Knockdown of zebrafish YY1a can downregulate the phosphatidylserine (PS) receptor expression, leading to induce the abnormal brain and heart development
© Shiu et al. 2016
Received: 16 July 2015
Accepted: 16 February 2016
Published: 29 February 2016
Yin Yang 1 (YY1) is a ubiquitously expressed GLI-Kruppel zinc finger-containing transcriptional regulator. YY1 plays a fundamental role in normal biologic processes such as embryogenesis, differentiation, and cellular proliferation. YY1 effects on the genes involved in these processes are mediated via initiation, activation, or repression of transcription depending upon the context in which it binds. The role of the multifunctional transcription factor Yin Yang 1 (YY1) in tissue development is poorly understood. In the present, we investigated YY1a role in developing zebrafish on PSR-mediated apoptotic cell engulfment during organic morphogenesis.
YY1a is first expressed 0.5 h post-fertilization (hpf), in the whole embryo 12 hpf, and in brain, eyes, and heart 72 hpf by in situ hybridization assay. The nucleotide sequence of zebrafish YY1a transcription factor (clone zfYY1a; HQ 166834) was found to be similar to that of zebrafish YY1a (99 % sequence identity; NM 212617). With the loss-of-function assay, YY1a knockdown by a morpholino oligonucleotide led to downregulation of the phosphatidylserine engulfing receptor zfPSR during embryonic segmentation and to the accumulation of a large number of dead apoptotic cells throughout the entire early embryo, especially in the posterior area. Up to 24 hpf, these cells interfered with embryonic cell migration and cell-cell interactions that normally occur in the brain, heart, eye, and notochord. Finally, with gain-of-function assay, defective morphants could be rescued by injecting both YY1a mRNA and PSR mRNA and trigger resumption of normal development.
Taken together, our results suggest that YY1a regulates PS receptor expression that linked to function of PSR-phagocyte mediated apoptotic cell engulfment during development, especially the development of organs such as the brain and heart. YY1a/PSR-mediated engulfing system may involve in diseases.
KeywordsYY1a Phosphatidylserine receptor Zebrafish Knockdown Brain Heart
Yin Yang 1 (YY1; also called delta, NF-E1, and UCRBP) is a ubiquitously expressed GLI-Kruppel zinc finger-containing transcriptional regulator [1–4]. YY1 plays a fundamental role in normal biologic processes such as embryogenesis, differentiation, and cellular proliferation . Its effects on the genes involved in these processes are mediated via initiation, activation, or repression of transcription depending upon the context in which it binds. The activation or repression can be direct via the disruption of binding sites or the causing of changes in DNA conformation or indirect via cofactor recruitment [6–12].
Since its original isolation, YY1 has been shown to regulate an ever-growing number of viral and cellular genes, including the human immunodeficiency virus type 1 and human papillomavirus oncogenes E6 and E7, several proto-oncogenes (c-myc, c-fos, and erb2), cdc-6, the DNA replication-dependent histone H3.2 gene, and others [13–16]. Transient transfection and/or in vitro approaches have been used to characterize YY1 target genes, most of which have yet to be validated as YY1 target genes in vivo. Nevertheless, these studies suggest important roles for YY1 in the control of cell growth, apoptosis, and oncogenic transformation.
Several lines of evidence suggest a critical requirement for YY1 in embryonic development, morphogenesis, and organogenesis. The Drosophila counterpart of YY1, pleiohomeotic (pho), participates in embryonic patterning; mutations of pho result in homeotic transformations associated with misexpression of homeotic genes [17–19]. YY1 is also essential for neural induction and patterning in Xenopus laevis . Constitutive ablation of YY1 in mice results in peri-implantation lethality . Mice heterozygous for the YY1 gene (yy1+/−) display a mild developmental delay and a subset of these animals exhibit neurulation defects and exencephaly . While these observations strongly suggest that YY1 acts during late embryogenesis, early embryonic lethality caused by constitutive loss of function has precluded investigation of the YY1 requirement at later developmental stages . Then, it was shown that YY1 overexpression can reduce fetal troponin (ssTn1) expression in neonatal myocardium  and promotes mesodermal cardiac differentiation . Recently, in the zebrafish system, YY1a have examined on promoting the zebrafish liver steatosis and lipotoxicity by inhibiting CHOP-10 exoression  and regulating the zebrafish hematopoiesis by beta-arrestin 1 via binding to YY1b and relieving polycomb group repression . Whether the YY1a involve in early embryonic development is still uncover.
