Innovative image-based assays for anti-malaria and toxoplasmosis drug discovery

Lawrence S. Ayong 한국실험동물학회 2012 한국실험동물학회 학술발표대회 논문집 Vol.2012 No.8

에너지헌장조약(ECT)의 잠정적용과 국제투자협정중재에의 동의

임아영(Lim Ayong) 법무부 국제법무정책과 2016 통상법률 Vol.- No.129

On April 20th 2016, The Hague District Court rendered a set-aside judgment of the investment treaty arbitral awards between Yukos Universal Limited, Hulley Enterprises, Veteran Petroleum Limited—shareholders of Yukos Oil Corporation OJSC—and the Russian Federation. The main reason of the set-aside was that Article 26 of the Energy Charter Treaty, which provides the basis of consent for investment treaty arbitration, cannot be provisionally applied vis-a-vis Russia. The Russian Federation had signed the Energy Charter Treaty on December 17th 1994, but had not ratified it until it eventually notified Portugal, the Depository State of the treaty, that Russia no longer intended to maintain a Signatory status to the Energy Charter Treaty. Thus, the Energy Charter Treaty had never formally entered into force in Russia. The key issue is whether Article 26 of the Energy Charter Treaty can be interpreted as to provisionally apply when Russia has signed, but has not ratified the treaty in question. The Hague District Court—unlike the Arbitral Tribunal in the Yukos Arbitration—was of the view that the ‘Piecemeal approach’ could be taken with respect to the Energy Charter Treaty, and that because the provisional application of Energy Charter Treaty Article 26 is incompatible with Russian domestic law, the provision cannot be provisionally applied in accordance with Article 45(1) of the Energy Charter Treaty; hence, the arbitral award must be set-aside since there is no valid consent to arbitration. This article will provide a critical analysis of The Hague District Court’s set-aside judgment of the Yukos arbitral award, and raises several questions of the Court’s rationale based on the following observations. The Piecemeal approach taken by the Court is compatible with the literal interpretation of the Energy Charter Treaty and discussions on the provisional application of treaties. On the issue of compatibility between Article 26 of the Energy Charter Treaty and Russian domestic law, questions can be raised as to (1) the categorization of investor-State disputes as public law disputes under domestic law; and (2) the broad range of ‘domestic law’ as interpreted and applied by the Court. As the implications of the Yukos set-aside judgment is expected to affect not only the pending enforcement proceedings of Yukos arbitral awards worldwide, but also subsequent investment dispute settlement cases under the Energy Charter Treaty, it is yet to be seen whether the appellate courts will put forth a decision properly addressing the tension between the provisional application of treaties and the ratification procedures under domestic law, as well as restoring the balance between the investor and the State.

Isolation and Proteomic Analysis of Extracellular Vesicles from Lactobacillus salivarius SNK-6

Huang Jiwen,Zhao Ayong,He Daqian,Wu Xiao,Yan Huaxiang,Zhu Lihui 한국미생물·생명공학회 2024 Journal of microbiology and biotechnology Vol.34 No.1

The proteins carried by the extracellular vesicles of Lactobacillus salivarius SNK-6 (LsEVs) were identified to provide a foundation for further explorations of the probiotic activities of L. salivarius SNK-6. LsEVs were isolated from the culture media of L. salivarius SNK-6 and morphological analysis was conducted by scanning electron microscopy. Subsequent transmission electron microscopy and nanoparticle tracking analysis were performed to assess the morphology and particle size of the LsEVs. In addition, the protein composition of LsEVs was analyzed using silver staining and protein mass spectrometry. Finally, internalization of the identified LsEVs was confirmed using a confocal microscope, and enzyme-linked immunosorbent assay was employed to analyze the levels of inflammatory cytokines in LPS-challenged RAW264.7 cells. The results revealed that the membraneenclosed LsEVs were spherical, with diameters ranging from 100–250 nm. The LsEVs with diameters of 111–256 nm contained the greatest amount of cargo. In total, 320 proteins (10–38 kD) were identified in the LsEVs and included anti-inflammatory molecules, such as PrtP proteinase, cochaperones, and elongation factor Tu, as well as some proteins involved in glycolysis/gluconeogenesis, such as fructose-1,6-bisphosphate aldolase. Enrichment analysis showed these proteins to be related to the terms “metabolic pathway,” “ribosome,” “glycolysis/gluconeogenesis,” “carbohydrate metabolism,” and “amino acid metabolism.” Furthermore, the LsEVs were internalized by host liver cells and can regulate inflammation. These findings confirm that LsEVs contain various functional proteins that play important roles in energy metabolism, signal transduction, and biosynthesis.

