Understanding whom in the community network to target with the relevant health knowledge is so far unclear and under debate. This research aims to investigate the effects of health intervention using different social network targeting methods. We recruited participants for delivery of the Dengue Fever knowledge from the Gangzai Village in Tainan, Taiwan. We randomized the 20 neighborhoods, separately for each intervention, to one of the two targeting methods. Findings show that random targeting method increased the redeem rate of mosquito repellent wristbands in the first six days of the experiment compared with high-indegree targeting, which the seeds are the head of the neighborhoods. Study of social network of peer-to-peer influence helps to provide better health intervention, as well as improve population health. This research hope to shed some light on the prevention of dengue fever in those communities with high prevalence rates in Southern Taiwan.
Comparative study between the biogeochemical records in Dongsha coral reef and environmental factors: Reconstructing the anthropogenic impact in the South China Sea
Over the past 250 years, atmospheric CO2 levels increase by nearly 40%, from pre-industrial levels of ~280 ppmv to ~400 ppmv in 2014. An increase in oceanic pCO2 is a predictable consequence of rising atmospheric CO2 over the next half of century1, which results in reduced pH and lower calcium carbonate saturation state in shallow oceanic environments. A substantial decrease in oceanic pH has raised much concern for the physiological and ecological response of marine calcifying organisms. However, the response of calcifying organisms to accelerating effects of declining seawater pH is still poorly constrained and uncertainties arise when involving the combined effects of climate change and ocean acidification (OA) on these biogenic calcifiers in natural environments. Insights into these issues can be gained by developing reliable paleo-proxies for marine carbonate chemistry. The boron (B) isotopic composition of marine biogenic carbonates is considered to be the most reliable proxy for seawater pH2,3. In order to evaluate if the coral reefs around the Taiwan islands are influenced by OA or other environmental changes, we conducted an in-situ calibration of the δ11B in coral skeleton (massive Porities spp.) against ambient seawater pH at their growth locations in Dongsha Atoll. Our results (2010-2013) show that low seawater pH (low δ11B value) was recorded in coral skeleton during the summer and high seawater pH (high
δ11B value) during the winter, consistent with the instrumental pH records at Dongsha Atoll. This suggests that coralline δ11B can faithfully reflect the seawater pH variation in our study site, and thus has a great potential to further elucidate how OA influences the coral reef ecosystem and the response of calcifying organisms in the Dongsha Atoll under climatic and environmental changes.
在過去的兩百五十年間，大氣二氧化碳濃度從工業革命前的280 ppmv 迅速增加至400 ppmv（2014 年），增加了將近40%。海洋中溶解的二氧化碳濃度也可預期地將於下一個世紀中大幅增加，進而導致海水pH 值和海洋碳酸鈣飽和程度的降低1。雖然國際上許多的研究已經注意並報導海洋酸化對海洋中鈣化生物於生理及生態上都有重大的影響，然而仍有許多具爭議之處，特別是當海洋酸化及其他氣候變遷共同影響的自然環境。為了進一步瞭解海洋酸化的影響，可靠的海洋碳酸鹽系統代用指標發展是必要的。生物性碳酸鈣的殼體或骨骼中硼同位素是目前被認為最佳的海水pH 值代用指標2,3，因此本研究團隊的主要目標為發展高精準硼同位素質譜分析術及校正東沙環礁內珊瑚骨骼硼同位素與海水的關係，作為未來重建東沙環礁附近海水pH 值變化的主要工具及參考依據。我們的研究發現（2010-2013 年間），珊瑚骨骼硼同位素所記錄的夏季海水pH 值較冬季時低。此結果與近期於東沙環礁內佈放的pH 探針所記錄到
1. Feely, R. A. et al., Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science 305, 362-366 (2004).
2. Hönisch, B. et al., Assessing scleractinian corals as recorders for paleo-pH: empirical calibration and vital effects. GCA 68, 3675-3685 (2004)
3. Anagnostou, E. et al., Evaluation of boron isotope ratio as a pH proxy in the deep-sea coral Desmophyllum dianthus: Evidence of physiological pH adjustment. EPSL 249-350, 251-260 (2012).
