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Recent advances in the field of tissue engineering and regenerative medicine have contributed to the repair of damaged tissues and organs. Renal dysfunctions such as chronic kidney disease (CKD) are considered intractable owing to its cellular heterogeneity. In addition, the absence of definitive treatment options other than dialysis or kidney transplantation in advanced CKD. In this study, we investigated therapeutic effects of a three-dimensional (3D) bio-printed omentum patch as treatment source. Because omentum contains a lot of biological sources for immune regulation and tissue regeneration, it has been used in clinic for100 years. By using autologous tissue as a bio-ink, the patch could minimize the immune response. The mechanically micronized omentum without any additives became small enough to print, but the original components could be preserved. Then, the 3D printed omentum patch was transplanted under renal subcapsular layer in unilateral ureteral obstruction (UUO) rat model. After 14 days of patch transplantation, the kidneys were analyzed through bulk RNA sequencing and histopathological staining. From the results, decreased tubular injury was observed in the omentum patch group. In addition, the omentum patch significantly altered biological process of gene ontology such as fibrosis-related gene and growth factors. RNA sequencing confirmed the antifibrotic effect by inhibiting fibrosis-inducing mechanisms within PI3K-AKT and JAK-STAT pathways. In conclusion, the omentum patch showed the effect of antitubular injury and antifibrosis on UUO kidneys. In particular, the omentum patch is expected to protect the organ from further degeneration and loss of function by inhibiting the progression of fibrosis. The omentum patch can be a novel therapeutic option for renal dysfunction. Impact statement Many studies and clinical trials are being conducted to develop new treatments for kidney disease. However, there are no newly developed renal replacement therapies. In this study, we developed a new treatment that can ameliorate renal interstitial fibrosis using three-dimensional (3D) bio-printed autologous omentum patch. The 3D printer enables precise patch printing, and the bio-ink made of autologous tissue minimizes the immune response after transplantation. The whole kidneys were analyzed by RNA sequencing and histopathological staining 14 days after transplantation. From the results, the omentum patch had the effect of relieving tubular injury in the injured state. Also, the omentum patch significantly altered biological process of gene ontology. In particular, genes related to fibrosis were observed to be downregulated by the omentum patch. RNA sequencing confirmed that the antifibrotic effect was owing to inducing mechanisms of PI3K-AKT and JAK-STAT pathways. The findings reported in this study represent a significant advancement in the application of 3D bio-printer to damaged organ treatments, especially fibrosis-related diseases.

BACKGROUND AND AIMS Renal fibrosis is one of the pathological hallmarks of chronic kidney disease (CKD) that can further progress to end-stage renal diseases (ESRD). Haemodialysis and kidney transplantation are the only feasible treatments of ESRD. In this study, a new treatment with autologous omental tissue was assessed to delay the progression of renal fibrosis. The omental tissue, a fatty structure, has been widely used in surgical procedures due to its immunologic, angiogenic characteristics and anti-fibrotic factors that enhance tissue repairs, cell proliferation and regeneration in a damaged tissue. Finally, we hypothesized that 3D printed omental patch can prevent renal fibrosis and help regenerate the damaged kidney in the animal model. The 3D printing can print cells and extracellular matrix uniformly to promote cell survival and differentiation during transplantation. The printing also can adjust the patch stiffness, providing a suitable environment for kidney recovery. METHOD Animal models of CKD were randomly divided into four groups: (A) sham-operated, (B) sham-operated + omental patch, (C) unilateral ureteral obstruction (UUO)-operated (Control) and (D) UUO-operated + omental patch. Micronized omental tissue as bio-ink was 3D printed in the form of a regular patch deformation. Then, the 3D printed omental patch was transplanted subcapsular layer of the kidney. All of the patches used in those groups were transplanted after 3 days of modelling. Kidneys were assessed histologically [Masson's Trichrome (MT), Sirius Red], by immunohistochemistry (α-SMA), molecular work (Bulk RNA sequencing, western blot) after 2 weeks of transplantation. RESULTS Omental patch treated group has been shown histologically less tubulointerstitial fibrosis and tubule atrophy by MT, Sirius Red staining. Particularly, the TGF beta mRNA expression level of the 3D printed omental treated group was decreased in the kidney, whereas the HGF level was significantly increased. Also, in terms of western blotting, pro-inflammatory and renal fibrosis markers (TGF-b, α-SMA) were all decreased. Moreover, in bulk RNA sequencing, we confirmed that STAT 3 kidney repair pathway and TF-beta signalling. From all those assessments we suggest that the induction of myofibroblasts and collagen deposition are all inhibited in the omental patch treated kidney model. The autologous 3D omental patch can prevent tubular atrophy and decreased tubulointerstitial fibrosis and this further delays the progression of ESRD. CONCLUSION The study demonstrated that the omental patch delays fibrosis and repairs tubule structure in the damaged kidney by providing protective effects to kidney disease. These studies indicate the feasibility of the use of the autologous omental patch in chronic kidney disease therapeutic effects and new treatment of renal replacement therapy (RRT).