Transcriptome analysis of the livers of ducklings hatched normally and with assistance

Yali Liu,Shishan He,Tao Zeng,Xue Du,Junda Shen,Ayong Zhao,Lizhi Lu 아세아·태평양축산학회 2017 Animal Bioscience Vol.30 No.6

Objective: “Hatchability” is an important economic trait in domestic poultry. Studies on poultry hatchability focus mainly on the genetic background, egg quality, and incubation conditions, whereas the molecular mechanisms behind the phenomenon that some ducklings failed to break their eggshells are poorly understood. Methods: In this study, the transcriptional differences between the livers of normally hatched and assisted ducklings were systematically analyzed. Results: The results showed that the clean reads were de novo assembled into 161,804 and 159,083 unigenes (≥200-bp long) by using Trinity, with an average length of 1,206 bp and 882 bp, respectively. The defined criteria of the absolute value of log2 fold-change ≥1 and false discovery rate≤0.05 were differentially expressed and were significant. As a result, 1,629 unigenes were identified, the assisted ducklings showed 510 significantly upregulated and 1,119 significantly down-regulated unigenes. In general, the metabolic rate in the livers of the assisted ducklings was lower than that in the normal ducklings; however, compared to normal ducklings, glucose- 6-phosphatase and ATP synthase subunit alpha 1 associated with energy metabolism were significantly upregulated in the assisted group. The genes involved in immune defense such as major histocompatibility complex (MHC) class I antigen alpha chain and MHC class II beta chain 1 were downregulated in the assisted ducklings. Conclusion: These data provide abundant sequence resources for studying the functional genome of the livers in ducks and other poultry. In addition, our study provided insight into the molecular mechanism by which the phenomenon of weak embryos is regulated.

황해 대륙붕에 서식하는 중형저서동물 군집 특성

정민규,김동성,강태욱,오제혁,신아영,오철웅,JUNG, MIN GYU,KIM, DONGSUNG,KANG, TEAWOOK,OH, JE HYEOK,SHIN, AYONG,OH, CHUL WOONG 한국해양학회 2022 바다 Vol.27 No.2

This study aims to identify the community characteristics of meiofauna inhabiting the Yellow Sea continental shelf. To this end an annual survey was conducted considering the seasons from 2018 to 2020 at 13 stations with a depth of 18~90 m of the Yellow Sea located at latitudes 35, 36 and 37 degrees north latitude. The survey was conducted in three seasons of spring, summer, and autumn at 5 stations in October 2018, 9 stations in April 2019, and 6 stations in August 2020 was used to collect 3 repetitions at each station. The habitat density of meiobenthos in the surveyed area was in the range of 45~1029 inds./10 cm², which was similar to the previous studies conducted in the Yellow Sea. The density of meiobenthos according to the seasons was 800±69 inds./10 cm² in autumn, the highest, and the lowest at 260±48 inds./10 cm² in summer. A total of 19 taxa appeared in meiobenthos, and the average value showed the number of nine taxa. Among the appearing taxa, the most dominant taxon was nematodes, accounting for 80.8% of the total density, followed by benthic copepods (8.8%) and benthic foraminifers (4.7%). As for the size distribution of medium benthic animals, the density of organisms corresponding to the size of 63~125 ㎛ was the highest, and 1~0.5 mm was the lowest. As for the vertical distribution in the sediments of medium benthic animals, the habitat density gradually decreased as the depth increased in the sediment surface layer. As a result of analysis of the N/C ratio, MPI, and ITD index using medium-sized benthic animals to identify the benthic environment, there were differences by season, but no values indicating pollution overall.

