Contractile and Tensile Measurement of Molecular Artificial Muscles for Biohybrid Robotics.

A printable artificial muscle assembled from biomolecular motors, which we have recently developed, showed great potential in overcoming the design limitations of conventional biohybrid robots as a new bio-actuator. Characterizing its contractility for extending its applicability is important. However, conventional measurement methods are composed of complex operations with poor reproducibility, flexibility, and real-time responsiveness. This study presents a new method for measuring the contractile force generated by artificial muscles. A measurement system was constructed, wherein artificial muscles were patterned by UV laser scanning in an oil-sealed microchamber, and the contractile force was measured in real time using a microforce sensor extended by a 3D-printed microcantilever. The measurement accuracy of the sensor was ensured through calibration and correction. For demonstration purposes, a series of contractile measurements were carried out using the proposed system. The relationship between contractile force and the dimensions of the activation space of the artificial muscles, as well as the tensile properties of the contracted muscle chain were evaluated. The results will help characterize the contractile properties of the artificial muscle and lay the foundations for its further application in biohybrid robotics.

Perioperative changes in ganglioside monosialodihexosylganglioside (GM3) molecular species for benign prostatic hyperplasia: a preliminary report.

Benign prostatic hyperplasia (BPH) progresses with age and is associated with chronic inflammation. We focused on the relationship between BPH and ganglioside monosialodihexosylganglioside (GM3), a sialic acid-containing glycosphingolipid that is involved in chronic inflammation. GM3 molecular species would have a significant role in regulating inflammatory processes. In this prospective study, preoperative and postoperative serum samples were obtained from patients who underwent holmium laser enucleation of the prostate (HoLEP) for BPH. Preoperative and postoperative measurements of serum GM3 species were performed one month before and three months after HoLEP. Twenty-three patients were included in the study. The average patient age was 75 years, and the average prostate volume was 66 mL. The average weight of the surgically resected prostate tissue was 42 g. At three months after HoLEP, the serum concentration of GM3 species was found to have decreased after HoLEP compared with the preoperative concentration of GM3 species. Six GM3 species such as d18:1-17:0 [C17 acyl chain (-17:0) linked to a C18 sphingosine base with a double bond (d18:1-) by an amide linkage], were significantly reduced. The sample size was small; therefore, this study showed only preliminary results and could not evaluate prostate tissue inflammation. This study showed that the serum concentrations of several GM3 species, which indicate chronic inflammation, may be significantly reduced after BPH surgery.

Sialyltransferase Activity Assay for Ganglioside GM3 Synthase.

GM3 synthase (GM3S) is a sialyltransferase that transfers sialic acid from CMP-sialic acid to lactosylceramide. This reaction results in formation of ganglioside GM3 and is essential for biosynthesis of its downstream derivatives, which include a- and b-series gangliosides. Here, we describe a method for GM3S enzymatic assay using fluorescence-labeled alkyl lactoside as acceptor substrate, followed by HPLC for separation of enzymatic product. The method allows quantitative assay of GM3S sialyltransferase activity in cultured cells and mouse brain tissues.

The rate of microtubule breaking increases exponentially with curvature.

Microtubules, cylindrical assemblies of tubulin proteins with a 25 nm diameter and micrometer lengths, are a central part of the cytoskeleton and also serve as building blocks for nanobiodevices. Microtubule breaking can result from the activity of severing enzymes and mechanical stress. Breaking can lead to a loss of structural integrity, or an increase in the numbers of microtubules. We observed breaking of taxol-stabilized microtubules in a gliding motility assay where microtubules are propelled by surface-adhered kinesin-1 motor proteins. We find that over 95% of all breaking events are associated with the strong bending following pinning events (where the leading tip of the microtubule becomes stuck). Furthermore, the breaking rate increased exponentially with increasing curvature. These observations are explained by a model accounting for the complex mechanochemistry of a microtubule. The presence of severing enzymes is not required to observe breaking at rates comparable to those measured previously in cells.

In situ integrated microrobots driven by artificial muscles built from biomolecular motors.

