Publications by authors named "Yu-Chen Hu"

152 Publications

A Systematic Review of Artificial Intelligence Techniques in Cancer Prediction and Diagnosis.

Arch Comput Methods Eng 2021 Sep 27:1-28. Epub 2021 Sep 27.

Department of Computer Science and Information Management, Providence University, Taichung City, Taiwan, ROC.

Artificial intelligence has aided in the advancement of healthcare research. The availability of open-source healthcare statistics has prompted researchers to create applications that aid cancer detection and prognosis. Deep learning and machine learning models provide a reliable, rapid, and effective solution to deal with such challenging diseases in these circumstances. PRISMA guidelines had been used to select the articles published on the web of science, EBSCO, and EMBASE between 2009 and 2021. In this study, we performed an efficient search and included the research articles that employed AI-based learning approaches for cancer prediction. A total of 185 papers are considered impactful for cancer prediction using conventional machine and deep learning-based classifications. In addition, the survey also deliberated the work done by the different researchers and highlighted the limitations of the existing literature, and performed the comparison using various parameters such as prediction rate, accuracy, sensitivity, specificity, dice score, detection rate, area undercover, precision, recall, and F1-score. Five investigations have been designed, and solutions to those were explored. Although multiple techniques recommended in the literature have achieved great prediction results, still cancer mortality has not been reduced. Thus, more extensive research to deal with the challenges in the area of cancer prediction is required.
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http://dx.doi.org/10.1007/s11831-021-09648-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8475374PMC
September 2021

Polyplex nanomicelle delivery of self-amplifying RNA vaccine.

J Control Release 2021 Oct 10;338:694-704. Epub 2021 Sep 10.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan; Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu 300, Taiwan. Electronic address:

Self-amplifying RNA (SaRNA) is a burgeoning platform that exploits the replication machinery of alphaviruses such as Venezuelan equine encephalitis (VEE) virus or Sindbis virus (SIN). SaRNA has been used for development of human vaccines, but has not been evaluated for porcine vaccine development. Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the worldwide pork industry, but current vaccines trigger delayed neutralizing antibody response and confer only partial protection. Here we first compared two SaRNA systems based on VEE and SIN, and demonstrated that in vitro transcribed VEE-based SaRNA conferred prolonged reporter gene expression and RNA amplification in pig cells with low cytotoxicity, but SIN-based SaRNA imparted evident cytotoxicity and limited gene expression in pig cells. Transfection of VEE-based SaRNA that encodes the major PRRSV antigen dNGP5 (SaRNA-dNGP5) conferred persistent expression for at least 28 days in pig cells. We next complexed SaRNA-dNGP5 with the polyaspartamide block copolymer PEG-PAsp(TEP) to form polyplex nanomicelle with high packaging efficiency and narrow size distribution. The polyplex nanomicelle enabled sustained dNGP5 expression and secretion in vitro. Compared with the commercial PRRS vaccine, nanomicelle delivery of SaRNA-dNGP5 into animal models accelerated the induction of potent neutralizing antibodies with minimal side effects, and elicited stronger IL-4 and IFN-γ responses against homologous and heterologous PRRSV. These properties tackle the problems of current vaccines and implicate the potential of SaRNA-dNGP5 nanomicelle as an effective PRRS vaccine.
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http://dx.doi.org/10.1016/j.jconrel.2021.09.008DOI Listing
October 2021

Bi-directional gene activation and repression promote ASC differentiation and enhance bone healing in osteoporotic rats.

Mol Ther 2021 Aug 25. Epub 2021 Aug 25.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan; Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Calvarial bone healing is challenging, especially for individuals with osteoporosis because stem cells from osteoporotic patients are highly prone to adipogenic differentiation. Based on previous findings that chondrogenic induction of adipose-derived stem cells (ASCs) can augment calvarial bone healing, we hypothesized that activating chondroinductive Sox Trio genes (Sox5, Sox6, Sox9) and repressing adipoinductive genes (C/ebp-α, Ppar-γ) in osteoporotic ASCs can reprogram cell differentiation and improve calvarial bone healing after implantation. However, simultaneous gene activation and repression in ASCs is difficult. To tackle this problem, we built a CRISPR-BiD system for bi-directional gene regulation. Specifically, we built a CRISPR-AceTran system that exploited both histone acetylation and transcription activation for synergistic Sox Trio activation. We also developed a CRISPR interference (CRISPRi) system that exploited DNA methylation for repression of adipoinductive genes. We combined CRISPR-AceTran and CRISPRi to form the CRISPR-BiD system, which harnessed three mechanisms (transcription activation, histone acetylation, and DNA methylation). After delivery into osteoporotic rat ASCs, CRISPR-BiD significantly enhanced chondrogenesis and in vitro cartilage formation. Implantation of the engineered osteoporotic ASCs into critical-sized calvarial bone defects significantly improved bone healing in osteoporotic rats. These results implicated the potential of the CRISPR-BiD system for bi-directional regulation of cell fate and regenerative medicine.
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http://dx.doi.org/10.1016/j.ymthe.2021.08.024DOI Listing
August 2021