Cell-type specific structural and functional studies of MD-InC thalamocortical neurons in neuropathic pain
Chronic pain is one of the severe disorder that generate discomfort for most people in moden society. However, many chronic pain including CPSP are drug-resistant. There is a urgent need to understand the initiation and regulatory mechanism of CPSP in order to treat this disorder. Here we want to use optogenetic method to specificall stimulate medial dorsal nucleus in the thalamus to activate cortex recipient region. By combining single cell labeling method, we could build a functional neuronal map for pain pathway between thalamus and cortex. Here we successfull stimiate MD in the thalamus and recorded the activation in the cortex using optogenetic method. In addition, we found that neuron in MD can be divdied into at least 2 different population according to their morphlogy. In the future, we hope to build a complete neuronal circiutry to study the chronic pain induction and potentially block the transduction for chronic pain treatment.
Investigate the biological effect of DNA methylation on G-quadruplex of hTERT gene
DNA甲基化(methylation)修飾為重要的表觀遺傳(epigenectic modification)調控方式，特別是啟動子(promoter)區域受DNA甲基化調控與癌症發展有極高的關聯性。DNA上的non-B二級結構如G-quadruplex被認為具會影響基因的表現的可能。但G-quadruplex結構形成序列上存在甲基化修飾對DNA結構及基因表現調控的可能影響，則仍不清楚。細胞癌化的重要特徵之一為其具端粒酶（telomerase）的活性，人類端粒酶反轉錄酶hTERT (human telomerase reverse transcriptase)作為端粒酶的主要組成之一，其基因過度表現對於端粒酶活性的影響扮演主要角色。文獻中已知hTERT 轉錄調控與DNA甲基化的位置有關。第一個外顯子(exon) 之高度甲機化(hypermethylation)可阻止轉錄阻遏因子(repressor) CTCF (CCCTC-binding factor)結合，為大部分癌細胞之hTERT基因表現所需。以基因序列分析，該CTCF結合區域也存在具有G-quadruplex結構的可能性，故合適以之探討G-quadruplex和DNA甲基化兩者可能的交互作用及影響轉錄因子結合調控基因表現機制。本研究以核磁共振(Nuclear Magnetic Resonance, NMR)確認受到甲基化調控的CTCF結合位序列會形成G-quadruplex結構。我們建構了含hTERT 啟動子區域且包含不同甲基化修飾之報導基因質體(reporter gene plasmids)，從報導基因表現結果證實位於CTCF結合位之G-quadruplex結構，會和hTERT甲基化調控有交互影響而改變基因表現。也利用染色質免疫沉澱（Chromatin Immunoprecipitation）及凝膠遷移滯後實驗（electrophoretic mobility shift assays）確認CTCF結合能力會因為此交互作用影響而改變。本研究證明了G-quadruplex結構和甲基化之間的互相影響會透過影響轉錄因子CTCF的結合而改變hTERT基因表現，此為一過去未被報導過的hTERT基因調控分子機制。
DNA methylation is one of important epigenetic hallmark that regulating gene expression, especially for methylated CpG dinucleotide wildly spread in proximal promoter region leads to reverse gene activation between cancer and normal cell. G-quadruplex structure is a non-B DNA secondary structure formed in G-rich regions and involves in regulating gene expression. However, it remains unclear whether the status of cytosine methylation in the G-quadruplex will affect the DNA structure and further modulate gene expression. Cell immortalization is a key event in carcinogenesis, which requires activation of telomerase. Human telomerase reverse transcriptase (hTERT) is the major component of the catalytic subunit of telomerase and over-expressed in the majority of cancers. It has been shown that CTCF binding to the proximal exonic region of the hTERT gene could suppress its transcription in telomerase-negative cells, while the methylation of the first exon of hTERT prevents CTCF binding and results in hTERT gene expression in telomerase-positive cells. The CTCF binding site of hTERT first exon is capable of forming G-quadruplex structure via sequence analysis as well. In this project, we found that the methylation in the CpG islands could stabilize the G-quadruplex structure via NMR analysis. By results of reporter assays with different methylation pattern and mutant reporter plasmids, we found that the methylation promoted G-quadruplex structures in this CTCF binding region could alter the binding of CTCF and following hTERT expression. The results of ChIP and EMSA were also demonstrated that the relationship between CTCF binding and DNA methylation within G-quardruplex. Our results not only provide the possible explanation for transcriptional activation of hTERT but also offer a new example of how CpG dinucleotide methylation modulated DNA secondary structural formation.
