Comparison of anaerobic digestion of starch- and petro-based bioplastic under hydrogen-rich conditions.

To identify an economically viable waste management system for bioplastics, thermoplastic starch (TPS) and poly(butylene adipate-co-terephthalate) (PBAT) were anaerobically digested under hydrogen (H2)/carbon dioxide (CO2) and nitrogen (N2) gas-purged conditions to compare methane (CH4) production and biodegradation. Regardless of the type of bioplastics, CH4 production was consistently higher with H2/CO2 than with N2. The highest amount of CH4 was produced at 307.74 mL CH4/g volatile solids when TPS digested with H2/CO2. A stepwise increased in CH4 yield was observed, with a nominal initial increment followed by accelerated methanogenesis conversion as H2 was depleted. This may be attributed to a substantial shift in the microbial structure from hydrogenotrophic methanogen (Methanobacteriales and Methanomicrobiales) to heterotrophs (Spirochaetia). In contrast, no significant change was observed with PBAT, regardless of the type of purged gas. TPS was broken down into numerous derivatives, including volatile fatty acids. TPS produced more byproducts with H2/CO2 (i.e., 430) than with N2 (i.e., 320). In contrast, differential scanning calorimetry analysis on PBAT revealed an increase in crystallinity from 10.20 % to 12.31 % and 11.36 % in the H2/CO2- and N2-purged conditions, respectively, after 65 days of testing. PBAT surface modifications were characterized via Fourier transform infrared spectroscopy and scanning electron microscopy. The results suggest that the addition of H2/CO2 can enhance the CH4 yield and increase the breakdown rate of TPS more than that of PBAT. This study provides novel insights into the CH4 production potential of two bioplastics with different biodegradabilities in H2/CO2-mediated anaerobic digestion systems.

Development of a Novel and Simple Anti-Metastatic Cancer Treatment Targeting Vasohibin-2.

Vasohibin-2 (VASH2), a homologue of vasohibin-1 (VASH1), is overexpressed in various cancer cells and promotes tumor progression. We therefore regard VASH2 as a molecular target for cancer treatment. Here we applied vaccine technology to develop a therapy against VASH2. We selected two amino acid sequences of VASH2 protein; the MTG and RRR peptides, which contain possible B cell epitopes. These sequences are identical between the human and murine VASH2 proteins and distinct from those of the VASH1 protein. We conjugated these peptides with the carrier protein keyhole limpet hemocyanin, mixed with an adjuvant, and injected subcutaneously twice at a 2-week interval in mice. Both vaccines increased antibodies against the antigen peptide; however, only the MTG peptide vaccine increased antibodies that recognized the recombinant VASH2 protein. When Lewis lung cancer (LLC) cells were subcutaneously inoculated, tumors isolated from mice immunized with the MTG peptide vaccine showed a significant decrease in the expression of epithelial-to-mesenchymal transition (EMT) markers. EMT is responsible for cancer cell invasion and metastasis. When the LLC cells were injected into the tail vein, the MTG peptide vaccine inhibited lung metastasis. Moreover, the MTG peptide vaccine inhibited the metastasis of pancreatic cancer cells to the liver in an orthotopic mouse model, and there was a significant inverse correlation between the ELISA titer and metastasis inhibition. Therefore, we propose that the MTG peptide vaccine is a novel anti-metastatic cancer treatment that targets VASH2 and can be applied even in the most malignant and highly metastatic pancreatic cancer.

Preparation of Waste PP/Fly Ash/Waste Stone Powder Composites and Evaluation of Their Mechanical Properties.

