Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity

Lin Chih-Hsin,Hsieh Yu-Shao,Sun Ying-Chieh,Huang Wun-Han,Chen Shu-Ling,Weng Zheng-Kui,Lin Te-Hsien,Wu Yih-Ru,Chang Kuo-Hsuan,Huang Hei-Jen,Lee Guan-Chiun,Hsieh-Li Hsiu Mei,Lee-Chen Guey-Jen 한국응용약물학회 2023 Biomolecules & Therapeutics(구 응용약물학회지) Vol.31 No.1

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer’s disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine’s screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.

Mesenchymal Stem Cell Secreted-Extracellular Vesicles are Involved in Chondrocyte Production and Reduce Adipogenesis during Stem Cell Differentiation

Tsai Yu-Chen,Cheng Tai-Shan,Liao Hsiu-Jung,Chuang Ming-Hsi,Chen Hui-Ting,Chen Chun-Hung,Zhang Kai-Ling,Chang Chih-Hung,Lin Po-Cheng,Huang Chi-Ying F. 한국조직공학과 재생의학회 2022 조직공학과 재생의학 Vol.19 No.6

BACKGROUND: Extracellular vesicles (EVs) are derived from internal cellular compartments, and have potential as a diagnostic and therapeutic tool in degenerative disease associated with aging. Mesenchymal stem cells (MSCs) have become a promising tool for functional EVs production. This study investigated the efficacy of EVs and its effect on differentiation capacity. METHODS: The characteristics of MSCs were evaluated by flow cytometry and stem cell differentiation analysis, and a production mode of functional EVs was scaled from MSCs. The concentration and size of EVs were quantitated by Nanoparticle Tracking Analysis (NTA). Western blot analysis was used to assess the protein expression of exosomespecific markers. The effects of MSC-derived EVs were assessed by chondrogenic and adipogenic differentiation analyses and histological observation. RESULTS: The range of the particle size of adipose-derived stem cells (ADSCs)- and Wharton’s jelly -MSCs-derived EVs were from 130 to 150 nm as measured by NTA, which showed positive expression of exosomal markers. The chondrogenic induction ability was weakened in the absence of EVs in vitro. Interestingly, after EV administration, type II collagen, a major component in the cartilage extracellular matrix, was upregulated compared to the EV-free condition. Moreover, EVs decreased the lipid accumulation rate during adipogenic induction. CONCLUSION: The results indicated that the production model could facilitate production of effective EVs and further demonstrated the role of MSC-derived EVs in cell differentiation. MSC-derived EVs could be successfully used in cell-free therapy to guide chondrogenic differentiation of ADSC for future clinical applications in cartilage regeneration.

Combined Assessment of Serum Alpha-Synuclein and Rab35 is a Better Biomarker for Parkinson’s Disease

Hung-Li Wang,Chin-Song Lu,Tu-Hsueh Yeh,Yu-Ming Shen,Yi-Hsin Weng,Ying-Zu Huang,Rou-Shayn Chen,Yu-Chuan Liu,Yi-Chuan Cheng,Hsiu-Chen Chang,Ying-Ling Chen,Yu-Jie Chen,Yan-Wei Lin,Chia Chen Hsu,Huang-Li 대한신경과학회 2019 Journal of Clinical Neurology Vol.15 No.4

Background and Purpose It is essential to develop a reliable predictive serum biomarker for Parkinson’s disease (PD). Te accumulation of alpha-synuclein (αSyn) and up-regulated expression of Rab35 participate in the etiology of PD. Te purpose of this investigation was to determine whether the combined assessment of serum αSyn and Rab35 is a useful predictive biomarker for PD. Methods Serum levels of αSyn or Rab35 were determined in serum samples from 59 sporadic PD patients, 19 progressive supranuclear palsy (PSP) patients, 20 multiple system atrophy (MSA) patients, and 60 normal controls (NC). Receiver operating characteristics (ROC) curves were calculated to determine the diagnostic accuracy of αSyn or/and Rab35 in discriminating PD patients from NC or atypical parkinsonian patients. Results The levels of αSyn and Rab35 were increased in PD patients. The serum level of Rab35 was positively correlated with that of αSyn in PD patients. Compared to analyzing αSyn or Rab35 alone, the combined analysis of αSyn and Rab35 produced a larger area under the ROC curve and performed better in discriminating PD patients from NC, MSA patients, or PSP patients. When age was dichotomized at 55, 60, 65, or 70 years, the combined assessment of αSyn and Rab35 for classifying PD was better in the group below the cutof age than in the group above the cutof age. Conclusions Combined assessment of serum αSyn and Rab35 is a better biomarker for discriminating PD patients from NC or atypical parkinsonian patients, and is a useful predictive biomarker for younger sporadic PD patients.

