Multiscale engineered human skeletal muscles with perfusable vasculature and microvascular network recapitulating the fluid compartments.

Creating a vasculature in engineered human skeletal muscle tissues (ehSMTs) enables us to create thick tissues, increase cell survival in implantation, provide models of blood-organ barriers for drug testing, and enhance muscle differentiation through paracrine signaling. Here, contractile ehSMTs with a central perfusable vascular channel and microvascular networks growing from this central vasculature into the surrounding skeletal muscle tissue were newly demonstrated. Because coculturing muscle cells and endothelial cells requires incompatible media, we recapitulated thein vivoextracellular fluid compartments between blood plasma and interstitial fluid by creating anin vitroperfusable vasculature running through skeletal muscle tissue with a physiologic cell density. By using this model, we constructed large vascularized ehSMTs and showed the potential to be utilized for drug testing platforms. Also, we found that coculturing with two separate media from an early stage of muscle differentiation led to increased contractile force, thicker myotubes, and improved muscle differentiation.

Tri-culture of spatially organizing human skeletal muscle cells, endothelial cells, and fibroblasts enhances contractile force and vascular perfusion of skeletal muscle tissues.

Constructing engineered human skeletal muscle tissues that resemble the function and microstructure of human skeletal muscles is key to utilizing them in a variety of applications such as drug development, disease modeling, regenerative medicine, and engineering biological machines. However, current in vitro skeletal muscle tissues are far inferior to native muscles in terms of contractile function and lack essential cues for muscle functions, particularly heterotypic cell-cell interactions between myoblasts, endothelial cells, and fibroblasts. Here, we develop an engineered muscle tissue with a coaxial three-layered tubular structure composed of an inner endothelial cell layer, an endomysium-like layer with fibroblasts in the middle, and an outer skeletal muscle cell layer, similar to the architecture of native skeletal muscles. Engineered skeletal muscle tissues with three spatially organized cell types produced thicker myotubes and lowered Young's modulus through extracellular matrix remodeling, resulting in 43% stronger contractile force. Furthermore, we demonstrated that fibroblasts localized in the endomysium layer induced angiogenic sprouting of endothelial cells into the muscle layer more effectively than fibroblasts homogeneously distributed in the muscle layer. This layered tri-culture system enables a structured spatial configuration of the three main cell types of skeletal muscle and promotes desired paracrine signaling, resulting in improved angiogenesis and increased contractile force. This research offers new insights to efficiently obtain new human skeletal muscle models, transplantable tissues, and actuators for biological machines.

A computational modeling of invadopodia protrusion into an extracellular matrix fiber network.

Invadopodia are dynamic actin-rich membrane protrusions that have been implicated in cancer cell invasion and metastasis. In addition, invasiveness of cancer cells is strongly correlated with invadopodia formation, which are observed during extravasation and colonization of metastatic cancer cells at secondary sites. However, quantitative understanding of the interaction of invadopodia with extracellular matrix (ECM) is lacking, and how invadopodia protrusion speed is associated with the frequency of protrusion-retraction cycles remains unknown. Here, we present a computational framework for the characterization of invadopodia protrusions which allows two way interactions between intracellular branched actin network and ECM fibers network. We have applied this approach to predicting the invasiveness of cancer cells by computationally knocking out actin-crosslinking molecules, such as α-actinin, filamin and fascin. The resulting simulations reveal distinct invadopodia dynamics with cycles of protrusion and retraction. Specifically, we found that (1) increasing accumulation of MT1-MMP at tips of invadopodia as the duration of protrusive phase is increased, and (2) the movement of nucleus toward the leading edge of the cell becomes unstable as duration of the retractile phase (or myosin turnover time) is longer than 1 min.

TeachBot: Towards teaching robotics fundamentals for human-robot collaboration at work.

