Vaccinia-related kinase 2 modulates role of dysbindin by regulating protein stability.

Vaccinia-related kinase 2 (VRK2) is a serine/threonine kinase that belongs to the casein kinase 1 family. VRK2 has long been known for its relationship with neurodegenerative disorders such as schizophrenia. However, the role of VRK2 and the substrates associated with it are unknown. Dysbindin is known as one of the strong risk factors for schizophrenia. The expression of dysbindin is indeed significantly reduced in schizophrenia patients. Moreover, dysbindin is involved in neurite outgrowth and regulation of NMDA receptor signaling. Here, we first identified dysbindin as a novel interacting protein of VRK2 through immunoprecipitation. We hypothesized that dysbindin is phosphorylated by VRK2 and further that this phosphorylation plays an important role in the function of dysbindin. We show that VRK2 phosphorylates Ser 297 and Ser 299 of dysbindin using in vitro kinase assay. In addition, we found that VRK2-mediated phosphorylation of dysbindin enhanced ubiquitination of dysbindin and consequently resulted in the decrease in its protein stability through western blotting. Over-expression of VRK2 in human neuroblastoma (SH-SY5Y) cells reduced neurite outgrowth induced by retinoic acid. Furthermore, a phosphomimetic mutant of dysbindin alleviated neurite outgrowth and affected surface expression of N-methyl-d-aspartate 2A, a subunit of NMDA receptor in mouse hippocampal neurons. Together, our work reveals the regulation of dysbindin by VRK2, providing the association of these two proteins, which are commonly implicated in schizophrenia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Fabrication of In Vitro Cancer Microtissue Array on Fibroblast-Layered Nanofibrous Membrane by Inkjet Printing.

In general, a drug candidate is evaluated using 2D-cultured cancer cells followed by an animal model. Despite successful preclinical testing, however, most drugs that enter human clinical trials fail. The high failure rates are mainly caused by incompatibility between the responses of the current models and humans. Here, we fabricated a cancer microtissue array in a multi-well format that exhibits heterogeneous and batch-to-batch structure by continuous deposition of collagen-suspended Hela cells on a fibroblast-layered nanofibrous membrane via inkjet printing. Expression of both Matrix Metalloproteinase 2 (MMP2) and Matrix Metalloproteinase 9 (MMP9) was higher in cancer microtissues than in fibroblast-free microtissues. The fabricated microtissues were treated with an anticancer drug, and high drug resistance to doxorubicin occurred in cancer microtissues but not in fibroblast-free microtissues. These results introduce an inkjet printing fabrication method for cancer microtissue arrays, which can be used for various applications such as early drug screening and gradual 3D cancer studies.

Fabrication of a nanofibrous mat with a human skin pattern.

A number of studies on skin tissue regeneration and wound healing have been conducted. Electrospun nanofibers have numerous advantages for use in wound healing dressings. Here, we present an electrospinning method for alteration of the surface morphological properties of electrospun mats because most previous studies focused on the materials used or the introduction of bioactive healing agents. In this study, a micromachined human skin pattern mold was used as a collector in an electrospinning setup to replicate the pattern onto the surface of the electrospun mat. We demonstrated the successful fabrication of a nanofibrous mat with a human skin pattern. To verify its suitability for wound healing, a 14-day in vitro cell culture was carried out. The results indicated that the fabricated mat not only induces equivalent cell viability to the conventional electrospun mat, but also exhibits guidance of cells along the skin pattern without significant deterioration of pattern geometry.

Spatiotemporal Niche Separation among Passeriformes in the Halla Mountain Wetland of Jeju, Republic of Korea: Insights from Camera Trap Data.

This study analyzed 5322 camera trap photographs from Halla Mountain Wetland, documenting 1427 independent bird sightings of 26 families and 49 species of Passeriformes. Key observations include morning activities in Cyanoptila cyanomelana and Horornis canturians and afternoon activity in Muscicapa dauurica and Phoenicurus auroreus. Wetlands were significantly preferred (P_i = 0.398) despite their smaller area, contrasting with underutilized grasslands (P_i = 0.181). Seasonal activity variations were notable, with overlap coefficients ranging from 0.08 to 0.81 across species, indicating diverse strategies in resource utilization and thermoregulation. Population density was found to be a critical factor in habitat usage, with high-density species showing more consistent activity patterns. The study's results demonstrate the ecological adaptability of Passeriformes in the Halla Mountain Wetland while highlighting the limitations of camera trapping methods. These limitations include their fixed field of view and intermittent recording capability, which may not fully capture the spectrum of complex avian behaviors. This research underlines the need for future studies integrating various methodologies, such as direct observation and acoustic monitoring, to gain a more comprehensive understanding of avian ecology.

