Background: Xenogenous bone has been proposed as an alternative to overcome the disadvantages of autogenous grafting. The aim of the present study was to study bone dynamics at inlay and onlay xenografts used for bone augmentation applying a ring technique. Methods: The bone at the lateral surface of the mandibular angle of 12 adult male New Zealand White rabbits was exposed bilaterally. The cortical layer received multiple perforations at one side of the mandible and a xenograft block of collagenated cancellous equine bone, 7 mm in diameter and 3 mm in width, was fixed on the prepared surface using an implant (onlay group). On the opposite side, a defect 7 mm in diameter and 3 mm in depth was prepared, the xenograft block was adapted to the defect and fixed with an implant (inlay group). Results: After ten weeks of healing, in the onlay grafts, new bone was mainly formed on the trabeculae surface, reaching in some specimens the most coronal regions of the block. In the inlay grafts, new bone was found arranged on the trabecular surfaces but also occupying the spaces among the trabeculae. The entrance of the defect was often found close at the top of the block by newly formed bone. A higher percentage of new bone was found in the inlay (19.0 ±9.3%) compared to the onlay (10.4 ±7.4%) groups (p=0.031). The mean gain in osseointegration at the implant in relation to the base of the original 3 mm deep defect was 0.95 ±1.05% at the onlay group and 0.78 ±0.71% at the inlay group (p=0.603). Conclusion: The inlay grafts exhibited a higher new bone percentage than the onlay grafts possibly due to the defect conformation that presented more sources for bone formation. The trabecular conformation and the composition of the grafts made possible the expression of the osteoconductive properties of the material used. This resulted, in several specimens, in growth of bone on the graft trabeculae toward the most superior regions in both groups, and in the closure of the coronal entrance of the defects in the inlay group.

Personal Mobility Vehicle (PMV) which has an inward tilting angle turns with the lateral force due to large camber angle, so it is necessary to consider the lateral movement of the tire vertical load axis during turning. Although the steering torque mechanism are very different from those of automobiles, there are not many examples which studied the steering torque mechanism of PMV. In this paper, based on the effects of six components force acting on the tires, a method for setting the steering axis specifications is derived, including the geometrical minimization of steering moment disturbance due to the vertical load reaction force during turning. Although, automobile tires have a significant ground camber angle when traveling on rutted roads, they do not have it on slant roads because the vehicle body tilts along the road surface. On the other hand, in PMV, the vehicle body always keeps upright when traveling both on slanted roads and on rutted roads. Therefore, the tires have ground camber angles on both types of road surface. We study the straight running ability under such road surface disturbances based on the geometrical minimization of steering moment disturbance due to the vertical load reaction force during turning. This straight running ability can be a remarkable strong point of PMV with an inward tilt mechanism.

Artifact metrics is a technology for authenticating artifacts based on their unique characteristics. The artifact-metric system offers "clone resistance," i.e., it makes it highly improbable to create another object exhibiting the same measured values as a genuine artifact. However, determined adversaries may still attempt to create imitations or clones with close physical characteristics to those of registered products, even if they cannot perfectly replicate the genuine product. Such clones serve to deceive users into believing they are genuine rather than counterfeits. Thus, in this study, we consider a scenario in which we measure raster-scanning-generated clones via a non-raster scanning method. Further, we employ image processing techniques to generate image data representing the clones and theoretically assess the filtering effects on these images. Our findings reveal that applying filters to specific frequency components in the spatial frequency domain can effectively highlight differences between raster-scanning-created clones and the corresponding genuine artifacts. Thus, we demonstrate directional emphasis filtering in artifact metrics as an effective approach for rejecting raster-scanning-created clones.

The realization of a perfect absorber A = 1 with transmittance and reflectance T=R=0 by a thin metasurface is one of the hot topics in recent nanophotonics prompted by energy harvesting and sensor applications (A + R + T =1 is the energy conservation). Here we tested optical properties of over 400 structures of metal-insulator-metal (MIM) metasurfaces for a range of variation in thickness of insulator, the diameter of a disc and intra-disc distance experimentally and numerically. Conditions of a near-perfect absorption A > 95% with simultaneously occurring anti-reflection property (R < 5%) were experimentally determined. Differences between the bulk vs. nano-thin film properties at mid-IR of the used materials can be of interest for plasmonic multi-metal alloys and high entropy metals.

The contactless coalescence of a droplet is of paramount importance for physical and industrial applications. This paper describes a coalescence method in mid-air via acoustic levitation using an ultrasonic phased array system. Acoustic levitation using ultrasonic phased arrays provides promising lab-on-a-drop applications, such as transportation, coalescence, mixing, separation, evaporation, and extraction in a continuous operation. The mechanism of droplet coalescence in mid-air may be better understood by experimentally and numerically exploring the droplet dynamics immediately before the coalescence. In this study, water droplets were experimentally levitated, transported, and coalesced by controlling acoustic fields. We observed that the edge of droplets deformed and attracted each other immediately before the coalescence. Through image processing, the radii of curvature of the droplets were quantified and the pressure difference between the inside and outside the droplet was simulated to obtain the pressure and velocity information on the droplet surface. The results revealed that the sound pressure acting on the droplet clearly decreased before the impact of the droplets. This pressure on the droplets was quantitatively analyzed from the experimental data. Our experimental and numerical results provide deeper physical insights into contactless droplet manipulation for futuristic lab-on-a-drop applications.

High-performance properties of interpenetration polymer network (IPN) hydrogels, based on physically crosslinked chitosan (CS) and chemically crosslinked poly(N-isopropylacrylamide) (PNiPAM), have been successfully developed. The IPN of CS/PNiPAM is proposed to overcome the limited mechanical properties of the single CS network. In this study, the viscoelastic behaviors of prepared materials in both solution and gel states have been extensively examined, considering the UV exposure time and crosslinker concentration as key factors. The effect of these factors on gel formation, hydrogel structures, thermal stabilities of networks, and HeLa cell adhesion were studied sequentially. The sol-gel transition was effectively demonstrated through the scaling law, which agrees well with Winter and Chambon's theory. By subjecting the CS hydrogel to the process operation in an ethanol solution, its properties can be significantly enhanced with increased crosslinker concentration, including the shear modulus, crosslinking degree, gel strength, and thermal stability in its swollen state. The IPN samples exhibit a smooth and dense surface with irregular pores, allowing for much water absorption. The HeLa cells were adhered to and killed using the CS surface cationic derivative, then released through hydrolysis by utilizing the hydrophilic/hydrophobic switchable property or thermo-reversible gelation of the PNiPAM network. The results demonstrated that IPN is a highly attractive candidate for anti-fouling materials.

(1) Background: Double-strand breaks (DSBs) in a single nucleus are usually measured using the sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) test, and comet assay (CA). We developed mono-dimensional single-cell pulsed-field gel electrophoresis (1D-SCPFGE) and angle-modulated two-dimensional single-cell pulsed-field gel electrophoresis (2D-SCPFGE) to observe DNA fragmentation in separated motile sperm. (2) Methods: We set up comparative standards, calibration curves, the required sensitivity, and eligibility criteria for test sperm to validate their measurement principles. (3) Results: We revealed that the conventional methods overlooked the interference of nucleoproteins in their measurements. In-gel proteolysis improves the measurement accuracies of 1D- and 2D-SCPFGE. We recommend using naked DNA for comparative standards and test specimens. Moreover, we have observed several dysfunctions that may induce DNA damage in the separated motile sperm. Overall, our discussion highlights the need to revisit the conventional univariable analyses based on the SCSA, SCD test, and CA. (4) Conclusions: Human infertility is a complex syndrome, and the aim of quality control in intracytoplasmic sperm injection is to identify the underlying dysfunctions remaining in the separated motile sperm that render them ineligible for injection. Well-designed, multivariable analyses with special consideration to confounding factors are necessary in future cohort studies.

In this study, based on our previous study, we examined the mathematical properties, especially the stability of the equilibrium for our proposed mathematical model. By means of the results of the stability in this study, we also used actual data representing transient booms and resurgent booms, and conducted parameter estimation for our proposed model using Bayesian inference. In addition, we conducted a model fitting to five actual data. By this study, we reconfirmed that we can express the resurgences or minute vibrations of actual data by means of our proposed model.

Eutherians have 11 retrotransposon Gag-like (RTL)/sushi-ichi retrotransposon homolog (SIRH) genes presumably derived from a certain retrovirus. Accumulating evidence indicates that the RTL/SIRH genes play a variety of roles in the current mammalian developmental system, such as in the placenta, brain and innate immune system in a eutherian-specific manner. It has been shown that the functional role of Paternally Expressed 10 (PEG10) in placental formation is unique to the therian mammals, as are the eutherian-specific roles of PEG10 and PEG11/RTL1 in maintaining the fetal capillary network and the endocrine regulation of RTL7/SIRH7 (aka Leucine Zipper Down-Regulated in Cancer 1 (LDOCK1)) in the placenta. In the brain, PEG11/RTL1 is expressed in the corticospinal tract and hippocampal commissure, mammalian-specific structures, and in the corpus callosum, a eutherian-specific structure. Unexpectedly, at least three RTL/SIRH genes, RTL5/SIRH8, RTL6/SIRH3 and RTL9/SIRH10, play important roles in combating a variety of pathogens, viruses, bacteria and fungi, respectively, suggesting that the innate immunity system of the brain in eutherians has been enhanced by the emergence of these new components. In this review, we will summarize the function of 10 out of the 11 RTL/SIRH genes and discuss their roles in eutherian development and evolution.

The steady streaming (SS) phenomenon is gaining increased attention in the microfluidics community, because it can generate net mass flow from the zero-mean vibration. We developed numerical simulation and experimental measurement tools to analyze this vibration induced flow, which has been challenging due to its unsteady nature. Validity of these analysis methods is confirmed by comparing the three-dimensional (3D) flow field induced around a cylindrical micropillar under circular vibration. In the numerical modeling, we directly solved the flow in the Lagrangian frame so that the substrate with a micropillar becomes stationary, and the result was converted to the Eulerian frame to compare them with the experimental results. The present approach enables to avoid the introduction of moving boundary or small perturbation approximation. The flow field obtained by the micro particle image velocimetry (PIV) measurement supported the three-dimensionality observed in the numerical results, which could be important for controlling the mass transport and manipulating particulate objects in the microfluidic systems.

Tunable hydrogels have gained significant attention in the bioengineering field due to their designer preparation approach. Towards this end, gelatine stands out as a promising candidate owing to the desirable attributes, such as biocompatibility, ability to support cell adhesion and proliferation, biodegradability, along with being cost-effective. This study presents the preparation of a robust gelatine hydrogel employing sacran dialdehyde (SDA) as a natural crosslinker. The resulting SDA-crosslinked gelatine hydrogels (GSDA) display an optimal compressive stress of 0.15 MPa at 50% strain, five times higher as compared to pure gelatine hydrogel. As SDA crosslinking concentration is increased, swelling capacity of GSDA declines. Probing further with FTIR spectroscopy and SEM at the micron-scale unveiled a dual-crosslinking mechanism within the hydrogels. This mechanism encompasses both short- and long-range covalent crosslinking, along with thermo-reversible physical crosslinking, resulting in a significant enhancement of the load-bearing capacity of the fabricated hydrogels.

This paper deals with the possibility for construction of the database on friction coefficient on the actual roads from the viewpoint of traffic safety. A measurement algorithm is determined in order to construct road friction measuring device. Next, the tires used for measurement are selected and the characteristics are measured using bench tire characteristic tester, and the measuring device is designed and constructed based on the characteristics. Finally, using this device, the measurement results of the road friction characteristics for two types of road surfaces are shown.