S-Equol Protects Chondrocytes against Sodium Nitroprusside-Caused Matrix Loss and Apoptosis through Activating PIK/Akt Pathway.

Int J Mol Sci 2021 Jun 30;22(13). Epub 2021 Jun 30.

Department of Biochemistry, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.

Osteoarthritis (OA) is a common chronic disease with increasing prevalence in societies with more aging populations, therefore, it is causing more concern. S-Equol, a kind of isoflavones, was reported to be bioavailable and beneficial to humans in many aspects, such as improving menopausal symptoms, osteoporosis and prevention of cardiovascular disease. This study investigated the effects of S-Equol on OA progress in which rat primary chondrocytes were treated with sodium nitroprusside (SNP) to mimic OA progress with or without the co-addition of S-Equol for the evaluation of S-Equol's efficacy on OA. Results showed treatment of 0.8 mM SNP caused cell death, and increased oxidative stress (NO and HO), apoptosis, and proteoglycan loss. Furthermore, the expressions of MMPs of MMP-2, MMP-3, MMP-9, and MMP-13 and p53 were increased. The addition of 30 μM S-Equol could lessen those caused by SNP. Moreover, S-Equol activates the PIK/Akt pathway, which is an upstream regulation of p53 and NO production and is associated with apoptosis and matrix degradation. As a pretreatment of phosphoinositide -kinases (PIK) inhibitor, all S-Equol protective functions against SNP decrease or disappear. In conclusion, through PIK/Akt activation, S-Equol can protect chondrocytes against SNP-induced matrix degradation and apoptosis, which are commonly found in OA, suggesting S-Equol is a potential for OA prevention.
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http://dx.doi.org/10.3390/ijms22137054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268809PMC
June 2021

3-year follow-up of half-dose verteporfin photodynamic therapy for central serous chorioretinopathy with OCT-angiography detected choroidal neovascularization.

Sci Rep 2021 06 24;11(1):13286. Epub 2021 Jun 24.

Department of Ophthalmology, Changhua Christian Hospital, Changhua, Taiwan, ROC.

To assess the 3-year outcome of half-dose verteporfin photodynamic therapy (PDT) in central serous chorioretinopathy (CSC) with optical coherence tomography angiography (OCT-A) detected choroidal neovascularization (CNV), we performed a retrospective, interventional study. Patients were divided into 2 groups according to the fluorescein angiography: point source leakage in group 1 and diffuse oozing in group 2. Data were collected from patients including changes of best-corrected visual acuity (BCVA), size of CNV, central macular thickness (CMT), choroidal thickness (CT), reabsorption of subretinal fluid (SRF), sessions of half-dose PDT, and the number of intravitreal injections (IVI) of anti-vascular endothelial growth factor (anti-VEGF). There was a total of 34 eyes in 32 patients included. The mean sessions of half-dose PDT was 1.50 ± 0.75. The mean number of IVI of anti-VEGF was 1.38 ± 3.34. BCVA improved from 0.38 ± 0.33 to 0.20 ± 0.22 (p < 0.001). Mean CMT was significantly reduced along with reduced CT and increased size of CNV. SRF was totally reabsorbed in 31 eyes. Patients in group 1 had significant less sessions of PDT and better final BCVA. In conclusion, half-dose PDT treatment was effective for CSC with CNV. Patients with diffuse oozing in FA may fare less well with half-dose PDT.
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http://dx.doi.org/10.1038/s41598-021-92693-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225805PMC
June 2021

CRISPR activation of long non-coding RNA DANCR promotes bone regeneration.

