28 results on '"Kaewsapsak P"'
Search Results
2. mTORC2 interactome and localization determine aggressiveness of high-grade glioma cells through association with gelsolin
- Author
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Chantaravisoot, Naphat, Wongkongkathep, Piriya, Kalpongnukul, Nuttiya, Pacharakullanon, Narawit, Kaewsapsak, Pornchai, Ariyachet, Chaiyaboot, Loo, Joseph A., Tamanoi, Fuyuhiko, and Pisitkun, Trairak
- Published
- 2023
- Full Text
- View/download PDF
3. Deep transcriptome profiling reveals limited conservation of A-to-I RNA editing in Xenopus
- Author
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Tram Anh Nguyen, Jia Wei Joel Heng, Yan Ting Ng, Rui Sun, Shira Fisher, Gokce Oguz, Pornchai Kaewsapsak, Shifeng Xue, Bruno Reversade, Adaikalavan Ramasamy, Eli Eisenberg, and Meng How Tan
- Subjects
RNA editing ,ADAR ,Xenopus ,Biology (General) ,QH301-705.5 - Abstract
Abstract Background Xenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored. Results Here, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals. Conclusions Collectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal’s unique physiology or environmental adaptation.
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- 2023
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- View/download PDF
4. mTORC2 interactome and localization determine aggressiveness of high-grade glioma cells through association with gelsolin
- Author
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Naphat Chantaravisoot, Piriya Wongkongkathep, Nuttiya Kalpongnukul, Narawit Pacharakullanon, Pornchai Kaewsapsak, Chaiyaboot Ariyachet, Joseph A. Loo, Fuyuhiko Tamanoi, and Trairak Pisitkun
- Subjects
Medicine ,Science - Abstract
Abstract mTOR complex 2 (mTORC2) has been implicated as a key regulator of glioblastoma cell migration. However, the roles of mTORC2 in the migrational control process have not been entirely elucidated. Here, we elaborate that active mTORC2 is crucial for GBM cell motility. Inhibition of mTORC2 impaired cell movement and negatively affected microfilament and microtubule functions. We also aimed to characterize important players involved in the regulation of cell migration and other mTORC2-mediated cellular processes in GBM cells. Therefore, we quantitatively characterized the alteration of the mTORC2 interactome under selective conditions using affinity purification-mass spectrometry in glioblastoma. We demonstrated that changes in cell migration ability specifically altered mTORC2-associated proteins. GSN was identified as one of the most dynamic proteins. The mTORC2-GSN linkage was mostly highlighted in high-grade glioma cells, connecting functional mTORC2 to multiple proteins responsible for directional cell movement in GBM. Loss of GSN disconnected mTORC2 from numerous cytoskeletal proteins and affected the membrane localization of mTORC2. In addition, we reported 86 stable mTORC2-interacting proteins involved in diverse molecular functions, predominantly cytoskeletal remodeling, in GBM. Our findings might help expand future opportunities for predicting the highly migratory phenotype of brain cancers in clinical investigations.
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- 2023
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5. Direct identification of A-to-I editing sites with nanopore native RNA sequencing
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Nguyen, Tram Anh, Heng, Jia Wei Joel, Kaewsapsak, Pornchai, Kok, Eng Piew Louis, Stanojević, Dominik, Liu, Hao, Cardilla, Angelysia, Praditya, Albert, Yi, Zirong, Lin, Mingwan, Aw, Jong Ghut Ashley, Ho, Yin Ying, Peh, Kai Lay Esther, Wang, Yuanming, Zhong, Qixing, Heraud-Farlow, Jacki, Xue, Shifeng, Reversade, Bruno, Walkley, Carl, Ho, Ying Swan, Šikić, Mile, Wan, Yue, and Tan, Meng How
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- 2022
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6. An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
- Author
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Kean Hean Ooi, Mengying Mandy Liu, Jie Wen Douglas Tay, Seok Yee Teo, Pornchai Kaewsapsak, Shengyang Jin, Chun Kiat Lee, Jingwen Hou, Sebastian Maurer-Stroh, Weisi Lin, Benedict Yan, Gabriel Yan, Yong-Gui Gao, and Meng How Tan
- Subjects
Science - Abstract
As the COVID-19 pandemic continues, variants of the virus are emerging. Here the authors present a diagnostic assay that can detect wildtype and known variants using engineered Cas12a.
