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Background Driver genes of GBM may be crucial for the onset of isocitrate dehydrogenase (IDH)-wildtype (WT) glioblastoma (GBM). However, it is still unknown whether the genes are expressed in the identical cluster of cells. Here, we have examined the gene expression patterns of GBM tissues and patient-derived tumorspheres (TSs) and aimed to find a progression-related gene. Methods We retrospectively collected primary IDH-WT GBM tissue samples (n = 58) and tumor-free cortical tissue samples (control, n = 20). TSs are isolated from the IDH-WT GBM tissue with B27 neurobasal medium. Associations among the driver genes were explored in the bulk tissue, bulk cell, and a single cell RNAsequencing techniques (scRNAseq) considering the alteration status of TP53, PTEN, EGFR, and TERT promoter as well as MGMT promoter methylation. Transcriptomic perturbation by temozolomide (TMZ) was examined in the two TSs. Results We comprehensively compared the gene expression of the known driver genes as well as MGMT, PTPRZ1, or IDH1. Bulk RNAseq databases of the primary GBM tissue revealed a significant association between TERT and TP53 (p TERT and TP53 expressing cells are in a same single cell cluster. The driver-enriched cluster dominantly expressed the glioma-associated long noncoding RNAs. Most of the driver-associated genes were downregulated after TMZ except IGFBP5. Conclusions GBM tissue level expression patterns of EGFR, TERT, PTEN, IDH1, PTPRZ1, and MGMT are observed in the GBM TSs. The driver gene-associated cluster of the GBM single cells were enriched with the glioma-associated long noncoding RNAs.

As stem cells show great promise in regenerative therapy, stem cell-mediated therapeutic efficacy must be demonstrated through the migration and transplantation of stem cells into target disease areas at the pre-clinical level. In this study, we developed manganese-based magnetic nanoparticles with hollow structures (MnOHo) and modified them with the anti-human integrin β1 antibody (MnOHo-Ab) to enable the minimal-invasive monitoring of transplanted human stem cells at the pre-clinical level. Compared to common magnetic resonance imaging (MRI)-based stem cell monitoring systems that use pre-labeled stem cells with magnetic particles before stem cell injection, the MnOHo-Ab is a new technology that does not require stem cell modification to monitor the therapeutic capability of stem cells. Additionally, MnOHo-Ab provides improved T1 MRI owing to the hollow structure of the MnOHo. Particularly, the anti-integrin β1 antibody (Ab) introduced in the MnOHo targets integrin β1 expressed in the entire stem cell lineage, enabling targeted monitoring regardless of the differentiation stage of the stem cells. Furthermore, we verified that intravenously injected MnOHo-Ab specifically targeted human induced pluripotent stem cells (hiPSCs) that were transferred to mice testes and differentiated into various lineages. The new stem cell monitoring method using MnOHo-Ab demonstrates whether the injected human stem cells have migrated and transplanted themselves in the target area during long-term stem cell regenerative therapy.

Background Gastric cancer is a considerable burden for worldwide patients. And diffuse gastric cancer is the most insidious subgroup with poor survival. The phenotypic characterization of the diffuse gastric cancer cell line can be useful for gastric cancer researchers. In this article, we aimed to characterize the diffuse gastric cancer cells with MRI and transcriptomic data. We hypothesized that gene expression pattern is associated with the phenotype of the cells and that the heterogeneous enhancement pattern and the high tumorigenicity of SNU484 can be modulated by the perturbation of the highly expressed gene. Methods We evaluated the 9.4 T magnetic resonance imaging and transcriptomic data of the orthotopic mice models from diffuse gastric cancer cells such as SNU484, Hs746T, SNU668, and KATO III. We included MKN74 as an intestinal cancer control cell. After comprehensive analysis integrating MRI and transcriptomic data, we selected CD34 and validated the effect by shRNA in the BALB/c nude mice models. Results SNU484, SNU668, Hs746T, and MKN74 formed orthotopic tumors by the 5 weeks after cell injection. The diffuse phenotype was found in the SNU484 and Hs746T. SNU484 was the only tumor showing the heterogeneous enhancement pattern on T2 images with a high level of CD34 expression. Knockdown of CD34 decreased the round-void shape in the H&E staining (P = 0.028), the heterogeneous T2 enhancement, and orthotopic tumorigenicity (100% vs 66.7%). The RNAseq showed that the suppressed CD34 is associated with the downregulated gene-sets of the extracellular matrix remodeling. Conclusion Suppression of CD34 in the human-originated gastric cancer cell suggests that it is important for the round-void histologic shape, heterogeneous enhancement pattern on MRI, and the growth of gastric cancer cell line.