The research was carried out to analyze the combined and mechanical properties of polypropylene (PP)/fly ash (FA)/waste stone powder (WSP) composite materials. PP, FA and WSP were mixed and prepared into PP100 (pure PP), PP90 (90 wt% PP + 5 wt% FA + 5 wt% WSP), PP80 (80 wt% PP + 10 wt% FA + 10 wt% WSP), PP70 (70 wt% PP + 15 wt% FA + 15 wt% WSP), PP60 (60 wt% PP + 20 wt% FA + 20 wt% WSP) and PP50 (50 wt% PP + 25 wt% FA + 25 wt% WSP) composite materials using an injection molding machine. The research results indicate that all PP/FA/WSP composite materials can be prepared through the injection molding process and there are no cracks or fractures found on the surface of the composite materials. The research results of thermogravimetric analysis are consistent with expectations, indicating that the preparation method of the composite materials in this study is reliable. Although the addition of FA and WSP powder cannot increase the tensile strength, it is very helpful to improve the bending strength and notched impact energy. Especially for notched impact energy, the addition of FA and WSP results in an increase in the notched impact energy of all PP/FA/WSP composite materials by 14.58-22.22%. This study provides a new direction for the reuse of various waste resources. Moreover, based on the excellent bending strength and notched impact energy, the PP/FA/WSP composite materials have great application potential in the composite plastic industry, artificial stone, floor tiles and other industries in the future.

Gene expression of Toll-like receptors, cytokines and a nuclear factor and cytokine secretion in DH82 canine macrophage cells infected with Brucella canis.

Canine brucellosis caused by Brucella canis infection occurs mainly in dogs, and is a zoonotic disease that also has the possibility of infection in humans. Many studies have been conducted to understand the immunopathological mechanism of B. canis infection. However, the precise immune mechanism remains to be elucidated because compared to other Brucella spp., B. canis has different immune evasion mechanisms. In this study, gene expression levels of Toll-like receptors (TLRs) and TLR-associated molecules and cytokine production were analyzed to figure out the roles of immune-related host factors in B. canis infection. Time-dependent gene expression of TLRs (1-10) and TLR-related molecules (TNF-α, IL-5, IL-23, CCL4, CD40 and NFκ-B) and release of Th1, Th2 and Th17-related cytokines (IFN-γ, IL-1ß, IL-4, IL-6, IL-10 and IL-17A) were investigated in DH82 canine macrophages infected with B. canis. Time-dependent induction of TLRs 3, 7 and 8 was observed, and TLR 7 had the highest expression level (p <0.05). The expression levels of all TLR-related genes were significantly increased after infection. In particular, the expression of the CCL4 and IL-23 genes was highly induced. The amounts of IL-1ß, IL-6 and IL-10 were significantly increased by B. canis infection, but the amounts of IL-4 and IL-17A were not. The production of IL-1ß and IL-6 was the highest at 24 hr after B. canis infection (p <0.05). This study demonstrates that TLRs 3, 7 and 8 are prominent sites of to immune response induction with the production of related cytokines and a nuclear factor in DH82 cells infected with B. canis. These results suggest a sequential immune mechanism of B. canis infection, involving TLRs, cytokines and their associated factors.

A cell surface-reducing microenvironment induces early osteogenic commitment.

Stem cell-based therapy has been highlighted as a potential avenue to promote tissue regeneration, where stimulation of stem cells to differentiate into the targeted cell type is essential. One of the factors that induce stem cells to differentiate is their surrounding microenvironment. In this study, the correlation between mild reductant and early osteogenic commitment was evaluated. A cell surface-reducing microenvironment significantly silenced the transforming growth factor (TGF)-ß signaling pathway of mesenchymal stem cells (MSCs), followed by increased focal adhesion and inhibition of cell membrane protein dimerization. Furthermore, in vivo transplantation of MSCs exposed to the reducing microenvironment resulted in an early osteogenic commitment and neobone formation. Thus, these results highlight the potential of cell surface-reducing microenvironment to influence early osteogenic commitment.

An autophagy enhancer ameliorates diabetes of human IAPP-transgenic mice through clearance of amyloidogenic oligomer.