Streptomyces Telomeres Contain a Promoter

Lin, Yuh-ru,Hahn, Mi-Young,Roe, Jung-Hye,Huang, Tzu-Wen,Tsai, Hsiu-Hui,Lin, Yung-Feng,Su, Tsung-Sheng,Chan, Yu-Jiun,Chen, Carton W. American Society for Microbiology 2009 Journal of Bacteriology Vol.191 No.3

<B>ABSTRACT</B><P>Bidirectional replication of the linear chromosomes and plasmids of <I>Streptomyces</I> spp. results in single-strand overhangs at their 3′ ends, which contain extensive complex palindromic sequences. The overhangs are believed to be patched by DNA synthesis primed by a terminal protein that remains covalently bound to the 5′ ends of the telomeres. We discovered that in vitro a conserved 167-bp telomere DNA binds strongly to RNA polymerase holoenzyme and exhibits promoter activities stronger than those of an rRNA operon. In vivo, the telomere DNA exhibited promoter activity in both orientations on a circular plasmid in <I>Streptomyces</I>. The telomere promoter is also active on a linear plasmid during exponential growth. Such promoter activity in a telomere has not hitherto been observed in eukaryotic or prokaryotic replicons. <I>Streptomyces</I> telomere promoters may be involved in priming the terminal Okazaki fragment (during replication) replicative transfer (during conjugation), or expression of downstream genes (including a conserved <I>ttrA</I> helicase-like gene involved in conjugal transfer). Interestingly, the <I>Streptomyces</I> telomeres also function as a promoter in <I>Escherichia coli</I> and as a transcription enhancer in yeast.</P>

Development of the Mandarin Phoneme Detection Score Sheet as a Screening Tool

Lin Chun-Yi,Hung Yu-Chen,Chang Hsiu-Wen 대한청각학회 2022 Journal of Audiology & Otology Vol.26 No.1

Background and Objectives: Hearing thresholds across frequencies must be obtained for hearing aid fitting. Narrow-band noise (NBN) and speech sounds are often used as stimuli in pediatric audiologic assessments to elicit children’s attention due to their wider frequency ranges as compared to pure tones. However, obtaining complete responses across frequency ranges is challenging in pediatric practice. Therefore, we developed a frequency-specific phoneme screening tool, the Mandarin Phoneme Detection Score Sheet, to help clinicians evaluate aided performance in pediatric practice.Subjects and Methods: A total of 30 adults with typical hearing and 30 children aged 3-12 years with hearing loss were recruited. Threshold ranges for the aided detection of Mandarin phonemes and NBN were measured using 95% confidence intervals. A stepwise regression analysis was then performed to identify the Mandarin phonemes that can predict NBN detection performance.Results: The Mandarin Phoneme Detection Score Sheet was developed based on the results of the regression analysis. It was shown that the phonemes /ɤ, a, tɕh/ could predict detection performance at different frequencies.Conclusions: The Mandarin Phoneme Detection Score Sheet can allow audiologists and early intervention professionals to determine the benefits of hearing aids for pediatric patients in the early stage of hearing loss conditions.

Efficiency of Hurdle Technology Applied to Raw Cured Meat (Si-Raw)Processing

Chen, Ming-Tsao,Lin, Young-Sun,Tsai, Hung-Tsung,Kuo, Hsiu-Lan Asian Australasian Association of Animal Productio 2002 Animal Bioscience Vol.15 No.11

Si-Raw is a raw cured meat (raw, cured meat fermented with steamed rice) produced by the aboriginal people of Taiwan. In order to prevent food poisoning or intoxication from botulism, new methods of monitoring the production base on hurdle technology were investigated. New methods investigated incorporated citric acid, sodium hypophosphite, Monascus anka mash, plum paste or lactic acid bacteria inoculum added separately to meat with steamed rice and salt to lower the Aw (water activity) and pH values of the products to control the microbial growth. Results showed that anaerobic bacterial counts, lactic acid bacterial counts and aerobic bacterial counts for the products of all treatments were less than $10^6$, $10^5$ and $10^2cfu/g$, respectively. Sodium chloride content of all products was above 5.46%, water activity was below 0.939 and pH value was below 4.27. IMP was lower and ATP and hypoxanthine were higher. ATP concentrations were higher in the samples which contained the anka mash. Result of sensory panel test indicated that most people preferred the products with added sodium hypophosphite. Except for the fact that the content of tryptamine in the sample with Monascus anka mash was higher, the amine concentrations for all treatments were lower than those of other fermented meat products. The amino acid nitrogen content was higher in the product made from raw meat treated with citric acid, but lower in the other products. Neither Clostridium botulinum nor Trichinella spiralis were detected in any of the treatments. The result may indicate that hurdle technology is effective for hygiene and safe producing Si-Raw.