Isolation and identification of goose skeletal muscle satellite cells and preliminary study on the function of C1q and tumor necrosis factor-related protein 3 gene

Wang Han,He Ke,Zeng Xuehua,Zhou Xiaolong,Yan Feifei,Yang Songbai,Zhao Ayong 아세아·태평양축산학회 2021 Animal Bioscience Vol.34 No.6

Objective: Skeletal muscle satellite cells (SMSCs) are significant for the growth, regeneration, and maintenance of skeletal muscle after birth. However, currently, few studies have been performed on the isolation, culture and inducing differentiation of goose muscle satellite cells. Previous studies have shown that C1q and tumor necrosis factor-related protein 3 (CTRP3) participated in the process of muscle growth and development, but its role in the goose skeletal muscle development is not yet clear. This study aimed to isolate, culture, and identify the goose SMSCs in vitro. Additionally, to explore the function of CTRP3 in goose SMSCs. Methods: Goose SMSCs were isolated using 0.25% trypsin from leg muscle (LM) of 15 to 20 day fertilized goose eggs. Cell differentiation was induced by transferring the cells to differentiation medium with 2% horse serum and 1% penicillin streptomycin. Immunofluorescence staining of Desmin and Pax7 was used to identify goose SMSCs. Quantitative realtime polymerase chain reaction and western blot were applied to explore developmental expression profile of CTRP3 in LM and the regulation of CTRP3 on myosin heavy chains (MyHC), myogenin (MyoG) expression and Notch signaling pathway related genes expression. Results: The goose SMSCs were successfully isolated and cultured. The expression of Pax7 and Desmin were observed in the isolated cells. The expression of CTRP3 decreased significantly during leg muscle development. Overexpression of CTRP3 could enhance the expression of two myogenic differentiation marker genes, MyHC and MyoG. But knockdown of CTRP3 suppressed their expression. Furthermore, CTRP3 could repress the mRNA level of Notch signaling pathway-related genes, notch receptor 1, notch receptor 2 and hairy/ enhancer-of-split related with YRPW motif 1, which previously showed a negative regulation in myoblast differentiation. Conclusion: These findings provide a useful cell model for the future research on goose muscle development and suggest that CTRP3 may play an essential role in skeletal muscle growth of goose. Objective: Skeletal muscle satellite cells (SMSCs) are significant for the growth, regeneration, and maintenance of skeletal muscle after birth. However, currently, few studies have been performed on the isolation, culture and inducing differentiation of goose muscle satellite cells. Previous studies have shown that C1q and tumor necrosis factor-related protein 3 (CTRP3) participated in the process of muscle growth and development, but its role in the goose skeletal muscle development is not yet clear. This study aimed to isolate, culture, and identify the goose SMSCs <i>in vitro</i>. Additionally, to explore the function of CTRP3 in goose SMSCs.Methods: Goose SMSCs were isolated using 0.25% trypsin from leg muscle (LM) of 15 to 20 day fertilized goose eggs. Cell differentiation was induced by transferring the cells to differentiation medium with 2% horse serum and 1% penicillin streptomycin. Immunofluorescence staining of Desmin and Pax7 was used to identify goose SMSCs. Quantitative realtime polymerase chain reaction and western blot were applied to explore developmental expression profile of CTRP3 in LM and the regulation of CTRP3 on myosin heavy chains (MyHC), myogenin (MyoG) expression and Notch signaling pathway related genes expression.Results: The goose SMSCs were successfully isolated and cultured. The expression of <i>Pax7</i> and Desmin were observed in the isolated cells. The expression of CTRP3 decreased significantly during leg muscle development. Overexpression of CTRP3 could enhance the expression of two myogenic differentiation marker genes, <i>MyHC</i> and <i>MyoG</i>. But knockdown of CTRP3 suppressed their expression. Furthermore, CTRP3 could repress the mRNA level of Notch signaling pathway-related genes, notch receptor 1, notch receptor 2 and hairy/enhancer-of-split related with YRPW motif 1, which previously showed a negative regulation in myoblast differentiation.Conclusion: These findings provide a useful cell model for the future research on goose muscle development and suggest that CTRP3 may play an essential role in skeletal muscle growth of goose.