This research deals with the possibility for construction of the database on the braking friction coefficient for actual roads from the viewpoint of traffic safety especially for automated driving such as level 4 or higher. In an automated driving such levels, the controller needs to control the vehicle, but the road surface condition, especially the road friction coefficient on wet roads, snowy or icy roads, changes greatly, and in some cases, changes by almost one order. Therefore, it is necessary for the controller to constantly collect environment information such as the road friction coefficients and prepare for emergencies such as obstacle avoidance. However, at present, the measurement of the road friction coefficients is not systemically performed, and a method for accurately measuring has not been established. In order to improve this situation, this study examines a method for continuously measurement for the road friction characteristics such as μ-s characteristics.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic systemic inflammation causing progressive joint damage that can lead to lifelong disability. The pathogenesis of RA involves a complex network of various cytokines and cells that trigger synovial cell proliferation and cause damage to both cartilage and bone. Involvement of the cytokines TNF- and IL-6 is central to the pathogenesis of RA, but recent research has revealed that other cytokines such as IL-17, IL-23, GM-CSF, IL-33, and IL-2 also play a role. Clarification of RA pathology has led to the development of therapeutic agents such as biological disease-modifying anti-rheumatic drugs (DMARDs) and Janus kinase (JAK) inhibitors, and further details of the immunological background to RA are emerging. This review covers existing knowledge regarding the roles of cytokines, related immune cells and the immune system in RA, manipulation of which may offer the potential for even safer and more effective treatments in the future.

Keywords: Y RNA; Canine hepatocellular carcinoma, canine hepatocellular adenoma

Glaucoma secondary to Peters' anomaly (PA) is an important factor affecting visual prognosis, but there are few reports on it. This study aimed to investigate the characteristics of glaucoma asso-ciated with PA and glaucoma surgery outcomes. This retrospective study included 31 eyes of 20 patients with PA. PA was classified into three stages: Stage 1, with a posterior corneal defect only; Stage 2, a corneal defect with iridocorneal adhesion; and Stage 3, a corneal defect with lens ab-normalities. The associations between glaucoma and anterior segment dysgenesis severity, visual prognosis, and glaucoma surgery outcomes were analyzed. Sixteen eyes of 10 patients developed glaucoma. Stage 1 PA had no glaucoma, 52% of Stage 2 had glaucoma, and 75% of Stage 3 had glaucoma. Of the 16 eyes with glaucoma, 11 underwent surgery. Eight of these 11 eyes achieved intraocular pressure (IOP) control. Five of the nine eyes that underwent trabeculotomy (TLO) succeeded, and none had corneal staphyloma. Three of the four eyes for which TLO was ineffective had corneal staphyloma (P=0.0331). Patients with PA are more likely to develop glaucoma as anterior segment dysgenesis progresses, and the effect of TLO is limited if corneal staphyloma is present.

In this paper, we propose a novel approach named EmbedHDP to enhance the evaluation models used to assess sentence suggestions within nursing care record applications. The key focus is determining whether these suggestions garner assessments that align with caregivers as human evaluators. It is crucial due to the direct relevance of the information provided to the health or condition of the elderly. The motivation behind this proposal stems from challenges observed in previous models, such as BERTScore, which encountered difficulties in effectively evaluating the nurse care record domain, consistently providing quality assessments of generated sentence suggestions above 60%. Additionally, while widely used, cosine similarity exhibits limitations concerning word order, leading to potential misjudgments of semantical differences within similar word sets. Similarly, relying on lexical overlap, ROUGE tends to overlook semantic accuracy. Furthermore, despite its utility, BLEU neglects semantic coherence in its evaluations. EmbedHDP excels in evaluating nurse care records by effectively handling a variety of sentence structures and medical terminology and providing differentiated and contextually relevant assessments. We used a dataset comprising 320 pairs of sentences with correspondingly equivalent lengths. The results revealed that EmbedHDP outperformed other evaluation models, achieving a coefficient score of 61%, followed by cosine similarity with a score of 59%, and BERTScore with 58%. This shows the effectiveness of our proposed approach in improving the evaluation of sentence suggestions in nursing care record applications.

We previously developed a highly sensitive and specific anti-PDPN mAb, LpMab-23 (mouse IgG1, kappa). In this study, we produced a humanized IgG1 version (humLpMab-23) and its defucosylated form (humLpMab-23-f) of an anti‑PDPN mAb to potentiate the ADCC activity. The humLpMab-23 could recognize PDPN‑overexpressed Chinese hamster ovary (CHO)-K1 (CHO/PDPN) and PDPN-positive PC-10 and LN319 cells by flow cytometry. Furthermore, we found that humLpMab-23-f exerted ADCC and complement-dependent cytotoxicity against CHO/PDPN, PC-10 and LN319 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that humLpMab-23-f could be useful for an antibody treatment regimen for PDPN-positive human cancers.

Formation of Fe- and Mg-rich smectite and zeolite under alkaline conditions is concerned as secondary minerals after alkaline alteration of bentonite in a repository of radioactive wastes. It might be crucial for safety assessment whether smectite will be formed or not as secondary minerals after alkaline alteration of bentonite. In present paper, Fe- and Mg-rich smectite which are currently interacting with hyperalkaline groundwater was found at Narra in Palawan, Philippines. Mineralogical and geochemical investigation was conducted to understand formation process of smectite and factors determined secondary mineral species. Our study revealed a certain amount of smectite may have been produced under hyperalkaline conditions, altered from amorphous or poorly crystalline material such as M-S-H and F-S-H. Key factor which decides smectite or zeolite as secondary minerals after alkaline alteration of bentonite might be whether nuclei of M-S-H and/or F-S-H will be formed or not. This might be decided by the presence of dissolved Mg2+ and Fe2+ in the system. Our suggested formation process of smectite under alkaline conditions is analogue with generally-accepted model of smectite formation that might have been occurred on early Mars.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs can be beneficial in reducing the adverse effects. In this study, we established a novel cancer-specific anti-HER2 antibody, named H₂Mab-250/H₂CasMab-2 (IgG₁, kappa). H₂Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H₂Mab-250 did not react with non-transformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs including H₂Mab-119 (IgG₁, kappa) reacted with both cancer and normal epithelial cells. Furthermore, a core-fucose deleted IgG_2a-type H₂Mab-250 (H₂Mab-250-mG_2a-f) could trigger the antibody-dependent cellular cytotoxicity activity to BT-474, but not to HaCaT cells. Furthermore, H₂Mab-250-mG_2a-f exhibited an in vivo antitumor effect against BT-474 xenograft. Immunohistochemical analysis demonstrated that H₂Mab-250 possesses much higher reactivity to the HER2-positive breast cancer tissues compared to H₂Mab-119, and did not react with normal tissues, including heart, breast, stomach, lung, colon, kidney, and esophagus. The epitope mapping demonstrated that the Trp614 of HER2 domain IV mainly contributes to the recognition by H₂Mab-250. H₂Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

Podoplanin (PDPN) is a cell-surface mucin-like glycoprotein that plays a critical role in tumor development and normal development of the lung, kidney, and lymphatic vascular systems. PDPN is overexpressed in several tumors and is involved in their malignancy. PDPN induces platelet aggregation through binding to platelet receptor C-type lectin-like receptor 2. Furthermore, PDPN modulates signal transductions that regulate cell proliferation, differentiation, migration, invasion, epithelial to mesenchymal transition, and stemness, all of which are crucial for the malignant progression of tumor. In the tumor microenvironment (TME), PDPN expression is up-regulated in the tumor stroma, including cancer-associated fibroblasts (CAFs) and immune cells. CAFs play significant roles in the extracellular matrix remodeling and the development of immunosuppressive TME. Additionally, PDPN functions as a co-inhibitory molecule on T cells, indicating the involvement with immune evasion. In this review, we describe the mechanistic basis and diverse roles of PDPN in malignant progression of tumor and discuss the possibility of the clinical application of PDPN-targeted cancer therapy, including cancer-specific monoclonal antibodies, and chimeric antigen receptor T technologies.

The effect of cooling rate on the magnetorheological response was investigated for magnetic hydrogels consisting of carrageenan and carbonyl iron particles with a concentration of 50 wt.%. For magnetic gels prepared by natural cooling, the storage moduli at 0 and 50 mT were 3.7×104 Pa and 5.6×104 Pa, respectively, and the change in the modulus was 1.9×104 Pa. For magnetic gels prepared by rapid cooling, the storage moduli at 0 and 50 mT were 1.2×104 Pa and 1.8×104 Pa, respectively, and the change in the modulus was 6.2×103 Pa, which was 3.1 times higher than that for the magnetic gel of rapid cooling. The critical strains, where G‘=G“ on the strain dependence of the storage modulus, for magnetic gels of natural cooling and rapid cooling were 0.023 and 0.034, respectively, indicating that the magnetic gel of rapid cooling has a hard structure compared to that of natural cooling. Oppositely to this, the change in the storage modulus at 500 mT for the magnetic gel of rapid cooling was 1.6×105 Pa, which was 2.5 times higher than that of natural cooling. SEM images revealed that many small aggregations of carrageenan network were found in the magnetic gel of natural cooling and continuous phases of carrageenan network with large sizes were found in the magnetic gel of rapid cooling. It was revealed that, magnetic particles in the magnetic gel of rapid cooling can move and form a chain structure at high magnetic fields by breaking the restriction from the continuous phases of carrageenan.

Metal-organic frameworks (MOFs) represent the largest class of materials among the crystalline porous materials ever developed and have attracted attention as core materials for separation technology. Their extremely uniform pore aperture and nearly unlimited structural and chemical characteristics have attracted great interest and promise in applying MOFs to adsorptive and membrane–based separations. This paper reviews the recent research and development of MOF membranes for gas separation. Strategies for polycrystalline membranes and mixed matrix membranes are discussed, with a focus on separation systems involving hydrocarbon separation and CO2 capture. Challenges and opportunities for the industrial deployment of MOF membranes are also be discussed, providing guidance for the design and fabrication of future high-performance membranes. The contributions of the underlying mechanism to separation performances, and the adopted strategies and membrane processing technologies for breaking the selectivity/permeability trade–off are discussed.

Agricultural drainage canals that connect upstream fish spawning areas to downstream rivers and lakes serve as crucial habitats for migrating fish. However, disconnections, such as drops and chutes, have been constructed in these canals due to agricultural modernization and flood control measures, hindering the movement of fish with limited swimming abilities. Portable fishways offer a promising solution to reconnect water bodies in agricultural canals, as they can be easily removed during high water discharges to avoid impeding the canals' drainage function. In addition to experimental assessments of fishway functionality, employing a hydrodynamic model to explore effective placement strategies for portable fishways is essential to maximize their effectiveness. This study presents a method to determine the best horizontal location of a portable fishway in an agricultural drainage canal using two-dimensional hydrodynamic simulations within the specified cases. The applicability of this method is demonstrated by addressing the positioning challenge of a portable fishway on a chute in an agricultural drainage canal in Japan. Results indicate that the proposed method allows for the selection of a suitable location, considering preferable hydraulic conditions both within the portable fishway and around its entrance.

