Direct reprogramming of fibroblasts into diverse lineage cells by DNA demethylation followed by differentiating cultures

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods.In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

Evaluating the Use of Distilled Water for Washing Sodium Hydroxide in Mycobacterial Culture / 대한임상미생물학회지

Background@#Respiratory specimens subjected to mycobacterial detection were initially pretreated with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) to remove the mucus and normal flora. Next, they were washed and neutralized with phosphate-buffered solution (PBS).The effectiveness of distilled water (DW) compared to PBS as a washing neutralizer during identification of mycobacteria was evaluated in this study. @*Methods@#We analyzed the results of mycobacterial test conducted at a general hospital in Gwangju from October 2016 to September 2018. PBS and DW were used as a respiratory sample washing agent for one year each. @*Results@#The positive culture rate for the culture of mycobacteria was 12.7% (1,843/14,532) and 14.7% (2,095/14,291), when PBS and DW were used, respectively. The recovery rate of the mycobacteria growth indicator tubes (MGIT) and the separation rates of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM) showed no significant change.However, in 2% Ogawa medium, as the NTM culture increased from 47.4% (399/841) to 56.1% (630/1,122), the recovery rate increased from 45.6% (841/1,843) to 53.6% (1,122/2,095). The MGIT contamination rate decreased from 6.5% to 4.1%. @*Conclusion@#DW as a washing agent for NALC-NaOH increased the recovery rate of Ogawa media and reduced the contamination rate of MGIT. Therefore, use of DW instead of PBS as a washing neutralizer during identification of mycobacteria might be useful.

Evaluating the Use of Distilled Water for Washing Sodium Hydroxide in Mycobacterial Culture / 대한임상미생물학회지

Background@#Respiratory specimens subjected to mycobacterial detection were initially pretreated with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) to remove the mucus and normal flora. Next, they were washed and neutralized with phosphate-buffered solution (PBS).The effectiveness of distilled water (DW) compared to PBS as a washing neutralizer during identification of mycobacteria was evaluated in this study. @*Methods@#We analyzed the results of mycobacterial test conducted at a general hospital in Gwangju from October 2016 to September 2018. PBS and DW were used as a respiratory sample washing agent for one year each. @*Results@#The positive culture rate for the culture of mycobacteria was 12.7% (1,843/14,532) and 14.7% (2,095/14,291), when PBS and DW were used, respectively. The recovery rate of the mycobacteria growth indicator tubes (MGIT) and the separation rates of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM) showed no significant change.However, in 2% Ogawa medium, as the NTM culture increased from 47.4% (399/841) to 56.1% (630/1,122), the recovery rate increased from 45.6% (841/1,843) to 53.6% (1,122/2,095). The MGIT contamination rate decreased from 6.5% to 4.1%. @*Conclusion@#DW as a washing agent for NALC-NaOH increased the recovery rate of Ogawa media and reduced the contamination rate of MGIT. Therefore, use of DW instead of PBS as a washing neutralizer during identification of mycobacteria might be useful.

Direct reprogramming of fibroblasts into diverse lineage cells by DNA demethylation followed by differentiating cultures

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods.In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

Expression and Localization of Keap1 During Amelogenesis in the Developing Molar Germ of Rats

The objective of this study was to examine the expression pattern of Kelch-like ECH-associated protein 1 (Keap1) in the maxillary 2(nd) molar germs of rats. We used the maxillary 2(nd) molar germs in rats' pup at postnatal day 3 (bell stage), 6 (crown formation stage) and 9 (root formation stage). The investigation on mRNA and protein levels were done using reverse transcription - polymerase chain reaction and western blot. Localization of Keap 1 in the maxillary 2(nd) molar germs were revealed through immunofluorescence staining. Keap1 from the maxillary 2nd molar germs were mostly manifested on postnatal day 3 and dramatically decreased on postnatal day 6 and 9 at mRNA and protein levels, while amelogenin and ameloblastin increased during the development of maxillary 2nd molar germs. During immunofluorescence analysis, the strong immunoreactivity against Keap1 was detected in the apical side of ameloblasts at the presecretory and secretory stages. However, Keap1 expression was hardly observed in the ameloblasts at the maturation stage. These results shows that Keap1 is strongly expressed in the presecretory and secretory ameloblasts of amelogenesis, and suggest that Keap1 may be a crucial molecule for the regulatory mechanisms tasked with the formation of enamel layer.

Epigallocatechin-3-gallate rescues LPS-impaired adult hippocampal neurogenesis through suppressing the TLR4-NF-kappaB signaling pathway in mice

Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activatingTLR4-NF-kappaB signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-kappaB pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPSinduced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation.

Clinical Characteristics of Streptococcus agalactiae Bacteremia in Adults Living in Jeju Island / 대한임상미생물학회지

BACKGROUND: Streptococcus agalactiae (Group B streptococcus, GBS) is known to be the leading cause of neonatal sepsis and meningitis in the United States and Europe. In addition, GBS infection has been increasingly noted in adults, particularly in those with underlying diseases, such as diabetes mellitus, malignancy and liver disease. A few studies reported that resistances to antibiotics, such as erythromycin, clindamycin, tetracycline are increasing. We report clinical and microbiological characteristics of GBS bacteremic patients in Jeju Island. METHODS: We retrospectively analyzed medical records, such as age, sex, underlying disease, mortality, skin defects, laboratory results and antibiotic resistances of GBS in hospitalized adult patients who were diagnosed with GBS bacteremia from 2008 to 2013 in Jeju Island. RESULTS: Twenty two adult patients were diagnosed as GBS bacteremia from 2008 to 2013. The mean age of GBS bacteremic patients was 66.2 years old. Of 22 bacteremic patients, fifteen patients (68%) were older than 60. Twenty patients (91%) of bacteremic patients had underlying diseases such as diabetes mellitus, malignancy and liver disease. Ten (45%) patients had skin defects which were on the lower extremities and buttock, fifteen (68%) patients had fever at the time of admission, twenty one (95%) patients were admitted via the emergency department. Two (9%) patients died. The mean white blood cell (WBC) count, percentile of neutrophil count, and C-reactive protein (CRP) levels were 11,488/microL, 84.3 %, 13.5 mg/dL respectively. All GBS isolates from bacteremia showed sensitivities to penicillin, ampicillin, and vancomycin, and showed resistances to erythromycin (25%), clindamycin (30%), and tetracycline (55%). CONCLUSION: Bacteremia caused by GBS was prevalent in adult patients with underlying diseases. Most of the GBS bacteremic patients were emergency cases, with a high body temperature, WBC, CRP level, and neutrophil count. Half of them had skin defects, which are considered a source of GBS bacteremia.

