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Recently, various bioelectronic nose devices based on human receptors were developed for mimicking a human olfactory system. However, such bioelectronic nose devices could operate in an aqueous solution, and it was often very difficult to detect insoluble gas odorants. Here, we report a portable bioelectronic nose platform utilizing a receptor protein-based bioelectronic nose device as a sensor and odorant-binding protein (OBP) as a transporter for insoluble gas molecules in a solution, mimicking the functionality of human mucosa. Our bioelectronic nose platform based on I7 receptor exhibited dose-dependent responses to octanal gas in real time. Furthermore, the bioelectronic platforms with OBP exhibited the sensor sensitivity improved by ∼100% compared with those without OBP. We also demonstrated the detection of odorant gas from real orange juice and found that the electrical responses of the devices with OBP were much larger than those without OBP. Since our bioelectronic nose platform allows us to directly detect gas-phase odorant molecules including a rather insoluble species, it could be a powerful tool for versatile applications and basic research based on a bioelectronic nose.

Arginine deiminase (ADI) is a microbial-derived enzyme which catalyzes the conversion of L-arginine into L-citrulline. ADI originating from Mycoplasma has been reported to present anti-tumor activity against arginine-auxotrophic tumors, including melanoma. Melanoma cells are sensitive to arginine depletion due to reduced expression of argininosuccinate synthase 1 (ASS1), a key enzyme for arginine biosynthesis. However, clinical applications of recombinant ADI for melanoma treatment present some limitations. Since recombinant ADI is not human-derived, it shows instability, proteolytic degradation, and antigenicity in human serum. In addition, there is a problem of drug resistance issue due to the intracellular expression of once-silenced ASS1. Moreover, recombinant ADI proteins are mainly expressed as inclusion body forms in Escherichia coli and require a time-consuming refolding process to turn them back into active form. Herein, we propose fusion of recombinant ADI from Mycoplasma hominis and 30Kc19α, a cell-penetrating protein which also increases stability and soluble expression of cargo proteins, to overcome these problems. We inserted matrix metalloproteinase-2 cleavable linker between ADI and 30Kc19α to increase enzyme activity in melanoma cells. Compared to ADI, ADI-LK-30Kc19α showed enhanced solubility, stability, and cell penetration. The fusion protein demonstrated selective cytotoxicity and reduced drug resistance in melanoma cells, thus would be a promising strategy for the improved efficacy in melanoma treatment. KEY POINTS: • Fusion of ADI with 30Kc19α enhances soluble expression and productivity of recombinant ADI in E. coli • 30Kc19α protects ADI from the proteolytic degradation by shielding effect, helping ADI to remain active • Intracellular delivery of ADI by 30Kc19α overcomes ADI resistance in melanoma cells by degrading intracellularly expressed arginine.

An in vitro model of an eye-to-brain circuit, composed of human photoreceptors that are the short-wavelength opsins and rhodopsins, was created. Two types of photosensitive neural spheroids were transfected for selective reaction under bluish-purple and green lights. These samples were applied to two devices with intact neuron and neural-spheroid to study the interaction. By photostimulation, the photosensitive spheroid initiated photoactivation and phototransduction, and the signal generated from its body was transmitted to adjacent neural networks. Specifically, the signal traveled two millimeters through the axon bundle in the narrow gap from the photosensitive spheroid to the intact spheroid as the eye-to-brain model including optic nerves. The whole process with the photosensitive spheroid was monitored by calcium assay fluorescence images and analyzed by several immunostaining imaging methods. The results of this study can be applied to examine and understand vision restoration and novel photosensitive biological systems with spectral sensitivity.

Recently, it has been revealed that the physical microenvironment can be translated into cellular mechanosensing to direct human mesenchymal stem cell (hMSC) differentiation. Graphene oxide (GO), a major derivative of graphene, has been regarded as a promising material for stem cell lineage specification due to its biocompatibility and unique physical properties to interact with stem cells. Especially, the lateral size of GO flakes is regarded as the key factor regulating cellular response caused by GO. In this work, GO that had been mechanically created and had an average diameter of 0.9, 1.1, and 1.7 m was produced using a ball-mill process. When size-controlled GO flakes were applied to hMSCs, osteogenic differentiation was enhanced by GO with a specific average diameter of 1.7 μm. It was confirmed that osteogenic differentiation was increased due to the enhanced expression of focal adhesion and the development of focal adhesion subordinate signals via extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MEK) pathway. These results suggest that size-controlled GO flakes could be efficient materials for promoting osteogenesis of hMSCs. Results of this study could also improve our understanding of the correlation between hMSCs and cellular responses to GO.

Induced pluripotent stem cells (iPSCs) have intrinsic properties, such as self-renewal ability and pluripotency, which are also shown in embryonic stem cells (ESCs). The challenge of improving the iPSC generation efficiency has been an important issue and there have been many attempts to develop iPSC generation methods. In this research, we added Lin28, known as one of the reprogramming factors, in the form of a soluble recombinant protein from E. coli to improve the efficiency of human iPSC (hiPSC) generation, in respect of alkaline phosphatase (AP)-positive colonies. To deliver Lin28 inside the cells, we generated a soluble Lin28-30Kc19 fusion protein, with 30Kc19 at the C-terminal domain of Lin28. 30Kc19, a silkworm hemolymph-derived protein, was fused due to its cell-penetrating and protein-stabilizing properties. The Lin28-30Kc19 was treated to human dermal fibroblasts (HDFs), in combination with four defined reprogramming factors (Oct4, Sox2, c-Myc, and Klf4). After 14 days of cell culture, we confirmed the generated hiPSCs through AP staining. According to the results, the addition of Lin28-30Kc19 increased the number and size of generated AP-positive hiPSC colonies. Through this research, we anticipate that this recombinant protein would be a valuable material for increasing the efficiency of hiPSC generation and for enhancing the possibility as a substitute of the conventional method.

A photoreceptor on the retina acts as an optical waveguide to transfer an individual photonic signal to the cell inside, which is determined by the refractive index of internal materials. Under the photoactivation of photoreceptors making conformational and chemical variation in a visual cell, the optical signal modulation is demonstrated using an artificial photoreceptor-based waveguide with a controlling beam refraction. Two types of nanodiscs are made of human photoreceptor proteins, short-wavelength-sensitive opsin and rhodopsin, with spectral sensitivity. The refractive index and nonlinear features of those two photosensitive nanodiscs are investigated as fundamental properties. The photonanodiscs are photoactivated in such a way that allow refractive index tuning over 0.18 according to the biological function of the respective proteins with color-dependent response.