We previously designed and synthesized dehydroxyepoxyquinomicin (DHMEQ) as an inhibitor of NF-κB based on the structure of microbial secondary metabolite epoxyquinomicin C. DHMEQ showed anti-inflammatory and anticancer activity in various in vivo disease models without toxicity. Cell detachment from the primary tumor and subsequent invasion are considered to be early phase of metastasis, while tumor cell attachment to the tissue and secondary tumor formation the late phase. The assay system for late phase was set up with intra-portal-vein injection of pancreatic cancer cells. Administration of DHMEQ was found to inhibit the liver metastasis possibly by decreasing the expression of MMP-9 and IL-8. Also when the pancreatic cancer cells treated with DHMEQ was inoculated into the peritoneal cavity of mice, the metastatic foci formation was inhibited. These results indicate that DHMEQ is likely to inhibit the late phase of metastasis. Meanwhile, we have recently employed three-dimensional (3D) culture of breast cancer cells for the model of early phase metastasis. DHMEQ inhibited the 3D invasion of breast cancer cells without toxicity. In this way, DHMEQ was shown to inhibit the late and early phases of metastasis. Thus, DHMEQ is likely to be useful for the suppression of cancer metastasis.

Patients with neurodegenerative diseases (NDDs) are rapidly increasing all over the world. Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s disease (HD), frontotemporal lobar degeneration (FTLD) and many other diseases are classified as NDDs, however, no therapeutic drugs have been developed against NDDs yet. As common phenomenon, protein oligomers and aggregates (inclusion bodies) are formed in neurons, and they trigger neuronal cell death and/or axonal degeneration. We have investigated the mechanisms of NDDs, and discovered several chemicals and peptides have potential to cure NDDs. Here, we show phorbol 12-myristate 13-acetate (PMA) significantly inhibits aggregate formation and promotes neurite regeneration in human neuronal cell culture model of NDDs, FTLD and AD. We expressed R406W mutant tau protein found in FTLD patients in human neuroblastoma SH-SY5Y cells as FTLD cell culture model. These FTLD neuronal cells showed intracellular aggregate formation and impaired neurite growth. Addition of PMA to culture medium 60% suppressed aggregate formation. PMA also enhanced neurite growth to the normal level at nanomolar concentration. We cultured SH-SY5Y cells in the medium containing -amyloid (1-42) as AD cell culture model. In these cells, neurite growth was limited to less than 50% of controls by the treatment of -amyloid (1-42). Surprisingly, addition of PMA increased the number of cells with >10-fold neurite extension to seven times even in -amyloid (1-42) contained medium. Also, PMA increased the expression of several genes and proteins important for cell survival in both cell culture models. These results suggest PMA has unexpected potential for the drug development against NDDs.

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular ATP into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment (TME) attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (mCD39) mAb, C₃₉Mab-1 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of C₃₉Mab-1 using flow cytometry. We performed the PA tag (12 amino acids)-substituted analysis (named PA scanning) and RIEDL tag (5 amino acids)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C₃₉Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275^th to 279^th, 282^nd to 291^st, and 306^th to 323^rd amino acids of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest receptor tyrosine kinases family. EphB4 is essential for cell adhesion and motility during embryogenesis. Pathologically, EphB4 is overexpressed and contributes to poor prognosis in various tumors. Therefore, sensitive monoclonal antibodies (mAbs) should be developed to predict the prognosis for multiple tumors with high EphB4 expression, including breast and gastric cancers. This study aimed to develop highly sensitive and specific anti-EphB4 mAbs for several applications using the Cell-Based Immunization and Screening (CBIS) method. EphB4-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/EphB4) cells were immunized into mice, and we established an anti-EphB4 mAb (clone B4Mab-7), which is applicable for flow cytometry, western blotting, and immunohistochemistry. B4Mab-7 reacted with endogenous EphB4-positive breast cancer cell lines, MCF-7 and MDA-MB-468, but did not react with EphB4-knockout MCF-7 (BINDS-52) in flow cytometry. Dissociation constant (KD) values were determined to be 2.9 × 10‑9 M, 1.3 × 10‑9 M, and 3.3 × 10‑9 M by flow cytometric analysis for CHO/EphB4, MCF-7, and MDA-MB-468 cells, respectively. B4Mab-7 detected the EphB4 protein bands from breast cancer cells in western blotting, and stained breast cancer tissues immunohistochemistry. Altogether, B4Mab-7 demonstrated high sensitivity and specificity against EphB4 in various applications.

CD44 is known as a cancer stem cell marker of head and neck squamous cell carcinoma (HNSCC) and plays a critical role in cancer malignant progression. Splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer therapy. Several monoclonal antibodies (mAbs) against CD44 have been developed by immunizing mice with CD44v3–10-overexpressed cancer cells. In this study, we characterized a novel clone, C₄₄Mab-18 (IgM, kappa). C₄₄Mab-18 reacted with CHO/CD44v3–10, but not with CHO/CD44s by flow cytometry. Enzyme-linked immunosorbent assay revealed that the epitope of C₄₄Mab-18 is determined to be the border sequence between variant 10 and the constant exon 16-encoded sequence. Flow cytometry showed that C₄₄Mab-18 recognizes HSC-3, an oral squamous cell carcinoma(OSCC) cell line. The apparent dissociation constant (K_D) of C₄₄Mab-18 for CHO/CD44v3–10 and HSC-3 was determined to be 1.6 × 10⁻⁷ M and 1.7 × 10⁻⁷ M, respectively. C₄₄Mab-18 detected CD44v3–10, but not CHO/CD44s in western blotting. Furthermore, C₄₄Mab-18 detected endogenous CD44v10 in immunohistochemistry using OSCC tissues. Taken together, C₄₄Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.

Hypoxia contributes to tumor progression and metastasis, and hypoxically dysregulated RNA molecules may thus be implicated in poor outcomes. Canine oral melanoma (COM) has a par-ticularly poor prognosis, and some hypoxia-mediated miRNAs are known to exist in this cancer; however, equivalent data on hypoxically dysregulated other non-coding RNAs (ncRNAs) are lacking. Accordingly, we aimed to elucidate non-miRNA ncRNAs that may be mediated by hy-poxia, targeting primary-site and metastatic COM cell lines and clinical COM tissue samples in next generation sequencing (NGS), with subsequent qPCR validation and quantification in COM primary and metastatic cells, plasma and extracellular vesicles (EVs) for any identified ncRNA of interest. Findings suggested that a number of non-miRNA ncRNA species are hypoxically up- or downregulated in COM. We identified one ncRNA, the long ncRNA fragment EN-SCAFT00000084705.1, as a molecule of interest due to its consistent downregulation in COM tissues, hypoxically and normoxically cultured primary and metastatic cell lines, when compared to the oral tissues from healthy dogs. However, this molecule was undetectable in plasma and plasma EVs, suggesting that its expression may be tumor tissue-specific and it has little potential as a biomarker. Here, we provide evidence of hypoxic transcriptional dysregulation for ncRNAs other than miRNA in COM for the first time, and suggest that ncRNA ENSCAFT00000084705.1 is a molecule of interest for future research on the role of the transcriptome in hypoxia-mediated progression of this aggressive cancer.

This study characterizes the microstructure and its associated crystallographic features of bulk maraging steels fabricated by selective laser melting (SLM) combined with a powder bed technique. The fabricated sample exhibited characteristic melt pools in which the regions had locally melted and rapidly solidified. A major part of these melt pools corresponded with the ferrite (alpha) matrix, which exhibited a lath martensite structure with a high density of dislocations. A number of fine retained austenite (gamma) with a <001> orientation along the build direction was often localized around the melt pool boundaries. The orientation relationship of these fine gamma grains with respect to the adjacent alpha grains in the martensite structure was (111) gamma // (011) alpha and [-101] gamma // [-1-11] apha (Kurdjumov–Sachs orientation relationship). Using the obtained results, we inferred the microstructure development of maraging steels during the SLM process. The results depict that new and diverse high-strength materials can be used to develop industrial molds and dies.

C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C9Mab-24 (rat IgG2a, kappa), using a 1 × alanine (1 × Ala) and 2 × alanine (2 × Ala)-substitution method via enzyme-linked immunosorbent assay. We first performed the 1 × Ala-substitution method using one alanine-substituted peptides of the mCCR9 N-terminus (amino acids 1-19). C9Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C9Mab-24-binding to mCCR9. Furthermore, we conducted the 2 × Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C9Mab-24 did not react with four peptides (M13A–F14A, F14A–D15A, D16A–F17A, and F17A–S18A), indicating that 13-MFDDFS-18 is involved in C9Mab-24-binding to mCCR9. Overall, combining, the 1 × Ala or 2 × Ala scanning methods could be useful for understanding for target-antibody interaction.

Tandem mass tag (TMT)-based proteomics facilitate multiplexing in mass spectrometry (MS)-based quantification and identification of proteins and their post-translational modifications. The use of TMT isobaric tags can enable multiplexing of up to 18 samples using commercially available kits. A single TMT experiment can quantify proteome, serine, threonine phosphorylation, and tyrosine phosphorylation. Of note, tyrosine phosphorylation is of low abundance, and identification/quantification can be improved using two complementary strategies. First, by employing SH2 superbinder which increases the number of identified sites. The SH2 Superbinder is more cost-effective than the commonly used phosphotyrosine antibodies. Second, by employing phosphotyrosine booster strategy, a pervanadate-treated channel to boost the signal of low-abundant phosphotyrosine. Noteworthy, pervanadate boost increases the likelihood of low abundant peptide to be selected for MS2, and facilitating the detection of > 6000 proteins, 10,000 unique pS/T and 1000 unique pY sites.

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8⁺ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C₈Mab-2, using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of C₈Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C₈Mab-2 recognizes the N-terminal region (1–33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the _17-DFFTAP_-22 sequence is important for the recognition by C₈Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C₈Mab-2.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 has been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb; Cx₁Mab-1 (rat IgG₁, kappa), using the Cell-Based Immunization and Screening (CBIS) method. Cx₁Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx₁Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6×10^-9 M and 2.1×10^-8 M, respectively. Furthermore, Cx₁Mab-1 could detect mCXCR1 by western blot analysis. These results indicated that Cx₁Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

In small animal models of severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infection, ferrets (Mustela putorius furo) have been used to investigate the pathogenesis. Podoplanin (PDPN) is an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) against ferret PDPN (ferPDPN) are useful for the pathological analyses of those tissues. We previously established an anti-ferPDPN mAb, PMab-292 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of PMab-292 using flow cytometry. The N-terminal ferPDPN deletion mutants analysis revealed that the Val34 is located at the N-terminus of the PMab-292 epitope. Furthermore, the PA tag-substituted analysis (PA scanning) showed that Asp39 is located at the C-terminus of PMab-292 epitope. The epitope sequence (₃₄-VRPEDD-₃₉) also exists between Val26 and Asp31 of ferPDPN, indicating that PMab-292recognizes the tandem repeat of the VRPEDD sequence of ferPDPN.

Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various tumors. PODXL is involved in tumor malignant progression through the promotion of invasiveness and metastasis. Therefore, PODXL has been considered as a promising target of monoclonal antibody (mAb)-based therapy. However, PODXL also plays an essential role in normal cells such as vascular and lymphatic endothelial cells.Therefore, cancer-specificity or selectivity is required for the reduction of adverse effects on normal cells. Here, we developed an anti-PODXL cancer-specific mAb (CasMab), PcMab-6 (IgG₁, kappa), by immunizing mice with soluble PODXL ectodomain derived from a glioblastoma LN229 cell.PcMab-6 reacted with the PODXL-positive LN229 cells, but not with PODXL-knockout LN229 cells in flow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H), but did not react with normal lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47 showed high reactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG_2a type (PcMab-6-mG_2a) and a humanized IgG₁-type (humPcMab-6) mAbs, and further produced the core fucose-deficient types (PcMab-6-mG_2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG_2a-f and humPcMab-6-f exerted ADCC and complement-dependent cellular cytotoxicity in the presence of effector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG_2a-f and humPcMab-6-f exhibited potent antitumor effects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.