Biomaterials 2021 08 11;275:120965. Epub 2021 Jun 11.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 30013, Taiwan; Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu 30013, Taiwan. Electronic address:

Healing of large calvarial bone defects in adults adopts intramembranous pathway and is difficult. Implantation of adipose-derived stem cells (ASC) that differentiate towards chondrogenic lineage can switch the bone repair pathway and improve calvarial bone healing. Long non-coding RNA DANCR was recently uncovered to promote chondrogenesis, but its roles in rat ASC (rASC) chondrogenesis and bone healing stimulation have yet to be explored. Here we first verified that DANCR expression promoted rASC chondrogenesis, thus we harnessed CRISPR activation (CRISPRa) technology to upregulate endogenous DANCR, stimulate rASC chondrogenesis and improve calvarial bone healing in rats. We generated 4 different dCas9-VPR orthologues by fusing a tripartite transcription activator domain VPR to catalytically dead Cas9 (dCas9) derived from 4 different bacteria, and compared the degree of activation using the 4 different dCas9-VPR. We unveiled surprisingly that the most commonly used dCas9-VPR derived from Streptococcus pyogenes barely activated DANCR. Nonetheless dCas9-VPR from Staphylococcus aureus (SadCas9-VPR) triggered efficient activation of DANCR in rASC. Delivery of SadCas9-VPR and the associated guide RNA into rASC substantially enhanced chondrogenic differentiation of rASC and augmented cartilage formation in vitro. Implantation of the engineered rASC remarkably potentiated the calvarial bone healing in rats. Furthermore, we identified that DANCR improved the rASC chondrogenesis through inhibition of miR-203a and miR-214. These results collectively proved that DANCR activation by SadCas9-VPR-based CRISPRa provides a novel therapeutic approach to improving calvarial bone healing.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120965DOI Listing
August 2021

Treatment experience in a HIV-infected patient with Vogt-Koyanagi-Harada disease- a case report.

Ocul Immunol Inflamm 2021 Jun 10:1-5. Epub 2021 Jun 10.

Department of Ophthalmology, Changhua Christian Hospital, Changhua City, Taiwan.

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http://dx.doi.org/10.1080/09273948.2021.1900277DOI Listing
June 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Intracerebral Hemorrhage in Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy: Prevalence, Clinical and Neuroimaging Features and Risk Factors.

Stroke 2021 Mar 4;52(3):985-993. Epub 2021 Feb 4.

Department of Neurology, Taipei Veterans General Hospital, Taiwan (Y.-C. Liao., Y.-C.H., C.-P.C., Y.-F.W., Y.-C. Lee.).

Background And Purpose: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic cerebral small vessel disease. The role of intracerebral hemorrhage (ICH) in CADASIL remains elusive. The present study aims to investigate the prevalence, characteristics, and risk factors for ICH in CADASIL.

Methods: This retrospective cross-sectional study investigated ICH and cerebral microbleeds (CMBs) in brain susceptibility-weighted imaging or T2*-weighted gradient-recalled echo images of 127 Taiwanese patients with genetically confirmed CADASIL. We analyzed CMBs, lacunes, white matter hyperintensity, and perivascular space. The total small vessel disease score (range, 0-4) was calculated to estimate the overall magnetic resonance imaging burden of small vessel disease. Multivariate regression analysis was performed to identify factors related to ICH lesions in CADASIL.

Results: Thirty-seven ICH lesions, including 15 symptomatic and 22 asymptomatic lesions, were found in 27 (21.3% [95% CI, 14.0%-30.9%]) of the 127 patients with CADASIL. The thalamus and lobar regions were the most common ICH locations, and 72.7% of the lobar hemorrhages occurred silently. Patients with CADASIL with ICH lesions more often had hypertension and a higher total small vessel disease score than those without ICH (odds ratio [95% CI]: 3.22 [1.25-8.30] and 3.79 [1.51-9.51]). The presence of CMBs in the brain stem and a total CMB count >10 were independently associated with ICH lesions in patients with CADASIL, with odds ratio (95% CI) of 5.82 (1.80-18.80) and 3.83 (1.08-13.67), respectively.

Conclusions: ICH is an underestimated but important manifestation of CADASIL. The location and number of CMBs are associated with the presence of ICH lesions in patients with CADASIL.
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http://dx.doi.org/10.1161/STROKEAHA.120.030664DOI Listing
March 2021

CRISPR-Cas13d for Gene Knockdown and Engineering of CHO Cells.

ACS Synth Biol 2020 10 21;9(10):2808-2818. Epub 2020 Sep 21.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30003, Taiwan.