The shortage of skilled workers who can use robots is a crucial issue hampering the growth of manufacturing industries. We present a new type of workforce training system, TeachBot, in which a robotic instructor delivers a series of interactive lectures using graphics and physical demonstration of its arm movements. Furthermore, the TeachBot allows learners to physically interact with the robot. This new human-computer interface, integrating oral and graphical instructions with motion demonstration and physical touch, enables to create engaging training materials. Effective learning takes place when the learner simultaneously interacts with an embodiment of new knowledge. We apply this "Learning by Touching" methodology to teach basic concepts, e.g. how a shaft encoder and feedback control work. In a pilot randomized control test with a small number of human subjects, we find suggestive evidence that Learning by Touching enhances learning effectiveness in this robotic context for adult learners. Students whose learning experience included touching the robot as opposed to watching it delivers the lessons showed gains in their ability to integrate knowledge about robotics. The "touching" group showed statistically significant gains in self-efficacy, which is an important antecedent to further learning and successful use of new technologies, as well as gains in knowledge about robotic concepts that trend toward significance.

Constraints on Cosmic Strings Using Data from the Third Advanced LIGO-Virgo Observing Run.

We search for gravitational-wave signals produced by cosmic strings in the Advanced LIGO and Virgo full O3 dataset. Search results are presented for gravitational waves produced by cosmic string loop features such as cusps, kinks, and, for the first time, kink-kink collisions. A template-based search for short-duration transient signals does not yield a detection. We also use the stochastic gravitational-wave background energy density upper limits derived from the O3 data to constrain the cosmic string tension Gµ as a function of the number of kinks, or the number of cusps, for two cosmic string loop distribution models. Additionally, we develop and test a third model that interpolates between these two models. Our results improve upon the previous LIGO-Virgo constraints on Gµ by 1 to 2 orders of magnitude depending on the model that is tested. In particular, for the one-loop distribution model, we set the most competitive constraints to date: Gµâ‰²4×10^{-15}. In the case of cosmic strings formed at the end of inflation in the context of grand unified theories, these results challenge simple inflationary models.

Inherent Haptic Feedback From Supernumerary Robotic Limbs.

Supernumerary Robotics Limbs, or SuperLimbs for short, are wearable extra limbs for augmenting the wearer. SuperLimbs are attached directly to a human and, thereby, transmit a force from the environment to the human body. This inherent haptic feedback allows the human to perceive the interaction between the robot and the environment, monitor its actions, and effectively control the robot. This article addresses basic properties and the usefulness of the inherent haptic feedback from SuperLimbs in two exemplary cases. First, we show that the inherent haptic feedback allows the wearer to close the loop and manually regulate the force output of the SuperLimb. Second, we show that the inherent haptic feedback is sufficient for the wearer to supervise the autonomous actions of the SuperLimb. This ability is a critical requirement for safely and effectively performing multiple tasks simultaneously with the natural limbs and SuperLimbs. Together, these findings suggest the importance of designing SuperLimbs to take advantage of the inherent haptic feedback.

Reduced induction of human ß-defensins is involved in the pathological mechanism of cutaneous adverse effects caused by epidermal growth factor receptor monoclonal antibodies.

Epidermal growth factor receptor inhibitors (EGFRIs) frequently cause cutaneous adverse effects such as papulopustular eruptions. However, the mechanism of the reactions remains unclear. To assess the pathological mechanism of cutaneous adverse reactions caused by EGFRIs, we investigated whether EGFRIs have an influence on the innate immune response of the skin. Levels of human ß-defensins (hBDs), which serve as the first line of defence against infection by pathogenic microorganisms, in the stratum corneum samples of patients treated with EGFR. monoclonal antibodies were measured before and after starting therapy. There were no obvious trends in hBD production in patients without eruptions, whereas a significant decrease in hBD1 and hBD3 production and a nonsignficant decrease in hBD2 production were observed in patients who developed papulopustular eruptions. Our results suggest that a reduction in hBD contributes to the increased incidence of papulopustular eruptions.

Multi-cell ECM compaction is predictable via superposition of nonlinear cell dynamics linearized in augmented state space.