Microrobots have been developed for applications in the submillimeter domain such as the manipulation of micro-objects and microsurgery. Rapid progress has been achieved in developing miniaturized components for microrobotic systems, resulting in a variety of functional microactuators and soft components for creating untethered microrobots. Nevertheless, the integration of microcomponents, especially the assembly of actuators and mechanical components, is still time-consuming and has inherent restrictions, thus limiting efficient fabrications of microrobots and their potential applications. Here, we propose a method for fabricating microrobots in situ inspired by the construction of microsystems in living organisms. In a microfluidic chip, hydrogel mechanical components and artificial muscle actuators are successively photopatterned from hydrogel prepolymer and biomolecular motors, respectively, and integrated in situ into functional microrobots. The proposed method allows the fast fabrication of microrobots through simple operations and affordable materials while providing versatile functions through the precise spatiotemporal control of in situ integration and reconfiguration of artificial muscles. To validate the method, we fabricated microrobots to elicit different motions and on-chip robots with unique characteristics for microfluidic applications. This study may establish a new paradigm for microrobot integration and lead to the production of unique biohybrid microrobots with various advantages.

Functional validation of novel variants in B4GALNT1 associated with early-onset complex hereditary spastic paraplegia with impaired ganglioside synthesis.

Childhood-onset forms of hereditary spastic paraplegia are ultra-rare diseases and often present with complex features. Next-generation-sequencing allows for an accurate diagnosis in many cases but the interpretation of novel variants remains challenging, particularly for missense mutations. Where sufficient knowledge of the protein function and/or downstream pathways exists, functional studies in patient-derived cells can aid the interpretation of molecular findings. We here illustrate the case of a 13-year-old female who presented with global developmental delay and later mild intellectual disability, progressive spastic diplegia, spastic-ataxic gait, dysarthria, urinary urgency, and loss of deep tendon reflexes of the lower extremities. Exome sequencing showed a novel splice-site variant in trans with a novel missense variant in B4GALNT1 [NM_001478.5: c.532-1G>C/c.1556G>C (p.Arg519Pro)]. Functional studies in patient-derived fibroblasts and cell models of GM2 synthase deficiency confirmed a loss of B4GALNT1 function with no synthesis of GM2 and other downstream gangliosides. Collectively these results established the diagnosis of B4GALNT1-associated HSP (SPG26). Our approach illustrates the importance of careful phenotyping and functional characterization of novel gene variants, particularly in the setting of ultra-rare diseases, and expands the clinical and molecular spectrum of SPG26, a disorder of complex ganglioside biosynthesis.

Motility Resilience of Molecular Shuttles Against Defective Motors.

Myosin and kinesin are biomolecular motors found in living cells. By propelling their associated cytoskeletal filaments, these biomolecular motors facilitate force generation and material transport in the cells. When extracted, the biomolecular motors are promising candidates for in vitro applications such as biosensor devices, on account of their high operating efficiency and nanoscale size. However, during integration into these devices, some of the motors become defective due to unfavorable adhesion to the substrate surface. These defective motors inhibit the motility of the cytoskeletal filaments which make up the molecular shuttles used in the devices. Difficulties in controlling the fraction of active and defective motors in experiments discourage systematic studies concerning the resilience of the molecular shuttle motility against the impedance of defective motors. Here, we used mathematical modelling to systematically examine the resilience of the propulsion by these molecular shuttles against the impedance of the defective motors. The model showed that the fraction of active motors on the substrate is the essential factor determining the resilience of the molecular shuttle motility. Approximately 40% of active kinesin or 80% of active myosin motors are required to constitute continuous gliding of molecular shuttles in their respective substrates. The simplicity of the mathematical model in describing motility behavior offers utility in elucidating the mechanisms of the motility resilience of molecular shuttles.

Linking path and filament persistence lengths of microtubules gliding over kinesin.