Chinese hamster ovary (CHO) cells are the predominant cell chassis for biopharmaceutical production. Engineering cellular pathways related to cell death, metabolism, and glycosylation in CHO cells is desired but challenging. Here, we present a novel approach that exploits CRISPR-Cas13d for gene silencing and CHO cell engineering. CRISPR-Cas13d is a burgeoning system that exploits Cas13d nuclease and guide RNA (gRNA) for RNA cleavage and gene knockdown. We first showed that CRISPR-Cas13d effectively knocked down exogenous genes in CHO cell lines (K1, DG44, and DUXB11) commonly used for recombinant protein production. We next demonstrated that CRISPR-Cas13d robustly suppressed the expression of exogenous genes and various endogenous genes involved in gene amplification, apoptosis, metabolism, and glycosylation (.., GS, BAK, BAX, PDK1, and FUT8) in CHO cells with efficiencies ranging from 60% to 80%, simply by transient transfection. By integrating the entire CRISPR-Cas13d system with the system and optimal gRNA design, we further improved the knockdown efficiency and rapidly generated stable cells with ≈80%-90% knockdown. With this approach, we knocked down FUT8 expression for >90% and significantly attenuated the IgG fucosylation. These data altogether implicated the potentials of CRISPR-Cas13d for gene regulation, glycoengineering, and cell engineering of CHO cells.
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http://dx.doi.org/10.1021/acssynbio.0c00338DOI Listing
October 2020

Enterovirus Inhibition by Hinged Aromatic Compounds with Polynuclei.

Molecules 2020 Aug 22;25(17). Epub 2020 Aug 22.

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium.

The modern world has no available drugs for the treatment of enteroviruses (EV), which affect millions of people worldwide each year. The EV71 is a major causative disease for hand, foot, and mouth disease; sometimes it is associated with severe central nervous system diseases. Treatment for enteroviral infection is mainly supportive; treatment for aseptic meningitis caused by enteroviruses is also generally symptomatic. Upon the urgent request of new anti-enterovirus drugs, a series of hinged aromatic compounds with polynulei were synthesized through two different chemical pathways. Among these morpholine-furan/thiophene/pyrrole-benzene-pyrazole conjugates, three new agents exhibited inhibitory activity with EC = 2.29-6.16 μM toward EV71 strain BrCr in RD cells. Their selectivity index values were reached as high as 33.4. Their structure-activity relationship was deduced that a thiophene derivative with morpholine and trifluorobenzene rings showed the greatest antiviral activity, with EC = 2.29 μM.
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http://dx.doi.org/10.3390/molecules25173821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503712PMC
August 2020

Domino Reaction for the Synthesis of Polysubstituted Pyrroles and Lamellarin R.

J Org Chem 2020 08 17;85(15):9835-9843. Epub 2020 Jul 17.

Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan.

A three-component annulation reaction was developed for the synthesis of pyrroles, a class of compounds with various properties valuable to biomedical and polymer industries. Treatment of α-silylaryl triflates, Schiff bases, and alkynes generated polysubstituted pyrroles in good yields (61-86%) with regioselectivity. This domino reaction involved completion of five sequential steps in a single flask, which comprised aryne formation through 1,2-elimination, their alkylation by Schiff bases through 1,2-addition, 1,4-intramolecular proton transfer, Hüisgen 1,3-dipolar cycloaddition, and dehydrogenative aromatization. It was then successfully applied as the key step in the synthesis of the natural product lamellarin R. This new reaction represents an efficient, sustainable process for the production of chemical materials.
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http://dx.doi.org/10.1021/acs.joc.0c01134DOI Listing
August 2020

Asian Congress on Biotechnology 2019.

Biotechnol J 2020 06;15(6):e2000214

Department of Chemical Engineering, National Tsing Hua University, 101, Sec. 2, Kuang Fu Rd., Hsinchu, 30013, Taiwan.

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http://dx.doi.org/10.1002/biot.202000214DOI Listing
June 2020

CRISPR interference-mediated noggin knockdown promotes BMP2-induced osteogenesis and calvarial bone healing.

Biomaterials 2020 09 10;252:120094. Epub 2020 May 10.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan; Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Healing of large calvarial bone defects remains a challenging task in the clinical setting. Although BMP2 (bone morphogenetic protein 2) is a potent growth factor that can induce bone repair, BMP2 provokes the expression of antagonist Noggin that self-restricts its bioactivity. CRISPR interference (CRISPRi) is a technology for programmable gene suppression but its application in regenerative medicine is still in its infancy. We reasoned that Nog inhibition, concurrent with BMP2 overexpression, can promote the osteogenesis of adipose-derived stem cells (ASC) and improve calvarial bone healing. We designed and exploited a hybrid baculovirus (BV) system for the delivery of BMP2 gene and CRISPRi system targeting Nog. After BV-mediated co-delivery into ASC, the system conferred prolonged BMP2 expression and stimulated Nog expression while the CRISPRi system effectively repressed Nog upregulation for at least 14 days. The CRISPRi-mediated Nog knockdown, along with BMP2 overexpression, additively stimulated the osteogenic differentiation of ASC. Implantation of the CRISPRi-engineered ASC into the critical size defects at the calvaria significantly enhanced the calvarial bone healing and matrix mineralization. These data altogether implicate the potentials of CRISPRi-mediated gene knockdown for cell fate regulation and tissue regeneration.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120094DOI Listing
September 2020