In Vivo Biophysical-stimulation Platform Applied to Rodent Calvarial Bone-defect Models.

Biophysical strain has been applied widely for bone regeneration. However, application of low-magnitude strains to cells on small-thickness scaffolds is problematic, especially in rodent calvarial defect models, because general translation systems have limitations in terms of generating low-magnitude smooth signals. To overcome these limitations, we developed an in vitro biophysical-stimulation platform for stimulation of cells on small-thickness scaffolds for rodent calvarial bone defects. The customized flexure-based translational nanoactuator enables generation of low-magnitude smooth signals at the subnano- to micrometer-scale. This nanoactuator, which is equipped with a piezoelectric actuator, is suitable for biological applications because it can generate friction-free motion with a high resolution. Moreover, its operation without wear or deterioration eliminates contamination factors in cell culture environments. The developed in vitro biophysical-stimulation platform using these nanoactuators showed predictable operational characteristics. Also, a few-micrometer sinusoidal signal was generated successfully without any distortion. Three-dimensional scaffolds fitting the critical-size rat calvarial defect model were fabricated using poly(caprolactone), poly(lactic-co-glycolic acid), and tricalcium phosphate. Runt-related transcription factor 2 expression was increased upon stimulation of human adipose-derived stem cells (ASCs) on these scaffolds were stimulated in the in vitro biophysical-stimulation platform. Additionally, the use of this platform resulted in up-regulation of alkaline phosphate, osteopontin, and osterix expression compared to the non-stimulated group. These preliminary in vitro results suggest that the biophysical environment provided by the in vitro biophysical-stimulation platform influences the osteogenic differentiation of ASCs.

Microscale diffusion measurements and simulation of a scaffold with a permeable strut.

Electrospun nanofibrous structures provide good performance to scaffolds in tissue engineering. We measured the local diffusion coefficients of 3-kDa FITC-dextran in line patterns of electrospun nanofibrous structures fabricated by the direct-write electrospinning (DWES) technique using the fluorescence recovery after photobleaching (FRAP) method. No significant differences were detected between DWES line patterns fabricated with polymer supplied at flow rates of 0.1 and 0.5 mL/h. The oxygen diffusion coefficients of samples were estimated to be ~92%-94% of the oxygen diffusion coefficient in water based on the measured diffusion coefficient of 3-kDa FITC-dextran. We also simulated cell growth and distribution within spatially patterned scaffolds with struts consisting of either oxygen-permeable or non-permeable material. The permeable strut scaffolds exhibited enhanced cell growth. Saturated depths at which cells could grow to confluence were 15% deeper for the permeable strut scaffolds than for the non-permeable strut scaffold.

Fabrication of patterned nanofibrous mats using direct-write electrospinning.

Due to the numerous advantages of nanofibers, there is a strong demand in various fields for nanofibrous structures fabricated by electrospinning. However, the process is currently beset by troublesome limitations with respect to geometric and morphological control of electrospun nanofibrous mats. This study presents a direct-write electrospinning process and apparatus with improved focusing and scanning functionalities for the fabrication of various patterned thick mats and nanofibrous patterns with high geometric fidelity, supported by a number of experimental results. Consequently, various patterned nanofibrous mats were fabricated using the developed method. Additionally, the fabricated mat was successfully used for cell patterning as a bioengineering application. The proposed method is expected to significantly improve the properties and functionalities of nanofibrous mats in a variety of applications.

Molecular Evidence Reveals the Sympatric Distribution of Cervus nippon yakushimae and Cervus nippon taiouanus on Jeju Island, South Korea.

Non-native species threaten native ecosystems and species, particularly on islands where rates of endemism and vulnerability to threats are high. Understanding species invasion will aid in providing insights into ecological and evolutionary processes. To identify the non-native sika deer (Cervus nippon) population in Jeju, South Korea, and their phylogenetic affinities, we collected tissue samples from roadkill and the World Natural Heritage Headquarters in Jeju. Mitochondrial DNA cytochrome B (CytB) gene sequences were analyzed to determine two distinct CytB haplotypes. Phylogenetic analysis using maximum likelihood tree revealed two haplotypes of CytB clustered into two different groups representing two subspecies: C. n. yakushimae, native to Japan, and C. n. taiouanus, native to Taiwan. The tentative divergence time between the two subspecies was estimated at 1.81 million years. Our study confirmed that the two subspecies of sika deer are sympatric in the natural ecosystem of Jeju Island. This study provides valuable information to help government and conservation agencies understand alien species and determine control policies for conserving native biodiversity in South Korea.

