• International Journal of Stem Cells
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• v.16 no.2
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• pp.135-144
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• 2023

Background and Objectives: Melanocyte (MC), derived from neural crest stem cell (NCSC), are involved in the production of melanin. The mechanism by which NCSC differentiates to MC remains unclear. N6-methyladenosine (m6A) modification was applied to discuss the potential mechanism. Methods and Results: NCSCs were isolated from hair follicles of rats, and were obtained for differentiation. Cell viability, tyrosinase secretion and activity, and transcription factors were combined to evaluated the MC differentiation. RT-qPCR was applied to determine mRNA levels, and western blot were used for protein expression detection. Total m6A level was measured using methylated RNA immunoprecipitation (MeRIP) assay, and RNA immunoprecipitation was used to access the protein binding relationship. In current work, NCSCs were successfully differentiated into MCs. Fat mass and obesity associated gene (FTO) was aberrant downregulated in MCs, and elevated FTO suppressed the differentiation progress of NCSCs into MCs. Furthermore, microphthalmia-associated transcription factor (Mitf), a key gene involved in MC synthesis, was enriched by FTO in a m6A modification manner and degraded by FTO. Meanwhile, the suppression functions of FTO in the differentiation of NCSCs into MCs were reversed by elevated Mitf. Conclusions: In short, FTO suppressed the differentiating ability of hair follicle-derived NCSCs into MCs by m6A modifying Mitf.

• The Plant Pathology Journal
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• v.16 no.1
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• pp.43-47
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• 2000

Citrus tristeza virus (CTV) is the causal agent of one of the most important diseases of citrus. Recently, CTV has been detected in Cheju Island by ELISA. The coat protein (CP) gene of CTV isolated form Cheju Island was cloned by RT-PCR and the nucleotide was analyzed in this study. Citrus leaves were collected from trees showing decline symptoms from various region of Cheju Island in the summer of 1998 and 1999. The CP gene open reading frame is composed of 670 nucleotides and encodes a polypeptide of 223 amono acids. Sequence analysis the CP gene revealed that two CTV strains present in Cheju Island. Viruses collected form Sogwipo area and Cheju City area in 1999 ahowed 91-93% nucleotide sequence homology with CTV T36 strain. Viruses collected form Cheju City area in 1999 and Sogwipo City in 1998 showed 94-98% nucleotide sequence homology with CTV SY568 strain. A efficient viral RNA extraction methods was developed by modifying procedure for animal virus RNA purification methods and PCR product was detected form one tenth of RNA purified from as small as 45 mg fresh or frozen tissue.

• Journal of Applied Biological Chemistry
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• v.66
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• pp.128-137
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• 2023

Transcriptome analysis revealed that the sinR gene encoding a transition-state regulator of Bacillus pumilus, genetically close to B. subtilis, was expressed at high levels during growth. The sinR gene is the second gene of the sinIR operon consisting of three promoters and two structural genes in B. subtilis. This study used the sinIR promoter of B. subtilis DB104 to construct a recombinant protein expression system. First, the expression ability depending on the number of sinIR promoter was investigated using enhanced green fluorescent protein (eGFP). The expression level of eGFP was slightly higher when using two promoters (Psin2) than using original promoters. The Psin2 promoter was further engineered by modifying the repressor binding site and -35 and -10 regions. Shine-Dalgarno (SD) sequence of the sinI gene was modified to the consensus sequence. Finally, combining the engineered Psin2 promoter with the modified SD sequence increased the expression level of eGFP by about 13.4-fold over the original promoter. Our results suggest that the optimized sinIR promoter could be used as a novel tool for recombinant protein expression in B. subtilis.