Engineering Stable S12 by CRISPR for 2,5-Furandicarboxylic Acid (FDCA) Production.

ACS Synth Biol 2020 05 28;9(5):1138-1149. Epub 2020 Apr 28.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.

FDCA (2,5-furandicarboxylic acid) can be enzymatically converted from HMF (5-hydroxymethylfurfural). S12 is promising for FDCA production, but generating stable S12 is difficult due to its polyploidy and lack of genome engineering tools. Here we showed that coupling CRISPR and λ-Red recombineering enabled one-step gene integration with high efficiency and frequency, and simultaneously replaced endogenous genes in all chromosomes. Using this approach, we generated two stable S12 strains expressing HMF/furfural oxidoreductase (HMFH) and HMF oxidase (HMFO), both being able to convert 50 mM HMF to ≈42-43 mM FDCA in 24 h. Cosupplementation of MnO and CaCO to the medium drastically improved the cell tolerance to HMF and enhanced FDCA production. Cointegrating and (HMF transporter) genes further improved FDCA production, enabling the cells to convert 250 mM HMF to 196 mM (30.6 g/L) FDCA in 24 h. This study implicates the potentials of CRISPR for generating stable S12 strains for FDCA production.
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http://dx.doi.org/10.1021/acssynbio.0c00006DOI Listing
May 2020

Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration.

Mol Ther 2020 02 6;28(2):441-451. Epub 2019 Dec 6.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan; Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which are governed by the Wnt (Wingless-related integration site) signaling cascade. To promote BMSC differentiation toward osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers the canonical Wnt pathway) and forkhead c2 (Foxc2) (that elicits the noncanonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14 days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis in vitro. Furthermore, the robust and prolonged coactivation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.
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http://dx.doi.org/10.1016/j.ymthe.2019.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001053PMC
February 2020

[Iatrogenic injury caused by manual reduction of anterior dislocation of shoulder and humeral head compression: a case report].

Zhongguo Gu Shang 2019 09;32(9):866-868

Department of the Second Hospital of Jilin University, Changchun 130041, Jilin, China;

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http://dx.doi.org/10.3969/j.issn.1003-0034.2019.09.018DOI Listing
September 2019

High Expression of MicroRNA-196a is Associated with Progression of Hepatocellular Carcinoma in Younger Patients.

Cancers (Basel) 2019 Oct 13;11(10). Epub 2019 Oct 13.

Institute of Clinical Medicine, National Yang-Ming University, Taipei 11221, Taiwan.

MicroRNAs are small RNAs involved in various biological processes and cancer metastasis. miR-196a was associated with aggressive behaviors in several cancers. The role of miR-196a in hepatocellular carcinoma (HCC) metastasis remains unknown. This study aimed to examine the role of miR-196a in HCC progression. Expression of miR-196a was measured in 83 human HCC samples. The HCC patients with high miR-196a expression had younger ages, lower albumin levels, higher frequency with alpha-fetoprotein (AFP) levels ≥20 ng/mL, more macrovascular invasion, and non-early stages. Kaplan-Meier analysis showed that high miR-196a expression was associated with lower recurrence-free survival. Knockdown of miR-196a decreased transwell invasiveness, sphere formation, transendothelial invasion, and Slug, Twist, Oct4, and Sox2 expression, suppressed angiogenesis, and reduced sizes of xenotransplants and number of pulmonary metastasis. Down-regulation of miR-196a decreased Runx2 and osteopontin (OPN) levels. Knockdown of Runx2 in vitro resulted in comparable phenotypes with miR-196a down-regulation. Restoration of Runx2 in miR-196a-knockdown HCC reverted tumor phenotypes. This study showed that high expression of miR-196a is associated with HCC progression in a subset of younger patients. miR-196a mediates HCC progression via upregulation of Runx2, OPN, epithelial-mesenchymal transition (EMT) regulators, and stemness genes. We proposed that miR-196a can be used as a prognostic marker and a potential therapeutic target.
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http://dx.doi.org/10.3390/cancers11101549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826650PMC
October 2019

Zinc protects chondrocytes from monosodium iodoacetate-induced damage by enhancing ATP and mitophagy.

