Primitive Action Based Combined Task and Motion Planning for the Service Robot.

The need for combined task and motion planning (CTAMP) in robotics is well known as robotic technologies become more mature. The goal of CTAMP is to determine a proper sequence of a robot's actions based on symbolic and geometric reasoning. Because of the fundamental difference in symbolic and geometric reasoning, a CTAMP system often requires an interface module between the two reasoning modules. We propose a CTAMP system in which a symbolic action sequence is generated in task planning, and each action is verified geometrically in motion planning using the off-the-shelf planners and reasoners. The approach is that a set of action models is defined with PDDL in the interface module (action library) and the required information to each planner is automatically provided by the interface module. The proposed method was successfully implemented in three simulated experiments that involve manipulation tasks. According to our findings, the proposed method is effective in responding to changes in the environment and uncertainty with errors in recognition of the environment and the robot motion control.

Missense mutation of SPAST protein (I344K) results in loss of ATPase activity and prolonged the half-life, implicated in autosomal dominant hereditary spastic paraplegia.

The spastin protein (SPAST) contains an ATPase with diverse cellular activities (AAA) domain and regulates microtubule dynamics. Missense mutations of the SPAST gene are frequently detected in patients with hereditary spastic paraplegias (HSPs) and represent the main reason of loss of SPAST function; however, the pathogenicity of mutant SPAST is heterogeneous. Here, SPAST variant with an I344K mutation (I344K-SPAST) was identified in a Korean family with autosomal dominant-type HSP. We investigated the role of the I344K-SPAST in HSP to provide a therapeutic mechanism. The I344K-SPAST mutation prolonged the half-life of the protein compared to wild-type SPAST (WT-SPAST) in cells by modulating post-translational modifications for proteasomal degradation. I344K-SPAST was localized in microtubule but defective in microtubule severing and ATPase activity compared to WT-SPAST in vitro and in cells. Mutant M87 isoform harboring the same mutation with I344K-M1 SPAST also increased protein stability and loss of MT severing activity, but the pathogenicity was not stronger than I344K-M1 SPAST in neurite outgrowth. Overexpression of I344K-SPAST resulted in microtubule accumulation following inhibited neurite growth in neuroblastoma, neural progenitor cells and mouse primary cortical neurons. Conversely, these pathogenic effects of I344K-SPAST were reduced by overexpression of WT-M1 SPAST in a dose dependent manner since WT-SPAST could interact with I344K-SPAST. Our data therefore provide proof-of-concept that gene transfer of WT-M1 SPAST may serve as a valid therapeutic option for HSPs.

Turning behaviors of T cells climbing up ramp-like structures are regulated by myosin light chain kinase activity and lamellipodia formation.

T cells navigate diverse microenvironments to perform immune responses. Micro-scale topographical structures within the tissues, which may inherently exist in normal tissues or may be formed by inflammation or injury, can influence T cell migration, but how T cell migration is affected by such topographical structures have not been investigated. In this study, we fabricated ramp-like structures with a 5 µm height and various slopes, and observed T cells climbing up the ramp-like structures. T cells encountering the ramp-like structures exhibited MLC accumulation near head-tail junctions contacting the ramp-like structures, and made turns to the direction perpendicular to the ramp-like structures. Pharmacological study revealed that lamellipodia formation mediated by arp2/3 and contractility regulated by myosin light chain kinase (MLCK) were responsible for the intriguing turning behavior of T cells climbing the ramp-like structures. Arp2/3 or MLCK inhibition substantially reduced probability of T cells climbing sharp-edged ramp-like structures, indicating intriguing turning behavior of T cells mediated by lamellipodia formation and MLCK activity may be important for T cells to access inflamed or injured tissues with abrupt topographical changes.

Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo.

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial-mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

MicroRNA-150 modulates intracellular Ca 2+ levels in naïve CD8+ T cells by targeting TMEM20.

Regulation of intracellular Ca2+ signaling is a major determinant of CD8+ T cell responsiveness, but the mechanisms underlying this regulation of Ca2+ levels, especially in naïve CD8+ T cells, are not fully defined. Here, we showed that microRNA-150 (miR-150) controls intracellular Ca2+ levels in naïve CD8+ T cells required for activation by suppressing TMEM20, a negative regulator of Ca2+ extrusion. miR-150 deficiency increased TMEM20 expression, which resulted in increased intracellular Ca2+ levels in naïve CD8+ T cells. The subsequent increase in Ca2+ levels induced expression of anergy-inducing genes, such as Cbl-b, Egr2, and p27, through activation of NFAT1, as well as reduced cell proliferation, cytokine production, and the antitumor activity of CD8+ T cells upon antigenic stimulation. The anergy-promoting molecular milieu and function induced by miR-150 deficiency were rescued by reinstatement of miR-150. Additionally, knockdown of TMEM20 in miR-150-deficient naïve CD8+ T cells reduced intracellular Ca2+ levels. Our findings revealed that miR-150 play essential roles in controlling intracellular Ca2+ level and activation in naïve CD8+ T cells, which suggest a mechanism to overcome anergy induction by the regulation of intracellular Ca2+ levels.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

Geometrically controlled asymmetric division of CD4+ T cells studied by immunological synapse arrays.

