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Canine mammary gland tumors (MGT) have a poor prognosis in intact female canines, posing a clinical challenge. This study aimed to establish novel canine mammary cancer cell lines from primary tumors and characterize their cellular and molecular features to find potential therapeutic drugs. The MGT cell lines demonstrated rapid cell proliferation and colony formation in an anchorage-independent manner. Vimentin and α-SMA levels were significantly elevated in MGT cell lines compared to normal canine kidney (MDCK) cells, while CDH1 expression was either significantly lower or not detected at all, based on quantitative real-time PCR (qRT-PCR) analysis. Functional annotation and enrichment analysis revealed that epithelial-mesenchymal transition (EMT) phenotypes and tumor-associated pathways, particularly the PI3K/Akt signaling pathway, were upregulated in MGT cells. BYL719 (Alpelisib), a PI3K inhibitor, was also examined for cytotoxicity on the MGT cell lines. The results show that BYL719 can significantly inhibit the proliferation of MGT cell lines in vitro. Overall, our findings suggest that the MGT cell lines may be valuable for future studies on the development, progression, metastasis, and management of tumors.

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Epidemic diseases that arise from infectious RNA viruses, particularly influenza viruses, pose a constant threat to the global economy and public health. Viral evolution has undermined the efficacy of acquired immunity from vaccines and the antiviral effects of FDA-approved drugs. As such, there is an urgent need to develop new antiviral lead agents. Natural compounds, owing to their historical validation of application and safety, have become a promising solution. In this light, a novel marine bacterium, Pseudomonas sp. M20A4R8, has been found to exhibit significant antiviral activity [half maximal inhibitory concentration (IC50) = 1.3 µg/mL, selectivity index (SI) = 919.4] against influenza virus A/Puerto Rico/8/34, surpassing the activity of chloroquine. The antiviral response via M20A4R8 extract was induced during post-entry stages of the influenza virus, indicating suitability for post-application after the establishment of viral infection. Furthermore, post-treatment with M20A4R8 extract protected the host from virus-induced apoptosis, suggesting its potential use in acute respiratory disease complexes resulting from immune effectors' overstimulation and autophagy-mediated self-apoptosis. The extract demonstrated an outstanding therapeutic index against influenza virus A/Wisconsin/15/2009 (IC50 = 8.1 µg/mL, SI = 146.2) and B/Florida/78/2015 Victoria lineage (IC50 = 3.5 µg/mL, SI = 343.8), indicating a broad anti-influenza virus activity with guaranteed safety and effectiveness. This study provides a new perspective on mechanisms for preventing a broad spectrum of viral infections through antiviral agents from novel and natural origins. Future studies on a single or combined compound from the extract hold promise, encouraging its use in preclinical challenge tests with various influenza virus strains.

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Despite significant improvements in vaccines and chemotherapeutic drugs, pathogenic RNA viruses continue to have a profound impact on the global economy and pose a serious threat to animal and human health through emerging and re-emerging outbreaks of diseases. To overcome the challenge of viral adaptation and evolution, increased vigilance is required. Particularly, antiviral drugs derived from new, natural sources provide an attractive strategy for controlling problematic viral diseases. In this antiviral study, we discovered a previously unknown bacterium, Mameliella sp. M20D2D8, by conducting an antiviral screening of marine microorganisms. An extract from M20D2D8 exhibited antiviral activity with low cytotoxicity and was found to be effective in vitro against multiple influenza virus strains: A/PR8 (IC50 = 2.93 µg/mL, SI = 294.85), A/Phil82 (IC50 = 1.42 µg/mL, SI = 608.38), and B/Yamagata (IC50 = 1.59 µg/mL, SI = 543.33). The antiviral action was found to occur in the post-entry stages of viral replication and to suppress viral replication by inducing apoptosis in infected cells. Moreover, it efficiently suppressed viral genome replication, protein synthesis, and infectivity in MDCK and A549 cells. Our findings highlight the antiviral capabilities of a novel marine bacterium, which could potentially be useful in the development of drugs for controlling viral diseases.

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Gayadomonas joobiniege G7 is an agar-degrading bacterium, which produces various agarases that have been biochemically characterized recently. In this study, we biochemically characterized a new ß-agarase AgaJ10 belonging to the glycoside hydrolase (GH) 42 family from G. joobiniege G7. AgaJ10 is composed of 762 amino acids (89 kDa) and has the highest similarity (63% identity) to a putative ß-agarase from the agar-degrading bacterium Catenovulum sp. DS-2, which was obtained from the intestines of a Haliotis diversicolor. The optimal pH and temperature for AgaJ10 activity were determined to be 5.0 and 30 °C, respectively. AgaJ10 exhibited a cold tolerance, retaining more than 40% of its enzymatic activity at 5 °C. The Km and Vmax of AgaJ10 for agarose were 61.5 mg/mL and 294.1 U/mg, respectively. Notably, the activity of AgaJ10 was significantly enhanced by Mn2+ but was strongly inhibited by some metal ions, including Fe2+, Ni2+, and Cu2+. Agarose-liquefaction, mass spectrometry, and thin-layer chromatography analyses showed that AgaJ10 is an exo-type ß-agarase that hydrolyzes agarose only into neoagarobiose. Therefore, this study is the first report of a GH42 ß-agarase that catalyzes a neoagarobiose-producing exo-type reaction.