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.

Cartilage lesions are difficult to repair due to low vascular distribution and may progress into osteoarthritis. Despite numerous attempts in the past, there is no proven method to regenerate hyaline cartilage. The purpose of this study was to investigate the ability to use a 3D printed biomatrix to repair a critical size femoral chondral defect using a canine weight-bearing model. The biomatrix was comprised of human costal-derived cartilage powder, micronized adipose tissue, and fibrin glue. Bilateral femoral condyle defects were treated on 12 mature beagles staged 12 weeks apart. Four groups, one control and three experimental, were used. Animals were euthanized at 32 weeks to collect samples. Significant differences between control and experimental groups were found in both regeneration pattern and tissue composition. In results, we observed that the experimental group with the treatment with cartilage powder and adipose tissue alleviated the inflammatory response. Moreover, it was found that the MOCART score was higher, and cartilage repair was more organized than in the other groups, suggesting that a combination of cartilage powder and adipose tissue has the potential to repair cartilage with a similarity to normal cartilage. Microscopically, there was a well-defined cartilage-like structure in which the mid junction below the surface layer was surrounded by a matrix composed of collagen type I, II, and proteoglycans. MRI examination revealed significant reduction of the inflammation level and progression of a cartilage-like growth in the experimental group. This canine study suggests a promising new surgical treatment for cartilage lesions.

Full thickness cartilage defects in cases of knee osteoarthritis are challenging in nature and are difficult to treat. The implantation of three-dimensional (3D) biofabricated grafts into the defect site can be a promising biological one-stage solution for such lesions that can avoid different disadvantages of the alternative surgical treatment options. In this study, the short-term clinical outcome of a novel surgical technique that uses a 3D bioprinted micronized adipose tissue (MAT) graft for knee cartilage defects is assessed and the degree of incorporation of such graft types is evaluated via arthroscopic and radiological analyses. Ten patients received 3D bioprinted grafts consisting of MAT with an allogenic hyaline cartilage matrix on a mold of polycaprolactone, with or without adjunct high tibial osteotomy, and they were monitored until 12 months postoperatively. Clinical outcomes were examined with patient-reported scoring instruments that consisted of the Western Ontario and McMaster Universities Arthritis Index (WOMAC) score and the Knee Injury and Osteoarthritis Outcome Score (KOOS). The graft incorporation was assessed using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. At 12 months follow-up, cartilage tissue biopsy samples were taken from patients and underwent histopathological examination. In the results, at final follow-up, the WOMAC and KOOS scores were 22.39 ± 7.7 and 79.16 ± 5.49, respectively. All scores were significantly increased at final follow-up (p < 0.0001). MOCART scores were also improved to a mean of 82.85 ± 11.49, 12 months after operation, and we observed a complete incorporation of the grafts with the surrounding cartilage. Together, this study suggests a novel regeneration technique for the treatment of knee osteoarthritis patients, with less rejection response and better efficacy.

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.

