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The dense extracellular matrix of connective tissues impedes cell migration and subsequent matrix formation at sites of injury. We recently employed transient nuclear softening via histone deacetylase inhibition with trichostatin A (TSA) treatment to overcome the stiff nuclear impediments to cell migration through dense tissues and electrospun matrices. Despite these positive findings, the long-term implications of transient nuclear softening on cell transcriptional phenotype and matrix formation capacity are unknown. To address this, we investigated the influence of transient TSA treatment on porcine meniscal cell behavior, beginning with the efficacy and reproducibility of transient TSA treatment on histone acetylation and chromatin remodeling in vitro and cell migration through native meniscus tissue. Within 3 days after cessation of transient TSA treatment, histone acetylation and chromatin remodeling returned to control levels. Following TSA treatment, endogenous cell migration through native meniscus tissue increased greater than 3-fold compared to controls. Importantly, meniscal cells completely restored their transcriptional phenotype and maintained their capacity to respond transcriptionally and functionally to a secondary pro-matrix stimuli (i.e., transforming growth factor β3) within 7 days after cessation of TSA treatment. Towards translation, we also showed the feasibility of biomaterial-delivered TSA to increase endogenous cell migration to a wound edge ex vivo. Together, this work defines the efficacy, reproducibility, safety, and feasibility of future translational approaches for nuclear softening to treat dense connective tissue injuries.

SUMMARYStiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers the hypermigration and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation, but the mechanism of this trigger is not known. Our analyses of whole-transcriptome microarray data sets from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology and from injured mouse femoral arteries revealed 80 genes that were differentially regulated (74 upregulated and 6 downregulated) relative to expression in control VSMCs cultured on soft hydrogels and in uninjured femoral arteries. A functional enrichment analysis revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Furthermore, we found that survivin, a member of the inhibitor of apoptosis protein family, mediates stiffness-sensitive cell cycling and proliferationin vivoandin vitroas determined by gene network and pathway analyses, RT-qPCR, and immunoblotting. The stiffness signal is mechanotransduced via FAK and Rac signaling to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Our findings indicate that survivin is necessary for VSMC cycling and proliferation and regulates stiffness-responsive phenotypes.

Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin (an inhibitor of apoptosis) is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs and mouse embryonic fibroblasts decreased the stiffness-mediated expression of ECM components implicated in arterial stiffness, namely, collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these proteins was upregulated by the overexpression of survivin in human VSMCs cultured on soft hydrogels. Atomic force microscopy analysis showed that suppressed or enhanced expression of survivin decreases or increases intracellular stiffness, respectively. These findings suggest a novel mechanism by which survivin modulates arterial stiffness.

Various cell aggregate culture technologies have been developed and actively applied to tissue engineering and organ-on-a-chip. However, the conventional culture technologies are labor-intensive, and their outcomes are highly user dependent. In addition, the technologies cannot be used to produce three-dimensional (3D) complex tissues. In this regard, 3D cell aggregate printing technology has attracted increased attention from many researchers owing to its 3D processability. The technology allows the fabrication of 3D freeform constructs using multiple types of cell aggregates in an automated manner. Technological advancement has resulted in the development of a printing technology with a high resolution of approximately 20 μm in 3D space. A high-speed printing technology that can print a cell aggregate in milliseconds has also been introduced. The developed aggregate printing technologies are being actively applied to produce various types of engineered tissues. Although various types of high-performance printing technologies have been developed, there are still some technical obstacles in the fabrication of engineered tissues that mimic the structure and function of native tissues. This review highlights the central importance and current technical level of 3D cell aggregate printing technology, and their applications to tissue/disease models, artificial tissues, and drug-screening platforms. The paper also discusses the remaining hurdles and future directions of the printing processes.