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- 2021
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7. An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
- Author
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Ooi, Kean Hean, Liu, Mengying Mandy, Tay, Jie Wen Douglas, Teo, Seok Yee, Kaewsapsak, Pornchai, Jin, Shengyang, Lee, Chun Kiat, Hou, Jingwen, Maurer-Stroh, Sebastian, Lin, Weisi, Yan, Benedict, Yan, Gabriel, Gao, Yong-Gui, and Tan, Meng How
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- 2021
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8. In Silico Evaluation of CRISPR-Based Assays for Effective Detection of SARS-CoV-2
- Author
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Pornchai Kaewsapsak, Naphat Chantaravisoot, Pattaraporn Nimsamer, Oraphan Mayuramart, Suwanan Mankhong, and Sunchai Payungporn
- Subjects
CRISPR ,SARS-CoV-2 ,COVID-19 ,variants of concern ,Medicine - Abstract
Coronavirus disease (COVID-19) caused by the SARS-CoV-2 has been an outbreak since late 2019 up to now. This pandemic causes rapid development in molecular detection technologies to diagnose viral infection for epidemic prevention. In addition to antigen test kit (ATK) and polymerase chain reaction (PCR), CRISPR-based assays for detection of SARS-CoV-2 have gained attention because it has a simple setup but still maintain high specificity and sensitivity. However, the SARS-CoV-2 has been continuing mutating over the past few years. Thus, molecular tools that rely on matching at the nucleotide level need to be reevaluated to preserve their specificity and sensitivity. Here, we analyzed how mutations in different variants of concern (VOC), including Alpha, Beta, Gamma, Delta, and Omicron strains, could introduce mismatches to the previously reported primers and crRNAs used in the CRISPR-Cas system. Over 40% of the primer sets and 15% of the crRNAs contain mismatches. Hence, primers and crRNAs in nucleic acid-based assays must be chosen carefully to pair up with SARS-CoV-2 variants. In conclusion, the data obtained from this study could be useful in selecting the conserved primers and crRNAs for effective detections against the VOC of SARS-CoV-2.
- Published
- 2022
- Full Text
- View/download PDF
9. Identification and validation of circulating miRNAs as potential new biomarkers for severe liver disease in patients with leptospirosis.
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Natthaya Chuaypen, Umaporn Limothai, Pattapon Kunadirek, Pornchai Kaewsapsak, Patipark Kueanjinda, Nattachai Srisawat, and Pisit Tangkijvanich
- Subjects
Medicine ,Science - Abstract
BackgroundLeptospirosis, a global zoonotic infectious disease, has various clinical manifestations ranging from mild self-limiting illness to life-threatening with multi-organ damage, including liver involvement. This study was aimed at identifying circulating microRNAs (miRNAs) as novel biomarkers for predicting severe liver involvement in patients with leptospirosis.MethodsIn a discovery set, 12 serum samples of patients with anicteric and icteric leptospirosis at initial clinical presentation were used for miRNA profiling by a NanoString nCounter miRNA assay. In a validated cohort, top candidate miRNAs were selected and further tested by qRT-PCR in serum samples of 81 and 16 individuals with anicteric and icteric leptospirosis, respectively.ResultsThe discovery set identified 38 significantly differential expression miRNAs between the two groups. Among these, miR-601 and miR-630 were selected as the top two candidates significantly up-regulated expressed in the icteric group. The enriched KEGG pathway showed that these miRNAs were mainly involved in immune responses and inflammation. In the validated cohort, miR-601 and miR-630 levels were significantly higher in the icteric group compared with the anicteric group. Additionally, these two miRNAs displayed good predictors of subsequent acute liver failure with a high sensitivity of 100%. On regression analysis, elevated miR-601 and miR-630 expression were also predictive of multi-organ failures and poor overall survival.ConclusionOur data indicated that miRNA expression profiles were significantly differentiated between the icteric and anicteric groups. Serum miR-601 and miR-630 at presentation could potentially serve as promising biomarkers for predicting subsequent acute liver failure and overall survival in patients with leptospirosis.
