Complete mitochondrial genome of the southern painted turtle (Chrysemys dorsalis, Testudines: Emydidae) in Korea.

The complete mitochondrial genome of Chrysemys dorsalis in Korea was sequenced and characterized. The mitochondrial genome is 17,258 bp in length and the GC content is 39%. It is constituted of 37 genes, 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a noncoding region. Phylogenetic analysis reveals that C. dorsalis forms a monophyletic group with C. picta turtles but is distinctly separated from them, aligning with previous findings. In Korea, C. dorsalis forms a discrete clade, separate from both native and invasive turtle species. No evidence of genetic disturbance or intermingling is observed. This is the first case of a complete mitochondrial genome from C. dorsalis and provides crucial data for understanding C. dorsalis and managing invasive species effectively, emphasizing the need for continued mitochondrial genome data accumulation.

Age Group Classification of Dental Radiography without Precise Age Information Using Convolutional Neural Networks.

Automatic age estimation using panoramic dental radiographic images is an important procedure for forensics and personal oral healthcare. The accuracies of the age estimation have increased recently with the advances in deep neural networks (DNN), but DNN requires large sizes of the labeled dataset which is not always available. This study examined whether a deep neural network is able to estimate tooth ages when precise age information is not given. A deep neural network model was developed and applied to age estimation using an image augmentation technique. A total of 10,023 original images were classified according to age groups (in decades, from the 10s to the 70s). The proposed model was validated using a 10-fold cross-validation technique for precise evaluation, and the accuracies of the predicted tooth ages were calculated by varying the tolerance. The accuracies were 53.846% with a tolerance of ±5 years, 95.121% with ±15 years, and 99.581% with ±25 years, which means the probability for the estimation error to be larger than one age group is 0.419%. The results indicate that artificial intelligence has potential not only in the forensic aspect but also in the clinical aspect of oral care.

Analysis of Microstrip Line with Asymmetric Arch Type Cross-Sectional Structure Using Micro Pattern Transfer Printing Method.

This paper presents the manufacturing procedure and electrical properties of a microstrip line on flexible printed circuit boards (FPCBs) fabricated using the micro pattern transfer printing (MPTP) method for millimeter wave band application. The MPTP method presented herein is compared to the conventional FPCB process based on the degree of insertion loss as it pertains to the cross-sectional shape of the formed microstrip line. Electromagnetic field simulations were performed to confirm that the cross-sectional arch shape fabricated by the MPTP process reduces insertion loss in the high-frequency band. Based on the simulation, the microstrip transmission line was optimized to a width of 217 µm and a length of 30 cm, fabricated on a 50 µm thick poly-cyclohexylene dimethylene terephthalate (PCT) substrate to measure the insertion loss. The insertion loss fabricated using the MPTP method is measured as 0.37 dB/cm at 10 GHz, while the conventional FPCB is measured as 0.66 dB/cm. Through the analysis, it was confirmed that the FPCBs manufactured by the MPTP process show lower insertion loss compared to the conventional FPCBs.

Risk assessment for the native anurans from an alien invasive species, American bullfrogs (Lithobates catesbeianus), in South Korea.

The invasive species are of global concern, and the Invasive American Bullfrog (IAB; Lithobates catesbeianus) is one of the worst invasive amphibian species worldwide. Like other countries, South Korea is also facing challenges from IAB. Although many studies indicated impacts of IAB on native anurans in Korea, the actual risk at the specific level is yet to evaluate. Considering the putative invasiveness of IAB, it is hypothesized that any species with the possibility of physical contact or habitat sharing with them, will have a potential risk. Thus, we estimated and observed their home range, preferred habitats, morphology, behavior, and ecology. Then, comparing with existing knowledge, we assessed risks to the native anurans. We found a home range of 3474.2 ± 5872.5 m2 and identified three types of habitats for IAB. The analyses showed at least 84% of native anurans (frogs and toads) were at moderate to extreme risks, which included all frogs but only 33% of toads. Finally, we recommended immediate actions to conserve the native anurans based on our results. As this study is the first initiative to assess the specific risk level from the invasiveness of L. catesbeianus, it will help the managers to set conservation priorities and strategies.