Cells interacting through an extracellular matrix (ECM) exhibit emergent behaviors resulting from collective intercellular interaction. In wound healing and tissue development, characteristic compaction of ECM gel is induced by multiple cells that generate tensions in the ECM fibers and coordinate their actions with other cells. Computational prediction of collective cell-ECM interaction based on first principles is highly complex especially as the number of cells increase. Here, we introduce a computationally-efficient method for predicting nonlinear behaviors of multiple cells interacting mechanically through a 3-D ECM fiber network. The key enabling technique is superposition of single cell computational models to predict multicellular behaviors. While cell-ECM interactions are highly nonlinear, they can be linearized accurately with a unique method, termed Dual-Faceted Linearization. This method recasts the original nonlinear dynamics in an augmented space where the system behaves more linearly. The independent state variables are augmented by combining auxiliary variables that inform nonlinear elements involved in the system. This computational method involves a) expressing the original nonlinear state equations with two sets of linear dynamic equations b) reducing the order of the augmented linear system via principal component analysis and c) superposing individual single cell-ECM dynamics to predict collective behaviors of multiple cells. The method is computationally efficient compared to original nonlinear dynamic simulation and accurate compared to traditional Taylor expansion linearization. Furthermore, we reproduce reported experimental results of multi-cell induced ECM compaction.

Extracellular matrix remodelling induced by alternating electrical and mechanical stimulations increases the contraction of engineered skeletal muscle tissues.

Engineered skeletal muscles are inferior to natural muscles in terms of contractile force, hampering their potential use in practical applications. One major limitation is that the extracellular matrix (ECM) not only impedes the contraction but also ineffectively transmits the forces generated by myotubes to the load. In the present study, ECM remodelling improves contractile force in a short time, and a coordinated, combined electrical and mechanical stimulation induces the desired ECM remodelling. Notably, the application of single and combined stimulations to the engineered muscles remodels the structure of their ECM networks, which determines the mechanical properties of the ECM. Myotubes in the tissues are connected in parallel and in series to the ECM. The stiffness of the parallel ECM must be low not to impede contraction, while the stiffness of the serial ECM must be high to transmit the forces to the load. Both the experimental results and the mechanistic model suggest that the combined stimulation through coordination reorients the ECM fibres in such a way that the parallel ECM stiffness is reduced, while the serial ECM stiffness is increased. In particular, 3 and 20 minutes of alternating electrical and mechanical stimulations increase the force by 18% and 31%, respectively.

Multicell migration tracking within angiogenic networks by deep learning-based segmentation and augmented Bayesian filtering.

Cell migration is a key feature for living organisms. Image analysis tools are useful in studying cell migration in three-dimensional (3-D) in vitro environments. We consider angiogenic vessels formed in 3-D microfluidic devices (MFDs) and develop an image analysis system to extract cell behaviors from experimental phase-contrast microscopy image sequences. The proposed system initializes tracks with the end-point confocal nuclei coordinates. We apply convolutional neural networks to detect cell candidates and combine backward Kalman filtering with multiple hypothesis tracking to link the cell candidates at each time step. These hypotheses incorporate prior knowledge on vessel formation and cell proliferation rates. The association accuracy reaches 86.4% for the proposed algorithm, indicating that the proposed system is able to associate cells more accurately than existing approaches. Cell culture experiments in 3-D MFDs have shown considerable promise for improving biology research. The proposed system is expected to be a useful quantitative tool for potential microscopy problems of MFDs.

Prospects for observing and localizing gravitational-wave transients with Advanced LIGO, Advanced Virgo and KAGRA.