Immunohistochemistry staining is an essential method in pathological diagnoses. Podoplanin (PDPN) is a specific maker of alveolar epithelium, lymphatic vessels, and glomeruli. In this study, we established a novel anti-giraffe PDPN (girPDPN) mAb, PMab-301, using the Cell-Based Immunization and Screening (CBIS) method. PMab-301 (mouse IgG₁, kappa) detected girPDPN in various applications, such as flow cytometry, western blot, and immunohistochemistry. PMab-301 specifically stained type-I alveolar cells using formalin-fixed paraffin-embedded giraffe lung tissues. Our findings suggest the potential usefulness of PMab-301 for the pathophysiological analyses of giraffe tissues.

Immune checkpoint blockade therapy has shown successful clinical outcomes in multiple human cancers. In dogs, several types of tumors resemble human tumors in many respects. Therefore, several groups have developed the anti-dog programmed cell death ligand 1 (dPD-L1) monoclonal antibodies (mAbs) and showed efficacy in several canine tumors. To examine the abundance of dPD-L1 in canine tumors, anti-dPD-L1 diagnostic mAbs for immunohistochemistry are required. In this study, we immunized the peptide in the dPD-L1 intracellular domain, and established anti-dPD-L1 mAbs, L1Mab-352 (mouse IgG1, kappa) and L1Mab-354 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, L1Mab-352 and L1Mab-354 showed high binding affinity to the dPD-L1 peptide, and the dissociation constants (KD) were determined as 6.9 ×10-10 M and 7.2 ×10-10 M, respectively. Furthermore, L1Mab-352 and L1Mab-354 were applicable for the detection of dPD-L1 in immunohistochemical analysis in paraffin-embedded dPD-L1-expressed cells. These results indicated that L1Mab-352 and L1Mab-354 are useful for detecting dPD-L1 in immunohistochemical analysis.

Breast cancer patients with high levels of HER2 (human epidermal growth factor receptor 2) expression had worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa). This was then altered to produce H2Mab-77-mG2a-f, an afucosylated mouse IgG2a. In the present work, we examined the reactivity of H2Mab-77-mG2a-f and antitumor effects against breast cancers in vitro and in vivo. BT-474, an endogenously HER2-expressed breast cancer cell line, was identified by H2Mab-77-mG2a-f with a strong binding affinity [a dissociation constant (KD): 5.0 × 10-9 M]. H2Mab-77-mG2a-f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in western blot analysis. Furthermore, H2Mab-77-mG2a-f demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells. MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H2Mab-77-mG2a-f. Additionally, in the BT-474-bearing tumor xenograft model, H2Mab-77-mG2a-f substantially suppressed tumor development when compared to the control mouse IgG2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograft model showed no response to H2Mab-77-mG2a-f. These findings point to the possibility of H2Mab-77-mG2a-f as a treatment regimen by showing that it has antitumor effects on HER2-positive breast tumors.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab-deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) by flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) of H2Mab-300 and H2Mab-304 for CHO/mHER2 was 1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD of H2Mab-300 and H2Mab-304 for NMuMG was 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2Mab-300 and H2Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein, and has been shown as a cell surface marker of cancer stem-like cells in various cancers. Especially, the splicing variants of CD44 (CD44v) are overexpressed in cancers, and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for the CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed Chinese hamster ovary CHO-K1 (CHO/CD44v3–10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay, and characterize their applications to flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C₄₄Mab-1 (IgG₁, kappa) reacted with a peptide of the variant 9-encoded region, indicating that C₄₄Mab-1 recognizes CD44v9. C₄₄Mab-1 reacted with CHO/CD44v3–10 cells or colorectal cancer cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (K_D) of C₄₄Mab-1 for CHO/CD44v3–10, COLO201, and COLO205 was 2.5 × 10⁻⁸ M, 3.3 × 10⁻⁸ M, and 6.5 × 10⁻⁸ M, respectively. Furthermore, C₄₄Mab-1 was able to detect the CD44v3–10 in western blotting, and endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C₄₄Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.

CC chemokine receptor 6 (CCR6) is one of the members of G protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, including B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. Coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, autoimmune diseases, and psoriasis. Therefore, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for patients. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), applicable for flow cytometry by immunizing a rat with N-terminal peptide of mCCR6. This study investigated the binding epitope of C6Mab-13 using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methods with the synthesized point mutated-peptides within 1-20 amino acids region of mCCR6. In ELISA, C6Mab-13 lost the reaction to the alanine-substituted peptide of D11A. The epitope of C6Mab-13 was identified to be Asp11 in ELISA. Furthermore, in SPR analysis, the dissociation constants (KD) could not be calculated for G9A and D11A mutants due to lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.

CC chemokine receptor 6 (CCR6) is a member of the G protein-coupled receptor (GPCR) family that is highly expressed in B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. CCR6 has been revealed to have important functions in many pathological conditions, such as cancer, intestinal bowel disease, psoriasis, and autoimmune diseases. The only CCR6 chemokine ligand, CC motif chemokine ligand 20 (CCL20), is also involved in pathogenesis by interacting with CCR6. The CCL20/CCR6 axis is drawing attention as an attractive therapeutic target for various diseases. In this study, we developed novel monoclonal antibodies (mAbs) against human CCR6 (hCCR6) using the peptide immunization method, which are applicable to flow cytometry and immunohistochemistry. The established anti-hCCR6 mAb, clone C6Mab-19 (mouse IgG1, kappa), reacted with hCCR6-overexpressed Chinese hamster ovary-K1 (CHO/hCCR6), HepG2 (human liver carcinoma), and HuH-7 (human differentiated hepatoma) cells in flow cytometry. The dissociation constant (KD) of C6Mab-19 was determined as 3.0 × 10−10 M for CHO/hCCR6, 6.9 × 10−10 M for HepG2, and 1.8 × 10−10 M for HuH-7. Thus, C6Mab-19 could bind to exogenously and endogenously expressed hCCR6 with extremely high affinity. Furthermore, C6Mab-19 could stain formalin-fixed paraffin-embedded lymph node tissues from a patient with non-Hodgkin lymphoma by immunohistochemistry. Therefore, C6Mab-19 is suitable for detecting hCCR6-expressing cells and tissues, and could be useful for pathological analysis and diagnosis.

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker, and plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mice with a CD44 variant (CD44v3−10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide which covers both variant 7 and 8-encoded region, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO) cells or oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3–10 and HSC-3 was 1.4 × 10−9 M and 3.2 × 10−9 M, respectively. C44Mab-34 could detect CD44v3–10 in western blotting, and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications, and expected for the application of OSCC diagnosis and therapy.

Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker, and plays tumor promotion and drug resistance in various cancers. Especially, the splicing variants are overexpressed in many carcinomas, and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO) cells, and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant 5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3–10 and PK-1 was 7.1 × 10−10 M and 1.9 × 10−9 M, respectively. C44Mab-3 could detect the exogenous CD44v3–10 and endogenous CD44v5 in western blotting, and stained the formalin-fixed paraffin-embedded pancreatic cancer cells, but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications, and expected for the application of pancreatic cancer diagnosis and therapy.

Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer deaths worldwide. Surgery or surgery plus radiotherapy and/or chemotherapy for patients with metastatic CRC (mCRC) were accepted as the main therapeutic strategies until the early 2000s, when targeted drugs, like cetuximab and bevacizumab were developed. The use of targeted drugs in clinical practice has significantly increased patients’ overall survival. To date, the emergence of several types of targeted drugs has opened new possibilities and revealed new prospects for mCRC treatment. Therapeutic strategies are continually being updated to select the most suitable targeted drugs based on the results of clinical trials that are currently underway. This review discusses the up-to date molecular evidence of targeted therapy for mCRC and summarizes the Food and Drug Administration-approved targeted drugs including the results of clinical trials. We also explain their mechanisms of action and how these affect the choice of a suitable targeted therapy.

With a large number of annotated non-coding RNAs (ncRNAs), repetitive sequences are found to constitute functional components (termed as repetitive elements) in ncRNAs that perform specific biological functions. Bioinformatics analysis is a powerful tool for improving our understanding of the role of repetitive elements in ncRNAs. This chapter summarizes recent findings that reveal the role of repetitive elements in ncRNAs. Furthermore, relevant bioinformatics approaches are systematically reviewed, which promises to provide valuable resources for studying the functional impact of repetitive elements on ncRNAs.

We summarize how to obtain protein crystals from which better diffraction images can be obtained. In particular, we describe in detail the quality evaluation of the protein sample, the crystallization methods and crystallization conditions, the flash-cooling protection of the crystal, and the crystallization under a microgravity environment.

Background: We compared corneal visualization Scheimpflug technology (CST) parameters between eyes with primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG). Methods: A retrospective analysis was done on data from 89 eyes with POAG and 83 eyes with PACG that had CST examinations. CST parameters were compared between eyes with POAG and those with PACG using a linear mixed model (LMM). Results: No differences were observed in age, central corneal thickness, intraocular pressure, or use of antiglaucoma eye drops between the two groups. Patients with PACG had a significantly shorter axial length (AL), a higher proportion of females, CST parameters, longer applanation 2 (A2) time, deeper A2 deformation amplitude, shorter peak distance (PD), longer whole eye movement, and longer whole eye movement time than patients with POAG. Highest concavity (HC) length and PD showed a significant positive correlation with AL. However, A1 length, A1 deformation amplitude, A2 time, A2 velocity, A2 length, A2 deformation amplitude, HC time, whole eye movement, and whole eye movement time were negatively correlated with AL. Conclusions: Biomechanical properties of the cornea differed between PACG and POAG. In some parts, AL differences between the POAG and PACG groups might contribute to the variation in CST parameters.

This study aimed to describe the injury profiles of young Korean baseball players according to position and age as the proportion and distribution of injuries based on playing position and age remains unclear. A total of 271 elite youth baseball players aged 8 to 16 years were divided into two groups: elementary school (ES) (n=135) and middle school (MS) (n=136). The participants' basic, baseball practice, and injury information were collected. Injuries in the MS group were not limited to the elbow and shoulder, and the injury prevalence varied by age group and baseball position. The most injured body region in the ES group was the elbow joint, regardless of the position. In contrast, the most injured body region in the MS group was the lower back, except for infielders whose elbows were the most injured. Additionally, the MS group was more likely to experience injuries of the lower back (OR=4.27, 95% CI=2.47–7.40), shoulder (OR=1.93, 95% CI=1.08–3.43; P=0.024), and knee (OR=2.15, 95% CI=1.17–3.94; P=0.012). Our findings indicate that excessive practice and lack of rest during MS (growth spurt period) can significantly increase the risk of lower back problems in young baseball players.

: We evaluated the pharmaceutical properties of levofloxacin (LV) in the form of an orally disintegrating tablet (LVODT) to find a new usefulness of low frequency (LF) Raman spectroscopy. LVODT contained dispersed granules with diameters in the order of several hundred micrometers, which were composed of the active pharmaceutical ingredient (API), as confirmed by infrared (IR) microspectroscopy. On the contrary, the API and inactive pharmaceutical ingredients (non-APIs) were homogeneously distributed in LV tablet (LVT) formulations. Microscopic IR spectroscopy and thermal analyses showed that LVODT and LVT contained the API in different crystalline forms or environment around the API each other. Furthermore, powder X-ray diffraction showed that LVT contained a hemihydrate of the API, while LVODT showed a partial transition to the monohydrate form. This result was confirmed by microscopic LF Raman spectroscopy. Moreover, this method confirmed the presence of thin layers coating the outer edges of the granules that contained the API. Spectra obtained from these thin layers indicated the presence of titanium dioxide, suggesting that the layers coexisted with a polymer that masks the bitterness of API. The microscopic LF Raman spectroscopy results in this study indicated new applications of this method in pharmaceutical science.