Hydrogel Stamping for Rapid, Multiplexed, Point-of-Care Immunostaining of Cells and Tissues.

In the era of precision oncology, multicolor fluorescence imaging has become a core technology for multiplexed molecular analysis of cellular and tissue specimens. However, conventional solution-based staining is labor-intensive and time-consuming and requires considerable expertise to yield optimal results, which creates difficulties for employing this technology in resource-limited settings. Here, we report a new immunostaining method based on hydrogel stamping, which is simple, fast, easy to use, and reproducible. We showed that a hydrophilic hydrogel stamp could effectively transfer fluorescent antibodies to targets and withdraw an excess solution when the reaction is completed, obviating the need for extra washing. This unique property allows for quality immunostaining in 5 min for cells using one-eighth of antibody consumption compared to the conventional solution-based method. Furthermore, we implemented fluorescence quenching and immunocycling with hydrogel staining for multiplexed analysis of 9 protein markers at a single cell level. Finally, we applied the immunocycling method to human breast cancer tissue samples and showed quality immunostaining over a large area (∼2 cm2) in 30 min for molecular subtyping of breast cancer. The hydrogel immunostaining could open new opportunities for rapid, automated, and multiplexed profiling in compact point-of-care systems for molecular cancer diagnosis.

The Role of Lysobacter antibioticus HS124 on the Control of Fall Webworm (Hyphantria cunea Drury) and Growth Promotion of Canadian Poplar (Populus canadensis Moench) at Saemangeum Reclaimed Land in Korea.

Populus canadensis Moench forests established in Saemangeum-reclaimed land have been invaded by Hyphantria cunea Drury, causing defoliation and stunted growth. This study investigated the biocontrol potential of cuticle degrading chitinase and protease secreted by Lysobacter antibioticus HS124 against H. cunea larvae. In addition, L. antibioticus HS124 was examined for indole-3-acetic acid phytohormone production for plant growth promotion. To determine the larvicidal activity in the laboratory experiments, crude enzymes, bacteria culture, CY medium, and water (control) were sprayed on the larvae reared on natural diet in insect rearing dishes. Treatment with crude enzymes and bacteria culture caused 76.7% and 66.7% larvae mortality, respectively. The larvae cuticle, mainly composed of chitin and proteins, was degraded by cuticle-degrading enzymes, chitinase, and protease in both the bacteria culture and crude enzyme treatments, causing swelling and disintegration of the cuticle. Field application of the bacteria culture was achieved by vehicle-mounted sprayer. Bacterial treatment caused morphological damage on the larvae cuticles and subsequent mortality. Foliar application of the bacteria culture reduced tree defoliation by H. cunea and enhanced growth compared to the control. Especially, L. antibioticus HS124 produced auxins, and increased growth of poplar trees.

Hydrogel-Based Stamping Technology for Solution-Free Blood Cell Staining.

An accurate microscopical analysis of blood smears requires a reproducible and convenient method of staining. Solution-based staining procedures can be cumbersome. Especially in low- and middle-income countries, the lack of skilled technicians and adequate laboratory facilities, as well as insufficient water and reagent quality, often become confounding factors. To overcome these obstacles, we developed a new cell staining method based on sequential stamping of agarose gel patches that contain eosin, methylene blue/oxidized methylene blue, Azure B, and buffer, respectively. Our method, termed "hydrogel staining", provides a simple, reproducible, solution-free, and inexpensive approach to stain blood cells. We have optimized incubation times to achieve the optimal transfer of dyes to fixed blood cells on a glass slide, with outcomes comparable to conventional solution-based methods for white blood cells and malaria-infected red blood cells. This hydrogel staining method does not require special skills to produce excellent quality stained blood film slides. The new method could enhance the accuracy of microscopical examination of blood smears, especially in resource-limited settings.

Bacillus velezensis CE 100 Inhibits Root Rot Diseases (Phytophthora spp.) and Promotes Growth of Japanese Cypress (Chamaecyparis obtusa Endlicher) Seedlings.