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- 2021
- Full Text
- View/download PDF
10. Publisher Correction: Determination of isoform-specific RNA structure with nanopore long reads
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Aw, Jong Ghut Ashley, Lim, Shaun W., Wang, Jia Xu, Lambert, Finnlay R. P., Tan, Wen Ting, Shen, Yang, Zhang, Yu, Kaewsapsak, Pornchai, Li, Chenhao, Ng, Sarah B., Vardy, Leah A., Tan, Meng How, Nagarajan, Niranjan, and Wan, Yue
- Published
- 2021
- Full Text
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11. Author Correction: Determination of isoform-specific RNA structure with nanopore long reads
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Aw, Jong Ghut Ashley, Lim, Shaun W., Wang, Jia Xu, Lambert, Finnlay R. P., Tan, Wen Ting, Shen, Yang, Zhang, Yu, Kaewsapsak, Pornchai, Li, Chenhao, Ng, Sarah B., Vardy, Leah A., Tan, Meng How, Nagarajan, Niranjan, and Wan, Yue
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- 2021
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12. Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking
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Pornchai Kaewsapsak, David Michael Shechner, William Mallard, John L Rinn, and Alice Y Ting
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RNA localization ,subcellular transcriptome ,peroxidase ,APEX2 ,Horseradish peroxidase ,RNA sequencing ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The spatial organization of RNA within cells is a crucial factor influencing a wide range of biological functions throughout all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, cytosol, and endoplasmic reticulum (ER), with specificity and sensitivity that rival or exceed those of conventional approaches. We further identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.
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- 2017
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13. Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells.
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Kalpongnukul, Nuttiya, Bootsri, Rungnapa, Wongkongkathep, Piriya, Kaewsapsak, Pornchai, Ariyachet, Chaiyaboot, Pisitkun, Trairak, and Chantaravisoot, Naphat
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- 2022
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14. A Sensitive and Specific Fluorescent RT-LAMP Assay for SARS-CoV-2 Detection in Clinical Samples.
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Kean Hean Ooi, Mengying Mandy Liu, Jia Rong Moo, Pattaraporn Nimsamer, Sunchai Payungporn, Pornchai Kaewsapsak, and Meng How Tan
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- 2022
- Full Text
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15. Tissue and Plasma-Based Highly Sensitive Blocker Displacement Amplicon Nanopore Sequencing for EGFR Mutations in Lung Cancer.
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Akkhasutthikun P, Kaewsapsak P, Nimsamer P, Klomkliew P, Visedthorn S, Chanchaem P, Teerapakpinyo C, Payungporn S, and Luangdilok S
- Subjects
- Humans, DNA, Neoplasm, Mutation, Protein Kinase Inhibitors therapeutic use, Real-Time Polymerase Chain Reaction, Carcinoma, Non-Small-Cell Lung diagnosis, Carcinoma, Non-Small-Cell Lung genetics, Cell-Free Nucleic Acids genetics, ErbB Receptors genetics, Lung Neoplasms diagnosis, Lung Neoplasms genetics, Nanopore Sequencing methods
- Abstract
Purpose: The epidermal growth factor receptor (EGFR) mutation is a widely prevalent oncogene driver in non-small cell lung cancer (NSCLC) in East Asia. The detection of EGFR mutations is a standard biomarker test performed routinely in patients with NSCLC for the selection of targeted therapy. Here, our objective was to develop a portable new technique for detecting EGFR (19Del, T790M, and L858R) mutations based on Nanopore sequencing., Materials and Methods: The assay employed a blocker displacement amplification (BDA)-based polymerase chain reaction (PCR) technique combined with Nanopore sequencing to detect EGFR mutations. Mutant and wild-type EGFR clones were generated from DNA from H1650 (19Del heterozygous) and H1975 (T790M and L858R heterozygous) lung cancer cell lines. Then, they were mixed to assess the performance of this technique for detecting low variant allele frequencies (VAFs). Subsequently, formalin-fixed, paraffin-embedded (FFPE) tissue and cell-free DNA (cfDNA) from patients with NSCLC were used for clinical validation., Results: The assay can detect low VAF at 0.5% mutant mixed in wild-type EGFR. Using FFPE DNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and Cobas real-time PCR were 98.46%, 100%, and 100%, respectively. For cfDNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and droplet digital PCR were 94.74%, 100%, and 100%, respectively., Conclusion: The BDA amplicon Nanopore sequencing is a highly accurate and sensitive method for the detection of EGFR mutations in clinical specimens.