The meniscus plays a critical role in knee mechanical function but is commonly injured given its central load bearing role. In the adult, meniscus repair is limited, given the low number of endogenous cells, the density of the matrix, and the limited vascularity. Menisci are fibrocartilaginous tissues composed of a micro-/nano-fibrous extracellular matrix (ECM) and a mixture of chondrocyte-like and fibroblast-like cells. Here, we developed a fibrous scaffold system that consists of bioactive components (decellularized meniscus ECM (dME) within a poly(e-caprolactone) material) fashioned into a biomimetic morphology (via electrospinning) to support and enhance meniscus cell function and matrix production. This work supports that the incorporation of dME into synthetic nanofibers increased hydrophilicity of the scaffold, leading to enhanced meniscus cell spreading, proliferation, and fibrochondrogenic gene expression. This work identifies a new biomimetic scaffold for therapeutic strategies to substitute or replace injured meniscus tissue.STATEMENT OF SIGNIFICANCEIn this study, we show that a scaffold electrospun from a combination of synthetic materials and bovine decellularized meniscus ECM provides appropriate signals and a suitable template for meniscus fibrochondrocyte spreading, proliferation, and secretion of collagen and proteoglycans. Material characterization andin vitrocell studies support that this new bioactive material is susceptible to enzymatic digestion and supports meniscus-like tissue formation.

Changes in the micro-environment of fibrous connective tissue can lead to alterations in the phenotypes of tissue-resident cells, yet the underlying mechanisms are poorly understood. Here, by visualizing the dynamics of histone spatial reorganization in tenocytes and mesenchymal stromal cells from fibrous tissue of human donors via super-resolution microscopy, we show that physiological and pathological chemomechanical cues can directly regulate the spatial nanoscale organization and density of chromatin in these tissue-resident cell populations. Specifically, changes in substrate stiffness, altered oxygen tension and the presence of inflammatory signals drive chromatin relocalization and compaction into the nuclear boundary, mediated by the activity of the histone methyltransferase EZH2 and an intact cytoskeleton. In healthy cells, chemomechanically triggered changes in the spatial organization and density of chromatin are reversible and can be attenuated by dynamically stiffening the substrate. In diseased human cells, however, the link between mechanical or chemical inputs and chromatin remodelling is abrogated. Our findings suggest that aberrant chromatin organization in fibrous connective tissue may be a hallmark of disease progression that could be leveraged for therapeutic intervention.

Due to limited intrinsic healing capacity of the meniscus, meniscal injuries pose a significant clinical challenge. The most common method for treatment of damaged meniscal tissues, meniscectomy, leads to improper loading within the knee joint, which can increase the risk of osteoarthritis. Thus, there is a clinical need for the development of constructs for meniscal repair that better replicate meniscal tissue organization to improve load distributions and function over time. Advanced three-dimensional bioprinting technologies such as suspension bath bioprinting provide some key advantages, such as the ability to support the fabrication of complex structures using non-viscous bioinks. In this work, the suspension bath printing process is utilized to print anisotropic constructs with a unique bioink that contains embedded hydrogel fibers that align via shear stresses during printing. Constructs with and without fibers are printed and then cultured for up to 56 d in vitro in a custom clamping system. Printed constructs with fibers demonstrate increased cell and collagen alignment, as well as enhanced tensile moduli when compared to constructs printed without fibers. This work advances the use of biofabrication to develop anisotropic constructs that can be utilized for the repair of meniscal tissue.

To fundamentally solve the bottleneck of Von Neumann’s computing architecture, a neuromorphic thin-film transistor (NTFT) employing Pb(Zr, Ti)O3 (PZT) was investigated. The indium gallium zinc oxide (IGZO) channel back gate TFT structure was chosen to solve the diffusion of atoms that form a channel layer during the annealing process for crystallization of PZT. A post-deposition process with IGZO after annealing PZT and using an oxide-based material as a channel structure can minimize the diffusion phenomenon of junction materials and oxygen together, which leads to a high and reliable performance of the NTFT. The basic operations of synapses short-term memory (STM) and long-term memory (LTM) were also analyzed to confirm the application of a neuromorphic device. The high dielectric constant and polarization properties of Pb(Zr, Ti)O3 (PZT) allow the power consumption of spike signals used in spike dependent plasticity change to be reduced to 10 pJ. Moreover, a wide dynamic range of $\text{G}_{\mathrm {max}}/\text{G}_{\mathrm {min}} \cong ~1000$ was obtained, and the channel conductance was maintained over 40000 seconds. The optimized pulse achieved multi-level states (>32), which made the learning process efficient. This study verified that the PZT-TFT structure has a high potential and merits for neuromorphic devices.