BACKGROUND/OBJECTIVES Bitter taste receptors are taste signaling pathway mediators, and are also expressed and function in extra-gustatory organs. Skin aging affects the quality of life and may lead to medical issues. The purpose of this study was to better understand the anti-skin aging effects of bitter taste receptors in D-galactose (D-gal)-induced aged human keratinocytes, HaCaT cells. MATERIALS/METHODS Expressions of bitter taste receptors in HaCaT cells and mouse skin tissues were examined by polymerase chain reaction assay. Bitter taste receptor was overexpressed in HaCaT cells, and D-gal was treated to induce aging. We examined the effects of bitter taste receptors on aging by using β-galactosidase assay, wound healing assay, and Western blot assay. RESULTS TAS2R16 and TAS2R10 were expressed in HaCaT cells and were upregulated by D-gal treatment. TAS2R16 exerted protective effects against skin aging by regulating p53 and p21, antioxidant enzymes, the SIRT1/mechanistic target of rapamycin pathway, cell migration, and epithelial-mesenchymal transition markers. TAS2R10 was further examined to confirm a role of TAS2R16 in cellular senescence and wound healing in D-gal-induced aged HaCaT cells. CONCLUSIONS Our results suggest a novel potential preventive role of these receptors on skin aging by regulating cellular senescence and wound healing in human keratinocyte, HaCaT.

Background The number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptional regulators for transdifferentiation, octamer-binding transcription factor 4 (OCT4) is famous for its role in the regulation of pluripotency of stem cells. Bone morphogenetic protein 4 (BMP4) is another factor that is known to have a significant role in osteogenic differentiation. Previous studies have achieved transdifferentiation of cells into osteoblasts using viral and plasmid deliveries of these factors. Although these methods are efficient, viral and plasmid transfection have safety issues such as permanent gene incorporations and bacterial DNA insertions. Herein, we developed a cell penetrating protein-based strategy to induce transdifferentiation of endothelial cells into osteoblasts via nuclear delivery of OCT4 recombinant protein combined with the BMP4 treatment. For the nuclear delivery of OCT4 protein, we fused the protein with 30Kc19, a cell-penetrating and protein stabilizing protein derived from a silkworm hemolymph of Bombyx mori with low cytotoxic properties. This study proposes a promising cell-based therapy without any safety issues that existing transdifferentiation approaches had. Methods OCT4-30Kc19 protein with high penetrating activities and stability was synthesized for a protein-based osteogenic transdifferentiation system. Cells were treated with OCT4-30Kc19 and BMP4 to evaluate their cellular penetrating activity, cytotoxicity, osteogenic and angiogenic potentials in vitro. The osteogenic potential of 3D cell spheroids was also analyzed. In addition, in vivo cell delivery into subcutaneous tissue and cranial defect model was performed. Results OCT4-30Kc19 protein was produced in a soluble and stable form. OCT4-30Kc19 efficiently penetrated cells and were localized in intracellular compartments and the nucleus. Cells delivered with OCT4-30Kc19 protein combined with BMP4 showed increased osteogenesis, both in 2D and 3D culture, and showed increased angiogenesis capacity in vitro. Results from in vivo subcutaneous tissue delivery of cell-seeded scaffolds confirmed enhanced osteogenic properties of transdifferentiated HUVECs via treatment with both OCT4-30Kc19 and BMP4. In addition, in vivo mouse cranial defect experiment demonstrated successful bone regeneration of HUVECs pretreated with both OCT4-30Kc19 and BMP4. Conclusions Using a protein-based transdifferentiation method allows an alternative approach without utilizing any genetic modification strategies, thus providing a possibility for safer use of cell-based therapies in clinical applications.

Human mesenchymal stem cells (hMSCs) are a commonly used cell source for cell therapy and tissue engineering because of their easy accessibility and multipotency. Runt-related transcription factor 2 (RUNX2) is a master regulator of the osteogenic commitment of hMSCs. Either recombinant plasmid delivery or viral transduction has been utilized to activate RUNX2 gene expression for effective hMSC differentiation. In this study, recombinant RUNX2 fused with cell-penetrating 30Kc19α protein (30Kc19α-RUNX2) was delivered into hMSCs for osteogenic commitment. Fusion of recombinant RUNX2 with 30Kc19α resulted in successful delivery of the protein into cells and enhanced soluble expression of the protein. Intracellular delivery of the 30Kc19α-RUNX2 fusion protein enhanced the osteogenic differentiation of hMSCs in vitro. 30Kc19α-RUNX2 treatment resulted in increased ALP accumulation and elevated calcium deposition. Finally, implantation of hMSCs treated with 30Kc19α-RUNX2 showed osteogenesis via cell delivery into the subcutaneous tissue and bone regeneration in a cranial defect mouse model. Therefore, we suggest that 30Kc19α-RUNX2, an osteoinductive recombinant protein, is an efficient tool for bone tissue engineering.

As the acute lymphoblastic leukaemia (ALL) develops, expression of L-asparaginase (ASNase) protein is known to decrease. Therefore, deficiency of the ASNase protein would be regarded as one of the significant indications of the ALL. For the treatment of ALL, recombinant ASNase protein derived from bacterial origin is used which causes cytotoxicity by deprivation of Asn. However, short half-life of the protein is an obstacle for medical use. In order to overcome this limit, recombinant ASNase was fused to 30Kc19 with protein-stabilizing and cell-penetrating properties. As the 30Kc19 protein may induce steric hindrance, we further added a PLGLAG linker sequence (LK) between the ASNase and 30Kc19. The treatment of ASNase-LK-30Kc19 fusion protein demonstrated enhanced stability, cell-penetrating property, and anti-cancer activity. Intracellular delivery of both the non-cleaved and cleaved forms of the protein were observed, suggesting that ASNase acted both internally and externally, performing high anti-cancer activity by effective depletion of intracellular Asn. Additionally, ASNase-LK-30Kc19 showed high selectivity towards cancer cells. In terms of the dosage, releasable ASNase from ASNase-LK-30Kc19 reached the same half-maximal inhibitory concentration at a concentration five times lower than non-releasable ASNase-30Kc19. Altogether, the findings suggest that this fusion approach has potential applications in the treatment of ALL.