We present possible observing scenarios for the Advanced LIGO, Advanced Virgo and KAGRA gravitational-wave detectors over the next decade, with the intention of providing information to the astronomy community to facilitate planning for multi-messenger astronomy with gravitational waves. We estimate the sensitivity of the network to transient gravitational-wave signals, and study the capability of the network to determine the sky location of the source. We report our findings for gravitational-wave transients, with particular focus on gravitational-wave signals from the inspiral of binary neutron star systems, which are the most promising targets for multi-messenger astronomy. The ability to localize the sources of the detected signals depends on the geographical distribution of the detectors and their relative sensitivity, and [Formula: see text] credible regions can be as large as thousands of square degrees when only two sensitive detectors are operational. Determining the sky position of a significant fraction of detected signals to areas of 5-[Formula: see text] requires at least three detectors of sensitivity within a factor of [Formula: see text] of each other and with a broad frequency bandwidth. When all detectors, including KAGRA and the third LIGO detector in India, reach design sensitivity, a significant fraction of gravitational-wave signals will be localized to a few square degrees by gravitational-wave observations alone.

Computational modeling of three-dimensional ECM-rigidity sensing to guide directed cell migration.

Filopodia have a key role in sensing both chemical and mechanical cues in surrounding extracellular matrix (ECM). However, quantitative understanding is still missing in the filopodial mechanosensing of local ECM stiffness, resulting from dynamic interactions between filopodia and the surrounding 3D ECM fibers. Here we present a method for characterizing the stiffness of ECM that is sensed by filopodia based on the theory of elasticity and discrete ECM fiber. We have applied this method to a filopodial mechanosensing model for predicting directed cell migration toward stiffer ECM. This model provides us with a distribution of force and displacement as well as their time rate of changes near the tip of a filopodium when it is bound to the surrounding ECM fibers. Aggregating these effects in each local region of 3D ECM, we express the local ECM stiffness sensed by the cell and explain polarity in the cellular durotaxis mechanism.

Determination of Green's function for three-dimensional traction force reconstruction based on geometry and boundary conditions of cell culture matrices.

Cell migration plays a particular important role in the initiation and progression of many physical processes and pathological conditions such as tumor invasion and metastasis. Three-dimensional traction force microscopy (TFM) of high resolution and high accuracy is being developed in an effort to unveil the underlying mechanical process of cell migration in a vivo-like environment. Linear elasticity-based TFM (LETM) as a mainstream approach relies on the Green's function (that relates traction forces to matrix deformation), of which the inherent boundary conditions and geometry of the matrix could remarkably affect the result as suggested by previous 2D studies. In this study, we investigated this close linkage in 3D environment, via modeling of a cell sensing a close-by fixed boundary of a 3D matrix surrounding it, and comparing the reconstructed traction forces from three different solutions of the Green's function, including a fully matching solution derived using the adapted Mindlin's approach. To increase fidelity in the estimate of traction forces for extreme conditions such as a sparse sampling of deformation field or targeting small focal adhesions, we numerically solved the singularity problem of the Green's function in a non-conventional way to avoid exclusion of singular point regions that could contain representative deformation indicators for such extreme conditions. A single case experimental study was conducted for a multi-cellular structure of endothelial cells that just penetrated into the gel at the early stage of angiogenesis. STATEMENT OF SIGNIFICANCE: This study focused on the fundamental issue regarding extension of linear elasticity-based TFM to deal with physically realistic matrices (where cells are encapsulated), which concerns determination of the Green's function matching their geometry and boundary conditions. To increase fidelity in the estimate of traction forces for extreme conditions such as a sparse sampling of deformation field or targeting small focal adhesions, we numerically solved the singularity problem of the Green's function to avoid exclusion of singular point regions that could contain representative deformation indicators for such extreme conditions. The proposed approach to adapting the Green's function for the specific 3D cell culture situation was examined in a single case experimental study of endothelial cells in sprouting angiogenesis.

Automated tracking and quantification of angiogenic vessel formation in 3D microfluidic devices.