Apoptotic cell death occurs by a mechanism that has been evolutionarily conserved from nematodes to humans . In vivo, apoptotic cells are typically engulfed and degraded by phagocytes . Removal of apoptotic cells suppresses inflammation, modulates the macrophage-directed deletion of host cells, and regulates the immune response . The phagocyte engages the dying cells through surface receptors that include the phosphatidylserine receptor (PSR) [30–32], Fc receptor, complement receptors 3 and 4, the ABC1 transporter, members of the scavenger-receptor family [33–36].
Morpholinos are chemically modified oligonucleotides with base-stacking abilities similar to those of natural genetic material . Morpholinos have been shown to bind to and block translation of mRNA in zebrafish cells .
Little is known regarding the part played by YY1a in the clearance of cell corpses in zebrafish. In this present, our aim was to define the genetic requirements for a potentially distinct death paradigm involving YY1a in zebrafish during early embryonic development.
Maintenance of fish embryos in culture
Techniques for the care and breeding of zebrafish have been previously described in detail . Embryos were collected from natural matings and maintained in embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 0.15 mM, 0.05 mM Na2HPO4, 0.7 mM NaHCO3) at 28.5 °C. Embryos were staged according to standard morphological criteria .
Synthesis and amplification of cDNA was carried out using the SuperScript One-Step™ reverse transcriptase-polymerase chain reaction (RT-PCR) system kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. YY1 primers P1 and P2 were each added to a final concentration of 0.2 μM. PCR cycling conditions were 54 °C for 30 min, 2 min at 94 °C (to inactivate the reverse transcriptase), 95 °C for 30 s (DNA denaturation), 57 °C for 30 s (annealing), and 72 °C for 60 s (extension) for a total of 35–40 cycles. The RT-PCR primers—zebrafish YY1 P1: 5′-ATGGCGTCGGGCGAGACACTGT-3′ (22mer) and YY1 P2: 5′-TCACTGATTGTTCTTAGCTTTCGCGTGTGT-3′ (30mer)—were used to amplify a fragment covering the complete mRNA region of YY1a. The purity and size of the amplified product were verified by 1.5 % agarose gel electrophoresis and staining with ethidium bromide . The 1074-bp, double-stranded cDNA was purified using the QIAquick™ gel extraction system (Qiagen, Valencia, CA) and subcloned using a pGEMT-easy cloning system (Promega, Madison, WI). The cloned PCR products were sequenced by the dye termination method using an ABI PRISM 477 DNA sequencer (Applied Biosystems, Foster City, CA) and their sequences were scanned against the GenBank database BLAST (www.ncbi.nlm.nih.gov/) and PROSITE (psort.ims.u-tokyo.ac.jp/) programs.
GenBank was the source of the human homologue (accession number NM 003403) and mouse homologue (accession number NM 009537), and the complete sequences for Xenopus homologue (accession number NM 001093935), D. melanogaster homologue (accession number XM 002099609), and zebrafish homologue (accession numbers NM 212617 and HQ166834).
All morpholinos (Gene Tools, LLC, Corvallis, OR) were arbitrarily designed to bind to sequences flanking and including the initiating methionine. We selected sequences based on design parameters recommended by the manufacturer (21–25mer antisense), and looked for the presence of each designed sequence elsewhere in the genome [37, 38]. Sequences were as follows (the sequence complimentary to the predicted start codon is underlined in all cases): YY1-MO-5′-gTACAgTgTCTCgCCCgACgCCAT-3′.
(The five sites in the control-MO sequence that are subject to point mutation in the YY1-MO sequence are shown in shaded blocks.)
Injection of YY1a morpholino oligonucleotides
Morpholino oligonucleotides were dissolved in water at a concentration of 1 mM and diluted with water to 0.5, 0.25, and 0.125 mM prior to injection (1.5–3 nl) into the yolk .