Root rot diseases, caused by phytopathogenic oomycetes, Phytophthora spp. cause devastating losses involving forest seedlings, such as Japanese cypress (Chamaecyparis obtusa Endlicher) in Korea. Plant growth-promoting rhizobacteria (PGPR) are a promising strategy to control root rot diseases and promote growth in seedlings. In this study, the potential of Bacillus velezensis CE 100 in controlling Phytophthora root rot diseases and promoting the growth of C. obtusa seedlings was investigated. B. velezensis CE 100 produced ß-1,3-glucanase and protease enzymes, which degrade the ß-glucan and protein components of phytopathogenic oomycetes cell-wall, causing mycelial growth inhibition of P. boehmeriae, P. cinnamomi, P. drechsleri and P. erythoroseptica by 54.6%, 62.6%, 74.3%, and 73.7%, respectively. The inhibited phytopathogens showed abnormal growth characterized by swelling and deformation of hyphae. B. velezensis CE 100 increased the survival rate of C. obtusa seedlings 2.0-fold and 1.7-fold compared to control, and fertilizer treatment, respectively. Moreover, B. velezensis CE 100 produced indole-3-acetic acid (IAA) up to 183.7 mg/L, resulting in a significant increase in the growth of C. obtusa seedlings compared to control, or chemical fertilizer treatment, respectively. Therefore, this study demonstrates that B. velezensis CE 100 could simultaneously control Phytophthora root rot diseases and enhance growth of C. obtusa seedlings.

Tendon-Driven Jamming Mechanism for Configurable Variable Stiffness.

Stiffness transition of a soft continuum body is an essential feature for dexterous interaction with an unstructured environment. Softness ensures safe interaction, whereas rigidness generates high force for movement or manipulation. Vacuum-based granular jamming is a widely used technique for on-line stiffness transition because of its high reconfigurability and intuitive driving method. However, vacuum driving method produces limited force levels, and the heavy weight and bulky size are unfavorable for portable applications. In this work, we propose a tendon-driven jamming mechanism for configurable variable stiffness. Compared with a vacuum system, an electric motor-tendon drive system has the benefits of force, bandwidth, size, and weight, but has different force characteristics for distribution, directionality, and transmissibility. In this study, a long snake-like shape is chosen instead of a lump shape for compatibility with tendon-drive characteristics. The snake-like shape is likely to cause buckling under the tendon force as the length increases, making the system extremely unstable. Implanting skeletal disk nodes in the structure is our solution to the buckling phenomenon by maintaining the tendon path in the desired position and for distributing the force evenly, thereby achieving stable stiffness transition capabilities for long free-curved shapes. As a proof of concept, a soft wearable device for wrist support is presented using the proposed variable stiffness mechanism. The weight of the device is 184 g, including the actuators, and it can support 2 kgf. Furthermore, the stiffness transition is completed within 2 s, thus achieving quick responses.

Complete mitochondrial genome of the Small Salamander in Korea, Hynobius unisacculus (Anura: Hynobiidae).

The complete mitochondrial (mt) genome of Hynobius unisacculus was sequenced and characterized. The circular mt genome constituted of 37 genes (13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs) and a non-coding region (NCR). Phylogenetic analysis based on the full mt genome sequences confirmed that H. unisacculus was closely related to Hynobius leechii rather than other Hynobius species. This is the first completed mt genome from H. unisacculus, which provides data for further study of phylogeny in Hynobiidaes.

Neuroprotective and antioxidant activities of bamboo salt soy sauce against H2O2-induced oxidative stress in rat cortical neurons.