Inspired by an adaptive immune system, we have developed a bioelectronic sensing platform which relies on nanovesicles for a signal amplification and can be easily adapted for the detection of new food allergens. In this work, nanovesicles with anti-immunoglobulin E (anti-IgE) antibody receptors were extracted from immune cells and immobilized on a carbon nanotube-based transistor to build a highly sensitive and selective biosensing platform. Our sensor could detect peanut allergen, arachis hypogaea 2 (Ara h 2), down to 0.1 fM and selectively discriminate target allergens in real food samples such as peanut and egg white. As a proof of concept, we demonstrated the detection of different target molecules using the same nanovesicles linked with different antibodies. Our sensor platform was also utilized to quantitatively evaluate the effect of allergy drug such as cromolyn. In this regard, our strategy can be utilized for basic research and versatile applications in food and pharmacological industries.

Collagen is the most abundant protein in the extracellular matrix of mammals and has a great effect on various cell behaviors including adhesion, differentiation, and migration. However, it is difficult to utilize collagen gel as a physical scaffold in vitro because of its severe contraction. Decrease in the overall hydrogel volume induces changes in cell distribution, and mass transfer within the gel. Uncontrolled mechanical and physiological factors in the fibrous matrix result in uncontrolled cell behaviors in the surrounding cells. In this study, two strategies were used to minimize the contraction of collagen gel. A disk-shaped frame made of polydopamine-coated polydimethylsiloxane (PDMS) prevented horizontal contraction at the edge of the hydrogel. The sequentially cross-linked collagen gel with alginate outer shell (CA-shell) structure inhibited the vertical gel contraction. The combined method synergistically prevented the hydrogel from shrinkage in long-term 3D cell culture. We observed the shift in balance of differentiation from adipogenesis to osteogenesis in mesenchymal stem cells under the environment where gel contraction was prevented, and confirmed that this phenomenon is closely associated with the mechanotransduction based on Yes-associated protein (YAP) localization. Development of this contraction inhibition platform made it possible to investigate the influence of regulation of cellular microenvironments. The physical properties of the hydrogel fabricated in this study were similar to that of pure collagen gel but completely changed the cell behavior within the gel by inhibition of gel contraction. The platform can be used to broaden our understanding of the fundamental mechanism underlying cell-matrix interactions and reproduce extracellular matrix in vivo.

Monitoring food freshness/spoilage is important to ensure food quality and safety. Current methods of food quality monitoring are mostly time-consuming and labor intensive processes that require massive analytical equipment. In this study, we developed a portable bioelectronic nose (BE-nose) integrated with trace amine-associated receptor (TAAR) nanodiscs (NDs), allowing food quality monitoring via the detection of food spoilage indicators, including the biogenic amines cadaverine (CV) and putrescine (PT). The olfactory receptors TAAR13c and TAAR13d, which have specific affinities for CV and PT, were produced and successfully reconstituted in ND structures. TAAR13 NDs BE-nose-based side-gated field-effect transistor (SG-FET) system was constructed by utilizing a graphene micropattern (GM) into which two types of olfactory NDs (TAAR13c ND and TAAR13d ND) were introduced, and this system showed ultrahigh sensitivity for a limit of detection (LOD) of 1 fM for CV and PT. Moreover, the binding affinities between the TAAR13 NDs and the indicators were confirmed by a tryptophan fluorescence quenching assay and biosimulations, in which the specific binding site was confirmed. Gas-phase indicators were detected by the TAAR13 NDs BE-nose platform, and the LODs for CV and PT were confirmed to be 26.48 and 7.29 ppb, respectively. In addition, TAAR13 NDs BE-nose was fabricated with commercial gas sensors as a portable platform for the measurement of NH

It is known that a dog possesses much better sense of smell than a human. It has been reported that a dog has advantages (compared to a human) in the number of olfactory receptor (OR) genes, proteins and cells, and also nasal structure. However, a definitive reason for superior sensitivity of the canine olfactory system remains controversial. In this study, we compared sensitivity of human and canine olfactory receptors relative to the same condition. Human OR (hOR2W1) and canine OR (cfOR0312), previously identified to recognize hexanal, were inserted to the pcDNA3 vector. This vector was transfected to HEK293 cells. Hexanal-discriminating ability of ORs was confirmed using Fura-2 AM, a dye which illuminates when binding to calcium ions flowing into cells once olfactory signaling occurs. Consequently, cfOR0312 was more sensitive to hexanal than hOR2W1. Comparison of OR sensitivity would be one of the major factors in clarifying difference of sensitivity between human and canine olfactory systems.

Field-effect transistor (FET) devices based on conductive nanomaterials have been used to develop biosensors. However, development of FET-based biosensors that allow efficient stability, especially in the gas phase, for obtaining reliable and reproducible responses remains a challenge. In this study, we developed a nanodisc (ND)-functionalized bioelectronic nose (NBN) based on a nickel (Ni)-decorated carboxylated polypyrrole nanoparticle (cPPyNP)-FET that offers the detection of liquid and gaseous cadaverine (CV). The TAAR13c, specifically binding to CV, which is an indicator of food spoilage, was successfully constructed in NDs. The NBN was fabricated by the oriented assembly of TAAR13c-embedded NDs (T13NDs) onto the transistor with Ni/cPPyNPs. The NBN showed high performance in selectivity and sensitivity for the detection of CV, with excellent stability in both aqueous and gas phases. Moreover, the NBN allowed efficient measurement of corrupted real-food samples. It demonstrates the ND-based device can allow the practical biosensor that provides high stability in the gas phase.

Human pluripotent stem cells (hPSCs), such as human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), have been widely used in stem cell research. hPSCs form colonies in culture dishes and are passaged as clumps. If the cells are dissociated into single cells, epithelial structures are disrupted, and rapid apoptosis is induced. The low viability of single cells restricts the passaging and expansion of hPSCs. Here, in order to enhance the single cell viability of hPSCs, we adopted an apoptotic strategy using the 30 Kc6 gene which inhibits mitochondrial apoptosis. With the expression of the 30 Kc6 gene in hiPSCs, no issues related to pluripotency or differentiation occurred. The hiPSC-30 Kc6 showed higher viability after the induction of apoptosis compared with control hiPSCs. Furthermore, hiPSC-30 Kc6 formed more colonies when the cells were enzymatically dissociated into single cells. Taken together, this study demonstrates that the 30 Kc6 gene could facilitate the passage and expansion of hiPSCs, potentially preventing the dissociation-induced apoptosis of single hPSCs.