Microtubules and kinesin motor proteins are involved in intracellular transports in living cells. Such intracellular material transport systems can be reconstructed for utilisation in synthetic environments, and they are called molecular shuttles driven by kinesin motors. The performance of the molecular shuttles depends on the nature of their trajectories, which can be characterized by the path persistence length of microtubules. It has been theoretically predicted that the path persistence length should be equal to the filament persistence length of the microtubules, where the filament persistence length is a measure of microtubule flexural stiffness. However, previous experiments have shown that there is a significant discrepancy between the path and filament persistence lengths. Here, we showed how this discrepancy arises by using computer simulation. By simulating molecular shuttle movements under external forces, the discrepancy between the path and filament persistence lengths was reproduced as observed in experiments. Our close investigations of molecular shuttle movements revealed that the part of the microtubules bent due to the external force was extended more than it was assumed in the theory. By considering the extended length, we could elucidate the discrepancy. The insights obtained here are expected to lead to better control of molecular shuttle movements.

Effects of defective motors on the active transport in biosensors powered by biomolecular motors.

Motor proteins, such as myosin and kinesin, are biological molecular motors involved in force generation and intracellular transport in living cells. They were proposed to drive molecular shuttles for the active transport of analytes, thus significantly accelerating the sensing process of biosensors. Integrating motor proteins into biosensors requires their immobilisation on the operating surfaces. However, this process makes some motor proteins defective, slowing analyte detection. Here, we investigated the movements of molecular shuttles on surfaces in the presence of active and defective motors using a Brownian dynamics simulation, and elucidated the effects of defective motor proteins on the transport efficiency of the shuttles. We found that the motility of shuttles depends on the fraction of active motors relative to defective ones and that over 90% of the surface-bound motor proteins must remain active for efficient transport. The high fraction of active motors required for efficient transport can be attributed to the difference in the binding lifetimes of active and defective motors to shuttles. These results provide insights into how motors accumulate on sensor surfaces and set a guideline for the choice of polymer materials for biosensors powered by motor proteins.

Homeostatic and pathogenic roles of the GM3 ganglioside.

Two decades ago, we achieved molecular cloning of ganglioside GM3 synthase (GM3S; ST3GAL5), the enzyme responsible for initiating biosynthesis of complex gangliosides. The efforts of our research group since then have been focused on clarifying the physiological and pathological roles of gangliosides, particularly GM3. This review summarizes our long-term studies on the roles of GM3 in insulin resistance and adipogenesis in adipose tissues, cholesterol uptake in intestine, and leptin resistance in hypothalamus. We hypothesized that GM3 plays a role in innate immune function of macrophages and demonstrated that molecular species of GM3 with differing acyl-chain structures and modifications functioned as pro- and anti-inflammatory endogenous Toll-like receptor 4 (TLR4) modulators in macrophages. Very-long-chain and α-hydroxy GM3 species enhanced TLR4 activation, whereas long-chain and unsaturated GM3 species counteracted this effect. Lipidomic analyses of serum and adipose tissues revealed that imbalances between such pro- and anti-inflammatory GM3 species promoted progression of metabolic disorders. GM3 thus functions as a physiological regulatory factor controlling the balance between homeostatic and pathological states. Ongoing studies based on these findings will clarify the mechanisms underlying ganglioside-dependent control of energy homeostasis and innate immune responses.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

A printable active network actuator built from an engineered biomolecular motor.

Leveraging the motion and force of individual molecular motors in a controlled manner to perform macroscopic tasks can provide substantial benefits to many applications, including robotics. Nonetheless, although millimetre-scale movement has been demonstrated with synthetic and biological molecular motors, their efficient integration into engineered systems that perform macroscopic tasks remains challenging. Here, we describe an active network capable of macroscopic actuation that is hierarchically assembled from an engineered kinesin, a biomolecular motor, and microtubules, resembling the contractile units in muscles. These contracting materials can be formed in desired areas using patterned ultraviolet illumination, allowing their incorporation into mechanically engineered systems, being also compatible with printing technologies. Due to the designed filamentous assembly of kinesins, the generated forces reach the micronewton range, enabling actuation of millimetre-scale mechanical components. These properties may be useful for the fabrication of soft robotic systems with advanced functionalities.

Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity.

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression.

Adaptation of Patterns of Motile Filaments under Dynamic Boundary Conditions.

Boundary conditions are important for pattern formation in active matter. However, it is still not well-understood how alterations in the boundary conditions (dynamic boundary conditions) impact pattern formation. To elucidate the effect of dynamic boundary conditions on the pattern formation by active matter, we investigate an in vitro gliding assay of microtubules on a deformable soft substrate. The dynamic boundary conditions were realized by applying mechanical stress through stretching and compression of the substrate during the gliding assay. A single cycle of stretch-and-compression (relaxation) of the substrate induces perpendicular alignment of microtubules relative to the stretch axis, whereas repeated cycles resulted in zigzag patterns of microtubules. Our model shows that the orientation angles of microtubules correspond to the direction to attain smooth movement without buckling, which is further amplified by the collective migration of the microtubules. Our results provide an insight into understanding the rich dynamics in self-organization arising in active matter subjected to time-dependent boundary conditions.

A new mouse model for noninvasive fluorescence-based monitoring of mitochondrial UCP1 expression.

Mitochondrial uncoupling protein 1 (UCP1) is well known for its thermogenic function in brown adipose tissue (BAT). Since UCP1 expends energy on thermogenesis, UCP1 activation has been considered an approach to ameliorate obesity. As a tool for uncovering yet unknown mechanisms of UCP1 activation, we generated a transgenic mouse model in which UCP1 expression levels are reflected in fluorescence derived from monomeric red fluorescent protein 1 (mRFP1). In these UCP1-mRFP1 BAC transgenic mice, fluorescence intensity mimics the change in UCP1 expression levels evoked through physiological or pharmacological stimulation. This transgenic mouse model will be useful in the search for bioactive compounds with the ability to induce UCP1 and for revealing undiscovered mechanisms of BAT activation.

Serum GM3(d18:1-16:0) and GM3(d18:1-24:1) levels may be associated with lymphoma: An exploratory study with haematological diseases.

GM3 (monosialodihexosylganglioside) is a type of ganglioside, which is a molecule composed of ceramide and oligosaccharide containing one or more sialic acids. Since GM3 is abundantly expressed in blood cells, we investigated the association between GM3 molecular species and haematological diseases. We measured the serum levels of seven GM3 molecular species in subjects with various haematological diseases (n = 52) and healthy subjects (n = 24) using a liquid chromatography tandem-mass spectrometry technique as an exploratory study. In all the subjects with haematological diseases, GM3(d18:1-16:0) were inversely correlated with the erythrocytes counts. Regarding the difference in serum GM3 molecular species levels among each haematological diseases and healthy subjects, the levels of GM3(d18:1-16:0) and GM3(d18:1-24:1) were higher in the lymphoid neoplasm group than healthy subjects. Principal component analyses also revealed that the GM3(d18:1-16:0) and GM3(d18:1-24:1) levels were significant contributing factors for discriminating the lymphoid neoplasm group. Moreover, in the lymphoid neoplasm group, the GM3(d18:1-16:0) levels were significantly and positively correlated with the levels of C-reactive protein, soluble interleukin-2 receptor, and lactate dehydrogenase. In conclusion, in our exploratory study with haematological diseases, GM3 molecular species showed different distribution among disease groups, and serum GM3(d18:1-16:0) and GM3(d18:1-24:1) might be associated with lymphoma.

Globo-series glycosphingolipids enhance Toll-like receptor 4-mediated inflammation and play a pathophysiological role in diabetic nephropathy.