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- 2024
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16. "Nano COVID-19": Nanopore sequencing of spike gene to identify SARS-CoV-2 variants of concern.
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Nimsamer P, Sawaswong V, Klomkliew P, Kaewsapsak P, Puenpa J, Poovorawan Y, and Payungporn S
- Subjects
- Humans, SARS-CoV-2 genetics, Phylogeny, Mutation, COVID-19 diagnosis, Nanopore Sequencing
- Abstract
Coronavirus disease 2019 (COVID-19) is a worldwide pandemic infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). World Health Organization (WHO) has defined the viral variants of concern (VOC) which cause more severe disease, higher transmissibility, and reduced vaccine efficacy. In this study, the "Nano COVID-19" workflow based on Oxford nanopore sequencing of the full-length spike gene combined with flexible data analysis options was developed to identify SARS-CoV-2 VOCs. The primers were designed to cover the full-length spike gene and can amplify all VOC strains. The results of VOC identification based on phylogenetic analysis of the full-length spike gene were comparable to the whole genome sequencing (WGS). Compared to the standard VOC identification pipeline, the fast analysis based on Read Assignment, Mapping, and Phylogenetic Analysis in Real Time (RAMPART) and the user-friendly method based on EPI2ME yielded 89.3% and 97.3% accuracy, respectively. The EPI2ME pipeline is recommended for researchers without bioinformatic skills, whereas RAMPART is more suitable for bioinformaticians. This workflow provides a cost-effective, simplified pipeline with a rapid turnaround time. Furthermore, it is portable to point-of-care SARS-CoV-2 VOC identification and compatible with large-scale analysis. Therefore, "Nano COVID-19" is an alternative viral epidemic screening and transmission tracking workflow., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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- 2023
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17. In vitro evaluation of the anti‑breast cancer properties and gene expression profiles of Thai traditional formulary medicine extracts.
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Khamwut A, Klomkliew P, Jumpathong W, Kaewsapsak P, Chanchaem P, Sivapornnukul P, Chantanakat K, T-Thienprasert NP, and Payungporn S
- Abstract
Breast cancer is a leading cause of cancer-related deaths worldwide. Moreover, standard treatments are limited, so new alternative treatments are required. Thai traditional formulary medicine (TTFM) utilizes certain herbs to treat different diseases due to their dominant properties including anti-fungal, anti-bacterial, antigenotoxic, anti-inflammatory and anti-cancer actions. However, very little is known about the anti-cancer properties of TTFM against breast cancer cells and the underlying molecular mechanism has not been elucidated. Therefore, the present study, evaluated the metabolite profiles of TTFM extracts, the anti-cancer activities of TTFM extracts, their effects on the apoptosis pathway and associated gene expression profiles. Liquid chromatography with tandem mass spectroscopy analysis identified a total of 226 compounds within the TTFM extracts. Several of these compounds have been previously shown to have an anti-cancer effect in certain cancer types. The MTT results demonstrated that the TTFM extracts significantly reduced the cell viability of the breast cancer 4T1 and MDA-MB-231 cell lines. Moreover, an apoptosis assay, demonstrated that the TTFM extracts significantly increased the proportion of apoptotic cells. Furthermore, the RNA-sequencing results demonstrated that 25 known genes were affected by TTFM treatment in 4T1 cells. TTFM treatment significantly up-regulated Slc5a8 and Arhgap9 expression compared with untreated cells. Moreover, Cybb , and Bach2os were significantly downregulated after TTFM treatment compared with untreated cells. Reverse transcription-quantitative PCR demonstrated that TTFM extract treatment significantly increased Slc5a8 and Arhgap9 mRNA expression levels and significantly decreased Cybb mRNA expression levels. Moreover, the mRNA expression levels of Bax and Casp9 were significantly increased after TTFM treatment in 4T1 cells compared with EpH4-Ev cells. These findings indicated anti-breast cancer activity via induction of the apoptotic process. However, further experiments are required to elucidate how TTFM specifically regulates genes and proteins. This study supports the potential usage of TTFM extracts for the development of anti-cancer drugs., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Khamwut et al.)