Differences in bundle sheath and mesophyll cell development between C4 and C3 plants
在這個研究中我們建立了一套研究特定組織功能之系統，其中包含特定組織轉錄組之建立，生物訊息分析與鑑定，再利用病毒誘導基因沉默（VIGS）技術進行預測的調控因子初篩，最終利用CRISPR/Cas9基因編輯技術來做功能研究。首先我們建立冷凍切片系統，LCM 系統，low input RNA-seq library system。此系統可讓我們獲得高品質的RNA 跟轉錄組。我們總共擷取22組特定組織細胞包括(4組 BS cells; 3 組PM cells; 2 組M cells; 2組vascular cells)，每組兩重複。並完成18組轉錄組包括(4組 BS cells; 3組PM cells; 2 組M cells)， 每組兩重複。經由生物訊息的分析組間的差異，鑑定出組間表現差異的基因，更進一步篩選出調控花環結構或葉脈系統的發育上的基因。再利用病毒誘導基因沉默（VIGS）技術進行預測的調控因子之初篩，最終利用CRISPR/Cas9基因編輯技術來做功能研究。經由此系統之研究，鑑定出因基因功能喪失之植株影響維管束鞘細胞、葉肉細胞發育造成維管束的束型改變，進而影響葉片發育、生長遲緩、最終導致產量下降。
In this project we created a specific tissue transcriptome system in which we established a cryosection system, a LCM system, and a low input RNA-seq library system. Thess systems allow us to obtain high-quality RNA and transcriptome data. We collected 22 groups of tissue-specific cells, including 4 groups of BS cells, 3 groups of PM cells, 2 groups of M cells, and 2 groups of vascular cells. In addition, we completed 18 groups of transcriptiomes including 4 groups of BS cells, 3 groups of PM cells, and 2 groups of M cells, each group with two replicates. Bioinformatics analyses of the transcriptomes were conducted to identify genes differentially expressed between transcriptomes and especially transcription factor (TF) genes responsible for the differentiation in regulation between BS and M cells. Finally, through an approach by combining VIGS and CRISPR/Cas9 systems, knockout plants displaying a dramatic reduction in growth as well as disruptions in vascular patterning and spacing, leaf development, and BS and M cell patterning and development will be collected. In summary, this project used a combination of tissue specfic high-throughput technologies, bioinformatics skills, and molecular biology techniques to identify regulators involved in leaf and Kranz antomy development.
Figure 1. LCM-mediated isolation of Kranz tissue specific cells.
Sections before LCM (first column), sections after LCM (middle column), and captured cells on the cap (last column). (Notation: 4 stages of Kranz cells : 3, 4, 5, and 6 BS GM cells; 3 stages of palisade-like M cells (the same 3, 4, 5 BS cell stages PM cells); 2 stages of M cells (1 and 2 M cells); 2 stages of V cells (5 BS-V cells stage vascular cells; 10BS-Vcells stage vascular cells).
Figure 2 Whole-plant and leaf vascular phenotypes of knockout plants.