Synthetic PPAR Agonist DTMB Alleviates Alzheimer's Disease Pathology by Inhibition of Chronic Microglial Inflammation in 5xFAD Mice.

Abnormal productions of amyloid beta (Aß) plaque and chronic neuroinflammation are commonly observed in the brain of patients with Alzheimer's disease, and both of which induce neuronal cell death, loss of memory, and cognitive dysfunction. However, many of the drugs targeting the production of Aß peptides have been unsuccessful in treating Alzheimer's disease. In this study, we identified synthetic novel peroxisome proliferator-activating receptor (PPAR) agonist, DTMB, which can ameliorate the chronic inflammation and Aß pathological progression of Alzheimer's disease. We discovered that DTMB attenuated the proinflammatory cytokine production of microglia by reducing the protein level of NF-κB. DTMB also improved the learning and memory defects and reduced the amount of Aß plaque in the brain of 5xFAD mice. This reduction in Aß pathology was attributed to the changes in gliosis and chronic inflammation level. Additionally, bulk RNA-sequencing showed that genes related to inflammation and cognitive function were changed in the hippocampus and cortex of DTMB-treated mice. Our findings demonstrate that DTMB has the potential to be a novel therapeutic agent for Alzheimer's disease.

A transparent polymeric flexure-hinge nanopositioner, actuated by a piezoelectric stack actuator.

Nanopositioning using piezoelectric actuation and a flexure mechanism is one of most common methods for nanometre-scale positioning. Generally, flexure mechanism nanopositioners have been made from metal. Thus, their application to various environments needs careful consideration with regard to corrosion and circumference interference. In this study, we propose the concept of a chip-like polymeric flexure-based nanopositioner equipped with piezoelectric actuation. In its design, motion performance was predicted using finite element analysis of deformation and stress, and injection mouldability was considered through an injection moulding simulation to allow for fabrication by injection moulding. A cyclic olefin copolymer nanopositioner was fabricated using a mesoscale injection moulding process. Experiments demonstrated that the developed nanopositioner had a travel range of 15 µm with high linearity and it could be successfully controlled by a proportional-integral-derivative (PID) algorithm including a low-pass filter with a root mean square control error of 3 nm.

Fabrication of Thermochromic Membrane and Its Characteristics for Fever Detection.

Body temperature is an important indicator of the health status of the human body. Thus, numerous studies have been conducted in various fields to measure body temperature. In this study, a biocompatible thermochromic membrane that changes its color when the temperature becomes higher than the transition temperature for thermochromism was fabricated using an extrusion-based three-dimensional printing process. The printing material was prepared by mixing a thermochromic pigment and a thermoplastic polymer in various ratios. The effects of mixing ratio on the various properties of the fabricated membranes were experimentally investigated. It is presented that the fabricated lattice membrane had excellent thermochromic reaction, which was experimentally evaluated using a measurement of color brightness. The pigment content affected the diameter and surface morphology of the printed filament. The elastic modulus decreased, and thermochromic response became faster as the pigment concentration increased. Subsequently, a patch for fever detection was developed and then attached to the skin to demonstrate its color change according to body temperature. Results show that the fabricated thermochromic patch could be successfully applied to fever detection.

hnRNP K Supports High-Amplitude D Site-Binding Protein mRNA (Dbp mRNA) Oscillation To Sustain Circadian Rhythms.

Circadian gene expression is defined by the gene-specific phase and amplitude of daily oscillations in mRNA and protein levels. D site-binding protein mRNA (Dbp mRNA) shows high-amplitude oscillation; however, the underlying mechanism remains elusive. Here, we demonstrate that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key regulator that activates Dbp transcription via the poly(C) motif within its proximal promoter. Biochemical analyses identified hnRNP K as a specific protein that directly associates with the poly(C) motif in vitro Interestingly, we further confirmed the rhythmic binding of endogenous hnRNP K within the Dbp promoter through chromatin immunoprecipitation as well as the cycling expression of hnRNP K. Finally, knockdown of hnRNP K decreased mRNA oscillation in both Dbp and Dbp-dependent clock genes. Taken together, our results show rhythmic protein expression of hnRNP K and provide new insights into its function as a transcriptional amplifier of Dbp.

Identification of sagging in melt-electrospinning of microfiber scaffolds.

