population-level distributions, these distributions contained unique information not encoded in individual cells. Thus, final differentiated sub-populations could be used to deduce order, timing, and duration of transient chemical events.

Stereocontrolled Synthesis of a Possible Stereoisomer of Laurenidificin and a Formal Total Synthesis of (+)-Aplysiallene Featuring a Stereospecific Ring Contraction.

We report a highly stereocontrolled total synthesis of one of the possible stereoisomers of laurenidificin. Highlights of the synthesis include the formation of the 2,6-dioxabicyclo[3.3.0]octane framework by a stereospecific bromolactonization-α-bromination-ring contraction sequence, followed by a stereoselective propargylation, an insertion of the Z-enyne side chain by a hydroindation/cross coupling reaction, and ethylation at C13 with an organocuprate reagent. While the synthetic compound was not identical to the natural product, the absolute stereochemistry of the natural product was proposed on the basis of NMR analyses. Moreover, a formal total synthesis of (+)-aplysiallene was achieved by extending the ring contraction strategy.

Rapid cell-free forward engineering of novel genetic ring oscillators.

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the 'repressilator', a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior.

Intercellular delay regulates the collective period of repressively coupled gene regulatory oscillator networks.

Most biological rhythms are generated by a population of cellular oscillators coupled through intercellular signaling. Recent experimental evidence shows that the collective period may differ significantly from the autonomous period in the presence of intercellular delays. The phenomenon has been investigated using delay-coupled phase oscillators, but the proposed phase model contains no direct biological mechanism, which may weaken the model's reliability in unraveling biophysical principles. Based on a published gene regulatory oscillator model, we analyze the collective period of delay-coupled biological oscillators using the multivariable harmonic balance technique. We prove that, in contradiction to the common intuition that the collective period increases linearly with the coupling delay, the collective period turns out to be a periodic function of the intercellular delay. More surprisingly, the collective period may even decrease with the intercellular delay when the delay resides in certain regions. The collective period is given in a closed-form in terms of biochemical reaction constants and thus provides biological insights as well as guidance in synthetic-biological-oscillator design. Simulation results are given based on a segmentation clock model to confirm the theoretical predictions.

Comparative observation of skeletal-dental abnormalities in wild, domestic, and laboratory rabbits.

Dietary habits must be considered as one of the major potential factors resulting in acquired malocclusions in rabbits. Although the dentition of the wild rabbit and the domesticated laboratory rabbit are basically identical, dietary habits are noticeably different. Therefore, the prevalence of tooth problems between these lagomorph species were investigated anatomically and radiographically. Mean measurements of the skull and dental arches suggested that wild rabbits have slightly shorter and wider skulls and dental arches compared with domestic laboratory rabbits. Root elongation of incisors and check teeth, and periodontal disease were more frequently observed in domestic laboratory rabbits. Diagnostic radiographs from domestic pet rabbits showed relatively higher crowns, severe root elongation, and advanced periodontitis. These results do not provide definitive evidence that dietary habits cause malocclusions, however they suggest that diet is a major factor in the initiation of malocclusions in rabbits.