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motilityin vitroand zebrafish intersegmental vessel (ISV) morphogenesisin vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migrationin vitroand ISV morphogenesisin vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kineticsin vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of myosin II activity to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

Tendons transmit mechanical forces between muscle and bone, and their biomechanical function requires high tensile strength, which is provided by highly organized collagen fibers. Tenocytes mainly drive tendon growth via extracellular matrix (ECM) production and organization. The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well established, partly due to the lack of a reliable in vitro system. Recent scaffold-free, cell-based tissue engineering approaches developed several unique in vitro tendon-like constructs. However, the application of these constructs is limited to the study of embryonic tendon development due to high cell density and immature matrix organization. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells and a differentially adherent growth channel. The 3D tendon constructs exhibited tissue maturation similar to the postnatal mouse tendon, including decreased cell density, increased thickness, and elongated cells between highly aligned extracellular matrix. The 3D tendon culture system is also feasible for genetic manipulation using adenovirus. Overall, the results suggest that the 3D tendon culture system using mouse tendon cells is a reliable in vitro system to study underlying biological mechanisms that regulate cellular and matrix maturation in postnatal tendon development.

A thin-film transistor (TFT) with a color filter function achieved by designing periodic nanohole arrays in the metal electrodes was studied. Since this metal electrode is not opaque but transparen...

Direct application of the tunneling mechanism into the conventional field-effect transistor structure only results in inefficient switching behavior and high leakage current. Here, we report a vertical metal–insulator–metal (MIM) tunneling transistor employing a floating electrode to achieve an efficient switching behavior and substantially low leakage current simultaneously. This switching method utilizes a tunneling mechanism transition between direct and Fowler–Nordheim tunneling by placing the floating electrode into a vertical tunneling channel. Engineering the electrical potential of the floating electrode with coplanar dual gates enables the efficient control of the tunneling mechanism transition. This particular arrangement of the gate and source/drain (no overlap) allows an extremely low gate leakage current (∼10–13 A). This value is significant in the tunneling transistor and promising for future electrical devices with low-power consumption. Furthermore, tunneling, whose operating principle is fundamentally different from the p–n junction and Schottky barrier in the Si transistor, has been proposed as a solution to tackle the issues such as high-frequency driving, power consumption, and so on. This structure is a strong candidate for ultrahigh-frequency driving because of its extremely low structural capacitance (∼1.5 zF). An additional electrical status can be shown to generate ternary or more statuses in a single transistor. Its electrical performance is expected to not only harmonize with THz communication systems, control process units, and high-speed electrical systems but also contributes a degree of integration. Moreover, the simple and low-temperature fabrication process of the vertical MIM tunneling transistor is advantageous for low-cost electrical devices and flexible electronic applications.

Background Oropharyngeal cancer (OPC) exhibits diverse immunological properties; however, their implications for immunotherapy are unknown. Methods We analysed 37 surgically resected and nine recurrent or metastatic anti-programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1)-treated OPC tumours. OPCs were classified into immune-rich (IR), mesenchymal (MS) and xenobiotic (XB) subtypes based on RNA-sequencing data. Results All IR type tumours were human papillomavirus (HPV) positive, most XB types were HPV negative, and MS types showed mixed HPV status. The IR type showed an enriched T cell exhaustion signature with PD-1+ CD8+ T cells and type I macrophages infiltrating the tumour nest on multiplex immunohistochemistry. The MS type showed an exclusion of CD8+ T cells from the tumour nest and high MS and tumour growth factor-β signatures. The XB type showed scant CD8+ T cell infiltration and focal CD73 expression. The IR type was associated with a favourable response signature during anti-PD-1/PD-L1 therapy and showed a high APOBEC mutation signature, whereas the MS and XB types showed resistance signature upregulation. Among anti-PD-1/PD-L1-treated OPC patients, the IR type showed a favourable clinical response (3/4 patients), whereas the XB type showed early progression (3/3 patients). Conclusion Our analysis classified OPCs into three subtypes with distinct immune microenvironments that are potentially related to the response to anti-PD-1/PD-L1 therapy.