Bamboo salt (BS) and soy sauce (SS) are traditional foods in Asia, which contain antioxidants that have cytoprotective effects on the body. The majority of SS products contain high levels of common salt, consumption of which has been associated with numerous detrimental effects on the body. However, BS may be considered a healthier substitute to common salt. The present study hypothesized that SS made from BS, known as bamboo salt soy sauce (BSSS), may possess enhanced cytoprotective properties; this was evaluated using a hydrogen peroxide (H2O2)-induced neuronal cell death rat model. Rat neuronal cells were pretreated with various concentrations (0.001, 0.01, 0.1, 1 and 10%) of BSSS, traditional soy sauce (TRSS) and brewed soy sauce (BRSS), and were subsequently exposed to H2O2 (100 µM). The viability of neuronal cells, and the occurrence of DNA fragmentation, was subsequently examined. Pretreatment of neuronal cells with TRSS and BRSS reduced cell viability in a concentration-dependent manner, whereas neuronal cells pretreated with BSSS exhibited increased cell viability, as compared with non-treated neuronal cells. Furthermore, neuronal cells pretreated with 0.01% BSSS exhibited the greatest increase in viability. Exposure of neuronal cells to H2O2 significantly increased the levels of reactive oxygen species (ROS), B-cell lymphoma 2-associated X protein, poly (ADP-ribose), cleaved poly (ADP-ribose) polymerase, cytochrome c, apoptosis-inducing factor, cleaved caspase-9 and cleaved caspase-3, in all cases. Pretreatment of neuronal cells with BSSS significantly reduced the levels of ROS generated by H2O2, and increased the levels of phosphorylated AKT and phosphorylated glycogen synthase kinase-3ß. Furthermore, the observed effects of BSSS could be blocked by administration of 10 µM LY294002, a phosphatidylinositol 3-kinase inhibitor. The results of the present study suggested that BSSS may exert positive neuroprotective effects against H2O2-induced cell death by reducing oxidative stress, enhancing survival signaling, and inhibiting death signals.

Influence of pH and Surface Chemistry on Poly(L-lysine) Adsorption onto Solid Supports Investigated by Quartz Crystal Microbalance with Dissipation Monitoring.

Poly(L-lysine) (PLL) adsorption onto various materials has been widely applied as a surface modification strategy and layer-by-layer fabrication method. Considering the role of electrostatic charges, a detailed understanding of the influence of solution pH on PLL adsorption process is important for optimization of PLL coating protocols. Herein, PLL adsorption onto different polar and hydrophilic substrates­silica, an amine-terminated self-assembled monolayer (SAM) on gold, and a carboxyl-terminated SAM on gold­across a range of pH conditions was investigated using the quartz crystal microbalance with dissipation. The adsorption kinetics consisted of an initial rapid phase, followed by a second phase where adsorption rate gradually decelerated. These features were interpreted by applying a mean-field kinetic model implying diffusion-limited adsorption in the first phase and reconfiguration of adsorbed PLL molecules in the second phase. The adsorption kinetics and uptake were found to be sensitive to the pH condition, surface chemistry, and flow rate. The strongest PLL adsorption occurred at pH 11 on all three surfaces while weak PLL adsorption generally occurred under acidic conditions. The surface morphology and roughness of adsorbed PLL layers were investigated using atomic force microscopy, and strong PLL adsorption is found to produce a uniform and smooth adlayer while weak adsorption formed a nonuniform and rough adlayer.

Elucidating how bamboo salt interacts with supported lipid membranes: influence of alkalinity on membrane fluidity.

Bamboo salt is a traditional medicine produced from sea salt. It is widely used in Oriental medicine and is an alkalizing agent with reported antiinflammatory, antimicrobial and chemotherapeutic properties. Notwithstanding, linking specific molecular mechanisms with these properties has been challenging to establish in biological systems. In part, this issue may be related to bamboo salt eliciting nonspecific effects on components found within these systems. Herein, we investigated the effects of bamboo salt solution on supported lipid bilayers as a model system to characterize the interaction between lipid membranes and bamboo salt. The atomic composition of unprocessed and processed bamboo salts was first analyzed by mass spectrometry, and we identified several elements that have not been previously reported in other bamboo salt preparations. The alkalinity of hydrated samples was also measured and determined to be between pH 10 and 11 for bamboo salts. The effect of processed bamboo salt solutions on the fluidic properties of a supported lipid bilayer on glass was next investigated by fluorescence recovery after photobleaching (FRAP) analysis. It was demonstrated that, with increasing ionic strength of the bamboo salt solution, the fluidity of a lipid bilayer increased. On the contrary, increasing the ionic strength of near-neutral buffer solutions with sodium chloride salt diminished fluidity. To reconcile these two observations, we identified that solution alkalinity is critical for the effects of bamboo salt on membrane fluidity, as confirmed using three additional commercial bamboo salt preparations. Extended-DLVO model calculations support that the effects of bamboo salt on lipid membranes are due to the alkalinity imparting a stronger hydration force. Collectively, the results of this work demonstrate that processing of bamboo salt strongly affects its atomic composition and that the alkalinity of bamboo salt solutions contributes to its effect on membrane fluidity.