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- 2023
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18. Classification of salivary bacteriome in asymptomatic COVID-19 cases based on long-read nanopore sequencing.
- Author
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Jitvaropas R, Mayuramart O, Sawaswong V, Kaewsapsak P, and Payungporn S
- Subjects
- Humans, Cross-Sectional Studies, COVID-19 diagnosis
- Abstract
The coronavirus (COVID-19) global pandemic has impacted the health of almost everyone, including changes in their salivary microbiota. Since 2019, there has been an increase in the number of new COVID-19 cases in Thailand. Therefore, COVID-19 active case finding is important for early detection and epidemic control. Moreover, the dynamic changes of salivary bacteriome in asymptomatic COVID-19 cases are largely unknown. This research aimed to investigate and compare the salivary bacteriome and the co-infectious bacterial pathogens in the asymptomatic COVID-19 positive group to the negative group, based on novel nanopore sequencing. This cohort was a cross-sectional study including saliva samples collected from 82 asymptomatic participants (39 COVID-19 positive and 43 COVID-19 negative cases). All samples were sequenced for the full-length bacterial 16S rDNA. The alpha and beta diversity analyses were not significantly different between groups. The three major species in salivary bacteriome including Veillonella parvula , Streptococcus mitis , and Prevotella melaninogenica were observed in both groups. Interestingly, Lautropia mirabilis was a significantly enriched species in the saliva of the asymptomatic COVID-19-positive cases based on linear discriminant analysis effect size (LEfSe) analysis. The results suggested that L. mirabilis was a co-infectious agent in the asymptomatic COVID-19 group. However, the potential role of L. mirabilis should be validated in further experimental studies.
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- 2022
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19. In Silico Evaluation of CRISPR-Based Assays for Effective Detection of SARS-CoV-2.
- Author
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Kaewsapsak P, Chantaravisoot N, Nimsamer P, Mayuramart O, Mankhong S, and Payungporn S
- Abstract
Coronavirus disease (COVID-19) caused by the SARS-CoV-2 has been an outbreak since late 2019 up to now. This pandemic causes rapid development in molecular detection technologies to diagnose viral infection for epidemic prevention. In addition to antigen test kit (ATK) and polymerase chain reaction (PCR), CRISPR-based assays for detection of SARS-CoV-2 have gained attention because it has a simple setup but still maintain high specificity and sensitivity. However, the SARS-CoV-2 has been continuing mutating over the past few years. Thus, molecular tools that rely on matching at the nucleotide level need to be reevaluated to preserve their specificity and sensitivity. Here, we analyzed how mutations in different variants of concern (VOC), including Alpha, Beta, Gamma, Delta, and Omicron strains, could introduce mismatches to the previously reported primers and crRNAs used in the CRISPR-Cas system. Over 40% of the primer sets and 15% of the crRNAs contain mismatches. Hence, primers and crRNAs in nucleic acid-based assays must be chosen carefully to pair up with SARS-CoV-2 variants. In conclusion, the data obtained from this study could be useful in selecting the conserved primers and crRNAs for effective detections against the VOC of SARS-CoV-2.