• Biomedical Science Letters
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• v.18 no.1
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• pp.79-82
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• 2012

Acinetobacter infections are of great concern in clinical settings because of multi-drug resistance (MDR) and high mortality of the infected patients. The MDR Acinetobacter baumannii has emerged as a significant infectious agent in hospitals worldwide. The purpose of this study was to determine for molecular characterization of MDR A. baumannii clinical isolates obtained from the Wonju Christian Hospital in Gangwon province of Korea. A total of seventy nonduplicate A. baumannii isolates were collected from the Wonju Christian Hospital in Korea from March to April in 2011. All of the MDR A. baumannii isolates were encoded by $bla_{OXA-23-like}$ gene and all isolates with the $bla_{OXA-23-like}$ gene had the upstream element ISAba1 to promote increased gene expression and subsequent resistance to carbapenem. 16S rRNA methylase gene (armA) was detected in 44 clinical isolates which were resistant to amikacin, and phosphotransferase genes encoding aac(3)-Ia and aac(6')-Ib were the most prevalent. A combination of 16S rRNA methylase and aminoglycoside-modifying enzyme genes (armA, aac(3)-Ia, aac(6')-Ib, and aph(3')-Ia) were found in 31 isolates. The sequencing results for the quinolone resistance-determining region (QRDR) of gyrA and parC revealed the presence of Ser (TCA) 83 Leu (TTA) and Ser (TCG) 80 Leu (TTG) substitutions in the respective enzymes for all MDR. Molecular typing for MDR A. baumannii could be helpful in confirming the identification of a common source or cross-contamination. This is an important step in enabling epidemiological tracing of these strains.

• Journal of Life Science
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• v.9 no.1
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• pp.5-8
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• 1999

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and a potent immunogen. Deletion mutageneses were performed in the H. pylori urease accessory genes by using combinations of restriction enzymes and other DNA modifying enzymes in order to assess the function of these accessory gene products in the expression of the active urease. Selective disruptions in the accessory gene regions resulted in complete abolishment of the urease activity, which is consistent with other bacterial ureases. Interestingly, deletions in ureE-containing regions caused reduced expression of the structural enzyme subunits.

• Genes and Genomics
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• v.40 no.12
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• pp.1301-1308
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• 2018

Background Adipocyte differentiation is completed by changing gene expression. Chromatin is closely related to gene expression. Therefore, its structure might be changed for adipocyte differentiation. Mouse 3T3-L1 preadipocytes have been used as a cell model to study molecular mechanisms of adipogenesis. Objective To examine changes of chromatin modification and expression of histone modifying enzymes during adipocyte differentiation. Methods Microscopic analysis and Oil Red O staining were performed to determine distinct phenotype of adipocyte differentiation. RT-PCR and Western blot analysis were used to examine expression levels of histone modifying enzymes during adipocyte differentiation. Histone modifications were examined by immunostaining analysis. Results Expression levels of P300 and cbp were increased during adipocyte differentiation. However, acetylation of histones was not quantitatively changed postdifferentiation of 3T3-L1 cells compared to that at pre-differentiation. RT-PCR and Western blot analyses showed that expression levels of hdac2 and hdac3 were increased during adipocyte differentiation, suggesting histone acetylation at chromatin level was homeostatically controlled by increased expression of both HATs and HDACs. Tri-methylation level of H3K9 (H3K9me3), but not that of H3K27me3, was significantly decreased during adipocyte differentiation. Decreased expression of setdb1 was consistent with reduced pattern of H3K9me3. Knock-down of setdb1 induced adipocyte differentiation. This suggests that setdb1 is a key chromatin modifier that modulates repressive chromatin. Conclusion These results suggest that there exist extensive mechanisms of chromatin modifications for homeostatic balance of chromatin acetylation and deconstruction of repressive chromatin during adipocyte differentiation.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.651-661
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• 2019