Alteration of glycosphingolipid (GSL) expression plays key roles in the pathogenesis and pathophysiology of many important human diseases, including cancer, diabetes and glycosphingolipidosis. Inflammatory processes are involved in development and progression of diabetic nephropathy, a major complication of type 2 diabetes mellitus. GSLs are known to play roles in inflammatory responses in various diseases, and levels of renal GSLs are elevated in mouse models of diabetic nephropathy; however, little is known regarding the pathophysiological role of these GSLs in this disease process. We studied proinflammatory activity of GSLs in diabetic nephropathy using spontaneously diabetic mouse strain KK. Mice were fed a high-fat diet (HFD) (60% kcal from fat) or normal diet (ND) (4.6% kcal from fat) for a period of 8 wk. HFD-feeding resulted in quantitative and qualitative changes of renal globo-series GSLs (particularly Gb3Cer), upregulation of TNF-α, and induction of renal inflammation. Gb3Cer/Gb4Cer treatment enhanced inflammatory responses via TLR4 in TLR4/MD-2 complex expressing cells, including HEK293T, mouse bone marrow-derived macrophages (BMDMs) and human monocytes. Our findings suggest that HFD-induced increase of Gb3Cer/Gb4Cer positively modulate TLR4-mediated inflammatory response, and that such GSLs play an important pathophysiological role in diabetic nephropathy.

Deficient ganglioside synthesis restores responsiveness to leptin and melanocortin signaling in obese KKAy mice.

GM3, a precursor for synthesis of a- and b-series gangliosides, is elevated in adipocytes of obese model animals and in sera of obese human patients with type 2 diabetes and/or dyslipidemia. GM3 synthase (GM3S)-KO C57BL/6 mice display enhanced insulin sensitivity and reduced development of high-fat diet-induced insulin resistance. However, the pathophysiological roles of GM3 and related gangliosides in the central control of feeding and metabolism remain unclear. We found that a mouse model (KKAy GM3S KO) generated by KO of the GM3S gene in the yellow obese strain, KKAy, displayed significant amelioration of obese phenotype. Whereas KKAy mice were hyperphagic and developed severe obesity, KKAy GM3S KO mice had significantly lower body weight and food intake, and greater glucose and insulin tolerance. The hypothalamic response to intraperitoneal administration of leptin was greatly reduced in KKAy mice, but was retained in KKAy GM3S KO mice. In studies of a cultured mouse hypothalamic neuronal cell line, enhanced leptin-dependent phosphorylation of ERK was observed in GM3S-deficient cells. Furthermore, KKAy GM3S KO mice did show altered coat color, suggesting that GM3S is also involved in melanocortin signaling. Our findings, taken together, indicate that GM3-related gangliosides play key roles in leptin and melanocortin signaling.

Mass Spectrometry of Gangliosides.

This chapter describes protocols for mass spectrometry (MS) applied to the characterization of ganglioside structures and the determination of ganglioside contents. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are often used to ionize biological materials and this chapter covers three protocols for atmospheric pressure MALDI MS (AP-MALDI MS), liquid chromatography-ESI MS (LC-ESI MS), and LC-ESI MS with multiple reaction monitoring (MRM). Purified gangliosides were used in AP-MALDI MS analyses while crude preparations of gangliosides were subjected to LC-ESI MS and LC-ESI MS with MRM. The LC protocol includes conditions for both reversed-phase and normal-phase column chromatography.

Comparison of riboflavin-derived flavinium salts applied to catalytic H2O2 oxidations.

A series of flavinium salts, 5-ethylisoalloxazinium, 5-ethylalloxazinium, and 1,10-ethylene-bridged alloxazinium triflates, were prepared from commercially available riboflavin. This study presents a comparison between their optical and redox properties, and their catalytic activity in H2O2 oxidations of sulfide, tertiary amine, and cyclobutanone. Reflecting the difference between the π-conjugated ring structures, the flavinium salts displayed very different redox properties, with reduction potentials in the order of: 5-ethylisoalloxazinium > 5-ethylalloxazinium > 1,10-ethylene-bridged alloxazinium. A comparison of their catalytic activity revealed that 5-ethylisoalloxazinium triflate specifically oxidises sulfide and cyclobutanone, and 5-ethylalloxazinium triflate smoothly oxidises tertiary amine. 1,10-Bridged alloxazinium triflate, which can be readily obtained from riboflavin in large quantities, showed moderate catalytic activity for the H2O2 oxidation of sulfide and cyclobutanone.