Bacterial infection of a wound is a major complication that can significantly delay proper healing and even necessitate surgical debridement and other complications. Conventional non-woven fabric dressings, including gauzes, bandages and cotton wools, often fail in treating wound infections in a timely manner due to their passive release mechanism of antibiotics. Here, we propose adhesive mechanically-activated microcapsules (MAMCs) capable of strongly adhering to a fibrous matrix to achieve a self-regulated release of antibiotics upon uniaxial stretching of non-woven fabric dressings. To achieve this, a uniform population of polydopamine (PDA)-coated MAMCs (PDA-MAMCs) are prepared using a microfluidics technique and subsequent oxidative dopamine polymerization. The PDA-MAMC allows for robust mechano-activation within the fibrous network through high retention and effective transmission of mechanical force under stretching. By validating the potential of a PDA-MAMCs-laden gauze to release antibiotics in a tensile strain-dependent manner, we demonstrate that PDA-MAMCs can be successfully incorporated into a woven material and create a smart wound dressing for control of bacterial infections. This new mechano-activatable delivery approach will open up a new avenue for a stretch-triggered, on-demand release of therapeutic cargos in skin-mountable or wearable biomedical devices.

Process and die design of cold forging for the asymmetric part, engine mount adapter has been studied. Forging of the asymmetric part frequently causes die failure because of the high forging load and local stress concentration of the die. Thus, performing process design of cold forging to minimize forging load is required. Preform for the engine mount adapter was chosen based on the forging load and filling rate of forgings by the finite element analysis. In the die design, number of stress rings, interface radius, and relative interference were investigated in several cases with maximum principle stress by the finite element method. The shape of the die was determined by comparing the load changing the radius of the flange area. Also, the life of the designed die was calculated using the Goodman theory by cyclic fatigue loading. As a result, it was confirmed that the calculation life and results of the test were identical. In this study, it is verified that stress concentration and fatigue life should be considered simultaneously in the design of cold forging die for the asymmetric part.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extra-skeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain-of-function mutation (R206H; c.617G>A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic-induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to biochemical ligand-receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1(R206H/+) mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1(R206H/+) cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1(R206H/+) cells is similar to the morphology of control Acvr1(+/+) cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1(R206H/+) cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers as compared to Acvr1(+/+) cells. We also identified increased YAP1 nuclear localization in Acvr1(R206H/+) cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish RhoA and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of Acvr1(R206H/+) progenitor cells to chondro/osteogenic lineages.

Hot deformation behavior of S32760 super austenitic stainless steel was studied in the temperature range of 950 ∼ 1250 °C and strain rate range of 0.1∼10 s-1 employing Gleeble 3800 equipment. The flow stress was modeled using Arrhenius equation and Zener-Hollomon parameter(Z). The microstructures of the specimens under various conditions were investigated, and dynamic recrystallization of austenite, dynamic recovery of ferrite and phase contents varied with temperature and strain rate. The difference of flow stress between experiment and constitutive equation was explained by the phase contents. Finite element analysis was performed to calculate forging load under the same conditions of compression test using modeled flow stress. It was validated that forging load can be predicted precisely with Arrhenius equation.

PURPOSE Standard treatment for cases of non-small cell lung cancer (NSCLC) exhibiting acquired drug resistance includes tumor rebiopsy, epidermal growth factor receptor (EGFR) mutation testing (e.g., for T790M mutations), and the subsequent administration of third-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, rebiopsies are typically invasive, costly, and occasionally not feasible. Therefore, the present study aimed to assess rebiopsy procedures by analyzing real-world data collected by the ASTRIS study of patients with resistant NSCLC. MATERIALS AND METHODS The present study used statistical models to evaluate data collected by the ASTRIS trial (NCT02474355) conducted at Yonsei Cancer Center, including the rebiopsy success rate, incidence of T790M mutations in collected tissue and plasma samples, and association of administered osimertinib treatment efficacy. RESULTS In a total of 188 screened patients, 112 underwent rebiopsy. An adequate tumor specimen was obtained in 95 of these patients, the greatest majority of whom (43.8%) were subjected to bronchoscopy. T790M mutations were detected in 53.3% of successfully EGFR-tested rebiopsy samples. A total of 88 patients received osimertinib treatment, and the objective response rate and median progression-free survival time was 44.3% and 32.7 weeks, respectively, among the treated patients overall, but 57.8% and 45.0 weeks, and 35.2% and 20.4 weeks among patients who exhibited T790M-positive tissue (n=45) and plasma (n=54) samples, respectively. CONCLUSION Approximately 60% of patients in the analyzed real-world cohort were eligible for tissue rebiopsy upon NSCLC progression. Osimertinib activity was higher in patients in whom T790M mutations were detected in tissues rather than in plasma samples.