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Critical limb ischemia, a severe manifestation of peripheral artery disease, is emerging as a major concern in aging societies worldwide. Notably, cell-based gene therapy to induce angiogenesis in ischemic tissue has been investigated as treatment. Despite many studies demonstrating the efficacy of this approach, better therapies are required to prevent serious sequelae such as claudication, amputation and other cardiovascular events. We have now established a simplified method to enhance the effects of therapeutic transgenes by selecting for and transplanting only transduced cells. Herein, mesenchymal stromal cells were transfected to co-express vascular endothelial growth factor as angiogenic factor and enhanced green fluorescent protein as marker. Transfected cells were then collected using flow cytometry based on green fluorescence and transplanted into ischemic hind limbs in mice. Compared with unsorted or untransfected cells, purified cells significantly improved blood perfusion within 21days, suggesting that transplanting only cells that overexpress vascular endothelial growth factor enhances therapeutic angiogenesis. Importantly, this approach may prove to be useful in cell-based gene therapy against a wide spectrum of diseases, simply by replacing the gene to be delivered or the cell to be transplanted.

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OBJECTIVE: Abdominal aortic aneurysm (AAA) is a common condition that may be life-threatening when it is unrecognized. The aim of this study is to evaluate and compare the efficacy of ramipril and carvedilol on limiting AAA expansion in mouse model. METHODS AND RESULTS: A total of 36 experimental AAA mouse model was induced with the continuous infusion of angiotensin II (Ang II) in 20-week-old male apolipoprotein E-deficient mice. They were randomly divided into 3 treatment groups and fed orally for 8 weeks; saline alone, ramipril (2.5 mg/30g/d), or carvedilol (3.125 mg/30g/d), respectively. Aortic diameter (AD) was measured by micro-computed tomography, and the level of biomarkers of aortic tissue such as monocyte chemoattractant protein-1 (MCP-1) and tissue inhibitor matrix metalloproteinase-1 (TIMP-1) was evaluated. After treatment, AD of both ramipril and carvedilol group was smaller than in the saline group. The percentage change of AD in both ramipril and carvedilol groups was significantly smaller than that of the saline group. Pathologic examination revealed relatively well-preserved aortic walls in the ramipril group compared to the carvedilol and saline groups. The level of MCP-1 was markedly decreased in both the ramipril and carvedilol groups compared to the saline group. The level of TIMP-1 was higher in the carvedilol group when compared to either the saline or ramipril groups. CONCLUSIONS: Ramipril and carvedilol treatment shows similar efficacy in limiting AAA expansion in mouse model. Future clinical research would be warranted to validate these results.

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The present study was conducted in order to improve gene expression efficiency of insulin­like growth factor­1 (IGF­1)­transfected mesenchymal stem cells (MSCs) using a non­viral carrier and a simplified method of dual gene selection. The therapeutic efficacy of this MSC­based IGF­1/enhanced green fluorescent protein (EGFP) dual gene sorting system was evaluated in a rat myocardial infarction (MI) model. IGF­1 and EGFP genes were expressed in MSCs in vitro. The purity of dual gene­expressing MSCs was 95.1% by fluorescence­activated cell sorting. Transfected MSCs injected into rats were identified based on green fluorescence, with an increased signal intensity observed in rats injected with sorted cells, compared with unsorted cells. IGF­1 expression levels were additionally increased in the sorted group, and decreases in infarct size, fibrotic area and fraction of apoptotic cells were observed. These results demonstrated that IGF­1 overexpression protects against fibrosis and apoptosis in the myocardium and reduces infarct size following MI. Additionally, the present vector sorting system may potentially be applied to other types of stem cell­based gene therapy.

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A novel ß-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be 30°C and 4.5, respectively. AgaJ5 was an acidic ß-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at 10°C. AgaJ5 required monovalent ions such as Na+ and K+ for its maximum activity, but its activity was severely inhibited by several metal ions. The Km and Vmax of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type ß-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing ß-agarase having specific biochemical features that may be useful for industrial applications.