We have reported that autophagy is crucial for clearance of amyloidogenic human IAPP (hIAPP) oligomer, suggesting that an autophagy enhancer could be a therapeutic modality against human diabetes with amyloid accumulation. Here, we show that a recently identified autophagy enhancer (MSL-7) reduces hIAPP oligomer accumulation in human induced pluripotent stem cell-derived ß-cells (hiPSC-ß-cells) and diminishes oligomer-mediated apoptosis of ß-cells. Protective effects of MSL-7 against hIAPP oligomer accumulation and hIAPP oligomer-mediated ß-cell death are significantly reduced in cells with knockout of MiTF/TFE family members such as Tfeb or Tfe3. MSL-7 improves glucose tolerance and ß-cell function of hIAPP+ mice on high-fat diet, accompanied by reduced hIAPP oligomer/amyloid accumulation and ß-cell apoptosis. Protective effects of MSL-7 against hIAPP oligomer-mediated ß-cell death and the development of diabetes are also significantly reduced by ß-cell-specific knockout of Tfeb. These results suggest that an autophagy enhancer could have therapeutic potential against human diabetes characterized by islet amyloid accumulation.

Mitochondria ubiquitin ligase, MARCH5 resolves hepatitis B virus X protein aggregates in the liver pathogenesis.

Infection of hepatitis B virus (HBV) increase the incidence of chronic liver disease and hepatocellular carcinoma (HCC). The hepatitis B viral x (HBx) protein encoded by the HBV genome contributes to the pathogenesis of HCC and thus, negative regulation of HBx is beneficial for the alleviation of the disease pathogenesis. MARCH5 is a mitochondrial E3 ubiquitin ligase and here, we show that high MARCH5 expression levels are correlated with improved survival in HCC patients. MARCH5 interacts with HBx protein mainly accumulated in mitochondria and targets it for degradation. The N-terminal RING domain of MARCH5 was required for the interaction with HBx, and MARCH5H43W lacking E3 ligase activity failed to reduce HBx protein levels. High expression of HBx results in the formation of protein aggregates in semi-denaturing detergent agarose gels and MARCH5 mediates the elimination of protein aggregates through the proteasome pathway. HBx-induced ROS production, mitophagy, and cyclooxygenase-2 gene expression were suppressed in the presence of high MARCH5 expression. These results suggest MARCH5 as a target for alleviating HBV-mediated liver disease.

Programmable Nuclease-Based Integration into Novel Extragenic Genomic Safe Harbor Identified from Korean Population-Based CNV Analysis.

Here, we found two genomic safe harbor (GSH) candidates from chromosomes 3 and 8, based on large-scale population-based cohort data from 4,694 Koreans by CNV analysis. Furthermore, estimated genotype of these CNVRs was validated by quantitative real-time PCR, and epidemiological data examined no significant genetic association between diseases or traits and two CNVRs. After screening the GSH candidates by in silico approaches, we designed TALEN pairs to integrate EGFP expression cassette into human cell lines in order to confirm the functionality of GSH candidates in an in vitro setting. As a result, transgene insertion into one of the two loci using TALEN showed robust transgene expression comparable to that with an AAVS1 site without significantly perturbing neighboring genes. Changing the promoter or cell type did not noticeably disturb this trend. Thus, we could validate two CNVRs as a site for effective and safe transgene insertion in human cells.

Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth.

KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.

Dense Granule Protein-7 (GRA-7) of Toxoplasma gondii inhibits viral replication in vitro and in vivo.

Dense granule protein-7 (GRA-7) is an excretory protein of Toxoplasma gondii. It is a potential serodiagnostic marker and vaccine candidate for toxoplasmosis. Previous reports demonstrated that GRA-7 induces innate immune responses in macrophages by interacting with TRAF6 via the MyD88-dependent pathway. In the present study, we evaluated the antiviral activity and induction of an antiviral state by GRA-7 both in vitro and in vivo. It was observed that GRA-7 markedly reduced the replication of vesicular stomatitis virus (VSV-GFP), influenza A virus (PR8-GFP), coxsackievirus (H3-GFP), herpes simplex virus (HSV-GFP), and adenovirus-GFP in epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. These antiviral activities of GRA-7 were attributed to the induction of type I interferon (IFN) signaling, resulting in the secretion of IFNs and pro-inflammatory cytokines. Additionally, in BALB/c mice, intranasal administration of GRA-7 prevented lethal infection by influenza A virus (H1N1) and exhibited prophylactic effects against respiratory syncytial virus (RSV-GFP). Collectively, these results suggested that GRA-7 exhibits immunostimulatory and broad spectrum antiviral activities via type I IFN signaling. Thus, GRA-7 can be potentially used as a vaccine adjuvant or as a candidate drug with prophylactic potential against viruses.