Adsorption of hyaluronic acid on solid supports: role of pH and surface chemistry in thin film self-assembly.

Owing to its biocompatibility, resistance to biofouling, and desirable physicochemical and biological properties, hyaluronic acid (HA) has been widely used to modify the surface of various materials. The role of various physicochemical factors in HA adsorption remains, however, to be clarified. Herein, we employed quartz crystal microbalance with dissipation (QCM-D) in order to investigate HA adsorption at different pH conditions onto three substrates-silicon oxide, amine-terminated self-assembled monolayer (SAM) on gold, and carboxylic acid-terminated SAM on gold. The QCM-D experiments indicated specific pH conditions where either strong or weak HA adsorption occurs. The morphology of the adsorbed HA layers was investigated by atomic force microscopy (AFM), and we identified that strong HA adsorption produced a complete, homogenous and smooth HA layer, while weak HA adsorption resulted in rough and inhomogeneous HA layers. The observed specifics of the kinetics of HA adsorption, including a short initial linear phase and subsequent long non-linear phase, were described by using a mean-field kinetic model taking HA diffusion limitations and reconfiguration in the adsorbed state into account. The findings extend the physicochemical background of design strategies for improving the use of passive HA adsorption for surface modification applications.

Peptide-induced formation of a tethered lipid bilayer membrane on mesoporous silica.

Tethered bilayer lipid membranes (tBLMs) on solid supports have substantial advantages as models of artificial cell membranes for such biomedical applications as drug delivery and biosensing. Compared with untethered lipid membranes, tBLMs have more space between substrate and the bilayer and greater stability. The purpose of this work was to use these properties to fabricate and characterize a zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid tBLM containing 2 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-maleimide(poly(ethylene glycol))-2000 (DSPE-PEG2000-NHS) lipid tethers on a 3-aminopropyltrimethoxysilane-modified mesoporous silica substrate. A quartz crystal microbalance with dissipation monitoring was used to monitor the process of vesicle adsorption and tBLM self-assembly, and atomic force microscopy was performed to characterize the structural properties of the tBLM obtained. Whereas tether-containing lipid vesicles ruptured neither spontaneously nor as a result of osmotic shock, introduction of an amphipathic α-helical (AH) peptide induced vesicle rupture and subsequent tBLM formation. Taken together, our findings suggest that the AH peptide is an efficient means of rupturing vesicles of both simple and complex composition, and is, therefore, useful for formation of tBLMs on solid and mesoporous materials for applications in biotechnology.

Bimodal tumor-targeting from microenvironment responsive hyaluronan layer-by-layer (LbL) nanoparticles.

Active targeting of nanoscale drug carriers can improve tumor-specific delivery; however, cellular heterogeneity both within and among tumor sites is a fundamental barrier to their success. Here, we describe a tumor microenvironment-responsive layer-by-layer (LbL) polymer drug carrier that actively targets tumors based on two independent mechanisms: pH-dependent cellular uptake at hypoxic tumor pH and hyaluronan-directed targeting of cell-surface CD44 receptor, a well-characterized biomarker for breast and ovarian cancer stem cells. Hypoxic pH-induced structural reorganization of hyaluronan-LbL nanoparticles was a direct result of the nature of the LbL electrostatic complex, and led to targeted cellular delivery in vitro and in vivo, with effective tumor penetration and uptake. The nanoscale drug carriers selectively bound CD44 and diminished cancer cell migration in vitro, while co-localizing with the CD44 receptor in vivo. Multimodal targeting of LbL nanoparticles is a powerful strategy for tumor-specific cancer diagnostics and therapy that can be accomplished using a single bilayer of polyamine and hyaluronan that, when assembled, produce a dynamic and responsive cell-particle interface.