Cells can 'sense' physical cues in the surrounding microenvironment and 'react' by changing their function. Previous studies have focused on regulating the physical properties of the matrix, such as stiffness and topography, thus changing the tension 'felt' by the cell as a result. In this study, by directly applying a quantified magnetic force to the cell, a correlation between differentiation and tension was shown. The magnetic force, quantified by magnetic tweezers, was applied by incorporating magnetotactic bacteria-isolated magnetic nanoparticles (MNPs) in human mesenchymal stem cells. As the applied tension increased, the expression levels of osteogenic differentiation marker genes and proteins were proportionally upregulated. Additionally, the translocation of YAP and RUNX2, deformation of nucleus, and activation of the MAPK signaling pathway were observed in tension-based osteogenic differentiation. Our findings provide a platform for the quantitative control of tension, a key factor in stem cell differentiation, between cells and the matrix using MNPs. Furthermore, these findings improve the understanding of osteogenic differentiation by mechanotransduction.

The human sweet taste receptor is a TAS1R2/TAS1R3 heterodimer. To investigate the correlation between the in vitro affinity of sweeteners with stably expressed human sweet taste receptor in HEK-293 cells and human sensory evaluation, the receptor-ligand activity of bulk (sucrose, D-fructose, and allulose) and high-intensity sweeteners (saccharin, rebaudioside A, rebaudioside M, and neohesperidin dihydrochalcone) was compared by analyzing the Ca(2+) release. The relative potency of the sweeteners was identified over a wide concentration range for EC(50)s. Relative to sucrose, bulk sweeteners showed similar concentration ranges and potency, whereas high-intensity sweeteners exhibited lower concentration ranges and higher potency. The log of the calculated EC(50) of each sweetener relative to sucrose by the in vitro affinity assay was positively correlated (r = 0.9943) with the molar relative sweetness reported in the previous literatures. These results suggested a good correlation between the in vitro activity assay of sweeteners and human sensory evaluation.

An extracellular matrix (ECM) utilized as a biomaterial can be obtained from organs of living organisms. Therefore, it has some limitations in its supply because of insufficient organs. Furthermore, therapeutic efficacy of ECMs varies depending on factors such as donor's health condition and age. For this reason, ECMs obtained from a cell line could be a good alternative because they can be produced under a controlled environment with uniform quality. Thus, the purpose of this study was to investigate the potential of the MC3T3-E1 cell line-derived ECM as bone graft. The optimized decellularization process was developed to separate the ECM from MC3T3-E1, osteoblast cell line, using Trypsin-EDTA and Triton X-100. The decellularized ECM was partially digested using pepsin. Also, human bone marrow-derived mesenchymal stem cells induced faster osteogenesis on the ECM-coated surface than on the collagen-coated surface. Partially digested ECM fragments were embedded on the polyethylene glycol scaffold without additional chemical modification or crosslinking. Micro-computed tomography and histological analysis results showed that the ECM in the scaffold promoted actual bone regeneration after in vivo implantation to a mouse calvarial defect model. This study suggests that the bone-specific ECM derived from the cell line can replace the ECM from organs for application in tissue engineering and regenerative medicine.

Endothelial progenitor cells (EPCs) can induce a pro-angiogenic response during tissue repair. Recently, EPC transplantations have been widely investigated in wound healing applications. To maximize the healing efficacy by EPCs, a unique scaffold design that allows cell retention and function would be desirable for in situ delivery. Herein, we fabricated an alginate/poly-l-ornithine/gelatin (alginate-PLO-gelatin) hydrogel sheet with a groove pattern for use as a cell delivery platform. In addition, we demonstrate the topographical modification of the hydrogel sheet surface with a groove pattern to modulate cell proliferation, alignment, and elongation. We report that the patterned substrate prompted morphological changes of endothelial cells, increased cell-cell interaction, and resulted in the active secretion of growth factors such as PDGF-BB. Additionally, we incorporated magnetic nanoparticles (MNPs) into the patterned hydrogel sheet for the magnetic field-induced transfer of cell-seeded hydrogel sheets. As a result, enhanced wound healing was observed via efficient transplantation of the EPCs with an MNP-embedded patterned hydrogel sheet (MPS). Finally, enhanced vascularization and dermal wound repair were observed with EPC seeded MPS.

We demonstrate the mechanism by which C3G, a major dietary anthocyanin, regulates energy metabolism and insulin sensitivity. Oral administration of C3G reduced hepatic and plasma triglyceride levels, adiposity, and improved glucose tolerance in mice fed high-fat diet. Hepatic metabolomic analysis revealed that C3G shifted metabolite profiles towards fatty acid oxidation and ketogenesis. C3G increased glucose uptake in HepG2 cells and C2C12 myotubes and induced the rate of hepatic fatty acid oxidation. C3G directly interacted with and activated PPARs, with the highest affinity for PPARα. The ability of C3G to reduce plasma and hepatic triglycerides, glucose tolerance, and adiposity and to induce oxygen consumption and energy expenditure was abrogated in PPARα-deficient mice, suggesting that PPARα is the major target for C3G. These findings demonstrate that the dietary anthocyanin C3G activates PPARs, a master regulators of energy metabolism. C3G is an agonistic ligand of PPARs and stimulates fuel preference to fat., Jia, Wu, Kim et al. show that cyanidin-3-O-glucoside (C3G), a major dietary anthocyanin, functions as a ligand for PPARalpha to regulate energy metabolism and insulin sensitivity in mouse models of obesity and diabetes. This study suggests that C3G slows down the metabolism of glucose, making it a promising therapeutic agent.

A bioelectronic nose device based on micelle-stabilized olfactory receptors is developed for the selective discrimination of a butter flavor substance in commercial fermented alcoholic beverages. In this work, we have successfully overexpressed ODR-10, a type of olfactory receptor, from Caenorhabditis elegans using a bacterial expression system at a low cost and high productivity. The highly-purified ODR-10 was stabilized in micelle structures, and it was immobilized on a carbon nanotube field-effect transistor to build a bioelectronic nose for the detection of diacetyl, a butter flavor substance, via the specific interaction between diacetyl and ODR-10. The bioelectronic nose device can sensitively detect diacetyl down to 10 fM, and selectively discriminate it from other substances. In addition, this sensor could directly evaluate diacetyl levels in a variety of real fermented alcoholic beverages such as beer, wine, and makgeolli (fermented Korean wine), while the sensor did not respond to soju (Korean style liquor without diacetyl). In this respect, our sensor should be a powerful tool for versatile food industrial applications such as the quality control of alcoholic beverages and foods.