We report minimal amount of hyaluronic acid (HA) conjugated magnetic nanocrystals (mHMs) for targeted imaging of CD44 abundant breast cancer cells via MRI. These mHMs lead to less induced cancer migration and drug resistance, which is distinct from conventional approaches using nanoplatforms (imaging or therapeutic systems) that are completely covered with HA. To synthesize mHMs, magnetic nanocrystals (MNCs), as MRI contrast agents, were encapsulated mostly with polysorbate 80 (P80, non-reactive to HA) and partially with aminated P80 (reactive to HA). This system enabled conjugation of an immensely diminished amount of HA onto MNCs. While these nanoparticles maintained good CD44 targeted imaging efficacy, they also showed no cytotoxicity and colloidal stability. We varied the HA ratios on an equal amount of MNCs and identified that when more HA was attached on nanoparticles, there was more facilitated cancer migration and drug resistant potentials. We chose the lowest amount of HA conjugated mHMs (mHM1) and demonstrated that mHM1 selectively diagnosed tumor regions in vivo. We believe that the technique described herein can be applied to various applications using HA to detect CD44 abundant cancer cell lines and offer a basis to understand the interaction between the cellular response and surface modification of nanoparticles.

The use of drug-eluting stents (DESs) is a promising strategy for non-vascular diseases, especially human biliary cancer. However, the implementation of DESs suffers from two major obstacles: the side effects of drugs and the difficulty of controlling the drug release. These problems can be overcome if the stent elutes targeting nanoparticles that release drugs at time intervals that are dictated by the mechanisms of those drugs. We designed temporally controlled polymeric multi-prodrug nanoparticles (TCMPNs) that can be eluted from stents comprising polyurethane (PU) nanofiber as a polymeric matrix and paclitaxel (PTX)-loaded, CD44-targeting, hyaluronic acid-conjugated poly(lactic-co-glycolic acid) and gemcitabine (GEM) (P-H-G). TCMPNs enable two different types of drugs to be released temporally; PTX is released first owing to the collapse of the structure in the endosomes, and GEM, which induces synergistic anticancer activities, is hydrolyzed from P-H-G later in response to low pH. Embedded in the PU nanofiber, the TCMPNs demonstrate low initial burst behavior and sustainable release of the prodrug in vitro. Furthermore, TCMPN-eluting stents (TESs) exhibit continuous synergistic efficacy as available targeted cellular uptake prodrug delivery systems in tumor-bearing mice. These results demonstrate that this technology will open up cancer therapy by combining localized delivery and functional multi-drug-loaded nanoparticles.

A portable and nonlabeled plasmonic biosensor was advanced to enable the sensitive and selective detection of microRNA (miRNA) in a biological sample. miRNAs can act on several key cellular processes, including cell differentiation, cell cycle progression, and function as oncogenes. Detection of circulating miRNAs, especially in blood or urine samples, allows noninvasive and simple diagnosis of diseases. Herein, we report a localized surface plasmon resonance sensor (LSPR) based on an enzyme-assisted target recycling system and a developed LSPR probe for the detection of gastric cancer relevant miRNAs, miR-10b. The sensitivity of the sensor was improved by increasing the concentration of the signal-amplifying agent using the duplex-specific nuclease and by strongly binding the developed LSPR probe, tannic acid capping gold nanoparticles, to the DNA. Under optimal conditions, miR-10b detection could be realized in the range of 5 pM-10 nM with a detection limit of 2.45 pM. This integrated detection system represents an approach to sensitive detection of miRNAs and offers great applications in personalized medicine and monitoring of cancer.

The cancer stem cell (CSC) hypothesis postulates that cancer cells overexpressing CD44 are marked as CSCs that cause tumorigenesis and recurrence. This hypothesis suggests that CD44 is a potential therapeutic target that can interfere with CSCs qualities. MicroRNA-34a (miR-34a) is a promising candidate for CD44 repression-based cancer therapy as it has been reported to inhibit proliferation, metastasis, and survival of CD44-positive CSCs. Here, we used nanovesicles containing PLI/miR complexes (NVs/miR) to systemically deliver miR-34a and induce miR-34a-triggered CD44 suppression in orthotopically and subcutaneously implanted tumors in nude mice. Poly(l-lysine-graft-imidazole) (PLI) condenses miRs and is functionally modified to deliver miRs to the site of action by buffering effect of imidazole residues under endosomal pH. Indeed, NVs/miR consisting of PEGylated lipids enveloping PLI/miR complexes greatly reduced inevitable toxicity of polycations by compensating their surface charge and markedly improved their in vivo stability and accumulation to tumor tissue compared to PLI/miR polyplexes. Our NVs-mediated miR-34a delivery system specifically increased endogenous target miR levels, thereby attenuating proliferation and migration of gastric cancer cells by repressing the expression of CD44 with decreased levels of Bcl-2, Oct 3/4 and Nanog genes. Our strategy led to a greater therapeutic outcome than PLI-based delivery with highly selective tumor cell death and significantly delayed tumor growth in CD44-positive tumor-bearing mouse models, thus providing a fundamental therapeutic window for CSCs.