Similar to stem cells, naïve T cells undergo asymmetric division following activation. While asymmetric division of T cells has been shown to be an important mechanism for the generation of lymphocyte fate diversity during immune responses, key factors that influence whether T cells will undergo symmetric or asymmetric divisions are not completely understood. Here, we utilized immunological synapse arrays (ISAs) to begin to dissect mechanisms of asymmetric T lymphocyte division. ISAs are protein micropatterned surfaces composed of two segregated regions, activation sites and adhesion fields. Activation sites are small spots presenting activation signals such as anti-CD3 and anti-CD28, and adhesion fields are the remaining regions surrounding activation sites immobilized with interintercel adhesion molecule 1 (ICAM-1). By varying the size and the distance between the activation sites and measuring the incidence of asymmetric cell divisions, we found that the distance between activation sites is an important regulator of asymmetric division. Further analysis revealed that more symmetric divisions occurred when two nascent daughter cells stably interacted with two distinct activation sites throughout and following cytokinesis. In contrast, more asymmetric divisions occurred when only one daughter cell remained anchored on an activation site while the other daughter became motile and moved away following cytokinesis. Together, these results indicate that TCR signaling events during cytokinesis may repolarize key molecules for asymmetric partitioning, suggesting the possibility that the density of antigen presenting cells that interact with T cells as they undergo cytokinesis may be a critical factor regulating asymmetric division in T cells.

Photopatterning with a printed transparency mask and a protein-friendly photoresist.

Microscope projection photolithography (MPP) based on a protein-friendly photoresist is a versatile tool for the fabrication of protein- and cell-micropatterned surfaces. Photomasks containing various features can be economically produced by printing features on transparency films. Features in photomasks are projected by the objective lens of a microscope, resulting in a significant reduction of the feature size to as small as ~1 µm, close to the practical limit of light-based microfabrication. A fluorescence microscope used in most biology labs can be used for the fabrication process with some modifications. Using such a microscope, multistep MPP can be readily performed with precise registration of each micropattern on transparency film masks. Here, we describe methods of the synthesis and characterization of a protein-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) and the setups of fluorescence microscopes and the MPP procedures. In addition, we describe the protocols used in the micropatterning of multiple lymphocytes and the dynamic micropatterning of adherent cells.

Dynamic modulation of small-sized multicellular clusters using a cell-friendly photoresist.

Dynamics of small-sized multicellular clusters is important for many biological processes including embryonic development and cancer metastasis. Previous methods to fabricate multicellular clusters depended on stochastic adhesion and proliferation of cells on defined areas of cell-adhering islands. This made precise control over the number of cells within multicellular clusters impossible. Variation in numbers may have minimal effects on the behavior of multicellular clusters composed of tens of cells but would have profound effects on groups with fewer than ten cells. Herein, we report a new dynamic cell micropatterning method using a cell-friendly photoresist film by multistep microscope projection photolithography. We first fabricated single cell arrays of partially spread cells. Then, by merging neighboring cells, we successfully fabricated multicellular clusters with precisely controlled number, composition, and geometry. Using this method, we generated multicellular clusters of Madin-Darby canine kidney cells with various numbers and initial geometries. Then, we systematically investigated the effect of multicellular cluster sizes and geometries on their motility behaviors. We found that the behavior of small-sized multicellular clusters was not sensitive to initial configurations but instead was determined by dynamic force balances among the cells. Initially, the multicellular clusters exhibited a rounded morphology and minimal translocation, probably due to contractility at the periphery of the clusters. For 2-cell and 4-cell clusters, single leaders emerged over time and entire groups aligned and comigrated as single supercells. Such coherent behavior did not occur in 8-cell clusters, indicating a critical group size led by a single leader may exist. The method developed in the study will be useful for the study of collective migration and multicellular dynamics.

Microfabricated platforms to modulate and monitor T cell synapse assembly.

Immunological synapse (IS) is a complex supramolecular structure formed at the interface between T cells and antigen presenting cells (APCs) during T cell antigen recognition. Microfabricated platforms have made great contributions to our understanding of the assembly dynamics and functional roles of the T cell synapses over the last decade. Here, we review three different types of microfabricated platforms developed to modulate and monitor the T cell synapse assembly. Firstly, multi-protein micropatterned surfaces presenting key ligands for T cell activation that can modulate the spatial distribution of receptors/signaling molecules in T cell synapses are described. Secondly, micropatterned supported bilayers that can modulate the dynamics of T cell receptor (TCR) microclusters are introduced. Lastly, T-APC pair arrays that allow for improved fluorescence live cell imaging are discussed.

Addressable micropatterning of multiple proteins and cells by microscope projection photolithography based on a protein friendly photoresist.

We report a new method for the micropatterning of multiple proteins and cells with micrometer-scale precision. Microscope projection photolithography based on a new protein-friendly photoresist, poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP), was used for the fabrication of multicomponent protein/cell arrays. Microscope projection lithography allows precise registration between multiple patterns as well as facile fabrication of microscale features. Thin films of PDMP became soluble in near-neutral physiological buffer solutions upon UV exposure and exhibited excellent resistance to protein adsorption and cell adhesion. By harnessing advantages in microscope projection photolithography and properties of PDMP thin films, we could successfully fabricate protein arrays composed of multiple proteins. Furthermore, we could extend this method for the patterning of two different types of immune cells for the potential study of immune cell interactions. This technique will in general be useful for protein chip fabrication and high-throughput cell-cell communication study.