Core binding factor β (Cbfβ) is a non-DNA binding cofactor of Runx2 that potentiates DNA binding. Previously, it has been reported that Cbfβ plays an essential role in osteogenic differentiation and skeletal development by inhibition adipogenesis. Here, we delivered the recombinant Cbfβ protein into human mesenchymal stem cells (MSCs) and triggered osteogenic lineage commitment. The efficient delivery of Cbfβ was achieved by fusing 30Kc19 protein, which is a cell-penetrating protein derived from the silkworm. After the production of the recombinant Cbfβ-30Kc19 protein in the Escherichia coli expression system, and confirmation of its intracellular delivery, MSCs were treated with the Cbfβ-30Kc19 once or twice up to 300 µg/ml. By investigating the upregulation of osteoblast-specific genes and phenotypical changes, such as calcium mineralization, we demonstrated that Cbfβ-30Kc19 efficiently induced osteogenic differentiation in MSCs. At the same time, Cbfβ-30Kc19 suppressed adipocyte formation and downregulated the expression of adipocyte-specific genes. Our results demonstrate that the intracellularly delivered Cbfβ-30Kc19 enhances osteogenesis in MSCs, whereas it suppresses adipogenesis by altering the transcriptional regulatory network involved in osteoblast-adipocyte lineage commitment. Cbfβ-30Kc19 holds great potential for the treatment of bone-related diseases, such as osteoporosis, by allowing transcriptional regulation in MSCs, and overcoming the limitations of current therapies.

A bioelectronic skin device based on nociceptive ion channels in nanovesicles is developed for the detection of chemical cold-pain stimuli and cold environments just like human somesthetic sensory systems. The human transient receptor potential ankyrin 1 (hTRPA1) is involved in transmission and modulation of cold-pain sensations. In the bioelectronic skin, the nanovesicles containing the hTRPA1 nociceptive ion channel protein reacts to cold-pain stimuli, and it is electrically monitored through carbon nanotube transistor devices based on floating electrodes. The bioelectronic skin devices sensitively detect chemical cold-pain stimuli like cinnamaldehyde at 10 fm, and selectively discriminate cinnamaldehyde among other chemical stimuli. Further, the bioelectronic skin is used to evaluate the effect of cold environments on the response of the hTRPA1, finding that the nociceptive ion channel responds more sensitively to cinnamaldehyde at lower temperatures than at higher temperatures. The bioelectronic skin device could be useful for a basic study on somesthetic systems such as cold-pain sensation, and should be used for versatile applications such as screening of foods and drugs.

Hormones within very low levels regulate and control the activity of specific cells and organs of the human body. Hormone imbalance can cause many diseases. Therefore, hormone detection tools have been developed, particularly over the last decade. Peptide hormones have a short half-life, so it is important to detect them within a short time. In this study, we report two types of peptide hormone sensors using human hormone receptor-carrying nanovesicles and graphene field-effect transistors (FETs). Parathyroid hormone (PTH) and glucagon (GCG) are peptide hormones present in human blood that act as ligands to G protein-coupled receptors (GPCRs). In this paper, the parathyroid hormone receptor (PTHR) and the glucagon receptor (GCGR) were expressed in human embryonic kidney-293 (HEK-293) cells, and were constructed as nanovesicles carrying the respective receptors. They were then immobilized onto graphene-based FETs. The two hormone sensors developed were able to detect each target hormone with high sensitivity (ca. 100 fM of PTH and 1 pM of GCG). Also, the sensors accurately recognized target hormones among different types of peptide hormones. In the development of hormone detection tools, this approach, using human hormone receptor-carrying nanovesicles and graphene FETs, offers the possibility of detecting very low concentrations of hormones in real-time.

Monitoring food spoilage is one of the most effective methods for preventing food poisoning caused by biogenic amines or microbes. Therefore, various analytical techniques have been introduced to detect low concentrations of cadaverine (CV) and putrescine (PT), which are representative biogenic polyamines involved in food spoilage (5-8 ppm at the stage of initial decomposition after storage for 5 days at 5 °C and 17-186 ppm at the stage of advanced decomposition after storage for 7 days at 5 °C). Although previous methods showed selective CV and PT detection even at low concentrations, the use of these methods remains challenging in research areas that require in-situ, real-time, on-site monitoring. In this study, we demonstrated for the first time an in-situ high-performance chemical receptor-conjugated graphene electronic nose (CRGE-nose) whose limits of detection (LODs), 27.04 and 7.29 ppb, for CV and PT are up to 10

Elucidating molecular mechanisms of rhodopsin signal transduction is crucial for understanding photo-reaction process and vision. In particular, a conformational change of retinal from 11-cis to all-trans configuration initiates a chain reaction. Earlier studies on photoinduced isomerization provided that chromophore retinal has torsional vibration modes related to the photoisomerization dynamics at very low frequency regime, including terahertz (THz) frequency. However, limited success has been made in detecting such change using low frequency electromagnetic wave, due to its extremely small absorption cross section within allowed signal-to-noise level. Here, we developed an ultrasensitive terahertz molecule sensor that allows us to detect the conformational change from 11-cis to all-trans state of retinal embedded in photoreceptor-nanovesicles. Nano-slot antenna array fabricated onto SiO2/Si substrate was used as a sensing chip for enhancing THz reflection signal change under photo excitation. In particular, THz top-down reflection system in normal geometry enabled us to measure extremely small changes in signal by dramatically reducing undesired light paths through several substrates, cover glass, and the absorptive sample itself, all of which comprise unavoidable parts in typical transmission measurement cases. Moreover, a direct monitoring of a photo-induced conformational change in photoreceptors could be performed at room temperature and in a label-free and a non-contact manner.