Digoxigenin-labeled antisense RNA probes were synthesized from linearized DNA templates containing YY1a, psr, pax2a, and nkx2.5, using T7 RNA polymerase (Boehringer Mannheim, Mannheim, Germany). Whole-mount in-situ hybridizations were performed as previously described . In-situ hybridization was used to analyze YY1a mRNA expression in embryos injected with YY1-MO or control-MO at 0.5 h post-fertilization (hpf), 12 hpf, 24 hpf, 48 hpf, and three days pf (3 dpf).
Apoptotic cell staining during embryonic development
Embryos at the one- or two-cell stage were injected with YY1-MO (25 ng) or control-MO (25 ng) or YY1-MO (25 ng) plus PSR mRNA (1 ng), harvested at 18.5 hpf, fixed with 4 % paraformaldehyde in PBS (pH 7.4) at room temperature for 30 min, stained with acridine orange (1 μg.ml−1; 3–5 min), washed twice in PBS, and evaluated under a fluorescence microscope (using a 488-nm filter for excitation and a 515-nm long-pass filter for detection) . For the TdT-dUTP labeling step, the embryos were fixed in paraformaldehyde at the end of the incubation period (12 and 24 hpf), dechorionated, incubated in blocking solution (0.1 % H2O2 in methanol, 30 min, room temperature), rinsed with PBS, incubated on ice in 0.1 % Triton X-100 in 0.1 % sodium citrate for 30 min to increase permeability, rinsed twice with PBS, treated with TUNEL reaction mixture (50 μl; in-situ cell death-detection Kit, Boehringer Mannheim), incubated in a humidified chamber for 60 min at 37 °C, and evaluated for fluorescence-positive apoptotic cells under a fluorescence microscope equipped with a spot II CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
Embryos were injected with YY1-MO or control-MO at the one- or two-cell stage, harvested 24 hpf, and lysed in 150–200 μl of sodium dodecyl sulfate (SDS) sample buffer (0.63 ml of 1 M Tris–HCl, pH 6.8, 1.0 ml of glycerol, 0.5 ml of ß-mercaptoethanol, 1.75 ml of 20 % SDS, 6.12 ml of H2O in 10 ml total). Protein (40 μg) from 24-hpf embryos was loaded onto each lane. Standard western-blot analysis was conducted using human anti-YY1 Mab (Cascade Bioscience, Winchester, MA), anti-zebrafish PSR N-terminus polyAb (self-made), and mouse anti-actin Mab (Chemicon, Temecula, CA). PSR was visualized using horseradish peroxidase-conjugated anti-mouse immunoglobulin (IgG) and the ECL detection kit (Amersham Pharmacia Biotech, Denmark) .
Microinjection of PSR mRNA rescues YY1a-mediated defective morphants
Zebrafish YY1a and PSR  were cloned into the pCDNA3 vector, which contains a T7 RNA polymerase promoter site. Linearized plasmid DNA was used as a template for in vitro transcription carried out using the Message Machine Kit (Ambion Inc, Austin, TX), according to the manufacturer’s instructions. For rescue of defective morphants, either 0.1 nl of 200 ng/μl mRNA encoding soluble YY1a mRNA or 0.1 nl of 80 and 40 ng/μl mRNA encoding soluble PSR mRNA and YY1-MO (0.5 μM) were injected into the one-cell stage of each embryo using a gas-driven microinjector (Medical System Corp, Greenvale, NY) .
Embryos at the one- or two-cell stage were injected with YY1-MO (25 ng) or control-MO (25 ng) or YY1-MO (25 ng) plus PSR mRNA (1 ng), harvested at 18.5 hpf. The mRNA expression levels were measured by a qRT-PCR with a Roche Lightcycler Nano (Roche, Penzberg, Germany). The final volume in a well was 20 ul, and contained 4 ul of OmicsGreen qPCR 5X master mix (Omics Bio, Taipei, Taiwan), 3.2 ng of cDNA, and 50 nM of primer pairs. Cycling parameters were as follows: 95 °C × 900 s, then 45 cycles of the following 95 °C × 15 s, 60 °C × 20 s and 72 °C × 20 s. The standard curve of each gene was checked in the linear range with ß-actin as internal controls. The primer sets are given as follows: P53 primer-forward 5′-ACCACT GGGACCAAACGT AG-3′; Primer-reversed - 5′-CAGAGTCGCTTCTTCCTTCG-3′.