Background: In schizophrenia (SZ), social cognition mediates the relationship between neurocognition and social functioning. Although people with major depressive disorder (MDD) also exhibit cognitive impairments, which are often prolonged, little is known about the role of social cognition in MDD. Methods: Using data obtained through an internet survey, 210 patients with SZ or MDD were selected using propensity score matching based on their demographic information and illness duration. Social cognition, neurocognition, and social functioning were evaluated using the Self-Assessment of Social Cognition Impairments, Perceived Deficits Questionnaire, and Social Functioning Scale, respectively. The mediation effects of social cognition on the relationship between neurocognition and social functioning were examined in each disease group. Invariances of the mediation model across the two groups were then analyzed. Results: The SZ and MDD groups had mean ages of 44.49 and 45.35 years, contained 42.0% and 42.8% women, and had mean illness durations of 10.76 and 10.45 years, respectively. In both groups, social cognition had significant mediation effects. Configural, measurement, and structural invariances across the groups were established. Conclusion: The role of social cognition in patients with chronic depression was similar to that in schizophrenia. Social cognition could be a common endophenotype for various psychiatric disorders.

Rett syndrome (RTT), a neurodevelopmental disorder, is mainly caused by mutations in methyl CpG-binding protein 2 (MECP2), which alter the functions of domains to either bind to methylated DNA or interact with a transcriptional co-repressor complex. It has been established that alterations in cyclin-dependent kinase-like 5 (CDKL5) or forkhead box protein G1 (FOXG1) correspond to distinct neurodevelopmental disorders, given that a series of studies have indicated that RTT is also caused by alterations in either one of these genes. We tried to elucidate RTT through evolution and structure assessment of MeCP2, CDKL5, and FOXG1, by focusing on their binding partners and disordered structures. Here, we provide insight into the similarities of the FOXG1 and MECP2 binding partners evolution and function. On the other hand, we suggest that CDKL5 could be a potential candidate for a classical RTT treatment, particularly based on its disordered structure that spans after the catalytic domain to the C-terminus, which shows abundant linear motifs that can bind to molecules with divergent structures of similar affinity. Additionally, we provide insight into the relationship between disordered structure and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cell receptor angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in ACE2-expressing cell surface induces cell–cell membrane fusion, thus forming syncytia. To exert its fusiogenic activity, the spike protein is typically processed at a specific site (S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike–ACE2 interacting surface, is critical for the fusiogenic and infectious roles of the SARS-CoV-2 spike protein. We further demonstrated that the C488 residue of spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin A-mediated endoplasmic reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing.

Urinary tract diseases are common in cats, and often require surgical reconstruction. Here, to explore the possibility of urinary tract reconstruction in cats using in-vivo tissue architecture (iBTA), biosheets fabricated using iBTA technology were implanted into the feline bladder and the regeneration process was histologically evaluated. The biosheets were prepared by embedding molds into the dorsal subcutaneous pouches of six cats for 2 months. A section of the bladder wall was removed, and the biosheets were sutured to the excision site. After 1 and 3 months of implantation, the biosheets were harvested and evaluated histologically. Implantable biosheets were formed with a success rate of 67%. There were no major complications following implantation, including tissue rejection, severe inflammation, or infection. Urinary incontinence was also not observed. Histological evaluation revealed the bladder lumen was almost entirely covered by urothelium after 1 month, with myofibroblast infiltration into the biosheets. After 3 months, the urothelium became multilayered, and mature myocytes and nerve fibers were observed at the implantation site. In conclusion, this study showed that tissue reconstruction using iBTA can be applied to cats, and that biosheets have the potential to be useful in both the structural and functional regeneration of the feline urinary tract.

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors, and is expressed on myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy. Although CCR1 is an attractive target for solid tumors and hematological malignancies, anti-CCR1 therapeutic agents have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody (mAb), C₁Mab-6 (rat IgG_2b, kappa), using the Cell-Based Immunization and Screening (CIBS) method. Flow cytometry and Western blot analyses showed that C₁Mab-6 recognizes mCCR1 specifically. The dissociation constant of C₁Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9×10^-9 M, indicating that C₁Mab-6 possesses a high affinity to mCCR1. These results suggest that C₁Mab-6 could be a useful tool for targeting CCR1 in preclinical mouse models.

C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 signaling induces chemotaxis of immune cells to inflammation sites and promotes inflammation in inflammatory diseases. Various mouse models to mimic the pathogenesis of each disease have been developed to understand mechanisms and evaluate therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx₃Mab-4 (rat IgG₁, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx₃Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx₃Mab-4 was determined as 1.3 × 10^-9 M, indicating that Cx₃Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx₃Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.

Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuzumab and pertuzumab, were clinically approved. We previously developed a highly sensitive and specific anti-HER2 mAb, H2Mab-139 (mouse IgG1, kappa). In this study, we produced a defucosylated IgG2a version of anti‑HER2 mAb (H2Mab-139-mG2a-f) to enhance ADCC-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with the dissociation constant (KD) determined to be 3.9 × 10‑9 M and 7.7 × 10‑9 M against HER2‑overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent cytotoxicity against CHO/HER2 and BT-474 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and could be useful for an antibody treatment regimen for HER2-positive human cancers.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of diagnostic biomarkers and effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeting monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells in immunohistochemistry. These results indicate that C44Mab-94 is useful for detecting CD44v8 in various applications and is expected to be useful for the application of GC diagnosis and therapy.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent KD of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and 2.3 × 10−8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

CD44 has been known as a marker of tumor initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in malignant progression of cancers by promoting the stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and establishment of the therapy. However, the function of the variant 4-encoded region has not to be elucidated. Therefore, specific monoclonal antibodies (mAbs) against the variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) monoclonal antibodies (mAbs) by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterized them. One of the established clones (C₄₄Mab-108; IgG₁, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The K_D of C₄₄Mab-108 for CHO/CD44 v3-10 was 3.4 × 10⁻⁷ M. In western blot analysis, C₄₄Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C₄₄Mab-108 stained formalin-fixed paraffin-embedded oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C₄₄Mab-108 is useful to detect CD44v4 in various applications.

CD44 is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo-resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10 ectodomain and screening using enzyme-linked immunosorbent assay. We then characterized them using flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-46; IgG1, kappa) reacted with CD44s-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The KD of C44Mab-46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1×10-8 M, 4.9×10-8 M, and 4.1×10-8 M, respectively. C44Mab-46 detected CD44s of CHO/CD44s and KYSE70, and CD44v of KYSE770 in western blot analysis. Furthermore, C44Mab-46 strongly stained esophageal squamous carcinoma cells in immunohistochemistry using formalin-fixed paraffin-embedded ESCC tissues. Taken together, C44Mab-46 is very useful for detecting CD44 in various applications.

Rett syndrome (RTT) is mainly caused by mutations in methyl CpG-binding protein 2, cyclin-dependent kinase-like 5, or forkhead box protein G1. These RTT-causing proteins harbor an intrinsically disordered region (IDR) whose conformation exhibits spatiotemporal heterogeneity, which not only confer versatility to the protein, but also implicates them in diseases. The IDR generally evolves more rapidly than an ordered structure. In this study, we examined the relationship between pathogenic RTT-associated point mutations in RTT-causing proteins and the evolutionary dynamics of sequence features including structural order–disorder, phosphorylation sites, and evolutionary rates. We also analyzed the molecular properties and evolution of proteins that interact with RTT-causing proteins in terms of phylogenetic profiles, tissue specificity, subcellular localization, expression level, and functions. The results indicate that constrained IDRs may function by forming contacts with other regions in the protein sequence causing pathogenic missense mutations likely to arise in the rapidly evolving IDR and affect molecular networks, leading to disease. The results also provide novel insights into the genetic basis for RTT and the evolution of the neocortex in higher vertebrates.

Quartz Crystal Microbalance (QCM) is a well-known method to measure the mass of ligands bound to receptor-covered surfaces in various fields, ranging from materials science to biology. Actin molecule is essential in various cellular processes through its function of monomer-to-filament polymerization, forming a double-stranded filament with a 75 nm pitch. In this study, QCM was applied to measure the physical properties of actin, finding that the frequency shifted negatively when monomeric actin bound to the QCM surface, whereas the frequency shifted positively when filaments bound. Using fluorescence light microscopic observations, we have examined whether negative and positive frequency shifts originated from their length. In order to control this process, a severing protein, fragmin was used to change the length of actin filament. When the average length of filaments shortened, the magnitude of positive shift decreased in a concentration-dependent manner. However, shorter filaments showed a negative frequency shift depending on their concentration, showing that the weight derived from the loaded concentration and the function derived from actin polymerization dynamics are detectable. Interestingly, the transition from positive to negative occurred when the average length of actin was ~33 nm, or approximately equal to the half-pitch of filaments. Thus, actin filaments can serve as an excellent standard to measure the mechanical properties of biopolymers. Our results show that the QCM sensor could be both a "weight" and a "function" sensor of biomolecules in vitro.

Background: Intradialytic hypotension (IDH) is a critical pathological condition associated with all−cause mortality in patients undergoing hemodialysis (HD). However, few studies have investigated IDH-related changes in hepatic and cerebral regional tissue oxygen saturation (rSO2). This study investigated IDH-induced changes in hepatic and cerebral rSO2. Methods: Hepatic and cerebral rSO2 during HD were measured using an INVOS 5100C oxygen saturation monitor, and their percentage (%) changes during IDH development were analyzed. Ninety-one patients undergoing HD were investigated, including 20 with IDH development. Results: In patients with IDH, % changes in hepatic and cerebral rSO2 decreased at IDH onset. Additionally, the % change in hepatic rSO2 was significantly larger than those in cerebral rSO2 (P < 0.001). In patients without IDH, no significant differences were found between the % changes in hepatic and cerebral rSO2 at time of the lowest systolic blood pressure during HD. Multivariable linear regression analysis showed that the difference between the % change in cerebral and hepatic rSO2 was significantly associated with IDH development (P < 0.001) and ultrafiltration rate (P = 0.010). Conclusion: Hepatic and cerebral rSO2 significantly decreased in IDH development and hepatic rSO2 was more largely decreased than cerebral rSO2 at IDH onset.

Hydroxyapatite and b-tricalcium phosphate have been clinically applied as artificial bone materials due to their high biocompatibility. The development of artificial bones requires the verification of safety and efficacy through animal experiments; however, from the viewpoint of animal welfare, there is necessary to reduce the number of animal experiments. In this study, we utilized machine learning to construct a model that estimates the bone-forming ability of bioceramics from material fabrication conditions, material properties, and in vivo experimental conditions. We succeeded in constructing the two models as follows: ‘Model 1’, which predicts material properties from their fabrication conditions, and ‘Model 2’, which predicts the bone-formation rate from material properties and in vivo experimental conditions. By an inverse analysis of the two models, we proposed candidates for material fabrication conditions to achieve target values of bone-formation rate. Under the proposed conditions, the material properties of the fabricated material were consistent with the estimated material properties. Furthermore, a comparison between bone-formation rates after 12 weeks of implantation in the porcine tibia and the estimated bone-formation rate. This result showed that the actual bone-formation rates existed within the error range of the estimated bone-formation rates, indicating that machine learning consistently predicts the results of animal experiments using material fabrication conditions. We believe that these findings will lead to the establishment of alternative animal experiments to replace animal experiments in the development of artificial bones.