Health-focused apps with chatbots (“healthbots”) have a critical role in addressing gaps in quality healthcare. There is limited evidence on how such healthbots are developed and applied in practice. Our review of healthbots aims to classify types of healthbots, contexts of use, and their natural language processing capabilities. Eligible apps were those that were health-related, had an embedded text-based conversational agent, available in English, and were available for free download through the Google Play or Apple iOS store. Apps were identified using 42Matters software, a mobile app search engine. Apps were assessed using an evaluation framework addressing chatbot characteristics and natural language processing features. The review suggests uptake across 33 low- and high-income countries. Most healthbots are patient-facing, available on a mobile interface and provide a range of functions including health education and counselling support, assessment of symptoms, and assistance with tasks such as scheduling. Most of the 78 apps reviewed focus on primary care and mental health, only 6 (7.59%) had a theoretical underpinning, and 10 (12.35%) complied with health information privacy regulations. Our assessment indicated that only a few apps use machine learning and natural language processing approaches, despite such marketing claims. Most apps allowed for a finite-state input, where the dialogue is led by the system and follows a predetermined algorithm. Healthbots are potentially transformative in centering care around the user; however, they are in a nascent state of development and require further research on development, automation and adoption for a population-level health impact.

Sickle cell disease (SCD) is a monogenic red blood cell (RBC) disorder with high morbidity and mortality. Here, we report, for the first time, the impact of SCD on the bone marrow (BM) vascular niche, which is critical for hematopoiesis. In SCD mice, we find a disorganized and structurally abnormal BM vascular network of increased numbers of highly tortuous arterioles occupying the majority of the BM cavity, as well as fragmented sinusoidal vessels filled with aggregates of erythroid and myeloid cells. By in vivo imaging, sickle and control RBCs have significantly slow intravascular flow speeds in sickle cell BM but not in control BM. In sickle cell BM, we find increased reactive oxygen species production in expanded erythroblast populations and elevated levels of HIF-1α. The SCD BM exudate exhibits increased levels of proangiogenic growth factors and soluble vascular cell adhesion molecule-1. Transplantation of SCD mouse BM cells into wild-type mice recapitulates the SCD vascular phenotype. Our data provide a model of SCD BM, in which slow RBC flow and vaso-occlusions further diminish local oxygen availability in the physiologic hypoxic BM cavity. These events trigger a milieu that is conducive to aberrant vessel growth. The distorted neovascular network is completely reversed by a 6-week blood transfusion regimen targeting hemoglobin S to

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.

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.

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.

The importance of anti-apoptosis in mammalian cell culture has been widely recognized. We reported previously that expression of Bombyx mori 30 K genes in Chinese hamster ovary (CHO) cells increases recombinant protein production by inhibiting apoptosis and enhancing specific productivity. However, previous studies have shown expression of the anti-apoptotic 30Kc6 protein as inclusion bodies in Escherichia coli. 30Kc19 protein, another silkworm hemolymph protein, has cell-penetrating and recombinant protein productivity-improving properties, and we found that it improves soluble expression of its partner. In this study, we fused 30Kc6 with 30Kc19 as an expression partner to express a soluble fused protein and to deliver the protein to cells. Supplementing the recombinant 30Kc19-30Kc6 fusion protein in cell culture medium increased viability, effectively penetrated the cells, and inhibited CHO cell apoptosis by 68%. Moreover, the mitochondrial membrane potential and ATP generation also increased by 50% and 33%, respectively. Erythropoietin (EPO) productivity increased by > 30% because of the anti-apoptotic effect and increased specific productivity. These results demonstrate the potential use of this fusion protein as a supplement in mammalian cell culture during production of biopharmaceutical proteins.

The aims of the present study were to prepare different-sized red ginseng powders and investigate the particle size effect on the release property of ginsenosides in in vitro digestion conditions. Ultrafine powder showed bimodal particle size distribution with a large peak at around 100 μm and small peak at around 10 μm, differently from fine powder showing unimodal distribution at 100 μm. The specific surface areas of fine- and ultrafine powders were 48.72 ± 6.41 and 86.74 ± 5.96 m2/g, respectively. Time-dependent release property of the powders in the simulated gastrointestinal fluids was determined by quantifying ginsenoside Rg1 released. The initial and final concentrations of ginsenoside Rg1 released was higher in ultrafine powder than fine one. It is expected that particle size reduction and corresponding increase in the specific surface area have a potential to improve the release of ginsenosides in the gastrointestinal tract and enhance the chances to be absorbed in human body.