- Published
- 2022
- Full Text
- View/download PDF
20. MicroRNA-223 Suppresses Human Hepatic Stellate Cell Activation Partly via Regulating the Actin Cytoskeleton and Alleviates Fibrosis in Organoid Models of Liver Injury.
- Author
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Ariyachet C, Chuaypen N, Kaewsapsak P, Chantaravisoot N, Jindatip D, Potikanond S, and Tangkijvanich P
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- Cell Proliferation, Humans, Liver Cirrhosis metabolism, Organoids metabolism, Signal Transduction, Actin Cytoskeleton metabolism, Hepatic Stellate Cells metabolism, MicroRNAs metabolism
- Abstract
MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate target mRNA expression, and altered expression of miRNAs is associated with liver pathological conditions. Recent studies in animal models have shown neutrophil/myeloid-specific microRNA-223 (miR-223) as a key regulator in the development of various liver diseases including fibrosis, where hepatic stellate cells (HSCs) are the key player in pathogenesis. However, the precise roles of miR-223 in human HSCs and its therapeutic potential to control fibrosis remain largely unexplored. Using primary human HSCs, we demonstrated that miR-223 suppressed the fibrogenic program and cellular proliferation while promoting features of quiescent HSCs including lipid re-accumulation and retinol storage. Furthermore, induction of miR-223 in HSCs decreased cellular motility and contraction. Mechanistically, miR-223 negatively regulated expression of smooth muscle α-actin (α-SMA) and thus reduced cytoskeletal activity, which is known to promote amplification of fibrogenic signals. Restoration of α-SMA in miR-223-overexpressing HSCs alleviated the antifibrotic effects of miR-223. Finally, to explore the therapeutic potential of miR-233 in liver fibrosis, we generated co-cultured organoids of HSCs with Huh7 hepatoma cells and challenged them with acetaminophen (APAP) or palmitic acid (PA) to induce hepatotoxicity. We showed that ectopic expression of miR-223 in HSCs attenuated fibrogenesis in the two human organoid models of liver injury, suggesting its potential application in antifibrotic therapy.
- Published
- 2022
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21. COVID-19 active case findings based on self-collected saliva samples with CRISPR-Cas12a detection.
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Chantaravisoot N, Kaewsapsak P, Mayuramart O, Nimsamer P, Mankhong S, Chomta N, Bootsri R, Alee I, Wongkongkathep P, Treeprasertsuk S, and Payungporn S
- Subjects
- CRISPR-Cas Systems genetics, Humans, Pandemics prevention & control, SARS-CoV-2 genetics, Saliva, Sensitivity and Specificity, COVID-19 diagnosis
- Abstract
COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus affecting the world population. Early detection has become one of the most successful strategies to alleviate the epidemic and pandemic of this contagious coronavirus. Surveillance testing programs have been initiated in many countries worldwide to prevent the outbreak of COVID-19. In this study, we demonstrated that our previously established clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay could detect variants of concern during 2021 in Thailand, including Alpha, Beta, and Delta strains as well as Omicron strain in early 2022. In combination with the newly designed saliva collection funnel, we established a safe, simple, economical, and efficient self-collection protocol for the COVID-19 screening process. We successfully utilized the assay in an active case finding with a total number of 578 asymptomatic participants to detect the SARS-CoV-2 in saliva samples. We finally demonstrated that the validation and evaluation in a large-scale setting could provide valuable information and elaborate the practicality of the test in real-world settings. Our optimized protocol yielded effective results with high sensitivity, specificity, and diagnostic accuracy (96.86%). In addition, this study demonstrates COVID-19 active case findings in low-resource settings, which would be feasible and attractive for surveillance and outbreak prevention in the future.
- Published
- 2022
- Full Text
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22. A Sensitive and Specific Fluorescent RT-LAMP Assay for SARS-CoV-2 Detection in Clinical Samples.