The objective of this study was to investigate the nutritional composition and antioxidant activity of a mixture of rice bran and bodies of Sparassis crispa fermented with lactic acid bacteria (LAB). LAB-fermented S. crispa mixture had higher water, crude lipid and crude ash content than that of S. crispa. Insoluble dietary fiber contents of the dried powder of S. crispa and LAB-fermented S. crispa mixture were 46.13% and 33.46%, respectively. β-glucan was higher in dried S. crispa (38.03%) than in LAB-fermented S. crispa mixture (5.44%). Dried S. crispa contained mainly fructose and glucose instead of containing sucrose in LAB-fermented S. crispa mixture. No significant differences in the total polyphenol contents were found in between dried S. crispa and LAB-fermented S. crispa mixture. Total flavonoid content was significantly higher in LAB-fermented S. crispa mixture than in dried S. crispa. No significant differences were found in the DPPH radical scavenging activity and in the antioxidant index between dried S. crispa and LAB-fermented S. crispa mixture. Finally, ABTS radical scavenging activity of LAB-fermented S. crispa mixture was significantly higher than that of dried S. crispa. These results may provide the basic data for future studies for a better understanding of the biological activities of LAB-fermented S. crispa mixture.

Exosomal messenger RNA (mRNA) has emerged as a valuable biomarker for liquid biopsy-based disease diagnosis and prognosis due to its stability in body fluids and its biological regulatory function. Here, we report a rapid one-step isothermal gene amplification reaction based on three-way junction (3WJ) formation and the successful detection of urinary exosomal mRNA from tumor-bearing mice. The 3WJ structure can be formed by the association of 3WJ probes (3WJ-template and 3WJ-primer) in the presence of target RNA. After 3WJ structure formation, the 3WJ primer is repeatedly extended and cleaved by a combination of DNA polymerase and nicking endonuclease, producing multiple signal primers. Subsequently, the signal primers promote a specially designed network reaction pathway to produce G-quadruplex probes under isothermal conditions. Finally, G-quadruplex structure produces highly enhanced fluorescence signal upon binding to thioflavin T. This method provides a detection limit of 1.23 pM (24.6 amol) with high selectivity for the target RNA. More importantly, this method can be useful for the sensing of various kinds of mRNA, including breast cancer cellular mRNA, breast cancer exosomal mRNA, and even urinary exosomal mRNA from breast cancer mice. We anticipate that the developed RNA detection assay can be used for various biomedical applications, such as disease diagnosis, prognosis, and treatment monitoring.

Germ cells are alternative sources for deriving pluripotent stem cells. Because embryonic germ cells (EGCs) possess physiological and developmental features similar to those of embryonic stem cells, pig EGCs are considered a potential tool for generating transgenic animals for agricultural usage. Therefore, in this study, we attempted to establish and characterize pig EGCs from fetal gonads. EGC lines were derived from the genital ridges of porcine fetuses in media containing leukemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and stem cell factor. After establishment, these cells were cultured and stabilized in LIF- or FGF2-containing media. The cell lines were maintained under both conditions over an extended time period and spontaneously differentiated into the three germ layers in vitro. Interestingly, expression of pluripotency markers showed different patterns between cell lines cultured in LIF or FGF2. SSEA4 was only expressed in FGF2-treated pig EGCs (FGF2-pEGCs), not LIF-treated pig EGCs (LIF-pEGCs). Pluripotency genes were upregulated in FGF2-pEGCs, and germline markers were highly expressed, indicating that FGF2 supplements are more efficient in supporting the pluripotency of pEGCs. In conclusion, we verified that FGF2 signaling plays an important role in reprogramming and maintaining pEGCs from fetal gonads.

