• Biomedical Science Letters
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• v.28 no.4
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• pp.247-259
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• 2022

Immunotherapy, which uses an immune mechanism in the body, has received considerable attention for cancer treatment. Suberoylanilide hydroxamic acid (SAHA), also known as a histone deacetylase inhibitor (HDACi), is used as a cancer treatment to induce active immunity by increasing the expression of T cell-induced chemokines. However, this SAHA treatment has the disadvantage of causing PD-L1 overexpression in tumor cells. In this study, we prevented PD-L1 overexpression by blocking the PD-1/PD-L1 pathway using PD-L1 siRNA. We designed two types of liposomes, the neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin (POPC) for SAHA, and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) for siRNA. To effectively target PD-L1 in cancer cells, we conjugated PD-L1 antibody with liposomes containing SAHA or PD-L1 siRNA. These immunoliposomes were also evaluated for cytotoxicity, gene silencing, and T-cell-induced chemokine expression in human non-small cell lung cancer A549 cells. It was confirmed that the combination of the two immunoliposomes increased the cancer cell suppression efficacy through Jurkat T cell induction more than twice compared to SAHA alone treatment. In conclusion, this combination of immunoliposomes containing a drug and nucleic acid has promising therapeutic potential for non-small-cell lung carcinoma (NSCLC).

• International Journal of Stem Cells
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• v.16 no.2
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• pp.156-167
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• 2023

Background and Objectives: Cellular reprogramming in regenerative medicine holds great promise for treating patients with neurological disorders. In this regard, small molecule-mediated cellular conversion has attracted special attention because of its ease of reproducibility, applicability, and fewer safety concerns. However, currently available protocols for the direct conversion of somatic cells to neurons are limited in clinical application due of their complex nature, lengthy process, and low conversion efficiency. Methods and Results: Here, we report a new protocol involving chemical-based direct conversion of human fibroblasts (HF) to matured neuron-like cells with a short duration and high conversion efficiency using temporal and strategic dual epigenetic regulation. In this protocol, epigenetic modulation by inhibition of histone deacetylase and bromodomain enabled to overcome "recalcitrant" nature of adult fibroblasts and shorten the duration of neuronal reprogramming. We further observed that an extended epigenetic regulation is necessary to maintain the induced neuronal program to generate a homogenous population of neuron-like cells. Conclusions: Therefore, our study provides a new protocol to produce neurons-like cells and highlights the need of proper epigenetic resetting to establish and maintain neuronal program in HF.

• Biomedical Science Letters
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• v.30 no.2
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• pp.37-48
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• 2024

Liposomes are one of the most actively studied and promising drug delivery systems for the treatment of various diseases. In this study, an aptamer-conjugated liposome called "aptamosome" was used, in which an anti-PD-L1 aptamer targeting cancer cells was conjugated to the liposome. These aptamosomes showed remarkable cellular uptake and efficient delivery to Lewis lung carcinoma 2 (LL/2) cancer cells. In addition, suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACi), was delivered through this aptamer to induce a strong anticancer immunotherapeutic effect. The results of this study showed that when LL/2 cells were treated with SAHA-entrapped aptamosome [SAHA] and liposome [SAHA] and free SAHA, aptamosome [SAHA] improved cell death compared with that of liposomes [SAHA] or free SAHA, and it has demonstrated anticancer efficacy. Moreover, aptamosome [SAHA] induce the secretion of chemokines that promote the migration of activated T cells into tumor tissues. Finally, in vivo experiments showed that aptamosome [SAHA] significantly inhibited the growth rate of LL/2 tumors. Therefore, liposomes combined with an anti-PD-L1 aptamer for efficient SAHA delivery are suggested as an excellent model for drug delivery systems suitable for targeting cancer cells.

• Biomolecules & Therapeutics
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• v.22 no.5
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• pp.406-413
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• 2014

to valproic acid (VPA) during pregnancy produces ASD-like core behavioral phenotypes as well as hyperactivity in offspring both in human and experimental animals, which makes it a plausible model to study ASD-related neurobiological processes. In this study, we examined the effects of two of currently available attention defecit hyperactivity disorder (ADHD) medications, methylphenidate (MPH) and atomoxetine (ATX) targeting dopamine and norepinephrine transporters (DAT and NET), respectively, on hyperactive behavior of prenatally VPA-exposed rat offspring. In the prefrontal cortex of VPA exposed rat offspring, both mRNA and protein expression of DAT was increased as compared with control. VPA function as a histone deacetylase inhibitor (HDACi) and chromatin immunoprecipitation experiments demonstrated that the acetylation of histone bound to DAT gene promoter was increased in VPA-exposed rat offspring suggesting epigenetic mechanism of DAT regulation. Similarly, the expression of NET was increased, possibly via increased histone acetylation in prefrontal cortex of VPA-exposed rat offspring. When we treated the VPA-exposed rat offspring with ATX, a NET selective inhibitor, hyperactivity was reversed to control level. In contrast, MPH that inhibits both DAT and NET, did not produce inhibitory effects against hyperactivity. The results suggest that NET abnormalities may underlie the hyperactive phenotype in VPA animal model of ASD. Profiling the pharmacological responsiveness as well as investigating underlying mechanism in multiple models of ASD and ADHD may provide more insights into the neurobiological correlates regulating the behavioral abnormalities.