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1-lineage cells. Normally, meniscal tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh agonist right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.

Microscale changes in tissue environment are translated to changes in cell behavior and phenotype, yet the mechanisms behind how these phenotypic changes occur are poorly understood. Here, we describe and model chromatin, which stores genetic information within the cell nucleus, as a dynamic nanomaterial whose configuration is modulated by chemo-mechanical cues in the microenvironment. Our findings indicate that physiologic chemo-mechanical cues can directly regulate chromatin architecture in progenitor cell populations. Via direct experimental observation and modeling that incorporates phase transitions and histone methylation kinetics, we demonstrate that soft environmental cues drive chromatin relocalization to the nuclear boundary and compaction. Conversely, dynamic stiffening attenuates these changes. Interestingly, in diseased human fibrous tissue cells, this link between mechanical inputs and chromatin nano-scale remodeling is abrogated. These data indicate that chromatin dynamics and plasticity may be hallmarks of disease progression and targets for therapeutic intervention.

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using Gli1-CreER tdTomato mice, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1+ cells. Normally, the tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh activator right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.

Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis.

Fermented foods have several advantages, including increased nutritional value, improved bioavailability, and functional health properties. We examined that these outcomes were also observed in fermented mixed grains (FMG) containing wheat germ, wheat bran, oats, brown rice, barley, quinoa, and lentils following solid-state fermentation (SSF) by Bacillus amyloliquefaciens 245. The metabolic profile during fermentation was screened using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). The amino acids were quantitatively measured for the validation of the changes in metabolites. The activity of enzymes (e.g., amylase, protease, and fibrinolysis) and antioxidant capacity was also assessed to elucidate the functionality of FMG. The essential amino acid contents gradually increased as fermentation progressed. As the metabolites involved in the urea cycle and polyamine pathway were changed by fermentation, arginine was used as a substance to produce citrulline, ornithine, and agmatine. FMG showed dramatic increases in enzyme activity. FMG incubated for 36 h also displayed higher total phenolic contents and free radical scavenging ability than MG. The data suggest that FMG produced by Bacillus amyloliquefaciens 245 possess improved nutritional and functional quality, leading to their potential use as dietary supplements.

In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2–4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-a-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca2+]i, in both physiologic and osmotically evoked fluidic environments in situ, illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca2+]i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration.

Exogenous mechanical cues are transmitted from the extracellular matrix to the nuclear envelope (NE), where mechanical stress on the NE mediates shuttling of transcription factors and other signaling cascades that dictate downstream cellular behavior and fate decisions. To systematically study how nuclear morphology can change across various physiologic microenvironmental contexts, we cultured mesenchymal progenitor cells (MSCs) in engineered 2D and 3D hyaluronic acid hydrogel systems. Across multiple contexts we observed highly 'wrinkled' nuclear envelopes, and subsequently developed a quantitative single-cell imaging metric to better evaluate how wrinkles in the nuclear envelope relate to progenitor cell mechanotransduction. We determined that in soft 2D environments the NE is predominately wrinkled, and that increases in cellular mechanosensing (indicated by cellular spreading, adhesion complex growth, and nuclear localization of YAP/TAZ) occurred only in absence of nuclear envelope wrinkling. Conversely, in 3D hydrogel and tissue contexts, we found NE wrinkling occurred along with increased YAP/TAZ nuclear localization. We further determined that these NE wrinkles in 3D were largely generated by actin impingement, and compared to other nuclear morphometrics, the degree of nuclear wrinkling showed the greatest correlation with nuclear YAP/TAZ localization. These findings suggest that the degree of nuclear envelope wrinkling can predict mechanotransduction state in mesenchymal progenitor cells and highlights the differential mechanisms of NE stress generation operative in 2D and 3D microenvironmental contexts.