Biochem Biophys Res Commun 2020 01 11;521(1):50-56. Epub 2019 Oct 11.

Department of Biochemistry, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan. Electronic address:

Osteoarthritis (OA) is characterized with articular cartilage degradation, and monosodium iodoacetate (MIA)-treated chondrocyte is the most commonly used model for mimicking OA progression. Zinc protects chondrocytes from MIA-induced damage. Here, we explored the protective effects of 25 μM zinc on 5 μM MIA-treated SW1353 cells (human chondrosarcoma cell line) through the analysis of energy metabolism- and autophagy-related parameters. We found that the exposure of SW1353 cells to MIA decreased ATP levels, expression of glycolysis-related proteins, including glucose transporter 1, hexokinase 2, and pyruvate dehydrogenase E1 component subunit alpha, and the levels of mitochondrial complex I, II, IV, and V subunits of the oxidative phosphorylation pathway. MIA treatment also decreased the expression of autophagy-related proteins, including autophagic elongation protein 5 (ATG5), ATG7, and microtubule-associated protein 1A/1B light chain 3B (LC3-II) and mitophagy-related proteins, including phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), ubiquitin, and p62. These results indicate that MIA interferes with energy metabolism and the autophagic clearance of dysfunctional mitochondria (so called mitophagy). Interestingly, zinc exposure could almost completely reverse the effects of MIA, suggesting its potential protective role against OA progression.
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http://dx.doi.org/10.1016/j.bbrc.2019.10.066DOI Listing
January 2020

Multi-walled carbon-nanotube-decorated tungsten ditelluride nanostars as anode material for lithium-ion batteries.

Nanotechnology 2020 Jan 27;31(3):035406. Epub 2019 Sep 27.

Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013 Taiwan.

Multi-walled carbon-nanotube (MWCNT)-decorated WTe nanostars ([email protected] nanocomposites) are to be employed for the first time as anode candidates in the development of lithium-ion (Li-ion) batteries. [email protected] nanocomposites deliver a high discharge capacity of 1097, 475, 439, 408, 395 and 381 mA h g with an increasing current density of 100, 200, 400, 600, 800 and 1000 mA g, respectively, while WTe nanostars exhibit a reversible capacity of 655, 402, 400, 362, 290 and 197 mA h g with the aforementioned current densities. Furthermore, [email protected] nanocomposites exhibit a superior reversible capacity of 592 mA h g at 500 mA g with a capacity retention of 100% achieved over 500 cycles, while bare WTe nanostars deliver ∼85 mA h g over 350 cycles. This remarkable Li cycling performance is attributed to MWCNTs interconnected with WTe nanostars. In addition, the exposed active interlayers of the WTe nanostars, which are responsible for maintaining the structural integrity of the electrodes, buffer the large volume expansion within the WTe nanostars, avoiding the agglomeration of the particles. The layered WTe nanostars were synthesized via the solution-phase method, and present extremely good possibilities for the scaling-up of Li-ion battery storage systems.
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http://dx.doi.org/10.1088/1361-6528/ab48b2DOI Listing
January 2020

CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration.

Theranostics 2019 14;9(21):6099-6111. Epub 2019 Aug 14.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, 300.

: Peripheral nerve regeneration requires coordinated functions of neurotrophic factors and neuronal cells. CRISPR activation (CRISPRa) is a powerful tool that exploits inactive Cas9 (dCas9), single guide RNA (sgRNA) and transcription activator for gene activation, but has yet to be harnessed for tissue regeneration. : We developed a hybrid baculovirus (BV) vector to harbor and deliver the CRISPRa system for multiplexed activation of 3 neurotrophic factor genes (, and ). The hybrid BV was used to transduce rat adipose-derived stem cells (ASC) and functionalize the ASC sheets. We further implanted the ASC sheets into sciatic nerve injury sites in rats. : Transduction of rat ASC with the hybrid BV vector enabled robust, simultaneous and prolonged activation of the 3 neurotrophic factors for at least 21 days. The CRISPRa-engineered ASC sheets were able to promote Schwann cell (SC) migration, neuron proliferation and neurite outgrowth . The CRISPRa-engineered ASC sheets further enhanced functional recovery, nerve reinnervation, axon regeneration and remyelination. : These data collectively implicated the potentials of the hybrid BV-delivered CRISPRa system for multiplexed activation of endogenous neurotrophic factor genes in ASC sheets to promote peripheral nerve regeneration.
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http://dx.doi.org/10.7150/thno.36790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735509PMC
September 2020

Obturator dislocation of the femoral head combined with ipsilateral femoral neck and pubic fractures: A rare case report.