• Journal of Life Science
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• v.27 no.4
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• pp.482-499
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• 2017

Allergic diseases have increased over the past several decade worldwide including developing countries. Allergic inflammatory responses are caused by Th (T helper)2 immune responses, triggered by allergen ingestion by antigen presenting cells such as dendritic cells (DCs). Intestinal microorganisms control the metabolism and physiological functions of the host, contribute to early immune system maturation during the early life, and homeostasis and epithelial integrity during life. Bifidobacteria have strain-specific immunostimulatory properties in the Th1/Th2 balance, inhibit TSLP (thymic stromal lymphopoietin) and IgE expression, and promote Flg (Filaggrin) and FoxP3 (Treg) expression to alleviate allergies. In addition, unmethylated CpG motif ODN (oligodeoxynucleotides) is recognized by TLR (toll-like receptors)9 of B cells and plasmacytoid dendritic cells (pDCs) to induce innate and adaptive immune responses, while the butyrate produced by Clostridium butyricum activates the GPR (G-protein coupled receptors)109a signaling pathway to induce the expression of anti-inflammatory gene of pDCs, and directly stimulates the proliferation of thymically derived regulatory T (tTreg) cells through the activation of GPR43 or inhibits the activity of HADC (histone deacetylase) to differentiate naive $CD4^+$ T cells into pTreg cells through the histone H3 acetylation of Foxp3 gene intronic enhancer.

• Journal of Life Science
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• v.28 no.8
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• pp.885-891
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• 2018

Clostridium difficile (C. difficile) toxin A is known to cause acute gut inflammation in humans and animals by triggering cytoskeletal disorganization in gut epithelial cells. In human colonocytes, toxin A blocks microtubule assembly by directly increasing the enzymatic activity of histone deacetylase-6 (HDAC-6), a tubulin-specific deacetylase, thereby markedly decreasing tubulin acetylation, which is essential for microtubule assembly. Microtubule assembly dysfunction-associated alterations (i.e., toxin A-exposed gut epithelial cells) are believed to trigger barrier dysfunction and gut inflammation downstream. We recently showed that potassium acetate blocked toxin A-induced microtubule disassembly by inhibiting HDAC-6. Herein, we tested whether acetic acid (AA), another small acetyl residue-containing agent, could block toxin A-induced tubulin deacetylation and subsequent microtubule assembly. Our results revealed that AA treatment increased tubulin acetylation and enhanced microtubule assembly in an HT29 human colonocyte cell line. AA also clearly increased tubulin acetylation in murine colonic explants. Interestingly, the AA treatment also alleviated toxin A-induced tubulin deacetylation and microtubule disassembly, and MTT assays revealed that AA reduced toxin A-induced cell toxicity. Collectively, these results suggest that AA can block the ability of toxin A to cause microtubule disassembly-triggered cytoskeletal disorganization by blocking toxin A-mediated deacetylation of tubulin.

• Journal of Life Science
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• v.13 no.1
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• pp.128-135
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• 2003

102 ESTs (Expressed Sequence Tags) were obtained by sequencing clones from a library of rainbow trout kidney cDNAs. Of the sequences generated, 55.8% of the ESTs were represented by 37 known genes. The 45 clones of unknown gene products potentially represent 40 novel genes. The genes involved in structural function (14.5%) and transcription/translation (11.6%) account for the major gene expression activities in the kidney Microarray experiment was conducted to compare gene expression of the unique ESTs in young and adult rainbow trout kidneys. While mitochondrion, cytochrome b, rho G, spastin protein, and three unknown genes were down-regulated in the mature fish kidney, calponin 1, calcium binding protein, histone deacetylase 1, and an unknown gene were up-regulated in the mature fish kidney. This research demonstrates the feasibility and power of functional genomics in rainbow trout.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.12
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• pp.1703-1712
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• 2015

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (-) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB- oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

• Tuberculosis and Respiratory Diseases
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• v.81 no.1
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• pp.80-87
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• 2018

Background: Asthma is a disease of chronic airway inflammation with heterogeneous features. Neutrophilic asthma is corticosteroid-insensitive asthma related to absence or suppression of $T_H2$ process and increased $T_H1$ and/or $T_H17$ process. Macrolides are immunomodulatory drug that reduce airway inflammation, but their role in asthma is not fully known. The purpose of this study was to evaluate the role of macrolides in neutrophilic asthma and compare their effects with those of corticosteroids. Methods: C57BL/6 female mice were sensitized with ovalbumin (OVA) and lipopolysaccharides (LPS). Clarithromycin (CAM) and/or dexamethasone (DXM) were administered at days 14, 15, 21, 22, and 23. At day 24, the mice were sacrificed. Results: Airway resistance in the OVA+LPS exposed mice was elevated but was more attenuated after treatment with CAM+DXM compared with the monotherapy group (p<0.05 and p<0.01). In bronchoalveolar lavage fluid study, total cells and neutrophil counts in OVA+LPS mice were elevated but decreased after CAM+DXM treatment. In hematoxylin and eosin stain, the CAM+DXM-treated group showed less inflammation additively than the monotherapy group. There was less total protein, interleukin 17 (IL-17), interferon ${\gamma}$, and tumor necrosis factor ${\alpha}$ in the CAM+DXM group than in the monotherapy group (p<0.001, p<0.05, and p<0.001). More histone deacetylase 2 (HDAC2) activity was recovered in the DXM and CAM+DXM challenged groups than in the control group (p<0.05). Conclusion: Decreased IL-17 and recovered relative HDAC2 activity correlated with airway resistance and inflammation in a neutrophilic asthma mouse model. This result suggests macrolides as a potential corticosteroid-sparing agent in neutrophilic asthma.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.269-279
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• 2013

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2'deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.