Numerous efforts have been made to measure tastes for various purposes. However, most taste information is still obtained by human sensory evaluation. It is difficult to quantify a degree of taste or establish taste standard. Although artificial taste sensors called electronic tongues utilizing synthetic materials such as polymers, semiconductors, or lipid membranes have been developed, they have limited performance due to their low sensitivity and specificity. Recently, bioelectronic tongues fabricated by integrating human taste receptors and nanomaterial-based sensor platforms have been found to have high performance for measuring tastes with human-like taste perception. However, human umami taste receptor is heterodimeric class C GPCR composed of human taste receptor type 1 member 1 (T1R1) and member 3 (T1R3). Such complicated structure makes it difficult to fabricate bioelectronic tongue. The objective of this study was to develop a protein-based bioelectronic tongue for detecting and discriminating umami taste with human-like performance using umami ligand binding domain called venus flytrap (VFT) domain originating from T1R1 instead of using the whole heterodimeric complex of receptors. Such T1R1 VFT was produced from Escherichia coli (E. coli) with purification and refolding process. It was then immobilized onto graphene-based FET. This bioelectronic tongue for umami taste (BTUT) was able to detect monosodium L-glutamate (MSG) with high sensitivity (ca. 1 nM) and specificity in real-time. The intensity of umami taste was enhanced by inosine monophosphate (IMP) that is very similar to the human taste system. In addition, BTUT allowed efficient reusable property and storage stability. It maintained 90% of normalized signal intensity for five weeks. To develop bioelectronic tongue, this approach using the ligand binding domain of human taste receptor rather than the whole heterodimeric GPCRs has advantages in mass production, reusability, and stability. It also has great potential for various industrial applications such as food, beverage, and pharmaceutical fields.

The curry compound, curcumin exerts multiple health-promotive functions; however, its poor solubility and stability limits its biological applications. In this study, we illuminate intermolecular binding mechanisms in the nano-sized complex of curcumin with silkworm protein, 30Kc19. The intrinsic fluorescence of 30Kc19 was gradually quenched by the increase of curcumin concentrations, which demonstrates molecule-molecule complexations mediated by the fluorophore amino acid residues (Tyr, Trp) in the protein. The fluorescence quenching showed that the binding occurred at 1:1 molar ratio with binding constant of 3.28 × 104 M-1. The results from scanning electron microscopy and dynamic light scattering indicate that the complexes were formed with cubicle shapes and sizes of 200–250 nm at pH 8.0 (zeta-potential < −20 mV). Along with Fourier transform infrared analysis, computational studies of protein-ligand docking simulation suggest a mechanism that curcumin and 30Kc19 forms complexes through specific amino acid residues (Trp174, Trp180, and Trp225) with minimum binding distance (4 A). The complexation of curcumin with 30Kc19 protein effectively suppressed the degradation of curcumin over 10 h and improved its antioxidant activity up to 30%. These findings suggest an application of 30Kc19 for the delivery of waterinsoluble bioactive medicines.

Nanoparticles (NPs) are an emerging strategy for drug delivery and have been studied for the delivery of various biomolecules, such as chemically synthesized drugs and therapeutic proteins. In particular, protein NPs are non-cytotoxic and biodegradable. Application of a full length recombinant 30Kc19 protein to human serum albumin (HSA) NPs has been shown to improve the cellular uptake and stability of the cargo enzyme. In this study, we demonstrate that drug delivery can be achieved with only the α-helix domain of the 30Kc19 protein (30Kc19α), and without the addition of HSA. Protein concentration and pH were crucial for NP generation. NPs had a uniformly spherical shape with an optimal diameter of 180–230 nm, and released β-galactosidase in a sustained manner. The 30Kc19α protein provided stability to the cargo enzyme, and helped maintain the specific activity of the enzyme. X-gal staining showed effective delivery of β-galactosidase into human dermal fibroblasts. Non-cytotoxic property of the 30Kc19α protein demonstrates that such NPs could be a resourceful tool for delivering drugs to cells.

Biomass forms a complex interwoven structure containing cellulose, hemicellulose and lignin that hinders enzymatic hydrolysis of cellulose. Enzymatic hydrolysis of the cellulose within yellow poplar (tulip tree) particles released only 9% of the total glucose in this study. To increase the accessibility of the cellulose component, wood particles were pretreated using hot compressed water and enzymatically-generated peracetic acid. The combined pretreatment started with hot compressed water (200 °C, 15 min), which selectively solubilized up to 90% of the xylan. The remaining solid was treated with peracetic acid (90 mM, 60 °C, 6 h), which solubilized up to 70% of the lignin. The remaining solid consisted of mainly glucan (∼75%) and corresponds to 87% of the glucan initially present in the yellow poplar particles. Hydrolysis of the remaining solid using a low loading of cellulase/β-glucosidase for 72 h released 90% of the glucose. The removal of the xylan and lignin structural barriers dramatically increased the cellulase accessibility to cellulose. The structural characteristics (crystallinity, functional group changes, morphology) of combined pretreated solid residue changed in a manner consistent with increased enzymatic digestibility. The combined pretreatment with hot compressed water and peracetic acid was more effective than either single pretreatment and more effective than the sum of the single pretreatments to remove xylan and lignin, thus demonstrating a cooperative effect of the two pretreatments. In addition, the combined pretreatment enhanced the accessibility of cellulases to the cellulose resulting in more efficient cellulose hydrolysis.

Biomass contains cellulose, xylan and lignin in a complex interwoven structure that hinders enzymatic hydrolysis of the cellulose. To separate these components in yellow poplar biomass, we sequentially pretreated with dilute sulfuric acid and enzymatically-generated peracetic acid. In the first step, the dilute acid with microwave heating (140°C, 5 min) hydrolyzed 90% of xylan. The xylose yield in hydrolysate after dilute acid pretreatment was 83.1%. In the second step, peracetic acid (60°C, 6 h) removed up to 80% of lignin. This sequential pretreatment fractionated biomass into xylan and lignin, leaving a solid residue enriched in cellulose (~80%). The sequential pretreatment enhanced enzymatic digestibility of the cellulase by removal of the other components in biomass. The glucose yield after enzymatic hydrolysis was 90.5% at a low cellulase loading (5 FPU/g of glucan), which is 1.6 and 18 times higher than for dilute acid-pretreated biomass and raw biomass, respectively. This novel sequential pretreatment with dilute acid and peracetic acid efficiently separates the three major components of yellow poplar biomass, and reduces the amount of cellulase needed.