Seaweeds have beneficial nutritional and medicinal properties. Several studies have examined the polysaccharides found in the extracts of Porphyra yezoensis (PPY), although the effects of particular proteins have not been reported, and peptides from the marine alga PPY function in antitumor cell signaling, although the precise mechanism is not well understood. Apoptosis plays an important role in cell death, which affects cell proliferation. Generally, regulation of apoptosis requires participation of the p53 and Bcl-2 family by the mammalian target of rapamycin (mTOR) pathway, which is activated in a variety of malignant cancers. Autophagy is another signaling pathway that leads to degradation of cellular components by lysosomal activity, and the relation- ship between autophagy and cancer has been of interest for several years. The present study investigated mTOR pathway activation in MCF-7 cells treated with 500 ng PPY for 24 h by assessing LC3 as a monitor of autophagy. We observed that the p53/NF-κB and mTOR pathways were affected by PPY, which contributes to our understanding of the functional relationship between the Bcl-2 family and mTOR under apoptotic condi- tions in MCF-7 cells.

Simple dynamic light scattering (DLS)-based methodologies were developed to determine primary particle size distribution of iron oxide particles in simulated gastrointestinal fluid. Iron oxide particles, which easily agglomerate in aqueous media, were converted into dispersed particles by modification of surface charge using citric acid and sodium citrate. After the modification, zeta-potential value decreased to -40mV at pH 7. Mean particle diameters in suspensions of iron oxide nano- and microparticles stabilized by the mixture of citric acid and sodium citrate were dramatically decreased to 166 and 358nm, respectively, which were close to the particle size distributions observed in the micrographs. In simulated gastrointestinal fluid, both iron oxide nano- and microparticles were heavily agglomerated with particle diameters of almost 2600 and 5200nm, respectively, due to charge shielding on the citrate-modified surface by ions in the media. For determining primary particle size distribution by using DLS-based approach, the iron oxide particles incubated in the simulated gastrointestinal fluid were converted to monodisperse particles by altering the pH to 7 and electrolyte elimination. The simple DLS-based methodologies are well suited to determine primary particle size distribution of mineral nanoparticles at various physical, chemical, and biological conditions.

Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have attracted increasing attention due to their high potential to deliver various, otherwise impermeable, bioactive agents, such as drugs and proteins across cell membranes. A number of CPPs have been discovered since then. Recently, 30Kc19 protein has attracted attention because it was the first cell-penetrating protein that has been found in insect hemolymph. Here, we report a cell-penetrating peptide derived from 30Kc19 protein, VVNKLIRNNKMNC, which efficiently penetrates cells when supplemented to medium for mammalian cell culture. Moreover, like other CPPs, this “Pep-c19” also efficiently delivered cell-impermeable cargo proteins, such as green fluorescent protein (GFP) into cells. In addition to the in vitro system, Pep-c19 exhibited the cell-penetrating property in vivo . When Pep-c19 was intraperitoneally injected into mice, Pep-c19 successfully delivered cargo proteins into various organ tissues with higher efficiency than the 30Kc19 protein itself, and without toxicity. Our data demonstrates that Pep-c19 has a great potential as a cell-penetrating peptide that can be used as a therapeutic tool to efficiently deliver different cell-impermeable cargo molecules into the tissues of various organs.

Nanoparticles have been widely used for delivering various chemical and biomolecular drugs, such as anti-cancer drugs and therapeutic proteins. Among nanoparticles, protein nanoparticles have advantages of non-cytotoxicity and biodegradability. In this study, a recombinant 30Kc19 protein was applied to human serum albumin (HSA) nanoparticles to enhance cellular uptake and stability of a nanoparticle cargo enzyme. The 30Kc19 protein, which originates from silkworm, has cell-penetrating and enzyme-stabilizing abilities. Therefore, 30Kc19-HSA nanoparticles were expected to enhance cellular uptake and stability of an enzyme loaded on the nanoparticles. Here, nanoparticles loaded with β-galactosidase were prepared using the desolvation method. The 30Kc19-HSA nanoparticles were uniformly spherical in shape, dispersed evenly in phosphate buffered saline and cell culture media, and released β-galactosidase in a sustained manner. The 30Kc19-HSA nanoparticles had negligible toxicity to animal cells and exhibited enhanced cellular uptake and intracellular stability of β-galactosidase in HeLa and HEK293 cells when compared with those of HSA nanoparticles. These results suggest that 30Kc19-HSA protein nanoparticles could be used as a versatile tool for drug delivery to various cells.