Objectives GX-H9 is a long-acting form of recombinant human GH under clinical development for both adults and children with GHD. In this report, 24-month efficacy and safety of once weekly and every other week (EOW) administration of GX-H9 were evaluated, in addition to Genotropin® switch-ability to GX-H9 after 12-month of treatment. Methods Subjects were randomly assigned to receive either one of three doses of GX-H9 (0.8 mg/kg/week, 1.2 mg/kg/week or 2.4 mg/kg every other week) or 0.03 mg/kg/day of Genotropin®. Treatment duration is 24-month for all patients in GX-H9 arms while patients in Genotropin® arm were re-randomized to one of three doses of GX-H9 at the completion of the first 12-month of treatment. Doses of GX-H9 were adjusted throughout the treatment period whenever necessary, based on IGF-1 levels. Results Out of 56 randomized, 54 received either GX-H9 or Genotropin®. Fifty subjects completed the 12-month treatment period. Of 50, 45 subjects completed the next 12-month, comprising 33 patients from GX-H9 and 12 patients who switched from Genotropin®. First year/second year mean±SD annualized height velocity (aHV) for 0.8 mg/kg/week, 1.2 mg/kg/week or 2.4 mg/kg every other week of GX-H9 were 10.50±2.54/9.14±1.96, 11.76±1.96/9.88±1.92 and 11.03±2.92/9.72±1.90 cm/year, respectively. First year mean±SD aHV for Genotropin® was 9.14±3.09 cm/year. Patients switched to one of the three doses of GX-H9 in the second year showed comparable aHV in the second year (8.73±2.69/7.60±0.90/9.13±1.07 cm/year for 0.8 mg/kg/week, 1.2 mg/kg/week and 2.4 mg/kg/EOW GX-H9, respectively). No significant slow-down of the growth was observed in the second year from patients who received GX-H9 throughout and patients who switched from Genotropin®. Mean change in height SDS after 12 months/24 months of GX-H9 treatment throughout from baseline treatment improved continuously (+1.10/+1.61 and +1.31/+1.89 and +1.15/+1.69 for 0.8 mg/kg/week, 1.2 mg/kg/week and 2.4 mg/kg EOW GX-H9, respectively). First year mean change in height SDS for Genotropin® was +0.92 SDS, and showed comparable improvement in height SDS after switching to GX-H9 weekly arms (+0.76 and +0.79 SDS for 0.8 mg/kg/week and 1.2 mg/kg/week, respectively). Most treatment-emergent adverse events were evaluated as unrelated to the study drug and were mild or moderate in severity. No new safety concerns were observed throughout 24 months of long-term GX-H9 treatment or after switching to GX-H9 from Genotropin®.Conclusions Growth response and safety profile of GX-H9 in children with GHD is comparable to those of daily GH, achieving robust growth rates after 24-month treatment. Subjects switched from Genotropin® in the second year, also showed substantial catch-up growth indicated by improvement in height SDS. GX-H9 has a unique potential to be a convenient long-term GH providing not only weekly but also twice-monthly treatment.

The effects of micro-hole arrays in the thin metal films were studied as a method to release bending stress in flexible electrodes and flexible thin film transistors (TFTs). Interest in flexible electronics is increasing, and many approaches have been suggested to solve the issue of the electrical failure of electrodes or electrical components such as TFTs after repeated bending. Here, we demonstrate a micro-hole array structure as a common solution to release bending stress. Although micro-size cracks were generated and propagated from the hole edges, the cracks stopped within a certain range when enough stress was released. Moreover, since the crack sites were predictable and controllable, a fatal electrical breakdown in a conductive layer such as a metal electrode or the semiconducting junction of a TFT can be prevented by specifically arranging the hole arrays. Thin film layers fabricated without holes suffered an electrical breakdown due to random crack propagation during bending tests. Aluminum thin film electrodes prepared with arrays of 3 μm diameter holes and 25% hole area showed excellent durability after 300,000 bending cycles. The change in resistance was below 3%. The electrical characteristics of an a-IGZO TFT with the micro-hole structure were almost equivalent to a standard a-IGZO TFT. After 10,000 bending cycles, ION and the ratio of ION/IOFF remained >107 A and ∼107, respectively. Since the effective hole diameter is micrometer in size, fabrication does not require additional process steps or expensive process equipment. Therefore, the approach can be an important way to enhance the reliability of various electrical devices in flexible and wearable applications.