Participation of GATA-3 in regulation of bone healing through transcriptional upregulation of bcl-xL expression

Mei-Hsiu Liao,Pei-I Lin,Wei-Pin Ho,Wing P Chan,Ta-Liang Chen,Ruei-Ming Chen 생화학분자생물학회 2017 Experimental and molecular medicine Vol.49 No.-

We have previously demonstrated the expression of GATA-DNA-binding protein (GATA)-3, a transcription factor, in osteoblasts and have verified its function in transducing cell survival signaling. This translational study was further designed to evaluate the roles of GATA-3 in regulating bone healing and to explore its possible mechanisms. A metaphyseal bone defect was created in the left femurs of male ICR mice. Analysis by micro-computed topography showed that the bone volume, trabecular bone number and trabecular thickness were augmented and that the trabecular pattern factor decreased. Interestingly, immunohistological analyses showed specific expression of GATA-3 in the defect area. In addition, colocalized expression of GATA-3 and alkaline phosphatase was observed at the wound site. As the fracture healed, the amounts of phosphorylated and non-phosphorylated GATA-3 concurrently increased. Separately, GATA-3 mRNA was induced during bone healing, and, levels of Runx2 mRNA and protein were also increased. The results of confocal microscopy and co-immunoprecipitation showed an association between nuclear GATA-3 and Runx2 in the area of insult. In parallel with fracture healing, Bcl-XL mRNA was significantly triggered. A bioinformatic search revealed the existence of a GATA-3-specific DNA-binding element in the promoter region of the bcl-xL gene. Analysis by chromatin immunoprecipitation assays further demonstrated transactivation activity by which GATA-3 regulated bcl-xL gene expression. Therefore, this study shows that GATA-3 participates in the healing of bone fractures via regulating bcl-xL gene expression, owing to its association with Runx2. In the clinic, GATA-3 may be used as a biomarker for diagnoses/prognoses or as a therapeutic target for bone diseases, such as bone fractures.

Relationship between Thermal Properties of Muscle Proteins and Pork Quality

Kuo, Hsiu-Lan,Chen, Ming-Tsao,Liu, Deng-Cheng,Lin, Lieh-Chin Asian Australasian Association of Animal Productio 2005 Animal Bioscience Vol.18 No.3

The purpose of this study was performed as model study using four animals to investigate the correction between the changes in Differential Scanning Calorimetry thermogram of muscle proteins during storage and meat freshness. M. longissimus dorsi of pork was obtained immediately after slaughter and chilled/stored at either $-2^{\circ}C$ or $25^{\circ}C$ for up to 96 h for analyses. DSC thermograms were determined and compared with pH values, ATP-related compounds, K-values, volatile basic nitrogen (VBN) levels, bacterial counts and electrophoretic behavior. Changes in pH, bacterial counts, VBN and K-values were associated with increased storage temperature and time. The levels of pH values, bacterial counts, VBN and K-values of pork samples stored at $25^{\circ}C$ were higher than those of the pork samples stored at $-2^{\circ}C$. ATP concentration decreased faster in samples stored at $25^{\circ}C$. Only IMP increased in samples stored at $-2^{\circ}C$, whereas the concentration of hypoxanthine and inosine increased in samples stored at $25^{\circ}C$. One exothermic peak and two endothermic peaks appeared on the thermograms of pork stored at either temperature. Lower transition temperature of myosin, sarcoplasmic protein and actin peaks were observed. The freshness parameters of K-value, VBN and hypoxanthine showed highly negative correlations (-0.742- -0.9980) to the changes in transition temperature. Therefore, the shift temperature on DSC thermogram can be used as an indicator of the freshness parameters of meat.

Current Indications for Spinal Endoscopic Surgery and Potential for Future Expansion

Kuo-Tai Chen,김진성,Abel Po-Hao Huang,Martin Hsiu-Chu Lin,Chien-Min Chen 대한척추신경외과학회 2023 Neurospine Vol.20 No.1

Endoscopic spine surgery (ESS) has evolved as a safe, effective, and efficient alternative for minimally invasive spine surgery (MISS). The innovation of full-endoscopic systems makes definitive decompression surgery through different approaches feasible. The approach can be determined according to the location of the target lesion or the surgeon's preference. During the past 2 decades, ESS has expanded its indications from lumbar to cervical spines. Except for decompression, endoscopy-assisted fusion surgery is also developing. However, ESS is still evolving and has a steep learning curve. The revolution of technologies and ESS techniques will enable surgeons to treat various spinal diseases more practically. In recent years, the application of the computer-assisted navigation system and augmented reality have reformed imaging quality and interpretation. The endoscopic rhizotomy techniques have opened a new way for MISS of chronic low back pain. This review introduces the current indications of ESS and its potential future expansion.

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages

Yuan-Shen Chen,Wei-Chu Chuang,Hsiu-Ni Kung,Ching-Yuan Cheng,Duen-Yi Huang,Ponarulselvam Sekar,Wan-Wan Lin 한국분자세포생물학회 2022 Molecules and cells Vol.45 No.4

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.