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A novel ß-agarase AgaJ11 belonging to the glycoside hydrolase (GH) 16 family was identified from an agar-degrading bacterium Gayadomonas joobiniege G7. AgaJ11 was composed of 317 amino acids (35 kDa), including a 26-amino acid signal peptide, and had the highest similarity (44 % identity) to a putative ß-agarase from an agarolytic marine bacterium Agarivorans albus MKT 106. The agarase activity of purified AgaJ11 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ11 activity were determined to be 4.5 and 40 °C, respectively. Notably, AgaJ11 is an acidic ß-agarase that was active only at a narrow pH range from 4 to 5, and less than 30 % of its enzymatic activity was retained at other pH conditions. The K m and V max of AgaJ11 for agarose were 21.42 mg/ml and 25 U/mg, respectively. AgaJ11 did not require metal ions for its activity, but severe inhibition by several metal ions was observed. Thin layer chromatography and agarose-liquefying analyses revealed that AgaJ11 is an endo-type ß-agarase that hydrolyzes agarose into neoagarohexaose, neoagarotetraose, and neoagarobiose. Therefore, this study shows that AgaJ11 from G. joobiniege G7 is a novel GH16 ß-agarase with an acidic enzymatic feature that may be useful for industrial applications.

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Gayadomonas joobiniege G7 is an agar-degrading marine bacterium belonging to a novel genus. Genomic sequencing of G. joobiniege revealed that AgaJ9 (formerly YjdB) belonging to the glycoside hydrolase (GH) 39 family. It showed the highest similarity (47% identity) to a putative ß-agarase from Catenovulum agarivorans DS-2, an agar-degrading marine bacterium sharing the highest similarity in the nucleotide sequence of 16s rRNA gene with G. joobiniege G7. The agaJ9 gene encodes a protein (134 kDa) of 1205 amino acids, including a 23-amino acid signal peptide. The agarase activity of purified AgaJ9 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ9 activity were determined as 5 and 25 °C, respectively. Notably, AgaJ9 is a cold-adapted ß-agarase retaining more than 80% of its activity even at a temperature of 5 °C. In addition, gel filtration chromatography revealed that AgaJ9 exists as two forms, dimer and monomer. Although the two forms had similar enzymatic properties, their kinetic parameters were different. The K m and V max of dimeric AgaJ9 for agarose was 0.68 mg/ml (5.7 × 10-6 M) and 17.2 U/mg, respectively, whereas the monomeric form had a K m of 1.43 mg/ml (1.2 × 10-5 M) and V max of 10.7 U/mg. Thin-layer chromatography and agarose-liquefying analyses revealed that AgaJ9 is an endo-type ß-agarase that hydrolyzes agarose into neoagarotetraose and neoagarobiose. This study is the first report of a GH39 ß-agarase with a cold-adapted enzymatic feature, a unique attribute, which may be useful for industrial applications.

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Developments of non-viral carriers have headed toward reducing cytotoxicity, which results from the use of conventional gene carriers, and enhancing gene delivery efficiency. Cys-(d-R9)-Cys repeated reducible poly(oligo-D-arginine) (rPOA) is one of the most efficient non-viral carriers for gene therapy; however, while its efficiency has been verified in the lung and brain, it is necessary to confirm its activity in each organ or tissue since there are differences of gene carrier susceptibility to among tissue types. We therefore tested the compatibility of rPOA in cardiac tissue by in vitro or in vivo experiments and confirmed its high transfection efficiency and low cytotoxicity. Moreover, substantial regenerative effects were observed following transfection with rPOA/pVEGF expression vector complexes (79% decreased infarct size) compared to polyethyleneimine (PEI) (34% decreased infarct size) in a rat myocardial infarction (MI) model. These findings suggest that rPOA efficiently enables DNA transfection in cardiac tissue and can be used as a useful non-viral therapeutic gene carrier for gene therapy in ischemic heart disease.

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Mesenchymal stem cell (MSC) implantation has emerged as a potential therapy for myocardial infarction (MI). However, the poor survival of MSCs implanted to treat MI has significantly limited the therapeutic efficacy of this approach. This poor survival is primarily due to reactive oxygen species (ROS) generated in the ischemic myocardium after the restoration of blood flow. ROS primarily causes the death of implanted MSCs by inhibiting the adhesion of the MSCs to extracellular matrices at the lesion site (i.e., anoikis). In this study, we proposed the use of graphene oxide (GO) flakes to protect the implanted MSCs from ROS-mediated death and thereby improve the therapeutic efficacy of the MSCs. GO can adsorb extracellular matrix (ECM) proteins. The survival of MSCs, which had adhered to ECM protein-adsorbed GO flakes and were subsequently exposed to ROS in vitro or implanted into the ischemia-damaged and reperfused myocardium, significantly exceeded that of unmodified MSCs. Furthermore, the MSC engraftment improved by the adhesion of MSCs to GO flakes prior to implantation enhanced the paracrine secretion from the MSCs following MSC implantation, which in turn promoted cardiac tissue repair and cardiac function restoration. This study demonstrates that GO can effectively improve the engraftment and therapeutic efficacy of MSCs used to repair the injury of ROS-abundant ischemia and reperfusion by protecting implanted cells from anoikis.

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