Porcine embryonic stem cells (pESCs) have become an advantageous experimental tool for developing therapeutic applications and producing transgenic animals. However, despite numerous reports of putative pESC lines, deriving validated pESC lines from embryos produced in vitro remains difficult. Here, we report that embryo aggregation was useful for deriving pESCs from in vitro-produced embryos. Blastocysts derived from embryo aggregation formed a larger number of colonies and maintained cell culture stability. Our derived cell lines demonstrated expression of pluripotent markers (alkaline phosphatase, Oct4, Sox2, and Nanog), an ability to form embryoid bodies, and the capacity to differentiate into the three germ layers. A cytogenetic analysis of these cells revealed that all lines derived from aggregated blastocysts had normal female and male karyotypes. These results demonstrate that embryo aggregation could be a useful technique to improve the efficiency of deriving ESCs from in vitro-fertilized pig embryos, studying early development, and deriving pluripotent ESCs in vitro in other mammals.

*GLP Center, Catholic University of Daegu, Keongsan 712-702, Korea. **Jeongseon Agricultural Extension Center, Jeongseon 233-852, Korea.***Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-701, Korea.****Department of Applied BioScience, Konkuk University, Seoul 143-701, Korea.*****Food Policy Cordination Division, Ministry of Korea Food & Drug Safety, Cheongwon 363-951, Korea.******Forest Practice Research Center, Korea Forest Reserach Institute, Pocheon 487-821, Korea.*******Department of Herbal Medicine Resource, Kangwon National University, Samcheok 245-905, Korea.ABSTRACT : Pharmacological studies and clinical practices have indicated that Radix Astragali, a dried root of Astragalusmembranaceus possesses a lot of biological activities, including antioxidant, hepatoprotective, anti-diabetic, tonic, diuretic,antimicrobial, antiviral, and immunological activities. These biological activities approved by the modern pharmacologicalstudies are mainly due to the constituents of Astragalus membranaceus including polysaccharides, saponins, flavonoids,amino acids, and trace elements. In resent, the main constituents in the root part showing a lot of biological activities hasbeen isolated also from the aboveground parts such as leaves and sprouts in our laboratory. However, the safety evaluationfor the aboveground parts of Astragalus membranaceus should be checked before expanding their application as one of food.In the study, a 90-day rat oral gavage study has been conducted with the extracts from Astragalus membranaceus-above-ground parts at doses of 1000, 3000, and 5000 ㎎/㎏/day. The following endpoints were evaluated: clinical observations, bodyweight, gross and microscopic pathology, clinical chemistry, and hematology. Based on the analysis of these endpoints, it wasestimated that NOEL (no observed effect level) for male rats and NOAEL (no observed adverse effect level) for female ratsare 5000 ㎎/㎏/day of the water-extracts from Astragalus membranaceus- aboveground parts. Key Words : Astragalus membranaceus, Aboveground Parts, NOAEL, Toxicity

Aim of the study Endothelial dysfunction is involved in stroke. Recent therapeutic options for stroke have focused on the combination therapy with a polyherbal mixture. This study was designed to provide insight into the effects of JP05, a water extract of 12 herbs, on the levels of regulators in bEnd.3 mouse brain endothelial cells. Materials and methods Production of endothelial nitric oxide synthase (eNOS)-mediated nitric oxide (NO), the expression of vascular endothelial growth factor (VEGF) and the phosphorylations of eNOS, phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated protein kinase (ERK) and cAMP response element binding protein (CREB) in JP05 were assayed in bEnd.3 cells, a mouse brain endothelial line. Results JP05 led to increase the levels of eNOS-mediated NO generation and VEGF expression in bEnd.3 cells. JP05 induced the phosphorylation of eNOS, Akt and ERK in bEnd.3 cells. As well, JP05 blocked the inhibition of PI3K/Akt and ERK activities by LY294002 (PI3K/Akt inhibitor) and PD98059 (mitogen-activated protein kinase inhibitor), respectively. JP05 also induced the phosphorylation of CREB, which plays an important role in endothelial cell function and blood vessel development. Conclusion Taken together, these results indicate that JP05 can upregulate eNOS-mediated NO generation and VEGF expression through the ERK and/or PI3K/Akt activation, an upstream event of angiogenesis. JP05 with vasoprotective properties has a potential therapy for human brain diseases including stroke.