Basic fibroblast growth factor (bFGF) has an established pivotal function in biomedical engineering, especially for the human pluripotent stem cells (iPSCs). However, the limitation of bFGF is the ease of denaturation under normal physiological conditions, inducing loss of its activity. In this study, we designed multi-trilayered nanofilm composed of a repeating polycation/polyanion/bFGF structure, which has high loading efficiency and short buildup time. We also investigated that the loading and release of bFGF from the nanofilm with two parameters (counter-polyanion and film architectures). Then, we prepared the optimized nanofilm which maintains a sustained bFGF level in physiological condition to apply the nanofilm to human iPSCs culture. The amount of bFGF release from 12 trilayer nanofilm was 36.4 ng/cm2, and activity of bFGF encapsulated into the nanofilm was maintained (60%) until 72 h during incubation at 37 °C. As a result, the iPSCs grown in the presence of the nanofilm with tridaily replacemen...

Human embryonic stem cells (hESCs) possess unique properties in terms of self-renewal and differentiation, which make them particularly well-suited for use in tissue engineering and regenerative medicine. The differentiation of hESCs in the form of human embryoid bodies (hEBs) recapitulates early embryonic development, and hEBs may provide useful insight into the embryological development of humans. Herein, cell-penetrating magnetic nanoparticles (MNPs) were utilized to form hEBs with defined sizes and the differentiation patterns were analyzed. Through intracellular delivery of MNPs into the hESCs, suspended and magnetized hESCs efficiently clustered in to hEBs driven by magnetic pin-based external magnetic forces. The hEB size was controlled by varying the suspended cell numbers that were applied in the magnetic pin system. After 3 days of differentiation in a suspended condition, ectodermal differentiation was observed to have been enhanced in the small hEBs (150 μm in diameter) while endodermal and mesodermal differentiation were enhanced in large hEBs (600 μm in diameter). This indicates that the size of the hEBs plays an important role in the early lineage commitment of hESCs, and MNP-based control of the hEB size would be a novel, useful methodology for lineage-specific hESC differentiation.

Technologies for degradation of three major components of lignocellulose (e.g. cellulose, hemicellulose and lignin) are needed to efficiently utilize lignocellulose. Here, we report Bacillus sp. 275 isolated from a mudflat exhibiting various lignocellulolytic activities including cellulase, xylanase, laccase and peroxidase in the cell culture supernatant. The complete genome of Bacillus sp. 275 strain contains 3832 protein cording sequences and an average G+C content of 46.32% on one chromosome (4045,581bp) and one plasmid (6389bp). The genes encoding enzymes related to the degradation of cellulose, xylan and lignin were detected in the Bacillus sp. 275 genome. In addition, the genes encoding glucosidases that hydrolyze starch, mannan, galactoside and arabinan were also found in the genome, implying that Bacillus sp. 275 has potentially a wide range of uses in the degradation of polysaccharide in lignocellulosic biomasses.

Odors are perceived differently as a function of individual human experience, and communicating about odors between individuals is therefore very difficult. There is a need to classify and standardize odors, but appropriate tools have not yet been developed. A bioelectronic nose mimics human olfaction and detects target molecules with high sensitivity and selectivity. This new tool has great potential in many applications and is expected to accelerate odor classification and standardization. In particular, a multiplexed bioelectronic nose can provide complex odor information using pattern recognition techniques, and could even reproduce odors via an integrated olfactory display system. We expect that a bioelectronic nose will be a useful tool for odor standardization by providing codes for odors that enable us to communicate odor information.

Ultraviolet (UV) irradiation induces skin photoaging by generating reactive oxygen species (ROS). ROS caused by UV-irradiation results in loss of skin cells and degradation of extracellular matrix. A number of antioxidants have been chemically synthesized or naturally extracted to prevent ROS-mediated skin photoaging. In our previous work, silkworm hemolymph extract (SHEX) was prepared, and its antioxidant activity was tested by free radical-scavenging assay. This study assessed the protective effects of SHEX on UV-induced photoaging of human immortalized keratinocytes (HaCaT). UVA (365 nm)-induced ROS generation was inhibited by supplementation of silkworm hemolymph (SH). Treatment with SHEX prepared by boiling SH inhibited death of HaCaT cells caused by UVB (315 nm) and UVA irradiation in a dose-dependent manner. Seven fractions were obtained by separating SHEX by gel permeation chromatography and the antioxidant activity of the fractions was examined. The fraction showing the highest protective efficacy on UV-induced cell damage corresponded to the lutein-containing fraction isolated in our previous study. Moreover, the SHEX fraction suppressed the expression of MMP-1 (matrix metalloproteinase-1), a matrix-degrading enzyme, suggesting that the active constituent of SHEX has the potential to inhibit skin photoaging. These results suggest that SHEX can be developed as a dietary and cosmetic supplement for prevention of skin photoaging.

A multiplexed bioelectronic sensor was developed for the purpose of rapid, on-site, and simultaneous detection of various target molecules. Olfactory and taste receptors were produced in Escherichia coli, and the reconstituted receptors were immobilized onto a multi-channel type carbon nanotube field-effect transistor. This device mimicked the human olfactory/taste system and simultaneously measured the conductance changes with high sensitivity and selectivity following treatment with various odor and taste molecules commonly known to be indicators of food contamination. Various pattern recognition of odorants and tastants was available with a customized platform for the simultaneous measurement of electrical signals. The simple portable bioelectronic device was suitable for efficient monitoring of food freshness and is expected to be used as a rapid on-site sensing platform with various applications.

In the neuroendocrine system, corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) play important roles in the regulation of the hypothalamic-pituitary-adrenal (HPA) system. Disorders of the HPA system lead to physiological problems, such as Addison's disease and Cushing's syndrome. Therefore, detection of CRH and ACTH is essential for diagnosing disorders related to the HPA system. Herein, receptors of the HPA axis were used to construct a bioelectronic sensor system for the detection of CRH and ACTH. The CRH receptor, corticotropin-releasing hormone receptor 1 (CRHR1), and the ACTH receptor, melanocortin 2 receptor (MC2R), were produced using an Escherichia coli expression system, and were reconstituted using nanodisc (ND) technology. The receptor-embedded NDs were immobilized on a floating electrode of a carbon nanotube field-effect transistor (CNT-FET). The constructed sensors sensitively detected CRH and ACTH to a concentration of 1 fM with high selectivity in real time. Furthermore, the reliable detection of CRH and ACTH in human plasma by the developed sensors demonstrated their potential in clinical and practical applications. These results indicate that CRHR1 and MC2R-based bioelectronic sensors can be applied for rapid and efficient detection of CRH and ACTH.