Photograph of 5-week-old plants. Knockout plants are smaller than W (wild-type), and have small grains (B) and thinner leaves (C). Light micrographs of Lugol’s staining leaf tissue (IKI-stained to reveal starch) shows that the regular parallel vein pattern (D-W) is disrupted in knockout leaves (D-2-8-7). Light microscope images of veins in knockout plant, a transverse section obtained two minor veins with merged bundle sheath (BS) cells (E).
Combination Nanotherapy of Liver Diseases
B型肝炎病毒(HBV)在感染人類的病原之中佔大宗。在本次實驗中，我們發現到在HBV表現的細胞中有兩個微小核醣核酸(micro-RNA; miR): miR-204及miR-1236的表現量下降，而且發現到這兩個miRNA能回過頭來抑制HBV的複製。我們利用生物資訊的方法以及reporter實驗找到了miR-1236透過結合到HBV的特定mRNA區域來抑制HBV的複製及蛋白質生成 。而另一方面，由生物資訊方法找到的miR-204則無法抑制HBV的RNA及蛋白質生成。但意外地，我們找到了miR-204能夠抑制HBV 前基因體(pregenomic) RNA的包裝(encapsidation)及病毒衣殼 (capsid) 的組裝。我們更進一步證實HBV透過活化宿主轉錄因子STAT3來抑制miR-204的表現量，驗證了HBV, miR-204及STAT3三者之間存在一個正向調控的路徑。有趣的是，miR-204已經在某些發表的文章中被認為是能夠抑制腫瘤的因子(tumor suppressor)，由於慢性B型肝炎的患者罹患肝癌的機率相對地高，我們推測HBV抑制miR-204的現象或許是促進肝癌發生的原因之一。在我們的研究中找到的兩個抑制HBV的miRNA: miR-204以及miR-1236，這兩者或許能透過新發展的微流體系統(microfluidic systems)技術，高通量地找尋出合適且能應用於治療HBV的合併療法。
Hepatitis B virus (HBV) is a major human pathogen. In this study, we found that miR-204 and miR-1236 were down-regulated in HBV-producing cells, and each could suppress HBV replication. Using a bioinformatic approach and a reporter assay, we identified miR-1236, which can reduce HBV replication and protein production by directly targeting at HBV specific mRNA. In contrast, miR-204 was identified by a microarray approach, and had no effect on HBV RNA and protein production. Surprisingly, miR-204 could inhibit HBV pregenomic RNA encapsidation and capsid assembly. We further demonstrated that HBV suppressed miR-204 expression via activating a host transcription factor STAT3. We established a positive feed-forward loop between HBV, miR-204 and STAT3. Interestingly, miR-204 has been considered as a tumor suppressor in some literature. Since the risk for hepatocellular carcinoma (HCC) is significantly increased in chronic HBV patients, it is possible that chronic suppression of miR-204 by HBV contributes to HCC incidence. Both miR-204 and miR-1236 might be useful for developing optimized combination therapy against HBV with the newly developed microfluidic systems for high throughput optimization.
Identification of the molecular mechanism of Klebsiella pneumoniae NTUH K2044-induced NETs formation and liver abscess
Klebsiella pneumoniae is an important pathogen causing community-acquired infections such as pneumonia and community-acquired pyogenic liver abscess (PLA). When encountered bacterial infection, neutrophils could engulf and kill the bacteria either by phagocytosis or form neutrophil extracellular traps (NETs) to bind and kill pathogens extracellularly. In this study, the K. pneumoniae NTUH-K2044 strain could induce NETs formation in vitro. Analysis of the genome of NTUH-K2044 strain revealed this strain harbors putative type VI secretion system (T6SS)-encoding genes which likely account for the host and pathogen interaction. Deletion of T6SS effector and structural proteins-encoding genes of the NTUH-K2044 all significantly reduced the activity of NETs formation. The PLA-associated K. pneumoniae utilized the T6SS-mediated competition to establish infection in mice. The NTUH-K2044 strain deleted for the T6SS genetic loci was profoundly attenuated in virulence as demonstrated in the mouse model of septicemia. These results obtained from this study will help us to understand the pathogenic mechanism of T6SS in K. pneumoniae and might identify potential targets for therapeutic design.