Expression of Tissue Inhibitors of Metalloproteinases in Developing Rat Tooth Germs / 체질인류학회지

Tissue inhibitors of metalloproteinases (TIMPs) are a family of secreted molecules that were identified as natural inhibitors of matrix metalloproteinases (MMPs). Tooth histomorphogenesis and cytodifferentiation are accompanied by rapid changes in cellular organization and remodeling of the extracellular matrix, in which MMPs and TIMPs might be expected to play significant roles. This study examined the expression and localization of TIMP-1 and TIMP-2 during the molar development of rats. The expression patterns of TIMPs were determined from Sprague-Dawley rat pups including the developing molars using RT-PCR, western blot and immunofluorescent staining. Gene and protein quantification analyses showed that both TIMPs increased from the cap stage to the root stage tooth germs. In contrast, the immunofluorescent data showed that they were expressed slight differentially. TIMP-1 was strongly expressed in secretory ameloblasts and moderate immunoreactivity was observed along the basement membrane. TIMP-2 expression was also detected in the basement membrane. Although strong immunoreactivity was observed in the secretory ameloblasts and enamel matrix itself, differentiated odontoblasts showed weak reactivity. However, little reactivity for both TIMPs were detected in the cap stage tooth germs and surrounding tissues. These distinct temporospatial expression patterns of TIMPs suggest that the TIMPs may play a variety of roles including dental hard tissue formation during molar tooth development.

Role of Nuclear Factor (NF)-kappaB Activation in Tumor Growth and Metastasis

BACKGROUND: Platelet-activating factor (PAF) induces nuclear factor (NF)-kappaB activation and angiogenesis and increases tumor growth and pulmonary tumor metastasis in vivo. The role of NF-kappaB activation in PAF-induced angiogenesis in a mouse model of Matrigel implantation, and in PAF-mediated pulmonary tumor metastasis were investigated. METHODS: Angiogenesis using Matrigel and experimental pulmonary tumor metastasis were tested in a mouse model. Electrophoretic mobility shift assay was done for the assessment of NF-kappaB translocation to the nucleus. Expression of angiogenic factors, such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were tested by RT-PCR and ELISA. RESULTS: PAF induced a dose- and time-dependent angiogenic response. PAF-induced angiogenesis was significantly blocked by PAF antagonist, CV6209, and inhibitors of NF-kappaB expression or action, including antisense oligonucleotides to p65 subunit of NF-kappaB (p65 AS) and antioxidants such as alpha-tocopherol and N-acetyl-L-cysteine. In vitro, PAF activated the transcription factor, NF-kappaB and induced mRNA expression of TNF-alpha, IL-1alpha, bFGF, VEGF, and its receptor, KDR. The PAF-induced expression of the above mentioned factors was inhibited by p65 AS or antioxidants. Also, protein synthesis of VEGF was increased by PAF and inhibited by p65 AS or antioxidants. The angiogenic effect of PAF was blocked when anti-VEGF antibodies was treated or antibodies against TNF-alpha, IL-1alpha, and bFGF was co-administrated, but not by antibodies against TNF-alpha, IL-1alpha, and bFGF each alone. PAF-augmented pulmonary tumor metastasis was inhibited by p65 AS or antioxidants. CONCLUSION: These data indicate that PAF increases angiogenesis and pulmonary tumor metastasis through NF-kappaB activation and expression of NF-kappaB-dependent angiogenic factors.

Regulation of Endotoxin - Induced TNF-alpha Gene Expression

It is well known that tumor necrosis factor (TNF-a), interleukin-1, platelet-activating factor (PAF) and arachidonic acid metabolites, such as thromboxane and leukotriens, are major mediators involved in the pathogenesis of endotoxic shock. In this study, we have investigated the effect of pentoxifylline (inhibitor of TNF-a release), BN50739 (PAF antagonist), indomethacin (cyclooxygenase inhibitor) and diethylcarbamazine (lipoxygenase inhibitor) on LPS- induced lethality as well as the relationship between major mediators in endotoxic shock. All inhibitors described above except diethylcarbamazine significantly protected mice against LPS- induced lethality. BN50739 and indomethacin were also effective in protection of TNF-a-induced lethality. The elevation of circulating TNF-a by LPS was significantly blocked by BN50739, but not affected by indomethacin. Convulsion appeared shortly after LPS injection was prevented by BN50739 but not by indomethacin, whereas diarrhea and limited movement was prevented by indomethacin but not by BN50739. These results indicate that i) TNF-a, PAF and cyclooxygenase products are important mediators involved in the pathogenesis of septic shock and ii) TNF-a directly influenced the release or production of PAF as well as cyclooxygenase products, and strongly suggest that i) TNF-a and PAF stimulate the release of each other via positive feedback network but TNF-a and cyclooxygenase products do not form the network and ii) PAF and cyclooxygenase product appear not to affect the release of each other.