A new polydiacetylene lipid/human olfactory receptor nano-assembly was fabricated for the visual detection of an odorant for the first time. The assembly consisted of phospholipid-mixed polydiacetylenes (PDAs) and human olfactory receptors (hORs) in detergent micelles. To overcome the limitations of bioelectronic noses, hOR-embedded chromatic complexes (PDA/hORs) were developed, introducing PDAs that showed color and fluorescence transitions against various stimuli. The chromatic nanocomplexes reacted with target molecules, showing a fluorescence intensity increase in a dose-dependent manner and target selectivity among various odorants. As a result, a color transition of the assembly from blue to purple occurred, allowing the visual detection of the odorant geraniol. Through circular dichroism (CD) spectroscopy and a tryptophan fluorescence quenching method, the structural and functional properties of the hORs embedded in the complexes were confirmed. Based on this first work, future array devices, integrating multiple nano-assemblies, can be substantiated and utilized in environmental assessment and analysis of food quality.

The instability of recombinant basic fibroblast growth factor (bFGF) is a major disadvantage for its therapeutic use and means frequent applications to cells or tissues are required for sustained effects. Originating from silkworm hemolymph, 30Kc19α is a cell-penetrating protein that also has protein stabilization properties. Herein, it was investigated whether fusing 30Kc19α to bFGF could enhance the stability and skin penetration properties of bFGF, which may consequently increase its therapeutic efficacy. The fusion of 30Kc19α to bFGF protein increased protein stability, as confirmed by ELISA. 30Kc19α-bFGF also retained the biological activity of bFGF as it facilitated the migration and proliferation of fibroblasts and angiogenesis of endothelial cells. It was discovered that 30Kc19α could improve the transdermal delivery of a small molecular fluorophore through the skin of hairless mice. Importantly, it increased the accumulation of bFGF and further facilitated its translocation into the skin through follicular routes. Finally, when applied to a skin wound model in vivo, 30Kc19α-bFGF penetrated the dermis layer effectively, which promoted cell proliferation, tissue granulation, angiogenesis, and tissue remodeling. Consequently, our findings suggest that 30Kc19α improves the therapeutic functionalities of bFGF, and would be useful as a protein stabilizer and/or a delivery vehicle in therapeutic applications.

Salmonella infection is the one of the major causes of food borne illnesses including fever, abdominal pain, diarrhea, and nausea. Thus, early detection of Salmonella contamination is important for our healthy life. Conventional detection methods for the food contamination have limitations in sensitivity and rapidity; thus, the early detection has been difficult. Herein, we developed a bioelectronic nose using a carbon nanotube (CNT) field-effect transistor (FET) functionalized with Drosophila odorant binding protein (OBP)-derived peptide for easy and rapid detection of Salmonella contamination in ham. 3-Methyl-1-butanol is known as a specific volatile organic compound, generated from the ham contaminated with Salmonella. We designed and synthesized the peptide based on the sequence of the Drosophila OBP, LUSH, which specifically binds to alcohols. The C-terminus of the synthetic peptide was modified with three phenylalanine residues and directly immobilized onto CNT channels using the π-π interaction. The p-type properties of FET were clearly maintained after the functionalization using the peptide. The biosensor detected 1 fM of 3-methyl-1-butanol with high selectivity and successfully assessed Salmonella contamination in ham. These results indicate that the bioelectronic nose can be used for the rapid detection of Salmonella contamination in food.

For several decades, significant efforts have been made in developing artificial taste sensors to recognize the five basic tastes. So far, the well-established taste sensor is an E-tongue, which is constructed with polymer and lipid membranes. However, the previous artificial taste sensors have limitations in various food, beverage, and cosmetic industries because of their failure to mimic human taste reception. There are many interactions between tastants. Therefore, detecting the interactions in a multiplexing system is required. Herein, we developed a duplex bioelectronic tongue (DBT) based on graphene field-effect transistors that were functionalized with heterodimeric human umami taste and sweet taste receptor nanovesicles. Two types of nanovesicles, which have human T1R1/T1R3 for the umami taste and human T1R2/T1R3 for the sweet taste on their membranes, immobilized on micropatterned graphene surfaces were used for the simultaneous detection of the umami and sweet tastants. The DBT platform led to highly sensitive and selective recognition of target tastants at low concentrations (ca. 100 nM). Moreover, our DBT was able to detect the enhancing effect of taste enhancers as in a human taste sensory system. This technique can be a useful tool for the detection of tastes instead of sensory evaluation and development of new artificial tastants in the food and beverage industry.

After drug administration, the drugs are absorbed, distributed, metabolized, and excreted (ADME). Because ADME processes affect drug efficacy, various in vitro models have been developed based on the ADME processes. Although these models have been widely accepted as a tool for predicting the effects of drugs, the differences between in vivo and in vitro systems result in high attrition rates of drugs during the development process and remain a major limitation. Recent advances in microtechnology enable more accurate mimicking of the in vivo environment, where cellular behavior and physiological responses to drugs are more realistic; this has led to the development of novel in vitro systems, known as "organ-on-a-chip" systems. The development of organ-on-a-chip systems has progressed to include the reproduction of multiple organ interactions, which is an important step towards "body-on-a-chip" systems that will ultimately predict whole-body responses to drugs. In this review, we summarize the application of microtechnology for the development of in vitro systems that accurately mimic in vivo environments and reconstruct multiple organ models.

Direct reprogramming of non-neural lineages to functional neurons holds great potential for neural development, neurological disease modeling, and regenerative medicine. Recent work has shown that single transcription factor Ascl1 can directly reprogram fibroblasts into neuronal cells under co-culture system with glial cells. It is confirmed that Ascl1 is the key driver in the reprogramming of induced neuronal cells (iNCs). However, most reprogramming methods use genetic materials and/or potentially mutagenic molecules to generate iNCs. Herein, we used 30Kc19 protein as a novel fusion partner of transcription factor Ascl1 to induce direct reprogramming of fibroblasts to neuronal cells. We demonstrated soluble expression and stability of Ascl1 protein was increased and maintained by co-expression with 30Kc19 protein, respectively. We confirmed that intracellular delivery of the fusion protein resulted in iNC generation. Protein-induced neuronal cells (p-iNCs) expressed neuronal protein markers (MAP2, Tuj1) and transcriptional genes (Ascl1, Brn2, and Myt1l). Protein-based direct reprogramming system eliminates the potential risk associated with the use of genetic materials. This method is anticipated to be useful for safe generation of patient-specific human neuronal cells for future applications in regenerative medicine.