YY1a cloning and determination of functional domains
The nucleotide sequence of zebrafish YY1a (zfYY1) (HQ 166834) had 99 %, 92 %, 98 %, 98 %, and 98 %, respectively, identity with zebrafish YY1a (NM 212617), and the YY1s for D. yakuba (XM 002099609), human (NM 003403), mice (NM 009537), and Xenopus laevis (NM 001093935). Here, we named zfYY1 (HQ 166834) gene is equal to YY1a (NM 212617) gene.
In addition, there were two consensus sequences for the nuclear localization signals within YY1a—a 7-amino-acid peptide (PSGRMKK; at 118–124 aa) and a 7-amino-acid peptide (PRKIKED; at 227–233 aa) (Fig. 1a, box A1-2). Domains of potential zinc finger proteins, CH2H2 type, corresponding to residues 241–263 (box B1; CPHKGCTKMFRDNSAMRKHLHTH, 23 aa), 270–290 (box B2; CAECGKAFVESSKLKRHQLVH, 21 aa), 298–320 (box B3; CTFEGCGKRFSLDFNLRTHVRIH, 23 aa), and 328–350 (box B4; CPFDGCNKKFAQSTNLKSHILTH, 23 aa), which are well-known DNA binding sequences, are shown in box B1–4 (Fig. 1a). The sequence shown in Fig. 1a, box C (residues 353–356; AKNN for KKXX domain) was identified as an endoplasmic reticulum membrane retention signal.
YY1a expression pattern
YY1a was expressed from the one-cell developmental stage (30 min) to the 3-day pf (3 dpf) larval stage (Fig. 1b, panels a–e). After somite segmentation, YY1a was apparent throughout the embryo (Fig. 1b, panels b–d) and the brain (Fig. 1b, panels b–d). At the 3-dpf larval stage, YY1a expression was detected in the brain, heart, and eye (Fig. 1b, panel e).
YY1a knockdown affects apoptotic cell accumulation and delays epiboly
Influence of cell corpses on normal embryonic development
The abnormalities at 48 hpf were both mild (just shown mild defects in brain and heart but can hatching out) and severe (severe defects in brain and heart, and did not hatching out) (Fig. 4c, c and d; cf. with Fig. 4c, a and b [wild type and control MO-injected]). The mild defects included abnormal mid- and hindbrain morphology or a slight delay in heart development prior to hatching (Fig. 4c, panel c; as indicated by arrows). The severe defects included marked delays in development manifesting as shrinkage of the brain, defects in heart development, and failure to hatch (Fig. 4c, panel d; as indicated by arrows).
Percentage of morphant phenotypes during knockdown of YY1a by YY1-morpholines
Number of embryos
YY1a knockdown downregulates PSR expression
We found that YY1-MO (25 ng) injection apparently reduced PSR expression throughout the embryo (Fig. 5b, c, as indicated by arrows; cf. with Fig. 5b, a [wild type] and Fig. 5B, b [control MO injection]) at 12 hpf and reduced PSR expression in brain and somites (Fig. 5b, f, as indicated by arrows; cf. with Fig. 5b, d [wild type] and Fig. 5b, e [control MO injection]) at 24 hpf. The changes at 24 hpf were still present at 48 hpf (Fig. 5b, i, as indicated by arrows; cf. with Fig. 5b, g and h [control]).
YY1a involvement in brain and heart organogenesis
In heart development study, probing with nkx 2.5 allowed the monitoring of heart morphogenesis. In-situ assay using nkx 2.5 as probe detected a severe delay in heart development (atria absent from a tube-like heart; Fig. 6b, c; cf with Fig. 6b, a [wild type] and b [control MO injection]) at 48 hpf and presence of a tube-like heart (twice the normal size in length) and abnormal blood circulation rate (Fig. 6b, f; cf. with Fig. 6b, d [wild type] and e [control MO injection]) at 3 dpf.
YY1a mRNA rescues defective morphants
The results of 2.5 ng of YY1a mRNA prevented the developmental block due to coinjection of YY1-MO with 25 ng of YY1-MO at the embryonic one- or two-cell stage. In the present study, the same result was obtained by injecting 25 ng of YY1-MO and 2.5 ng of PSR mRNA.