Porcine embryonic stem (ES) cells have a great potential as tools for transgenic animal production and studies of regulation of differentiation genes. Although several studies showed successful derivation of porcine ES-like cells, these cells were not maintained long-term in culture. Therefore, this study was conducted to establish porcine pluripotent ES-like cells using in vivo fertilized embryos and to maintain these cells in long term culture. Porcine ES-like cells from in vivo embryos obtained by immunosurgery or whole explant culture were successfully cultured for over 56 passages. Morphology of porcine ES-like cells was flat-shaped with a monolayer type colony. These cells stained for alkaline phosphatase throughout the culture. Furthermore, porcine ES-like cells reacted with antibodies against Oct-4, SSEA-1, SSEA-4, Tra-1-60, and Tra-1-81, which are typical markers of undifferentiated stem cells. To characterize the ability of porcine ES-like cells to differentiate into three germ layers, embryoid body formation was induced. After plating of these cells, porcine ES-like cells were spontaneously differentiated into various cell types of all three germ layers. In addition, porcine ES-like cells were successfully derived from IVF blastocysts in media containing human recombinant basic fibroblast growth factor.

Excessive production of inflammatory mediators, nitric oxide (NO) and proinflammatory cytokines from activated microglia has been implicated in neurodegeneration in human brain diseases. Recently, it seems possible that treatment with antiinflammatory agents, including Oriental medicinal plants, might delay the progression of neurodegeneration through the inhibition of microglial activation. The present study evaluated the effect of a methanol extract of Ficus religiosa leaf (MFL) on lipopolysaccharide (LPS)-induced production of NO and proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-beta (IL-1β) and IL-6 in BV-2 cells, a mouse microglial line. MFL inhibited LPS-induced production of NO and proinflammatory cytokines in a dose-dependent manner. MFL also attenuated the expression of mRNA and proteins of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines, suggesting the blockage of transcription levels, respectively. The molecular mechanism of MFL-mediated attenuation underlies the down-regulation of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathway, and suppresses the nuclear factor kappaB (NF-κB) activation. The results suggest that MFL exhibits antiinflammatory properties in LPS-induced activation of BV2 microglial cells, and that might have a therapeutic potential for various neurodegenerative diseases. Copyright © 2008 John Wiley & Sons, Ltd.

Regional air pollution in northeast Asia is an emerging environmental problem requiring long-term impact assessment of acidic deposition. In this study, the gridded distribution of nitrogen uptake led by both growing forests and harvested biomass for eight tree species: Japanese Larch, Red pine, Korean pine, Oak tree, Chestnut, Other Conifers, Other broad leaved trees, and Mixed forest was identified to estimate critical loads for nitrogen over South Korea. The gridded spatial distribution of averaged nitrogen uptake was mapped by 0.125° Latitude × 0.125° Longitude resolution. The results showed that net uptake of nitrogen led by both growth and harvested biomass was totaled at 438 molc ha−1 year−1 among which harvested biomass contribution was estimated to be 25 molc ha−1 year−1, yielding a very small fraction of total nitrogen uptake presumably due to the younger stages of forest in South Korea.

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P

Correction for ‘Stent containing CD44-targeting polymeric prodrug nanoparticles that release paclitaxel and gemcitabine in a time interval-controlled manner for synergistic human biliary cancer therapy’ by Dayeon Yun et al., J. Mater. Chem. B, 2017, 5, 6317–6324.

Some human embryonic stem cell lines have shown genomic instabilities over long-term culture. To study the controversial origin of the SCNT-hES-1 line, which was derived from autologous somatic cell nuclear transfer (SCNT), we compared the expression and methylation patterns of imprinted genes in the SCNT-hES-1 cells with the donor's somatic cells by semi-quantitative RT-PCR, real-time PCR and bisulfite sequencing. Examined imprinted genes were H19, GNAS, SLC22A18, UBE3A and ZNF264 for maternally expressed genes, and IGF2, SNRPN, PEG3, PEG10, MEST, MAGEL2 and ARHI for paternally expressed genes, respectively. We found that the expression of imprinted genes in the SCNT-hES-1 cell line is comparable to that in the donor's somatic cells, and that its methylation patterns are similar to those of other SCNT-products. Therefore, the present study indicates that the SCNT-hES-1 line was derived from SCNT.

We report a case of microcystic (glycogen-rich) adenoma of the whole pancreas with coexistent pancreatic low-grade malignant islet cell tumor in a 29-year-old woman. She complained of nausea, vomiting, and growing abdominal mass. Abdominal computed tomography showed multiple cysts in the whole pancreas and a calcified solid mass in the pancreatic head. A Whipple's operation and total pancreatectomy with splenectomy was performed to treat pancreatic cystic neoplasm. The pancreas was entirely replaced by variable-sized, multilocular cysts, which were lined by a flattened-to-cuboidal glycogen-rich epithelium. Furthermore, in the head of the pancreas, a focal yellowish solid mass showed a positive reaction for chromogranin A and neuron-specific enolase. Careful examination of the pancreas is warranted in cases of microcystic adenoma to rule out a possible coexistent pancreatic malignancy.