SUMMARYAtherosclerosis and vascular injury are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMCs would advance the effort to treat vascular disease. However, the response to treatments aimed at VSMCs is often different among patients with the same disease condition, suggesting patient-specific heterogeneity in VSMCs. Here, we present an experimental and computational method called HETEROID (Heterogeneous Spheroid), which examines the heterogeneity of the responses to drug treatments at the single-spheroid level by combining a VSMC spheroid model and machine learning (ML) analysis. First, we established a VSMC spheroid model that mimics neointima formation induced by atherosclerosis and vascular injury. We found that FAK-Rac/Rho, but not Cdc42, pathways regulate the VSMC spheroid formation through N-cadherin. Then, to identify the morphological subpopulations of drug-perturbed spheroids, we used an ML framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our ML approach reveals that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect the spheroid morphology, suggesting there exist multiple distinct pathways governing VSMC spheroid formation. Overall, our HETEROID pipeline enables detailed quantitative characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis of various drug treatments.

The meniscus plays a critical role in knee mechanical function but is commonly injured given its central load bearing role. In the adult, meniscus repair is limited, given the low number of endogenous cells, the density of the matrix, and the limited vascularity. Menisci are fibrocartilaginous tissues composed of a micro-/nano- fibrous extracellular matrix (ECM) and a mixture of chondrocyte-like and fibroblast-like cells. Here, we developed a fibrous scaffold system that consists of bioactive components (decellularized meniscus ECM (dME) within a poly(e-caprolactone) material) fashioned into a biomimetic morphology (via electrospinning) to support and enhance meniscus cell function and matrix production. This work supports that the incorporation of dME into synthetic nanofibers increased hydrophilicity of the scaffold, leading to enhanced meniscus cell spreading, proliferation, and fibrochondrogenic gene expression. This work identifies a new biomimetic scaffold for therapeutic strategies to substitute or replace injured meniscus tissue.

Background: We retrospectively investigated the treatment outcomes of second-line treatment with pazopanib or gemcitabine/docetaxel in patients with advanced soft tissue sarcoma (STS). Methods: Ninety-one patients who were treated with pazopanib or gemcitabine/docetaxel for advanced STS between 1995 and 2015 were analyzed. Results: Forty-six and 45 patients received pazopanib and gemcitabine/docetaxel, respectively. The median progression-free survival for the group treated with pazopanib was 4.5 months compared with 3.0 months for the gemcitabine/docetaxel group (p = 0.593). The median overall survival for the group treated with pazopanib was 12.6 months compared with 14.2 months for the gemcitabine/docetaxel group (p = 0.362). The overall response rates (ORRs) were 6.5 and 26.7% in the pazopanib and gemcitabine/docetaxel groups, respectively. The following parameters had ORRs favoring gemcitabine/docetaxel: age ≥50 years (31.6 vs. 2.9%, p = 0.006), histologic grade 1–2 (40.9 vs. 0%, p = 0.001), and poor first-line treatment response (23.3 vs. 3.0%, p = 0.022). Gemcitabine/docetaxel was associated with better ORRs for the following histologic subtypes: leiomyosarcoma (p = 0.624), malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma (p = 0.055), and angiosarcoma (p = 0.182). However, the ORR of synovial sarcoma favored pazopanib (p = 0.99). Conclusions: The efficacies of pazopanib and gemcitabine/docetaxel as second-line treatments after doxorubicin or ifosfamide failure differed among clinical and histologic subgroups and appeared to facilitate a more personalized treatment approach for advanced STS.