Survival and morphant phenotype rates were estimated in wild type (n = 215), control-MO (n = 136), YY1-MO plus YY1a mRNA (n = 171), and YY1-MO (n = 185) embryos at 3 dpf. The rate of severe defect occurrence was reduced from 27.5 % to 5 % and the rate of mild defect occurrence was reduced from 45 % to 11 % by injection of the YY1a mRNA (Fig. 7b). Rescue restored morphogenesis and brain-heart development to normal (Figs. 7c, d; cf. with wild type [Fig. 7c, a] and control-MO injected embryos [Fig. 7c, b]) at 3 dpf. The YY1-MO-injected embryos with slight reduction in brain size and heart development had patterns of morphogenesis and YY1a expression that reflected these mild deformities (Figs. 7c, c; as indicated by arrows; cf. with Fig. 7c, a [wild type] and Fig. 7c, b [control MO injection]).
PSR mRNA rescues YY1a-mediated defective morphants
To determine extra reloading PSR mRNA whether can rescue YY1a defected. The 0.5 ng or 1 ng of PSR mRNA were used to coinjection of YY1-MO with 25 ng of PSR-MO at the embryonic one- or two-cell stage.
YY1-MO-injected embryos had severe developmental defects (Fig. 8b:b, e and h) at 24 h, 48 h and 72 hpf, respectively. After rescue, embryos had just mild defects at 72 hpf in the heart (Fig. 8b:i; indicated by arrow). And no defects were evident in the controls (Fig. 8b, g [control MO injection]).
After rescued by PSR mRNA, the normal type (Fig. 8c; 3 dpf) and morphant phenotype rates were estimated in control MO (n = 210), YY1-MO (n = 178), and YY1-MO plus PSR mRNA (1 ng; n = 207) embryos at 3 dpf. The rate of normal type occurrence was significantly increased from 19 % to 42 % (1 ng of mPSR group), which can enhance up to 23 % in normal type. The rate of mild defect occurrence was the same from 38 % to 38 % and the rate of severe (strongly) defect occurrence was reduced from 43 % to 20 % by injection of the PSR mRNA (Fig. 8c), which can reduce up to 23 % that was shown up (YY1a)-and-downstream (PSR) relationship.
Discussion and conclusion
Phagocytosis plays an important role in inflammation and autoimmune responses , [47, 48], and in remodeling of tissue during development . The link between phagocytosis and development thus provides a useful system to identify genes involved in phagocytosis. These genes have been difficult to identify in mammalian systems . While some questions remain, PSR appears to play a central role in the clearance of apoptotic cells . In the present, transcriptional factor YY1a played a positively regulator in the PSR gene expression that PSR-mediated engulfment of cell corpses required for normal embryonic development and tissue morphogenesis.
PSR and others receptors involved in cell corpse engulfment during development
The phagocytic removal of apoptotic cells suppresses inflammation, modulates the macrophage-mediated deletion of host cells, and critically regulates the immune response  in higher organisms. In vertebrates, the phagocyte engages dying cells through specific receptors such as the phospatidylserine receptor (PSR) [30–32]—a professional receptor in zebrafish, but not in Drosophila system. Some receptors involved in phagocytosis (e.g., Fc receptors, complement receptors 3 and 4, the ABC1 transporter, members of the scavenger-receptor family , and BAI1  are not expressed in the early embryonic stage. Recently, the gene encoding Satblin-2 receptor  was found to be expressed very early in embryonic development (before 5.4 hpf), but whether regulation by YY1 influences its expression remains unknown. On the other hand, a new corpse engulfment receptor TIM4  was found to be expressed late (72 hpf) but not early in the embryonic development of zebrafish, so this receptor may not be involved in early corpse engulfment. In our system, we found that PSR-mediated death cell clearing is important for normal development and especially on brain and heart development that can regulate by YY1a in the up-streaming.
YY1a knockdown can affect dead cell engulfment and embryonic phenotype
In our present, first reported abnormal development after knockdown of YY1 was monitored over time. Three embryonic phenotypes (mildly defective, severely defective, and dead embryos) could be differentiated at 12, 24, 48, and 72 hpf. Mild defects (enlarged heart cavity and brain malformation) were apparent at 48 hpf but not at 12 hpf. Severe defects at 12 hpf were associated with accumulation of a large number of apoptotic corpses throughout the embryo that interfered with epiboly and posterior development and their persistence at 48 hpf led to failure to hatch and death at 3 dpf. This pattern resembled that of embryos subjected to PSR knock down . Interestingly embryos could be prevented from developing mild and severe defects by injecting YY1 mRNA at 48 h and 72 hpf (Fig. 7) and PSR mRNA at 24 h, 48 h and 72 hpf (Fig. 8b and c) as a downstream regulating gene, indicating that such an injection could potentially be used to correct YY1a gene defects.