Medicine (Baltimore) 2019 Sep;98(37):e17150

Department of Orthopedics, The Second Hospital of Jilin University, Nanguan District, Changchun City, Jilin, China.

Rationale: Obturator dislocation of the femoral head combined with ipsilateral femoral neck and pubic fracture is a rare injury. We used cannulated screws combined with a femoral neck medial plate for the first time to treat this type of injury and achieved good follow-up results.

Patient Concerns: The patient was hospitalized due to an accident resulting in swelling and deformity accompanied by limited mobility of the right hip and left knee.

Diagnoses: X-ray examination and computed tomography confirmed that the patient suffered from right hip obturator dislocation, femoral head and neck fracture, pubic fracture, as well as open fracture of the left femoral shaft.

Interventions: Open reduction and internal fixation with cannulated screws and a medial support plate of the femoral neck were performed for the right hip obturator dislocation, femoral head and neck fracture, and the pubic fracture. Artificial bone grafting was performed to support the femoral head defect. Debridement and the second phase of internal fixation were executed to cure the open fracture of the left femoral shaft.

Outcomes: The patient was followed-up for 6 months and showed good hip function. X-ray examination and computed tomography indicated that the fractures healed well without fracture displacement or loosening of the implants. Meanwhile, there were no signs of femoral neck valgus and femoral head necrosis observed.

Lessons: The combination of cannulated screws and medial support plate was suggested for the treatment of hip obturator dislocation and femoral head and neck fracture. Furthermore, partial weight loading exercise should be performed promptly to reduce the risk of muscular atrophy and myasthenia.
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http://dx.doi.org/10.1097/MD.0000000000017150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750302PMC
September 2019

CRISPR technologies for stem cell engineering and regenerative medicine.

Biotechnol Adv 2019 12 9;37(8):107447. Epub 2019 Sep 9.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan; Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu 300, Taiwan. Electronic address:

CRISPR/Cas9 system exploits the concerted action of Cas9 nuclease and programmable single guide RNA (sgRNA), and has been widely used for genome editing. The Cas9 nuclease activity can be abolished by mutation to yield the catalytically deactivated Cas9 (dCas9). Coupling with the customizable sgRNA for targeting, dCas9 can be fused with transcription repressors to inhibit specific gene expression (CRISPR interference, CRISPRi) or fused with transcription activators to activate the expression of gene of interest (CRISPR activation, CRISPRa). Here we introduce the principles and recent advances of these CRISPR technologies, their delivery vectors and review their applications in stem cell engineering and regenerative medicine. In particular, we focus on in vitro stem cell fate manipulation and in vivo applications such as prevention of retinal and muscular degeneration, neural regeneration, bone regeneration, cartilage tissue engineering, as well as treatment of diseases in blood, skin and liver. Finally, the challenges to translate CRISPR to regenerative medicine and future perspectives are discussed and proposed.
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http://dx.doi.org/10.1016/j.biotechadv.2019.107447DOI Listing
December 2019

Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle.

Int J Mol Sci 2019 Jun 24;20(12). Epub 2019 Jun 24.

National Medical Research Center of Cardiology, Russian Ministry of Health, 121552 Moscow, Russia.

Cell therapy remains a promising approach for the treatment of cardiovascular diseases. In this regard, the contemporary trend is the development of methods to overcome low cell viability and enhance their regenerative potential. In the present study, we evaluated the therapeutic potential of gene-modified adipose-derived stromal cells (ADSC) that overexpress hepatocyte growth factor (HGF) in a mice hind limb ischemia model. Angiogenic and neuroprotective effects were assessed following ADSC transplantation in suspension or in the form of cell sheet. We found superior blood flow restoration, tissue vascularization and innervation, and fibrosis reduction after transplantation of HGF-producing ADSC sheet compared to other groups. We suggest that the observed effects are determined by pleiotropic effects of HGF, along with the multifactorial paracrine action of ADSC which remain viable and functionally active within the engineered cell construct. Thus, we demonstrated the high therapeutic potential of the utilized approach for skeletal muscle recovery after ischemic damage associated with complex tissue degenerative effects.
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http://dx.doi.org/10.3390/ijms20123088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627773PMC
June 2019

CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.