The functional relationship between transglutaminase 2 and transforming growth factor ß1 in the regulation of angiogenesis and endothelial-mesenchymal transition.

The importance of transglutaminase 2 (TG2) in angiogenesis has been highlighted in recent studies, but other roles of this multi-functional enzyme in endothelial cell (EC) function still remains to be fully elucidated. We previously showed that the extracellular TG2 is involved in maintaining tubule formation in ECs by a mechanism involving matrix-bound vascular endothelial growth factor (VEGF) signalling. Here, by using the ECs and fibroblast co-culture and ECs 3D culture models, we demonstrate a further role for TG2 in both endothelial tubule formation and in tubule loss, which involves its role in the regulation of transforming growth factor ß1 (TGFß1) and Smad signalling. We demonstrate that inhibition of tubule formation by TG2 inhibitors can be restored by add-back of exogenous TGFß1 at pg/ml levels and show that TG2 -/- mouse ECs are unable to form tubules in 3D culture and display negligible Smad signalling compared to wild-type cells. Loss of tubule formation in the TG2 -/- ECs can be reconstituted by transduction with TG2. We demonstrate that extracellular TG2 also has an important role in TGFß1-induced transition of ECs into myofibroblast-like cells (endothelial-mesenchymal transition), resulting in loss of EC tubules and tubule formation. Our data also indicate that TG2 may have a role in regulating TGFß signalling through entrapment of active TGFß1 into the extracellular matrix. In conclusion, our work demonstrates that TG2 has multi-functional roles in ECs where its ability to fine-tune of TGFß1 signalling means it can be involved in both endothelial tubule formation and tubule rarefaction.

Bioreducible-Cationic Poly(amido amine)s for Enhanced Gene Delivery and Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

The development of efficient and safe gene delivery carriers has been a major challenge in the clinical application of non-viral gene therapy. Herein, we report novel bioreducible poly(amido amine)s for the efficient delivery of genetic material such as plasmid DNA. A library of 34 different bioreducible polymer compounds was synthesized and screened to find lead materials for in vitro gene transfection. Our lead material (CBA-106) allows effortless polyplex formation with genetic materials by electrostatic interactions at the weight ratio of 1:5 (DNA/polymer). Polyplexes were further characterized by DLS and AFM analysis. Enhanced serum stability and bioreducibility under physiological conditions were confirmed, in addition to low cellular cytotoxicity. When compared with a commercially available gene delivery carrier (Lipofectamine 2000), CBA-1 06 shows comparable or even surpassing gene transfection efficiency. Furthermore, BMP-2 plasmids were efficiently delivered to tonsil-derived mesenchymal stem cells (TMSCs) for osteogenic commitment in vitro and in vivo. Taken together, our results clearly demonstrate the potential of novel bioreducible polymeric systems for gene delivery applications. We suggest that our system can provide a valuable platform for the broad application of gene regulation in cell therapy and regenerative medicine.

Non-viral approaches for direct conversion into mesenchymal cell types: Potential application in tissue engineering.

Acquiring adequate number of cells is one of the crucial factors to apply tissue engineering strategies in order to recover critical-sized defects. While the reprogramming technology used for inducing pluripotent stem cells (iPSCs) opened up a direct path for generating pluripotent stem cells, a direct conversion strategy may provide another possibility to obtain desired cells for tissue engineering. In order to convert a somatic cell into any other cell type, diverse approaches have been investigated. Conspicuously, in contrast to traditional viral transduction method, non-viral delivery of conversion factors has the merit of lowering immune responses and provides safer genetic manipulation, thus revolutionizing the generation of directly converted cells and its application in therapeutics. In addition, applying various microenvironmental modulations have potential to ameliorate the conversion of somatic cells into different lineages. In this review, we discuss the recent progress in direct conversion technologies, specifically focusing on generating mesenchymal cell types.