Development of Novel Small Molecule Inhibitors and MRI Probes Targeting Alzheimer’s Amyloid-β Oligomers
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder affecting tens of millions people. The pathologic hallmarks are extracellular senile plaque composed of amyloid-β (Aβ) fibrils and intracellular neurofibrillary tangles comprising hyperphosphorylated tau proteins. Blocking Aβ self-assembly or disassembling Aβ insoluble plague by small molecules would be potential therapeutic strategies to ameliorate Aβ aggregation-induced neuronal toxicity in AD. In work presented here, we synthesized and examined the effect of a series of new rationally designed bivalent synthetic compounds on Aβ fibrillization by thioflavin T (ThT) assay and by transmission electron microscopy (TEM). One of these compounds, diCA, the divalent amide form of caffeic acid (CA), possessing a propylene diamine linker of ~5.0 Å length that is close to the distance of intermolecular β sheets in Aβ fibrils showed good potency to inhibit Aβ fibrillization and reduce Aβ toxicity in human neuroblastoma and the Aβ transgenic C. elegans. In addition, the specific binding of diCA to Aβ was demonstrated by using electrospray ionization-traveling wave ion mobility-mass spectrometry (ESI-TWIM-MS). Molecular simulation was also conducted to gain structural insights for Aβ and diCA complex. Our results present a new potential for AD therapeutics.
阿茲海默症（AD）是影響數千萬人的漸進性神經變性。病理特徵包含由類澱粉蛋白β（Aβ）纖維組成的細胞外老年斑塊和含高度磷酸化tau蛋白的細胞內神經纖維糾結。阻斷Aβ組裝或透過小分子拆解Aβ不溶性斑塊將是改善AD中Aβ聚集誘導之神經元毒性的潛在治療策略。在這裡，我們合成了一系列新穎符合邏輯性設計的二價合成化合物，並檢視其對Aβ由硫代黃素T（ThT）測定和透射電子顯微鏡（TEM）觀測纖維化的影響。這些化合物之一，diCA，其結構是以約5.0 Å長度的丙二胺連接二個以醯胺形式存在的咖啡酸（CA，此長度接近於Aβ纖維中分子間β片的距離）顯示出良好的抑制Aβ纖維化的能力，並且減少Aβ在人類神經母細胞瘤和Aβ轉殖線蟲中的毒性。此外，透過使用電噴灑離子化 – 行波離子遷移率 – 質譜（ESI-TWIM-MS）我們證明了diCA與Aβ的專一性結合，並且由分子模擬我們獲得Aβ和diCA複合物的細部結構。我們的成果提出了一個新的AD治療潛在策略。
Neurobiological basis of rewarding circuitry involved in itch relief-induced pleasure: a chemo-optogenetic approach
“搔癢生樂受，無癢更安樂”（龍樹菩薩）。我們為何會去抓癢？癢可以定義為一種不愉快的感覺會引發去抓癢的欲望。在日常生活經驗中抓癢可以止癢且得到快感。然而，仍有數以百萬的人深受無法根治的癢所苦。癢與止癢的關係是我們身體與大腦的秘密之一。迄今我們對於止癢及止癢得到的快感內在的分子與神經生物機制仍然所知不多。有趣的是這兩件事顯然是一體的兩面。由於相似的腦區可能牽涉了止癢的快感與得到酬賞，值得進一步去探究其內在神經生物機制。植基於台大心理系賴文崧教授與中研院生醫所陳志成研究員兩個團隊長期的合作與豐碩的成果，我們團隊結合了化學遺傳學與光遺傳學兩項特質的化學-光遺傳學進一步合作去探討止癢與決策所牽涉的酬賞迴路與機制。我們近期初步的研究結果揭示了止癢及止癢引發之酬賞效果的分子調控與神經迴路，特別是位在視丘 Parvalbumin 神經細胞的 Asic 1a。目前正增加樣本數及實驗來驗證我們的研究發現。
“There is pleasure when an itch is scratched, but to be without an itch is more pleasurable still (by Buddhist philosopher Nagarjuna)”. Why do we scratch an itch? Itch is defined as an unpleasant sensation that evokes a desire to scratch. Our daily experience tells that scratching relieves itch and is pleasurable. However, this may be not the case for the millions of people who suffer from intractable itch. The itch-scratch relationship is one of the mysteries in our bodies and brain. Up to date, little is known about the molecular and neurobiological basis of itch-relief and the following pleasure. Interestingly, itch and itch relief-induced pleasure appear to be two sides of the same coin. Giving the factors that similar brain circuits and areas might be involved in the processes of reward and pain relief, it is of great interest to investing neurobiological basis of rewarding circuitry involved in itch and itch relief-induced pleasure. Based on our long-term collaboration between Dr. Wen-Sung Lai at NTU and Dr. Chih-Cheng Chen at Academia Sinica, we move one step further to explore the rewarding circuitry involved in itch relief-induced pleasure and decision making from a chemo-optogenetic, a combination of both chemogenetics and optogenetics, approach. Our recent data reveal the molecular determinants and neurobiological basis that involved in anti-itch circuitry and itch relief-induced pleasure, especially for Asic1a in the parvalbumin neurons in the thalamus. Currently, we are trying to increase our sample size and to reassure our exciting findings.