Melt-electrospinning is a cost-effective and flexible process to fabricate micro-scaled polymeric fibers. Melt-electrospun microfiber structures have been receiving considerable attention from various fields due to their numerous advantages. However, the application of melt-electrospinning is limited by various factors, such as the sagging behavior and unstable whipping motion of microfibers. Here, we presented an experimental approach called beam bridge test to identify the sagging behavior of melt-electrospun microfibers for preparing 3D lattice structures with controllable architecture and well-defined pores in transverse direction. Consequently, the sagging behavior of melt-electrospun microfibers could be identified in a systematic manner. Moreover, the melt-electrospun 3D microfiber lattice structures with various grid sizes had sagging, which agreed well with the beam bridge test results. In addition, fibroblast cells (NIH-3T3) were cultured on the fabricated 3D microfiber lattice structures with various grid sizes. Cell culture results indicated that the cell growth was considerably influenced by microfiber sagging and the grid size of lattice structures. Also it was shown that the cell population for location could be controlled.

A Microfluidic Chip Embracing a Nanofiber Scaffold for 3D Cell Culture and Real-Time Monitoring.

Recently, three-dimensional (3D) cell culture and tissue-on-a-chip application have attracted attention because of increasing demand from the industries and their potential to replace conventional two-dimensional culture and animal tests. As a result, numerous studies on 3D in-vitro cell culture and microfluidic chip have been conducted. In this study, a microfluidic chip embracing a nanofiber scaffold is presented. A electrospun nanofiber scaffold can provide 3D cell culture conditions to a microfluidic chip environment, and its perfusion method in the chip can allow real-time monitoring of cell status based on the conditioned culture medium. To justify the applicability of the developed chip to 3D cell culture and real-time monitoring, HepG2 cells were cultured in the chip for 14 days. Results demonstrated that the cells were successfully cultured with 3D culture-specific-morphology in the chip, and their albumin and alpha-fetoprotein production was monitored in real-time for 14 days.

hnRNP Q Regulates Internal Ribosome Entry Site-Mediated fmr1 Translation in Neurons.

Fragile X syndrome (FXS) caused by loss of fragile X mental retardation protein (FMRP), is the most common cause of inherited intellectual disability. Numerous studies show that FMRP is an RNA binding protein that regulates translation of its binding targets and plays key roles in neuronal functions. However, the regulatory mechanism for FMRP expression is incompletely understood. Conflicting results regarding internal ribosome entry site (IRES)-mediated fmr1 translation have been reported. Here, we unambiguously demonstrate that the fmr1 gene, which encodes FMRP, exploits both IRES-mediated translation and canonical cap-dependent translation. Furthermore, we find that heterogeneous nuclear ribonucleoprotein Q (hnRNP Q) acts as an IRES-transacting factor (ITAF) for IRES-mediated fmr1 translation in neurons. We also show that semaphorin 3A (Sema3A)-induced axonal growth cone collapse is due to upregulation of hnRNP Q and subsequent IRES-mediated expression of FMRP. These data elucidate the regulatory mechanism of FMRP expression and its role in axonal growth cone collapse.

A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy.

Patient-specific ex vivo models of human tumours that recapitulate the pathological characteristics and complex ecology of native tumours could help determine the most appropriate cancer treatment for individual patients. Here, we show that bioprinted reconstituted glioblastoma tumours consisting of patient-derived tumour cells, vascular endothelial cells and decellularized extracellular matrix from brain tissue in a compartmentalized cancer-stroma concentric-ring structure that sustains a radial oxygen gradient, recapitulate the structural, biochemical and biophysical properties of the native tumours. We also show that the glioblastoma-on-a-chip reproduces clinically observed patient-specific resistances to treatment with concurrent chemoradiation and temozolomide, and that the model can be used to determine drug combinations associated with superior tumour killing. The patient-specific tumour-on-a-chip model might be useful for the identification of effective treatments for glioblastoma patients resistant to the standard first-line treatment.

Three-Dimensional Hepatocellular Carcinoma/Fibroblast Model on a Nanofibrous Membrane Mimics Tumor Cell Phenotypic Changes and Anticancer Drug Resistance.

Three-dimensional (3D) in vitro tissue or organ models can effectively mimic the complex microenvironment of many types of human tissues for medical applications. Unfortunately, development of 3D cancer models, which involve cancer/stromal cells in a 3D environment, has remained elusive due to the extreme complexity of the tumor microenvironment (TME) and the stepwise progression of human cancer. Here, we developed hepatocellular carcinoma (HCC) models, which consist of fibroblasts as stromal cells, HCC cells, and a nanofibrous membrane to mimic the complex TME. The 3D HCC models were fabricated using three distinct culture methods: cancer cells grown directly on the nanofibrous membrane (mono model), fibroblasts covering the nanofibrous membrane (layer model), and both cancer cells and fibroblasts grown on the nanofibrous membrane (mixed model). Interestingly, the mono model and layer model showed similar tissue structures, whereas the mixed model resulted in phenotypic changes to the cancer cells. Further analysis demonstrated that the mixed models promoted the expression of fibronectin and vimentin, and showed higher resistance to anticancer drugs compared with the other models. Thus, our 3D HCC model could be utilized for testing efficient anticancer therapies at various stages of cancer, with potential application to different tumor types.