Chemosensitization potential of P-glycoprotein inhibitors in malaria parasites

Alcantara, L.M.,Kim, J.,Moraes, C.B.,Franco, C.H.,Franzoi, K.D.,Lee, S.,Freitas-Junior, L.H.,Ayong, L.S. Academic Press 2013 Experimental parasitology Vol.134 No.2

Members of the ATP-binding cassette (ABC)-type transporter superfamily have been implicated in multidrug resistance in malaria, and various mechanistic models have been postulated to explain their interaction with diverse antimalarial drugs. To gain insight into the pharmacological benefits of inhibiting ABC-type transporters in malaria chemotherapy, we investigated the in vitro chemosensitization potential of various P-glycoprotein inhibitors. A fluorescent chloroquine derivative was synthesized and used to assess the efflux dynamics of chloroquine in MDR and wild type Plasmodium falciparum parasites. This novel BODIPY-based probe accumulated in the digestive vacuole (DV) of CQ-sensitive parasites but less so in MDR cells. Pre-exposure of the MDR parasites to non-cytocidal concentrations of unlabeled chloroquine resulted in a diffused cytoplasmic retention of the probe whereas a similar treatment with the CQR-reversing agent, chlorpheniramine, resulted in DV accumulation. A diffused cytoplasmic distribution of the probe was also obtained following treatment with the P-gp specific inhibitors zosuquidar and tariquidar, whereas treatments with the tyrosine kinase inhibitors gefitinib or imatinib produced a partial accumulation within the DV. Isobologram analyses of the interactions between these inhibitors and the antimalarial drugs chloroquine, mefloquine, and artemisinin revealed distinct patterns of drug synergism, additivity and antagonism. Taken together, the data indicate that competitive tyrosine kinase and noncompetitive P-glycoprotein ATPase-specific inhibitors represent two new classes of chemosensitizing agents in malaria parasites, but caution against the indiscriminate use of these agents in antimalarial drug combinations.

Discovery of Carbohybrid-Based 2-Aminopyrimidine Analogues As a New Class of Rapid-Acting Antimalarial Agents Using Image-Based Cytological Profiling Assay

Lee, Sukjun,Lim, Donghyun,Lee, Eunyoung,Lee, Nakyung,Lee, Hong-gun,Cechetto, Jonathan,Liuzzi, Michel,Freitas-Junior, Lucio H.,Song, Jin Sook,Bae, Myung Ae,Oh, Sangmi,Ayong, Lawrence,Park, Seung Bum American Chemical Society 2014 Journal of medicinal chemistry Vol.57 No.17

<P>New antimalarial agents that exhibit multistage activities against drug-resistant strains of malaria parasites represent good starting points for developing next-generation antimalarial therapies. To facilitate the progression of such agents into the development phase, we developed an image-based parasitological screening method for defining drug effects on different asexual life cycle stages of <I>Plasmodium falciparum</I>. High-throughput screening of a newly assembled diversity-oriented synthetic library using this approach led to the identification of carbohybrid-based 2-aminopyrimidine compounds with fast-acting growth inhibitory activities against three laboratory strains of multidrug-resistant <I>P. falciparum</I>. Our structure–activity relationship study led to the identification of two derivatives (<B>8aA</B> and <B>11aA</B>) as the most promising antimalarial candidates (mean EC<SUB>50</SUB> of 0.130 and 0.096 μM against all three <I>P. falciparum</I> strains, selectivity indices >600, microsomal stabilities >80%, and mouse malaria ED<SUB>50</SUB> values of 0.32 and 0.12 mg/kg/day, respectively), targeting all major blood stages of multidrug-resistant <I>P. falciparum</I> parasites.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jmcmar/2014/jmcmar.2014.57.issue-17/jm5009693/production/images/medium/jm-2014-009693_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/jm5009693'>ACS Electronic Supporting Info</A></P>