1) Background: Capturing patients’ medical histories significantly influences clinical decisions. Errors in this process lead to clinical errors, increase costs and dissatisfaction among physicians and patients. Physicians in developing countries are overloaded with patients and cannot always follow the proper history-taking procedure. The challenges are acknowledged; however, a comprehensive understanding of the status and the remedies remained unexplored. 2) Objective: This paper aims to investigate the workload, history-taking challenges, and the willingness of the physicians to accept digital solutions. 3) Methods: A cross-sectional online survey was conducted on 104 physicians across Bangladesh, featuring 22 questions regarding their professional environment, workload, digitization status of health records, challenges in history-taking, and attitudes toward adopting digital solutions for managing patient histories; 4) Results: 92.67% of the physicians face workloads, 88.46% struggle in medical history-taking, and only 4.81% use digital medical records. About 70% struggle to complete the necessary history-taking steps, emphasizing the urgent need for solutions. An AI-based Smart Health Gantt Chart (SHGC) system has been introduced for their instant feedback. 93.27% of physicians expressed their willingness to use such a system. 5) Conclusions: The proposed SHGC has the potential to enhance healthcare efficiency in developing nations, benefit physicians, and improve patient-centered care.

CD44 is a type I transmembrane glycoprotein, and possesses various isoforms which are largely classified into CD44 standard and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 mAb, C₄₄Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C₄₄Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10), and found that C₄₄Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C₄₄Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C₄₄Mab-108 includes Asp280 and Trp281 of CD44v3-10.

The Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling in cancers. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. However, a mAb against mouse EGFR (mEGFR) for flow cytometry has not been established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG1, kappa) reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10−8 M and 1.9 × 10−8 M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR by flow cytometry, and is expected in the use of preclinical study.

Conventional hydrogen production, as an alternative energy resource, has relied on fossil fuels to produce hydrogen, releasing CO2 into the atmosphere. Hydrogen production via the dry forming of methane (DRM) process is a lucrative solution to utilize greenhouse gases, such as carbon dioxide and methane, by using them as raw materials in the DRM process. However, there are a few DRM processing issues, with one being the need to operate at a high temperature to gain high conversion of hydrogen, which is energy intensive. In this study, bagasse ash, which contains a high percentage of silicon dioxide, was designed and modified for catalytic support. Modification of silicon dioxide from bagasse ash was utilized as a waste material and the performance was explored of bagasse ash-derived catalysts interacting with light irradiation and reducing the amount of energy used in the DRM process. The results showed that the performance of 3%Ni/SiO2 bagasse ash WI was higher than that of 3%Ni/SiO2 commercial SiO2 in terms of the hydrogen product yield, with hydrogen generation initiated in the reaction at 300 °C. Using the same synthesis method, the current results suggested that bagasse ash-derived catalysts had better performance than commercial SiO2-derived catalysts when exposed to an Hg-Xe lamp. This indicated that silicon dioxide from bagasse ash as a catalyst support could help improve the hydrogen yield while lowering the temperature in the DRM reaction, resulting in less energy consumption in hydrogen production.

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG1, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The KD of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10-8 M and 3.2 × 10-8 M, respectively. In western blot analysis, recEpMab-37 detected EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by CBIS method, is useful for detecting EpCAM in various applications.

The purpose of the present study was to investigate the effect of the laterally moving tactile stimuli to the sole on the anticipatory postural adjustment (APA) of the gait initiation. Thirteen healthy males participated in this study. A sound cue was provided at the beginning of each trial. The moving tactile stimuli were delivered to the sole after the start cue. The loci of the tactile stimuli moved from the left to right most side of the sole, and then moved from the right to left most side of that in a stimuli cycle. The duration of one stimuli cycle was 960 ms, and this cycle repeated 16 times in a trial. The participants made three steps forward from quiet stance at the participants' preferred time after the start cue. The APA did not onset at the specific direction or phase of the moving tactile stimuli, indicating that they did not use the stimuli as a trigger for initiating the gait. The moving tactile stimuli decreased the amplitude and increased the duration of the APA. The moving tactile stimuli increased the time between the APA onset and toe-off of the initial support leg, indicating that they moved slower when initiating gait during the moving tactile stimuli. Those findings are explained by the view that suppression of the APA induced by the moving tactile stimuli to the sole is caused by the decrease in the velocity of the gait initiation, and the slower velocity of the gait initiation is caused by masking tactile sensation of the sole.

Quarry waste fines and eggshells are unavoidable wastes which relentlessly contribute to environmental load and pollution. Although a number of studies have suggested various methods for recycling, these wastes remain underutilized due to some technical constraints. In addition, no study has yet been explored on the possibility of combining quarry waste fines (QWF) and eggshell powder (ESP) for tobermorite synthesis. Tobermorite is the main component which primarily provides strength to the autoclaved aerated concrete products. For this purpose, this study seeks to evaluate the potential of QWF- ESP mix at 10%, 15% and 20% amounts of cement, respectively. The XRF, XRD, and TGA–DTA techniques were used to characterize the waste materials while physical and mechanical property tests and XRD analysis were performed on the autoclaved samples. It was found that QWF contains 53.77% SiO2, while ESP contains 97.8% CaO which are key components for tobermorite synthesis. This study also revealed that the mixture with only 10% cement has the highest compressive strength among the QWF-ESP samples. Furthermore, the formation of tobermorite in the samples was confirmed through XRD analysis. Hence, the hydrothermal curing of QWF –ESP can be further developed to produce functional tobermorite – bearing materials.

Most pivotal clinical trials in advanced non-small cell lung cancer (NSCLC) have excluded patients with poor performance status (PS), and data on the efficacy and safety of pharmacotherapy have not been fully accumulated. For NSCLC patients with PS 2 and without druggable genetic alterations, monotherapy with cytotoxic agents or carboplatin-based combination therapy is usually administered based on the results of several randomized trials. However, the evidence of cytotoxic chemotherapy for patients with PS 2 is insufficient, with limited efficacy and toxicity concerns. Immune checkpoint inhibitors (ICIs) are a promising treatment for patients with PS 2 because of lower incidence of severe toxicity compared to cytotoxic chemotherapy. Meanwhile, several reports suggest that anti-PD-1 antibodies monotherapy is less effective for patients with PS 2, especially for those with PS 2 caused by disease burden. Although the combination therapy of nivolumab and ipilimumab is a promising treatment option, there is a divergence in efficacy data between clinical trials. The standard of care for advanced NSCLC with PS 2 has not been established, and future therapeutic strategies should take into account the heterogeneity of the PS 2 population.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors, and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, the elucidation of each CD44 isoform’s function in tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through immunization of CD44v3–10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3) determined by enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3–10) or some cancer cell lines (COLO205 and HSC-3) by flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3–10, COLO205, and HSC-3 was 1.5 × 10−9 M, 6.3 × 10−9 M, and 1.9 × 10−9 M, respectively. C44Mab-6 could detect the CD44v3–10 in western blotting, and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments, and expected for the application of tumor diagnosis and therapy.

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergy, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-3 (rat IgG2a, kappa), and C3Mab-4 (rat IgG2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. The CCR3 extracellular domain-substituted mutant analysis showed that C3Mab-3, C3Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, the alanine scanning was conducted in the N-terminal region. The results revealed that Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C3Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C3Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C3Mab-3, C3Mab-4, and J073E5.

To cope the high glycemic variability (GV) is crucial in the management of multiple daily insulin (MDI) in diabetes. We compared the effect of low dose metformin 750mg/d adding vildagliptin 100mg/d (DPP4+LMET) or the high dose metformin 1500mg/d (HMET), in type 2 diabetes (T2D) with MDI, evaluating GV by continuous glucose monitoring (CGM). Single center, open-label, 12 weeks - 2 period crossover design. Twenty T2D with inadequately controlled (7.0% <HbA1c ≦9.0%) with MDI + LMET were enrolled. Primary endpoints were GV and hypoglycemia derived from CGM performed after each 12 weeks treatment periods. There was no significant difference in HbA1c, body weight changes, total daily dose of insulin. DPP4+LMET compared to the HMET, significantly reduced the calculated GV value, mean (7.15±1.3 vs 7.82±1.6, p<0.05), standard deviation (1.78±0.55 vs 2.27±1.11, p=0.03), continuous overlapping net glycemic action (6.44±1.28 vs 7.12±1.69, p<0.05), J-Index (26.7±11.0 vs 34.9±19.8, p<0.05), high blood glucose index (3.01±1.96 vs. 6.73±4.85, p=0.02), and mean amplitude of glycemic excursions (4.53±1.35 vs 5.50±2.34, p=0.03). The GV metrics with hypoglycemia and nocturnal hypoglycemia were not significantly different. DPP4+LMET decreased GV associated with hyperglycemia. Adding DPP4 inhibitor to the lower dose of metformin is an alternative approach to the stable GV in MDI.

Friction is an important subject for sustainability due to problems associated with energy loss. Recent years, surface micro- and nanostructures have attracted much attention to reduce friction; however, suitable structures are still under consideration. Many functional surfaces are present in nature, such as the friction reduction surfaces of snake skins. In this study, we focused on firebrats, Thermobia domestica, living in narrow spaces such as under bark, so their surface frequently contacts with surrounding surfaces. We speculate that their body surface would be adapted to reduce friction. To investigate the firebrat surface functions, firebrat surfaces were observed by using a field-emission scanning electron microscope (FE-SEM) and a colloidal probe atomic force microscope (AFM), respectively. Results of surface observations by the FE-SEM revealed that firebrats are entirely covered with scales, whose surfaces have micro groove structures. Scale groove periods around the firebrat's head are almost uniform within a scale but vary between scales. AFM friction force measurements revealed that firebrat scale reduces friction by decreasing contact area between scales and a colloidal probe. The heterogeneity of groove periods of the scales suggest that it is difficult to fix the whole body in particular rough surfaces and that lead to be "fail-safe".

Non-alcoholic steatohepatitis (NASH), which is the most severe manifestation of non-alcoholic fatty liver disease (NAFLD), has been recognized as a major hepatocellular carcinoma (HCC) catalyst. However, the molecular mechanism of NAFLD-NASH-HCC sequence remains unclear and a specific and effective treatment for NASH has not yet been established. The progress in this field depends on the availability of reliable preclinical models which show the steady progress to NASH, liver cirrhosis, and HCC. However, most of NASH mouse models that have been described to date develop NASH generally for more than 24 weeks and there is an uncertainty of HCC development. To overcome such shortcomings of experimental NASH studies, we established a novel NASH-HCC mouse model with very high reproducibility, generality, and convenience. We treated male C57BL/6J mice with a newly developed choline-deficient and methionine-restricted high-fat diet (CDMRHFD) for 60 weeks. Treatment of CDMRHFD for 3 weeks revealed marked steatosis, lobular inflammation, and fibrosis, histologically diagnosed as NASH. Liver cirrhosis was observed in all mice with 36-week treatment. Liver nodule emerged at 12 weeks of the treatment, and diameter >2 mm liver tumors developed in all mice at 24 weeks of the treatment. In conclusion, our rapidly progressive and highly reproducible NASH-liver cirrhosis-HCC model is helpful for preclinical development and research on the pathogenesis of human NAFLD-NASH-HCC. Our mouse model would be useful for the development of novel chemistry for NASH-HCC-targeted therapies or HCC prevention strategies.