Sickle cell disease (SCD) is a monogenic red cell disorder with high morbidity and mortality due to significant end-organ disease. Here we investigated for the first time the impact of SCD on the bone marrow (BM) vascular niche, which is critical for hematopoiesis. In SCD mice, we find a disorganized and structurally abnormal BM vasculature with highly tortuous arterioles and fragmented sinusoidal vessels, the latter filled with aggregates of sickle erythroid and myeloid cells. By in vivo imaging, sickle and control red blood cells exhibit significantly slow intravascular flow speeds in sickle cell BM but not in control BM. These abnormalities lead to increased ROS production in expanded erythroblast populations, and to up-regulation in the BM of HIF-1a and key growth factors required to support angiogenesis, e.g. VEGF-A, Ang1, Ang2. Moreover, increased levels of VCAM-1, a marker for vascular dysfunction, are detected. SCD also impacts the BM perivascular niche, i.e. the number of CXCL12-abundant reticular cells, is markedly decreased, and is associated with mobilization of hematopoietic stem/progenitor cells as well as mature leukocytes to the periphery. Strikingly, the distorted vascular network is completely reversed by blood transfusion, which highlights the plasticity of the BM vascular niche. Thus, we provide molecular and cellular features of the pathologic BM microenvironment, which suggest that the local hypoxia-induced angiogenic network combined with inflammatory vascular dysfunction is responsible for the vasculopathy. In addition, the findings may serve to develop new biomarkers and therapeutic targets of BM end-organ damage. Figure Disclosures No relevant conflicts of interest to declare.

In polymeric nanoparticle preparation, despite similar conditions, large fluctuations in particle size distributions are usually observed. Herein, we demonstrate that the intermittent addition of a desolvating agent can improve reproducibility in the preparation of polymeric bovine serum albumin (BSA) nanoparticles. Using this modification, BSA nanoparticles of controlled size can be manufactured with narrow particle size distributions. In our study, ethanol as a desolvating agent was added intermittently to 1% BSA solutions at different pHs with stirring at 700rpm. The effect of the preparation parameters on size and optical density of the fabricated nanoparticles were studied. The average particles sizes of BSA nanoparticles prepared at pH values of 6, 7 and 9 were approximately 100, 200 and 300nm, respectively. As ethanol addition increased, desolvation of BSA molecules resulted in formation of loose-structured particles with pH-dependent size. Beyond that, only particle density increased, but size remained unchanged with further addition of ethanol. Consistently uniform particle size distribution was achieved by adding ethanol intermittently.

Acid food indicators can be used as pH indicators for evaluating the quality and freshness of fermented products during the full course of distribution. Iron oxide particles are hardly suspended in water, but partially or completely agglomerated. The agglomeration degree of the iron oxide particles depends on the pH. The pH-dependent particle agglomeration or dispersion can be useful for monitoring the acidity of food. The zeta potential of iron oxide showed a decreasing trend as the pH increased from 2 to 8, while the point of zero charge (PZC) was observed around at pH 6.0-7.0. These results suggested that the size of the iron oxide particles was affected by the change in pH levels. As a result, the particle sizes of iron oxide were smaller at lower pH than at neutral pH. In addition, agglomeration of the iron oxide particles increased as the pH increased from 2 to 7. In the time-dependent aggregation test, the average particle size was 730.4 nm and 1,340.3 nm at pH 2 and 7, respectively. These properties of iron oxide particles can be used to develop an ideal acid indicator for food pH and to monitor food quality, besides a colorant or nutrient for nutrition enhancement and sensory promotion in food industry.

Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme-degrading neutral glycosphingolipid that is transported to lysosome. Glycosphingolipid accumulation by this disease causes multi-organ dysfunction and premature death of the patient. Currently, enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. To maximize the efficacy of treatment, enhancement of cellular delivery and enzyme stability is a challenge in ERT using α-galactosidase. In this study, protein nanoparticles using human serum albumin (HSA) and 30Kc19 protein, originating from silkworm, were used to enhance the delivery and intracellular α-galactosidase stability. 30Kc19-HSA nanoparticles loaded with the α-galactosidase were formed by desolvation method. 30Kc19-HSA nanoparticles had a uniform spherical shape and were well dispersed in cell culture media. 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of delivered α-galactosidase in human foreskin fibroblast. Additionally, they showed enhanced globotriaosylceramide degradation in Fabry patients’ fibroblasts. It is expected that 30Kc19-HSA protein nanoparticles could be used as an effective tool for efficient delivery and enhanced stability of drugs.

Hizikia fusiformis, a brown alga that is widely consumed in Korea, Japan, and China, possesses a number of potentially beneficial compounds, including antioxidants and anticoagulants. However, the molecular mechanisms of H. fusiformis in hepatoma cells have not been elucidated. This study investigated the antiproliferative effect and mechanism of action of a glycoprotein from H. fusiformis (HFGP) in HepG2 human hepatoma cells. In an MTS assay, 25 HFGP inhibited the proliferation of HepG2 cells by . HFGP caused the dose-dependent growth inhibition of HepG2 cells by inducing apoptosis and a sub-G1 phase arrest. The antiproliferative activity of HFGP was confirmed based on the expression of several apoptosis-related proteins, which was assessed by Western blot analysis. The expressions of Fas, Fas-associated death domain protein, Bax, and Bad was significantly up-regulated in HFGP-treated cells, and HFGP induced the translocation of Bax to mitochondria and the release of cytochrome c into the cytosol. Therefore, HFGP might be useful in the treatment of liver cancer.

Encapsulation performance of Maillard reaction products (MRPs) produced by whey proteins and maltodextrin were examined for microencapsulation of conjugated linoleic acid (CLA) using spray-drying. CLA was encapsulated using 3 different wall materials containing a single constituent such as whey protein concentrate (WPC), whey protein isolate (WPI), and maltodextrin (MD), and 4 different MRPs. For the development of MRPs, ratios of WPC to MD were 1:2 and 1:3, whereas those of WPI to MD were 1:5 and 1:10. CLA and wall material were mixed and homogenized, and then spraydried. The physical properties of encapsulated CLA powders were characterized by particle size and morphology, ζ-potential, flowability, solubility, and dispersibility. The CLA powders coated with WPI-based MRPs have better encapsulation efficiency, water solubility, and smaller particle size than those coated with WPI, WPC, or MD alone. These encapsulated CLA powders have a number of possible utilities as ingredients in a variety of foods.

Red tides dominated by the harmful dinoflagellate Cochlodinium polykrikoides have caused annual losses of USD $5–60 million to the Korean aquaculture industry annually since 1995 and a loss of USD $3 million during a 1999 net-pen fish mortality event in Canada. In order to evaluate the potential to control C . polykrikoides red tides dominated by using mass-cultured heterotrophic protistan grazers, we monitored the abundance of Strombidinopsis jeokjo (a naked ciliate) and C . polykrikoides after mass-cultured S. jeokjo was introduced into mesocosms (ca. 60 l) deployed in situ and containing natural red tide waters dominated by C . polykrikoides . Water temperature, salinity, and pH, as well as the abundance of co-occurring other protists and metazooplankton were measured concurrently. To compare the growth and ingestion rates of S. jeokjo feeding on cultured versus natural populations of C . polykrikoides , we also monitored the abundance of cultured C . polykrikoides and S. jeokjo in bottles during laboratory grazing experiments. S. jeokjo introduced into the mesocosms grew well, effectively reducing natural populations of C. polykrikoides from approximately 1000 cells ml −1 to below 10 cells ml −1 within 2 days. The growth and ingestion rates of cultured S. jeokjo on natural populations of C. polykrikoides in the mesocosms for the first 30 h (0.72 day −1 and 51 ng C grazer −1 day −1 ) were 84% and 44%, respectively, of those measured in the laboratory during bottle incubations with similar initial prey concentrations. The calculated grazing impact of S. jeokjo on natural populations of C. polykrikoides suggests that large-scale cultures of this ciliate could be used for controlling red tides by C. polykrikoides in small areas.