Solvent-assisted lipid bilayer formation on silicon dioxide and gold.

Planar lipid bilayers on solid supports mimic the fundamental structure of biological membranes and can be investigated using a wide range of surface-sensitive techniques. Despite these advantages, planar bilayer fabrication is challenging, and there are no simple universal methods to form such bilayers on diverse material substrates. One of the novel methods recently proposed and proven to form a planar bilayer on silicon dioxide involves lipid deposition in organic solvent and solvent exchange to influence the phase of adsorbed lipids. To scrutinize the specifics of this solvent-assisted lipid bilayer (SALB) formation method and clarify the limits of its applicability, we have developed a simplified, continuous solvent-exchange version to form planar bilayers on silicon dioxide, gold, and alkanethiol-coated gold (in the latter case, a lipid monolayer is formed to yield a hybrid bilayer) and varied the type of organic solvent and rate of solvent exchange. By tracking the SALB formation process with simultaneous quartz crystal microbalance-dissipation (QCM-D) and ellipsometry, it was determined that the acoustic, optical, and hydration masses along with the acoustic and optical thicknesses, measured at the end of the process, are comparable to those observed by employing conventional fabrication methods (e.g., vesicle fusion). As shown by QCM-D measurements, the obtained planar bilayers are highly resistant to protein adsorption, and several, but not all, water-miscible organic solvents could be successfully used in the SALB procedure, with isopropanol yielding particularly high-quality bilayers. In addition, fluorescence recovery after photobleaching (FRAP) measurements demonstrated that the coefficient of lateral lipid diffusion in the fabricated bilayers corresponds to that measured earlier in the planar bilayers formed by vesicle fusion. With increasing rate of solvent exchange, it was also observed that the bilayer became incomplete and a phenomenological model was developed in order to explain this feature. The results obtained allowed us to clarify and discriminate likely steps of the SALB formation process as well as determine the corresponding influence of organic solvent type and flow conditions on these steps. Taken together, the findings demonstrate that the SALB formation method can be adapted to a continuous solvent-exchange procedure that is technically minimal, quick, and efficient to form planar bilayers on solid supports.

Influence of osmotic pressure on adhesion of lipid vesicles to solid supports.

The adhesion of lipid vesicles to solid supports represents an important step in the molecular self-assembly of model membrane platforms. A wide range of experimental parameters are involved in controlling this process, including substrate material and topology, lipid composition, vesicle size, solution pH, ionic strength, and osmotic pressure. At present, it is not well understood how the magnitude and direction of the osmotic pressure exerted on a vesicle influence the corresponding adsorption kinetics. In this work, using quartz crystal microbalance with dissipation (QCM-D) monitoring, we have experimentally studied the role of osmotic pressure in the adsorption of zwitterionic vesicles onto silicon oxide. The osmotic pressure was induced by changing the ionic strength of the solvent across an appreciably wider range (from 25 to 1000 mM NaCl outside of the vesicle, and 125 mM NaCl inside of the vesicle, unless otherwise noted) compared to that used in earlier works. Our key finding is demonstration that, by changing osmotic pressure, all three generic types of the kinetics of vesicle adsorption and rupture can be observed in one system, including (i) adsorption of intact vesicles, (ii) adsorption and rupture after reaching a critical vesicle coverage, and (iii) rupture just after adsorption. Furthermore, theoretical analysis of pressure-induced deformation of adsorbed vesicles and a DLVO-type analysis of the vesicle-substrate interaction qualitatively support our observations. Taken together, the findings in this work demonstrate that osmotic pressure can either promote or impede the rupture of adsorbed vesicles on silicon oxide, and offer experimental evidence to support adhesion energy-based models that describe the adsorption and spontaneous rupture of vesicles on solid supports.