Targeting the pathogen viability using drugs is associated with development of drug resistance due to selective pressure. Hence, there is an increased interest in developing agents that target bacterial virulence. In this study, the inhibitory effect of ciclopirox, an antifungal agent with iron chelation potential, on the microbial virulence factors was evaluated in 26 clinical MDR Pseudomonas aeruginosa isolates collected from Alexandria Main University Hospital, a tertiary hospital in Egypt. Treatment with 9 ㎍/ml ciclopirox inhibited the hemolytic activity in 70% isolates, reduced pyocyanin production, decreased protease secretion in 46% isolates, lowered twitching and swarming motility, and decreased biofilm formation by 1.5- to 4.5-fold. The quantitative real-time PCR analysis revealed that treatment with ciclopirox downregulated the expression levels of alkaline protease (aprA) and pyocyanin (phzA1). Ciclopirox is used to treat hematological malignancies and the systemic administration of ciclopirox is reported to have adequate oral absorption with a satisfactory drug safety profile. It is important to calculate the appropriate clinical dose and therapeutic index to reposition ciclopirox from a topical antifungal agent to a promising virulence-modifying agent agent against P. aeruginosa, a problematic Gram-negative pathogen.

• Journal of Periodontal and Implant Science
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• v.45 no.6
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• pp.223-228
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• 2015

Purpose: The goal of this study was to characterize the patterns of antimicrobial resistance and virulence genes in samples of Staphylococcus aureus (S. aureus) isolated from periodontitis patients. Methods: From July 2015 to August 2015, oral saliva was collected from a total of 112 patients diagnosed with periodontitis, including 80 outpatients in dental hospitals and 32 patients in dental clinics located in Seoul and Cheonan. The samples were subjected to a susceptibility test to evaluate the prevalence of antimicrobial resistance, and the pathogenic factors and antimicrobial resistance factors in the DNA of S. aureus were analyzed using polymerase chain reaction. Results: A susceptibility test against 15 antimicrobial agents showed that 88% of cultures were resistant to ampicillin, 88% to penicillin, and 2% to oxacillin. Resistance to at least two drugs was observed in 90% of cultures, and the most common pattern of multidrug resistance was to ampicillin and penicillin. Enterotoxins were detected in 65.9% of samples. The cell hemolysin gene hld was detected in 100% of cultures and hla was detected in 97.6% of samples. All strains resistant to penicillin and ampicillin had the blaZ gene. The aph(3')IIIa gene, which encodes an aminoglycoside modifying enzyme, was detected in 46.3% of samples. Conclusions: In the treatment of oral S. aureus infections, it is important to identify the pathogenic genes and the extent of antimicrobial resistance. Furthermore, it is necessary to study patterns of antimicrobial resistance and cross-infection in the context of periodontological specialties in which antimicrobials are frequently used, such as maxillofacial surgery, where the frequency of antimicrobial use for minor procedures such as implant placement is increasing.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.367-372
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• 2019

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'-dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ${\Delta}forP$ mutant (or ${\Delta}istP$ mutant strain) successfully restored the biosynthesis of 3',4'-dideoxy fortimicins and istamycins, thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

• Journal of Pharmacopuncture
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• v.22 no.4
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• pp.210-224
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• 2019

Rheumatoid arthritis is a severe autoimmune disorder, related to joints. It is associated with serious cartilage destruction. This causes disability and reduces the excellence of life. Numerous treatments are existed to combat this disease, however, they are not very efficient and possess severe side effects, higher doses, and frequent administration. Therefore, newer therapies are developed to overcome all these limitations. These include different monoclonal antibodies, immunoglobulins, small molecules used for immunotherapy and transgenes for gene therapy. One of the main goals of these new generation therapeutics is to address the underlying distressing biological processes by specifically targeting the causative agents with fewer systemic side effects and greater patient console. It is very fortuitous that loads of progressive investigations are going on in this field and many of them have entered into the successful clinical trial. But till date, a limited molecule has got FDA clearance and entered the market for treating this devastating disease. This review highlights the overview of conventional therapy and advancements in newer therapeutics including immunotherapy and gene therapy for rheumatoid arthritis. Further, different novel techniques for the delivery of these therapeutics of active and passive targeting are also described.