Co-targeting of Tiam1/Rac1 and Notch ameliorates chemoresistance against doxorubicin in a biomimetic 3D lymphoma model.

Lymphoma is a heterogeneous disease with a highly variable clinical course and prognosis. Improving the prognosis for patients with relapsed and treatment-resistant lymphoma remains challenging. Current in vitro drug testing models based on 2D cell culture lack natural tissue-like structural organization and result in disappointing clinical outcomes. The development of efficient drug testing models using 3D cell culture that more accurately reflects in vivo behaviors is vital. Our aim was to establish an in vitro 3D lymphoma model that can imitate the in vivo 3D lymphoma microenvironment. Using this model, we explored strategies to enhance chemosensitivity to doxorubicin, an important chemotherapeutic drug widely used for the treatment of hematological malignancies. Lymphoma cells grown in this model exhibited excellent biomimetic properties compared to conventional 2D culture including (1) enhanced chemotherapy resistance, (2) suppressed rate of apoptosis, (3) upregulated expression of drug resistance genes (MDR1, MRP1, BCRP and HIF-1α), (4) elevated levels of tumor aggressiveness factors including Notch (Notch-1, -2, -3, and -4) and its downstream molecules (Hes-1 and Hey-1), VEGF and MMPs (MMP-2 and MMP-9), and (5) enrichment of a lymphoma stem cell population. Tiam1, a potential biomarker of tumor progression, metastasis, and chemoresistance, was activated in our 3D lymphoma model. Remarkably, we identified two synergistic therapeutic oncotargets, Tiam1 and Notch, as a strategy to combat resistance against doxorubicin in EL4 T and A20 B lymphoma. Therefore, our data suggest that our 3D lymphoma model is a promising in vitro research platform for studying lymphoma biology and therapeutic approaches.

A cell-laden hybrid fiber/hydrogel composite for ligament regeneration with improved cell delivery and infiltration.

Ligament, a fibrous connective tissue between bones, is a unique tissue in human anatomy because it has complex viscoelastic properties and is very tough. Moreover, it is an important tissue for regeneration because frequent injuries occur, but there are limited types of substitutes that can be used as a tissue replacement. In this study, we present a stem cell-laden fiber/hydrogel composite structure with a layered fibrous structure, which can enhance cell infiltration, topographical cue and mechanical properties. It can promote cell viability, proliferation, and differentiation of the ligament phenotype with the help of a growth factor. The mechanical properties of the developed structure were experimentally identified using tensile tests, while cell viability and various functionalities were verified through culture tests using mesenchymal stem cells.

Fish Scale Collagen Peptides Protect against CoCl2/TNF-α-Induced Cytotoxicity and Inflammation via Inhibition of ROS, MAPK, and NF-κB Pathways in HaCaT Cells.

Skin diseases associated with inflammation or oxidative stress represent the most common problem in dermatology. The present study demonstrates that fish scale collagen peptides (FSCP) protect against CoCl2-induced cytotoxicity and TNF-α-induced inflammatory responses in human HaCaT keratinocyte cells. Our study is the first to report that FSCP increase cell viability and ameliorate oxidative injury in HaCaT cells through mechanisms mediated by the downregulation of key proinflammatory cytokines, namely, TNF-α, IL-1ß, IL-8, and iNOS. FSCP also prevent cell apoptosis by repressing Bax expression, caspase-3 activity, and cytochrome c release and by upregulating Bcl-2 protein levels in CoCl2- or TNF-α-stimulated HaCaT cells. In addition, the inhibitory effects of FSCP on cytotoxicity and the induction of proinflammatory cytokine expression were found to be associated with suppression of the ROS, MAPK (p38/MAPK, ERK, and JNK), and NF-κB signaling pathways. Taken together, our data suggest that FSCP are useful as immunomodulatory agents in inflammatory or immune-mediated skin diseases. Furthermore, our results provide new insights into the potential therapeutic use of FSCP in the prevention and treatment of various oxidative- or inflammatory stress-related inflammation and injuries.