New role of YY1a affect PSR expression on engulfing apoptotic cell function
Recently, it was shown that YY1 can direct targeting by miR-34a . Constitutive ablation of YY1 in mice has been reported to result in peri-implantation lethality . Heterozygous mice (yy1+/−) display a mild delay in development, and a subset of these animals exhibit neurulation defects and exencephaly . While these observations strongly suggest that YY1 acts during late embryogenesis, the early embryonic lethality caused by constitutive loss of function precludes investigation of the YY1 requirement at later developmental stages . Furthermore, DNA array analysis of the response to conditional allele knockout of YY1 gene in mouse embryonic fibroblasts  identified about 135 up- and downregulated genes involved in (1) cell cycle control, (2) mitosis and cytokinesis, (3) DNA replication and repair, (4) apoptosis, (5) cell growth and proliferation, and (6) development. In zebrafish system, we found that zfYY1a knockdown can reduce PSR mRNA and protein expression levels. Furthermore, we found that zfYY1a-mediated defected could be rescued by mPSR injection at 24 h, 48 h and 72 hpf (Fig. 8b and c). Thus, YY1 may play a new role that act as a positive regulator of PSR in the up-streaming during early embryonic development.
Why is important of YY1a-mediated PSR clearing system on brain and heart morphogenesis
The normal development is important for tissues or organs completely formation that involved in turn on the programmed cell death and engulfing of corpses cell system, those events were tightly control and regulated, if somewhere connection was mistaken or failed that also caused the abnormal development and diseases. We proposed that YY1a/PSR-mediated engulfing system is important for this role.
The above-mentioned evidence supports our idea that the accumulation of cell corpses interferes with normal embryonic development by altering cell movement (Figs. 3b and c) in early epiboly stage during normal genetic program process.
From the developmental view, at the mid-blastula stage (5.25 hpf), the gastrula starts to emerge, all cells acquire the ability to move  (ZFIN website), and programmed cell death/apoptosis is turned on . Development requires movement of cells that several types of movement occur including epiboly, involution, convergence, and extension [53–55]. If the apoptotic cell corpses are not quickly removed by professional engulfing PSR-mediated clearance system , they can impede cell movement and delay epiboly and thereby the onset of organ development such as on three germ-layers development for ectoderm, mesoderm and endoderm formations. Cells involved in the development of somites, primordial pharyngea, neuromeres, primary organs, and the tailbud are typically associated with one of three germ layers (ectoderm, mesoderm, or endoderm) [40, 53]. Cell corpses typically affect morphogenesis of normal organs at the pharyngula (24 h), hatching (48 h), and early larval (72 h) stages.
Recently, this is new emergence area that mechanic forces induced by accumulated dead cells [56–58] that the phagocyte engages the dying cells through phosphatidylserine receptors (PSR) and other receptors [30–32]. Here, we found that PSRs are professional receptors for phagocytosis in zebrafish  between at 5.4hpf and 6 hpf that PSR-mediated engulfing pathway regulated by YY1a.
Summary (Fig. 9)
The first traced the transcription factor YY1a as a material factor was expressed from 0.5 hpf to 3 dpf. This factor can regulate downstream molecule PS receptor expression for engulfing of corpse cells during priming the programmed cell death at about 6 hpf. With PSR-mediated engulfing system for completed to clean the death cells during early development that is important for three germ layers formation and then tissue and organs development. If it didn’t smooth process that defected embryos especially on brain and heart developments also found at late development stage. So YY1a/PSR-mediated engulfing system is very important for normal development and may cause diseases.
We thank Mr. H-Y Gong for providing us with the pax 2a plasmid and Dr. J-N Chen for providing us with the nkx 2.5 plasmid for use as probes. This work was supported by a grant (NSC-99-2321-B-006-010-MY3) awarded to Dr. Jiann-Ruey Hong from the National Science Council, Taiwan, Republic of China.
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