Induced myogenic commitment of human chondrocytes via non-viral delivery of minicircle DNA.

Lineage conversion from one somatic cell type to another is an attractive approach for deriving specific therapeutic cell generation. In order to bypass inducing pluripotent stage, transdifferentiation/direct conversion technologies have been recently developed. We report the development of a direct conversion methodology in which cells are transdifferentiated through a plastic intermediate state induced by exposure to non-integrative minicircle DNA (MCDNA)-based reprogramming factors, followed by differentiation into myoblasts. In order to increase the MCDNA delivery efficiency, reprogramming factors were delivered into the chondrocytes via electroporation followed by poly (ß-amino esters) (PBAE) transfection. We used this approach to convert human chondrocytes to myoblast, and with treatment of SB-431542, an inhibitor of the activin receptor-like kinase receptors, to enhance myogenic commitment. Differentiated cells exhibited expression of myogenic markers such as MyoD and Myog. This methodology for direct lineage conversion from chondrocytes to myoblast represents a novel non-viral Method to convert hard-to-transfect cells to other lineage.

Neovessel formation promotes liver fibrosis via providing latent transforming growth factor-ß.

AIM: Hepatic fibrosis and angiogenesis occur in parallel during the progression of liver disease. Fibrosis promotes angiogenesis via inducing vascular endothelial growth factor (VEGF) from the activated hepatic stellate cells (HSCs). In turn, increased neovessel formation causes fibrosis, although the underlying molecular mechanism remains undetermined. In the current study, we aimed to address a role of endothelial cells (ECs) as a source of latent transforming growth factor (TGF)-ß, the precursor of the most fibrogenic cytokine TGF-ß. METHODS: After recombinant VEGF was administered to mice via the tail vein, hepatic angiogenesis and fibrogenesis were evaluated using immunohistochemical and biochemical analyses in addition to investigation of TGF-ß activation using primary cultured HSCs and liver sinusoidal ECs (LSECs). RESULTS: In addition to increased hepatic levels of CD31 expression, VEGF-treated mice showed increased α-smooth muscle actin (α-SMA) expression, hepatic contents of hydroxyproline, and latency associated protein degradation products, which reflects cell surface activation of TGF-ß via plasma kallikrein (PLK). Liberating the PLK-urokinase plasminogen activator receptor complex from the HSC surface by cleaving a tethering phosphatidylinositol linker with its specific phospholipase C inhibited the activating latent TGF-ß present in LSEC conditioned medium and subsequent HSC activation. CONCLUSION: Neovessel formation (angiogenesis) accelerates liver fibrosis at least in part via provision of latent TGF-ß that activated on the surface of HSCs by PLK, thereby resultant active TGF-ß stimulates the activation of HSCs.

Ras-dependent induction of HIF-1alpha785 via the Raf/MEK/ERK pathway: a novel mechanism of Ras-mediated tumor promotion.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays crucial roles in tumor promotion by transactivating approximately 60 kinds of its target genes. Recently, we reported a novel splice variant HIF-1alpha(785), which is regulated primarily by phorbol ester. This variant can be stabilized under normoxic conditions because it loses an acetylation site Lys532. Its expression was found to promote xenografted tumor growth in nude mice. We here found that the Ras oncogene regulates HIF-1alpha(785) expression via the Raf/MEK/ERK pathway, and that both phorbol ester and epidermal growth factor also induced HIF-1alpha(785) via the same pathway. We also identified the nonhypoxic regulatory domain responsible for phorbol ester-induced HIF-1alpha(785) expression. These results imply that HIF-1alpha(785) may play an important role in tumor promotion mediated by the Ras oncogene, phorbol ester or tumor growth factors.