Nucleic Acids Res 2019 07;47(13):e74

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.

Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.
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http://dx.doi.org/10.1093/nar/gkz267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648329PMC
July 2019

Local magnetic activation of CRISPR.

Nat Biomed Eng 2019 02;3(2):83-84

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.

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http://dx.doi.org/10.1038/s41551-019-0354-yDOI Listing
February 2019

Chikungunya virus inhibition by synthetic coumarin-guanosine conjugates.

Eur J Med Chem 2019 Mar 21;166:136-143. Epub 2019 Jan 21.

Department of Chemistry, National Tsing Hua University, Hsinchu, 300, Taiwan; Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, 300, Taiwan; Department of Chemistry, National Central University, Jhongli City, Taoyuan, 320, Taiwan. Electronic address:

Since its discovery in Tanganyika, Africa in 1952, chikungunya virus (CHIKV) outbreaks have occurred in Africa, Asia, Europe, and America. Till now chikungunya fever has spread in nearly 40 countries. Because of lack of effective vaccines and antiviral drugs to intervene this disease, 21 new conjugated compounds were designed and synthesized by coupling of 6,8-dithioguanosine at its C-6 position with 3-(chloromethyl)coumarins bearing an F, Cl, Br, Me, or -OMe substituent through the -SCH- joint. Meanwhile, an organic "dummy" ligand (e.g., methyl, benzyl, and naphthylmethyl) or a coumarinyl moiety was attached at the C-8 position. By high through-put screening, three of these new conjugates were found to inhibit CHIKV in Vero cells with significant potency (EC = 9.9-13.9 μM) and showed low toxicity (CC = 96.5-212 μM). The selectivity index values were 9.37-21.7. Their structure-activity relationship was deduced, which indicates that the coumarin moiety is essential and the presence of a -OMe group enhances the antiviral activity.
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http://dx.doi.org/10.1016/j.ejmech.2019.01.037DOI Listing
March 2019

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.

Biotechnol Bioeng 2019 05 8;116(5):1066-1079. Epub 2019 Feb 8.

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.
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http://dx.doi.org/10.1002/bit.26915DOI Listing
May 2019

Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation.

Nucleic Acids Res 2019 02;47(3):e13

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.

CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing.
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http://dx.doi.org/10.1093/nar/gky1165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379646PMC
February 2019

Knockdown of Ubiquitin Associated Protein 2-Like (UBAP2L) Inhibits Growth and Metastasis of Hepatocellular Carcinoma.

Med Sci Monit 2018 Oct 6;24:7109-7118. Epub 2018 Oct 6.

Department of Pathology, First Affiliated Hospital of University of Science and Technology of China, Hefei, Anhui, China (mainland).

BACKGROUND The aim of this study was to explore the influence of ubiquitin associated protein 2-like (UBAP2L) on the growth and metastasis of hepatocellular carcinoma (HCC) and its potential underlying mechanism. MATERIAL AND METHODS UBAP2L gene was knocked down in SMMC-7721 by RNA interference and cell function experiments were performed. A subcutaneous xenograft tumor model was constructed to examine the effect of UBAP2L silence on HCC growth. Finally, the whole genomic microarrays were used to screen the potential mechanism of UBAP2L in regulating the biological function of HCC. RESULTS Compared with those in the control group, the cell proliferation and clone formation were significantly reduced, cell cycle was arrested in G2/M phase, the number of apoptotic cells was remarkably increased, and the abilities of vascular formation and cell migration and metastasis were dramatically weakened in the shUBAP2L group (All P<0.05). UBAP2L knockdown significantly suppressed the tumor growth of HCC in vivo. Moreover, a total of 320 genes changed significantly after UBAP2L knockdown, among which, 159 genes were upregulated and 161 genes were downregulated. Then, gene enrichment analysis revealed that PI3K/AKT and P53 signal pathway were the most significant in the top 10 enrichments. Finally, Western blot analysis verified that UBAP2L knockdown caused the increase of P21 and PTEN and decrease of CDK1, CCNB1, p-PI3K, and p-AKT. CONCLUSIONS UBAP2L plays an oncogenic role in HCC, and knockdown of its expression significantly inhibits HCC growth and metastasis, which may be related to the regulation of PI3K/AKT and P53 signaling pathways by UBAP2L.
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http://dx.doi.org/10.12659/MSM.912861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284357PMC
October 2018
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