Background and Aim: Zinc plays a pivotal role in various zinc enzymes, resulting in the maintenance of liver function. Patients with chronic liver diseases (CLDs) usually have lower concentrations of zinc, which decrease further as liver fibrosis progresses. It remains unknown whether long-term zinc supplementation improves liver function and reduces the risk of hepatocellular carcinoma (HCC) development. Patients and Methods: Two hundred sixty-seven patients with CLDs who received a zinc preparation (Zn-group; 196 patients), or who did not receive zinc (no Zn-treatment group; 71 patients) were retrospectively analyzed in this study. The Zn-group was divided into 4 groups according to their serum Zn concentrations at 6 months after the start of Zn treatment. Results: Liver function significantly deteriorated in the no Zn-treatment group, while no notable change was observed in the Zn-group. The cumulative incidence rates of events and HCC at 3 years were lower in the Zn-group (9.5%, 7.6%) than in the no Zn-treatment group (24.9%, 19.2%) (p<0.001). According to the serum Zn concentrations, the cumulative incidence rates of events and HCC were significantly decreased in patients with Zn concentrations ≥ 70 µg/dl (p<0.001). Conclusion: Zinc supplementation appears to be effective at maintaining liver function and suppressing events and HCC development, especially among patients whose Zn concentration is greater than 70 µg/dl.

Background: The aim of this study was to demonstrate involvement of whole-body compensatory alignment in different conditions of spinopelvic sagittal balance (compensated/decompensated). Methods: We enrolled 330 healthy subjects and divided them according to sagittal vertical axis (SVA): the compensated group, < 4 cm, (group C) and the decompensated group, ≥ 4 cm, (group D). The correlation between lack of ideal lumbar lordosis (iLL), which is calculated by using the Schwab formula, and the compensatory radiographic parameters in each group were analyzed. The threshold value of KF angle that indicates a spinopelvic sagittal imbalance (SVA ≥ 4) was determined by ROC curve analysis. Results: Correlation analysis of lack of iLL and each com-pensatory parameter showed a strong correlation for pelvic tilt (PT) (r = -.723), and a weak cor-relation for thoracic kyphosis (TK) (r = 275) in Group C. In Group D, the correlations were strong for PT (r = -.796), moderate for TK (r = .462), and KF (r = -.415). The optimal cutoff value for KF angle was determined to be 8.4 degrees (sensitivity 89%, specificity 46%). Conclusions: The pre-sent study showed differences between compensated/decompensated spinopelvic sagittal balance in the correlation strength between lack of iLL and whole-body compensatory parameters.

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti‐EpCAM monoclonal antibody (EpMab-37; mouse IgG1, kappa). In this study, we investigated the antibody‐dependent cellular cytotoxicity (ADCC), complement‐dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG2a-type of EpMab-37 (EpMab-37-mG2a-f) against an EpCAM‐expressing breast cancer cell line (BT-474). EpMab-37-mG2a-f recognized BT-474 cells with a moderate binding-affinity [a dissociation constant (KD): 2.9x10-8 M] by flow cytometry. EpMab-37-mG2a-f exhibited ADCC and CDC for BT-474 cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG2a-f significantly suppressed the BT-474 xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG2a-f exerts antitumor activities against the BT-474 xenograft, and could provide valuable therapeutic regimen for the breast cancers.

This study aimed to elucidate the similarities and differences between amyloid-forming corpora amylacea (CA) in the prostate and lung, examine the nature of CAs in cystic tumors of the atrioventricular node (CTAVN), and clarify the distinctions between amyloid-forming CA and spheroid-type amyloid deposition. We conducted proteomics analyses using liquid chromatography–tandem mass spectrometry with laser microdissection and immunohistochemistry to validate the characteristics of CAs in the lung and prostate. Our findings revealed that CAs in these organs primarily consist of common proteins (β2-microglobulin and lysozyme) and locally produced proteins. Moreover, we observed a discrepancy between the histopathological and proteomic analysis results in CTAVN-associated CAs. In addition, while the histopathological appearance of amyloid-forming CAs and spheroid-type amyloid deposits were nearly identical, the latter deposition lacked β2-microglobulin and lysozyme, and exhibited evident destruction of surrounding tissue. A literature review further supported these findings. These results suggest that amyloid-forming CAs in the lung and prostate are formed through a shared mechanism, serving as waste containers (wasteosomes) and/or storage for excess proteins (functional amyloids). In contrast, we hypothesize that while amyloid-forming CA and spheroid-type amyloid deposits are formed, in part, through common mechanisms, the latter are pathological.

CD44 is a type I transmembrane glycoprotein, which is associated with poor prognosis in various solid tumors. Since CD44 plays critical roles in tumor development by regulating cell adhesion, survival, proliferation, and stemness, it has been considered a target for tumor therapy. Anti-CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody-drug conjugates and chimeric antigen receptor-T cell therapy. We previously developed anti‐pan-CD44 mAbs, C₄₄Mab-5 and C₄₄Mab-46, which can recognize both CD44 standard (CD44s) and variant isoforms. In the present study, a defucosylated mouse IgG_2a version of the anti-pan-CD44 mAbs (5-mG_2a-f and C₄₄Mab-46-mG_2a-f) was generated to evaluate the antitumor activities against CD44-positive cells. Both 5-mG_2a-f and C₄₄Mab-46-mG_2a-f recognized CD44s-overexpressed CHO-K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5-mG_2a-f and C₄₄Mab-46-mG_2a-f could activate effector cells in the presence of CHO/CD44s cells and exhibited the complement‐dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5-mG_2a-f and C₄₄Mab-46-mG_2a-f significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control defucosylated mouse IgG_2a. These results indicate that 5-mG_2a-f and C₄₄Mab-46-mG_2a-f could exert antitumor activities against CD44-positive cancers and could be a promising therapeutic regimen for tumors.

Porous structures and micropatterning surfaces play a crucial role in the development of highly sensitive force sensors. However, achieving these two conductive architectures typically requires complex materials synthesis and expensive manufacturing processes. In this study, we introduce a novel conductive composite film featuring a porous/micropyramid hybrid conductive architecture, which is achieved through a straightforward process of materials mixing and one-step screen printing. By utilizing a deep eutectic solvent in the ink component, micropores are induced in the printed composite, while the mesh of the screen mask acts as a template, resulting in a micropyramid film surface. We have successfully realized highly sensitive flexible force sensors with multifunctional capabilities for perceiving normal force and shear force.

Hsp70.1 has dual functions as chaperone protein and lysosomal stabilizer. Previously, we reported that calpain-mediated cleavage of carbonylated Hsp70.1 causes ischemic neuronal death by inducing lysosomal rupture. Recently, we found that the consecutive injections of vegetable oil-peroxidation product ‘hydroxynonenal’ induces hepatocyte death via the similar cascade. As Hsp70.1 is related also to fatty acid β-oxidation in the liver, its deficiency is known to cause accumulation of fat. Genetic deletion of betaine-homocysteine S-methyltransferase (BHMT) was reported to perturb choline metabolism, inducing decrease of phosphatidylcholine with the resultant hepatic steatosis. Here, focusing on disorders of Hsp70.1 and BHMT, we studied the mechanism of hepatocyte degeneration and steatosis, using monkeys after the consecutive injections of synthetic hydroxynonenal. As these monkeys showed a significant impairment of liver functions, the liver tissues without and with hydroxynonenal injections were compared by proteomics, immunoblotting, immunohisto-chemical and electron microscopic analyses. Western blotting showed upregulation of neither Hsp70.1 nor BHMT, but an increased cleavage of both proteins. Proteomics showed downregulation of Hsp70.1, while 2-fold increments of carbonylated BHMT. Hsp70.1 carbonylation was negligible, showing a marked contrast to ischemic neurons which were associated with ~10-fold increments. The control liver histologically showed lipid droplets in Ito cells, but lipid depositions within hepatocytes were very little. In contrast, after hydroxynonenal injections, widespread fatty degeneration and focal coagulation necrosis were observed with accumulation of numerous tiny lipid droplets within and around the degenerating/dying hepatocytes. Electron microscopy showed lysosomal membrane permeabilization/rupture, remarkable dissolution of mitochondria and rough ER membrane, and proliferation of abnormal peroxisomes. It is probable that disruptions of rough ER caused impaired synthesis of Hsp70.1 and BHMT proteins, while impairments of mitochondria and peroxisomes presumably contributed to the sustained generation of reactive oxygen species. Since both Hsp70.1 and BHMT are vulnerable to the long-standing oxidative stressor, hydroxynonenal-induced disorders facilitated degeneration and steatosis of hepatocytes, respectively.

CD39 is involved in adenosine metabolism through conversion of extracellular ATP to adenosine. Because extracellular adenosine plays a critical role in the immune suppression of tumor microenvironment, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. In this study, we developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mCD39 mAbs, which were established by the CBIS method including C₃₉Mab-1 (rat IgG_2a, kappa) and C₃₉Mab-2 (rat IgG_2a, lambda), reacted with not only mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) but also endogenously mCD39-expressed cell lines, such as L1210 (mouse lymphocytic leukemia) and J774-1 (mouse macrophage-like) cell lines through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constant (K_D) of C₃₉Mab-1 and C₃₉Mab-2 for CHO/mCD39 was 7.3 × 10⁻⁹ M and 5.5 × 10⁻⁹ M, respectively. K_D of C₃₉Mab-1 and C₃₉Mab-2 for L1210 was 3.3 × 10⁻⁹ M and 3.6 × 10⁻¹⁰ M, respectively. Furthermore, C₃₉Mab-1 could detect the lysate of CHO/mCD39 by western blot analysis. These results indicate that C₃₉Mab-1 and C₃₉Mab-2 are useful for the detection of mCD39 in many functional studies.

For the diagnosis of disease, the ability to quantitatively detect trace amounts of the causal proteins from bacteria/viruses as biomarkers in patient specimens is highly desirable. Here we introduce a simple, rapid, and colorimetric assay as a de novo, ultrasensitive detection method. This ultrasensitive assay consists of sandwich enzyme-linked immunosorbent assay (ELISA) and thionicotinamide-adenine dinucleotide (thio-NAD) cycling, forming an ultrasensitive ELISA, in which the signal substrate (i.e., thio-NADH) accumulates in a triangular manner, and the accumulated thio-NADH is measured at its maximum absorption wavelength of 400 nm. We have successfully achieved a limit of detection of ca. 10–18 moles/assay for a target protein. As an example of infectious disease detection, HIV-1 p24 could be measured at 0.0065 IU/assay (i.e., 10⁻¹⁸ moles/assay), and as a marker for a lifestyle-related disease, adiponectin could be detected at 2.3 × 10⁻¹⁹ moles/assay. In particular, despite the long-held belief that the trace amounts of adiponectin in urine can only be detected using a radioisotope, our ultrasensitive ELISA was able to detect urinary adiponectin. This method is highly versatile, because simply changing the antibody enables the detection of various proteins. This assay system requires only the measurement of absorbance, thus it requires equipment that is easily obtained by medical facilities, which facilitates diagnosis in hospitals and clinics. Moreover, we describe an expansion of our ultrasensitive ELISA to a non-amplification nucleic acid detection method for nucleic acids using hybridization. These de novo methods will enable simple, rapid, and accurate diagnosis.

Quality-of-life (QOL) is important for cancer patients with poor prognosis. However, QOL survey is difficult using patients. We created cancer scenarios and investigated QOL utility values among the general public using vignette-based methods. On the other hand, some scenarios may have been difficult for the general public to image and understand. Therefore, we conducted a QOL survey among physicians. This survey was conducted by interviewing physicians adminis-tering chemotherapy to patients for recurrent/metastatic pancreatic cancer. Responses were eval-uated using Composite Time Trade-Off (cTTO) and Visual Analog Scale (VAS) for 11 pancreatic cancer status scenarios to be evaluated (survey scenarios). Survey scenarios consisted of health state, type and Grade of adverse events. Health status was classified into two categories: Stable disease (SD) and Progressive disease (PD). Twenty responders answered in this survey, however two responses were excluded because of inconsistent responses. The stable disease had the highest QOL value for both assessment method. Whereas progression disease (PD) had the lowest QOL value. The physicians scored higher QOL values on VAS and cTTO than the general public in all survey scenarios. The QOL values obtained by physicians were consistent with the degree of sta-tus in any assessment scenarios.