Transcription factors have been studied as an important drug candidate. Ever since the successful generation of induced pluripotent stem cells (iPSCs), there has been tremendous interest in reprogramming transcription factors. Because of the safety risks involved in a virus-based approach, many researchers have been trying to deliver transcription factors using nonintegrating materials. Thus, delivery of transcription factors produced as recombinant proteins in E. coli was proposed as an alternative method. However, the low level of soluble expression and instability of such recombinant proteins are potential barriers. We engineered a Bombyx mori 30Kc19 protein as a fusion partner for transcription factors to overcome those problems. We have previously reported that 30Kc19 protein can be produced as a soluble form in E. coli and has a cell-penetrating property and a protein-stabilizing effect. Transcription factors fused with 30Kc19 (Oct4-30Kc19, Sox2-30Kc19, c-Myc-30Kc19, L-Myc-30Kc19, and Klf4-30Kc19) were produced as recombinant proteins. Interestingly, Oct4 and L-Myc were expressed as a soluble form by conjugating with 30Kc19 protein, whereas Oct4 alone and L-Myc alone aggregated. The 30Kc19 protein also enhanced the stability of transcription factors both in vitro and in cells. In addition, 30Kc19-conjugated transcription factors showed rapid delivery into cells and transcriptional activity significantly increased. Overall, 30Kc19 protein conjugation simultaneously enhanced soluble expression, stability, and transcriptional activity of transcription factors. We propose that the conjugation with 30Kc19 protein is a novel approach to solve the technical bottleneck of gene regulation using transcription factors.

In this study, we examined the protective effects of porphyra-334 against UVA-irradiated cellular damage and elucidated the underlying mechanisms. Porphyra-334 prevented UVA-induced cell death and exhibited scavenging activities against intracellular oxidative stress induced by UVA irradiation in skin fibroblasts. We found that porphyra-334 significantly reduced the secretion and expression of IL-6 and TNF-α, reduced nuclear expression of Nuclear factor-κB (NF-κB), and sustained NF-E2-related factor 2 (Nrf2) activation. Further mechanism research revealed that porphyra-334 promoted the Nrf2 signaling pathway in UVA-irradiated skin fibroblasts. Our results show that the antioxidant effect of porphyra-334 is due to the direct scavenging of oxidative stress and its inhibitory effects on NF-κB-dependent inflammatory genes, such as IL-6 and TNF-κ. Therefore, we hypothesize that boosting the Nrf2- NF-κB-dependent response to counteract environmental stress is a promising strategy for the prevention of UVA-related damage.

Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

The significant increase in life expectancy is closely related to the growing interest in the impact of aging on the function and appearance of the skin. Skin aging is influenced by several factors, and solar ultraviolet (UV) irradiation is considered one of the most important causes of skin photoaging. The aim of this study was to examine the anti-photoaging role of porphyra-334 from Porphyra (P.) yezoensis, a mycosporine-like amino acid (MAA), using high-performance liquid chromatography (HPLC), and electrospray ionization-mass spectrometry (ESI-MS). In the present study, extracted UV-absorbing compounds from P. yezoensis included palythine, asterina-330 and porphyra-334. Porphyra-334 was the most abundant MAA in P. yezoensis, and it was therefore used for conducting antiphotoaging experiments. The effect of porphyra-334 on the prevention of photoaging was investigated by measuring reactive oxygen species (ROS) production and matrix metalloproteinase (MMP) levels, as well as extracellular matrix (ECM) components and protein expression in UVA-irradiated human skin fibroblasts. Porphyra-334 suppressed ROS production and the expression of MMPs following UVA irradiation, while increasing levels of ECM components, such as procollagen, type I collagen, elastin. These results suggest that porphyra-334 has various applications in cosmetics and toiletries because of its anti-photoaging activities and may serve as a novel anti-aging agent.