Using 1-D Spatially Resolved Silicon-Nanowire-Field-Effect-Transistor (SiNW-FET) Platform to Characterize Cell Membrane Proteins in Their Native Bilayer Environment.
Cell membranes are the gateway for the cells to send and receive signals. More than 60% of commercial drugs interact with membrane proteins to alter cellular biochemical pathways. Since membrane proteins are primary pharmaceutical targets, information about what ligands can bind to them is useful to facilitate drug discovery and development. Most of the current methods to identify the ligand binding to membrane proteins rely on immunodetection. However, immunodetection methods may alter biomolecular interactions. Here, we develop a label-free detection method based on field effect transistor (FET) technique, which can provide rapid and sensitive responses to the ligand binding. Supported lipid bilayers (SLBs) have been widely used as platforms for studying interactions between ligands and membrane proteins in vitro. To build a FET detection platform combined with SLBs, we chose graphene as the FET semiconductive material because the two-dimensionality is compatible with the planar bilayer platform and it has high sensitivity. We fabricated graphene field effect transistor (GFET) on silica substrates and investigated the formation of SLBs on the graphene/silica substrate. We discovered that the poly(methyl methacrylate) (PMMA) residues left on graphene surface can significantly influence the formation of SLBs on graphene and applying an extra annealing step to further clean the graphene surface is required for the robust SLB formation. In addition, because the fluorescent dyes in the SLB above graphene were almost completely quenched by graphene, which hinders us from using the conventional fluorescence recovery after photobleaching (FRAP) method to verify the formation of SLBs, we developed a modified FRAP method to examine whether SLBs successfully formed on the graphene/silica substrate. Furthermore, we discovered that ions in a regular buffer can gradually adsorb to the graphene surface and cause the Dirac point to shift significantly with time during our ligand binding detection. With careful control of the history of buffer addition and flow duration in a microfluidic device, we were able to eliminate the response caused by the ion adsorption and obtained the response only from the ligand binding. We used streptavidin-biotin-lipid as a model ligand-receptor to examine the GFET response to ligand binding on the SLB. Our results show that the SLB-GFET is capable to detect the streptavidin molecules binding onto the biotin-lipids embedded in the SLB, and the consistency between the electrical response and the fluorescent quantification result shows the quantitative detection capability of our GFET device. Incorporating this platform with the technique to obtain membrane vesicles from E. coli could allow us to study the interactions between potential drug candidates and interested membrane proteins in their native-like environment.