The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti‐EGFR and human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients’ survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti‐EGFR mAb (E134Bf) and a mouse-dog chimeric anti‐HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR and dHER2‐positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3x10-9 M). Furthermore, E134Bf-H77scFv exerted antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.

MicroRNAs (miRNAs) dysregulation contribute the cancer pathogenesis. However, the miRNA profile of canine oral melanoma (COM), one of the frequent malignant melanoma in dog is still unrevealed. The aim of this study is to reveal the miRNA profile in canine oral melanoma. MicroRNAs profile of oral tissues from normal healthy dogs and COM patients were compared by next-generation sequencing. Along with tumour suppressor microRNAs (miRNAs), we report 30 oncogenic miRNAs in COM. Expression of miRNAs were further confirmed by quantitative real-time PCR (qPCR). Pathway analysis showed that deregulated miRNAs impact on cancer and signalling pathways. Three oncogenic miRNAs targets (miR-450b, 301a, and 223) from human study also were down-regulated in COM and had significant negative co-relation with their respective miRNA. Furthermore, we found that miR-450b expression is higher in metastatic cells and regulated MMP9 expression through a PAX9-BMP4-MMP9 axis. In silico analysis indicated that miR-126, miR-20b, and miR-106a regulated the highest numbers of differentially expressed transcription factors in respect to human melanoma. Chromosomal enrichment analysis revealed the X chromosome was enriched with oncogenic miRNAs. We comprehensively analyzed the miRNA’s profile in COM which will be a useful resource for developing therapeutic interventions in both species.

Background: Tachycardia-induced cardiomyopathy (TIC) is caused by prolonged tachycardia leading to left ventricular dilatation and systolic dysfunction with heart failure. Although TIC is more common in adult, it is rare in early infant. Methods: Clinical testing was performed as part of medical evaluation and management. Next gene se-quencing (NGS) was performed for a patient with TIC. Literature review about TIC was con-ducted. Results: The case was a 5-month-old infant referred the hospital due to symptoms with heart failure at least lasting two months. Heart rate was 200 beats per minute, left ventricular ejection fraction fell below 14%, and electrocardiograms showed atrial flutter, suggesting TIC. After cardiover-sion, there was no recurrence of atrial flutter, and cardiac function was improved at 98 days after tachycardia arrest. There were no pathogenic variants identified according to NGS. Literature review identified 8 early infantile cases with TIC reported. However, there were no previous re-ports such as our case with longer duration of TIC. Conclusions: Improvement of cardiac function depends on the duration of TIC. TIC should be considered in all patients with a dilated cardiomyopathy of uncertain origin and who have tachycardias such as atrial flutter.

Observation of the ambient aerosol surface area concentrations is important to understand the aerosol toxicity because an increased surface area may be able to act as an enhanced reaction interface for certain reactions between aerosol particles and biological cells, as well as an augmented carrier surface for co-pollutants. In this study, the concentration of aerosol surface area was measured from April 2015 to March 2016 in Fukuoka, Japan. We investigated the monthly and diurnal variations in the correlations between the aerosol surface area and black carbon (BC) and sulfate concentrations. Throughout the year, aerosol surface area concentration was strongly correlated with the concentrations of BC, which has relatively large surface area since BC particles are usually submicron agglomerates consisting of much smaller (tens of nanometers) sized primary soot particles. The slopes of the regression between the aerosol surface area and BC concentrations was highest in August and September 2015. This appears to have been the results of an increase in the proportion of the airmass that originated on the main islands of Japan. This may enhance the introduction of the BC from the main islands of Japan that is relatively fresh (or “uncoated”), thereby maintaining its larger surface area.

This study examined the patterns of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) deposition in the small intestine and colon to evaluate the association between histopathological severity of EFEMP1 deposition and constipation and determine if amyloid transthyretin (ATTR) and EFEMP1 deposits can colocalize. In 40 older cases (≥80 years of age), EFEMP1 deposition in the small intestine initiated in the submucosal and subserous vessels, subserous interstitium, and serosa (early stage); progressing to the muscularis propria and peri-Auerbach plexus area (intermediate stage); and finally, spreading diffusely to areas, excluding the mucosa and muscularis mucosa (advanced stage). The colon had a similar pattern of progression. During the middle-to-advanced stages, amyloid formation was observed in some vascular and serous deposits. We identified a subgroup of cases in which EFEMP1 deposition was the only presumed cause of constipation. Additionally, we demonstrated the colocalization of ATTR and EFEMP1 deposition. Apple-green birefringence was detected under polarized light only in approximately one-half of the cases in the small intestine and one-third of the cases in the colon. These findings emphasize that EFEMP1 deposits may be associated with lower gastrointestinal pathological conditions. Given the challenges of histopathological diagnosis on Congo red-stained specimens, we recommend the combined use of elastic fiber staining and immunohistochemistry for EFEMP1 to prevent overlooking this deposition.

In many developed countries, mental health disorders have become problematic, and the economic loss due to treatment costs and interference with work is immeasurable. Therefore, we developed a method to assess individuals’ mental health using emotional components contained in their voice. We propose two indices of mental health: vitality, a short-term index, and mental activity, a long-term index capturing the trends in vitality. To evaluate our method, we used the voices of healthy individuals (n = 14) and patients with major depression (n = 30). The patients were also assessed by specialists using the Hamilton Rating Scale for Depression (HAM-D). A significant negative correlation existed between the vitality extracted from the voices and HAM-D scores (r = -0.33, p &lt; .05). We could discriminate the voice data of healthy individuals and patients having depression with a high accuracy using the vitality (p = .0085, area under the curve = 0.76). Further, we developed a method to estimate stress through emotion instead of analyzing stress directly from voice data. By daily monitoring of vitality using smartphones, we can encourage hospital visits for people before they become depressed or during the early stages of depression, to prevent adverse consequences of depression.

Clupeiformes, such as sardines and herrings, represent an important share of worldwide fisheries. Among those, the European sardine (Sardina pilchardus, Walbaum 1792) exhibits significant commercial relevance. While the last decade showed a steady and sharp decline in capture levels, recent advances in culture husbandry represent promising research avenues. Yet, the complete absence of genomic resources from sardine imposes a severe bottleneck to understand its physiological and ecological requirements. We generated 69 Gbp of paired-end reads using Illumina HiSeq X Ten and assembled a draft genome assembly with an N50 scaffold length of 25579 bp and BUSCO completeness of 82.1% (Actinopterygii). The estimated size of the genome ranges between 655 and 850 Mb. Additionally, we generated a relatively high-level liver transcriptome. To deliver a proof of principle of the value of this dataset, we established the presence and function of enzymes (elovl2, elovl5 and fads2) that have pivotal roles in the biosynthesis of long chain polyunsaturated fatty acids, essential nutrients particularly abundant in oily fish such as sardines. Our study provides the first omics dataset from a valuable economic marine teleost species, the European sardine, an essential resource for their effective conservation, management and sustainable exploitation.

Objective: Type I interferon receptor (IFNAR) signaling contributes to several autoimmune and vascular diseases such as atherosclerosis and stroke. The purpose of this study was to assess the influence of IFNAR1 deficiency on the formation and progression of experimental abdominal aortic aneurysms (AAAs). Methods: AAAs were induced in type I interferon receptor subunit 1 (IFNAR1) deficient and wild type control male mice via intra-infrarenal aortic infusion of porcine pancreatic elastase. Immunostaining for IFNAR1 was evaluated in experimental and clinical aneurysms. The initiation and progression of experimental AAAs was assessed via ultrasound imaging prior to (day 0) and 3-, 7-, and 14-days following elastase infusion. Aneurysmal histopathology was analyzed at sacrifice. Results: Increased aortic medial and adventitial IFNAR1 expression was present in both clinical AAAs harvested at surgery and experimental AAAs. Following AAA initiation, wild type mice experienced progressive, time-dependent infrarenal aortic enlargement. This progression was substantially attenuated in IFNAR1 deficient mice. On histological analyses, medial elastin degradation, smooth muscle cell depletion, leukocyte accumulation and neoangiogenesis were markedly diminished in IFNAR1 deficient as compared to wild type mice. Conclusion: IFNAR1 deficiency limited experimental AAA progression in response to intra-aortic elastase infusion. Combined with clinical observations, these results suggest a regulatory role for IFNAR1 activity in AAA pathogenesis.

Left-to-right differences in the histopathologic patterns of transthyretin-derived amyloid (ATTR) deposition in the atria of older adults have not yet been investigated. Hence, this study evaluated heart specimens from 325 serial autopsy subjects. The amount of ATTR deposits in the seven cardiac regions, including both sides of atria and atrial appendages, was evaluated semiquantitatively. Using digital pathology, we quantitatively evaluated the immunohistochemical deposition burden of ATTR in the myocardium. We identified 20 sporadic ATTR cardiac amyloidosis cases (9 males). All patients had ATTR deposition in the left atrial regions of the myocardium. In the semiquantitative analysis, 14 of the 20 cases showed more severe ATTR deposition on the left atrial regions than on the right side, with statistically significant differences in the pathology grading (p &lt; 0.01 for both the atrium and atrial appendage). Quantitative analysis further supported the difference. Moreover, six had ATTR deposition in the epineurium and/or neural fibres of the atria. Cluster analysis revealed that ATTR deposition in the myocardium was significantly more severe in males than in females. The heterogeneous distribution of amyloid deposits between atria revealed in this study may impair the orderly transmission of the cardiac conduction system and induce arrhythmias, which may be further aggravated by additional neuropathy in the advanced phase. This impairment could be more severe among males. These findings emphasise that atrial evaluation is important for individuals with sporadic ATTR cardiac amyloidosis, particularly for early detection.

Cancer-specific monoclonal antibodies (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy are innovative therapeutic strategies for minimizing the adverse effects. We previously established a cancer-specific anti-human epidermal growth factor receptor 2 (HER2)monoclonal antibody (mAb), H₂Mab-250/H₂CasMab-2. In flow cytometry and immunohistochemistry, H₂Mab-250 reacted with HER2-positive breast cancer cells but did not show the reactivity to normal epithelial cells. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, strongly recognizes both breast cancer and normal epithelial cells in flow cytometry. The human IgG₁ version of H₂Mab-250 (H₂Mab-250-hG₁) possesses compatible in vivo antitumor effects against breast cancer xenografts with trastuzumab despite the lower affinity and effector activation than trastuzumab in vitro. This study compared the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) between H₂Mab-250-hG₁ and trastuzumab. Both H₂Mab-250-hG₁ and trastuzumab showed ADCC activity against CHO/HER2 and breast cancer cell lines (BT-474 and SK-BR-3) in the presence of human natural killer cells. Some tendency was observed that trastuzumab showed a more significant ADCC effect compared to H₂Mab-250-hG₁. Importantly, H₂Mab-250-hG₁ exhibited superior CDC activity in these cells compared to trastuzumab. Similar results were obtained in mouse IgG_2a types of both H₂Mab-250 and trastuzumab. These results suggest the different contributions of ADCC and CDC activities to the antitumor effects of H₂Mab-250-hG₁ and trastuzumab and indicate the future direction for the clinical development of H₂Mab-250-hG₁ against HER2-positive tumors.