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a critical step in cancer invasion. Better understanding of the angiogenic mechanisms is required to develop effective antiangiogenic therapies for cancer treatment. We culture angiogenic vessels in 3D microfluidic devices under different Sphingosin-1-phosphate (S1P) conditions and develop an automated vessel formation tracking system (AVFTS) to track the angiogenic vessel formation and extract quantitative vessel information from the experimental time-lapse phase contrast images. The proposed AVFTS first preprocesses the experimental images, then applies a distance transform and an augmented fast marching method in skeletonization, and finally implements the Hungarian method in branch tracking. When applying the AVFTS to our experimental data, we achieve 97.3% precision and 93.9% recall by comparing with the ground truth obtained from manual tracking by visual inspection. This system enables biologists to quantitatively compare the influence of different growth factors. Specifically, we conclude that the positive S1P gradient increases cell migration and vessel elongation, leading to a higher probability for branching to occur. The AVFTS is also applicable to distinguish tip and stalk cells by considering the relative cell locations in a branch. Moreover, we generate a novel type of cell lineage plot, which not only provides cell migration and proliferation histories but also demonstrates cell phenotypic changes and branch information.

A process engineering approach to increase organoid yield.

Temporal manipulation of the in vitro environment and growth factors can direct differentiation of human pluripotent stem cells into organoids - aggregates with multiple tissue-specific cell types and three-dimensional structure mimicking native organs. A mechanistic understanding of early organoid formation is essential for improving the robustness of these methods, which is necessary prior to use in drug development and regenerative medicine. We investigated intestinal organoid emergence, focusing on measurable parameters of hindgut spheroids, the intermediate step between definitive endoderm and mature organoids. We found that 13% of spheroids were pre-organoids that matured into intestinal organoids. Spheroids varied by several structural parameters: cell number, diameter and morphology. Hypothesizing that diameter and the morphological feature of an inner mass were key parameters for spheroid maturation, we sorted spheroids using an automated micropipette aspiration and release system and monitored the cultures for organoid formation. We discovered that populations of spheroids with a diameter greater than 75 µm and an inner mass are enriched 1.5- and 3.8-fold for pre-organoids, respectively, thus providing rational guidelines towards establishing a robust protocol for high quality intestinal organoids.

Association of cigarette smoking with a past history and incidence of herpes zoster in the general Japanese population: the SHEZ Study.

Few studies have examined the impact of cigarette smoking on the risk for herpes zoster. The Shozu Herpes Zoster (SHEZ) Study is a community-based prospective cohort study over 3 years in Japan aiming to clarify the incidence and predictive and immunological factors for herpes zoster. We investigated the associations of smoking status with past history and incidence of herpes zoster. A total of 12 351 participants provided valid information on smoking status and past history of herpes zoster at baseline survey. Smoking status was classified into three categories (current, former, never smoker), and if currently smoking, the number of cigarettes consumed per day was recorded. The participants were under the active surveillance for first-ever incident herpes zoster for 3 years. We used a logistic regression model for the cross-sectional study on the association between smoking status and past history of herpes zoster, and a Cox proportional hazards regression model for the cohort study on the association with risk of incidence. The multivariable adjusted odd ratios (95% CI) of past history of herpes zoster for current vs. never smokers were 0·67 (0·54-0·80) for total subjects, 0·72 (0·56-0·93) for men and 0·65 (0·44-0·96) for women. The multivariable adjusted hazard ratios (95% CI) of incident herpes zoster for current vs. never smokers were 0·52 (0·33-0·81) for total subjects, 0·49 (0·29-0·83) for men and 0·52 (0·19-1·39) for women. Smoking status was inversely associated with the prevalence and incidence of herpes zoster in the general population of men and women aged ⩾50 years.

Three-Dimensional Characterization of Mechanical Interactions between Endothelial Cells and Extracellular Matrix during Angiogenic Sprouting.

We studied the three-dimensional cell-extracellular matrix interactions of endothelial cells that form multicellular structures called sprouts. We analyzed the data collected in-situ from angiogenic sprouting experiments and identified the differentiated interaction behavior exhibited by the tip and stalk cells. Moreover, our analysis of the tip cell lamellipodia revealed the diversity in their interaction behavior under certain conditions (e.g., when the heading of a sprout is switched approximately between the long-axis direction of two different lamellipodia). This study marks the first time that new characteristics of such interactions have been identified with shape changes in the sprouts and the associated rearrangements of collagen fibers. Clear illustrations of such changes are depicted in three-dimensional views.