- Author
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Ooi KH, Liu MM, Moo JR, Nimsamer P, Payungporn S, Kaewsapsak P, and Tan MH
- Subjects
- Humans, COVID-19 genetics, COVID-19 Nucleic Acid Testing, Molecular Diagnostic Techniques, Nucleic Acid Amplification Techniques, RNA, Viral genetics, SARS-CoV-2 genetics
- Abstract
The raging COVID-19 pandemic has created an unprecedented demand for frequent and widespread testing to limit viral transmission. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a promising diagnostic platform for rapid detection of SARS-CoV-2, in part because it can be performed with simple instrumentation. However, isothermal amplification methods frequently yield spurious amplicons even in the absence of a template. Consequently, RT-LAMP assays can produce false positive results when they are based on generic intercalating dyes or pH-sensitive indicators. Here, we report the development of a sensitive RT-LAMP assay that leverages on a novel sequence-specific probe to guard against spurious amplicons. We show that our optimized fluorescent assay, termed LANTERN, takes only 30 min to complete and can be applied directly on swab or saliva samples. Furthermore, utilizing clinical RNA samples from 52 patients with COVID-19 infection and 21 healthy individuals, we demonstrate that our diagnostic test exhibits a specificity and positive predictive value of 95% with a sensitivity of 8 copies per reaction. Hence, our new probe-based RT-LAMP assay can serve as an inexpensive method for point-of-need diagnosis of COVID-19 and other infectious diseases.
- Published
- 2022
- Full Text
- View/download PDF
23. Identification and validation of circulating miRNAs as potential new biomarkers for severe liver disease in patients with leptospirosis.
- Author
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Chuaypen N, Limothai U, Kunadirek P, Kaewsapsak P, Kueanjinda P, Srisawat N, and Tangkijvanich P
- Subjects
- Adult, Aged, Biomarkers blood, Female, Gene Expression Profiling, Gene Ontology, Humans, Leptospirosis blood, Leptospirosis genetics, Liver Diseases genetics, Male, Metabolic Networks and Pathways, MicroRNAs blood, Middle Aged, Molecular Diagnostic Techniques, Predictive Value of Tests, Real-Time Polymerase Chain Reaction, Survival Analysis, Circulating MicroRNA blood, Leptospirosis complications, Liver Diseases diagnosis, Liver Diseases etiology
- Abstract
Background: Leptospirosis, a global zoonotic infectious disease, has various clinical manifestations ranging from mild self-limiting illness to life-threatening with multi-organ damage, including liver involvement. This study was aimed at identifying circulating microRNAs (miRNAs) as novel biomarkers for predicting severe liver involvement in patients with leptospirosis., Methods: In a discovery set, 12 serum samples of patients with anicteric and icteric leptospirosis at initial clinical presentation were used for miRNA profiling by a NanoString nCounter miRNA assay. In a validated cohort, top candidate miRNAs were selected and further tested by qRT-PCR in serum samples of 81 and 16 individuals with anicteric and icteric leptospirosis, respectively., Results: The discovery set identified 38 significantly differential expression miRNAs between the two groups. Among these, miR-601 and miR-630 were selected as the top two candidates significantly up-regulated expressed in the icteric group. The enriched KEGG pathway showed that these miRNAs were mainly involved in immune responses and inflammation. In the validated cohort, miR-601 and miR-630 levels were significantly higher in the icteric group compared with the anicteric group. Additionally, these two miRNAs displayed good predictors of subsequent acute liver failure with a high sensitivity of 100%. On regression analysis, elevated miR-601 and miR-630 expression were also predictive of multi-organ failures and poor overall survival., Conclusion: Our data indicated that miRNA expression profiles were significantly differentiated between the icteric and anicteric groups. Serum miR-601 and miR-630 at presentation could potentially serve as promising biomarkers for predicting subsequent acute liver failure and overall survival in patients with leptospirosis., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
- Full Text
- View/download PDF
24. Determination of isoform-specific RNA structure with nanopore long reads.
- Author
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Aw JGA, Lim SW, Wang JX, Lambert FRP, Tan WT, Shen Y, Zhang Y, Kaewsapsak P, Li C, Ng SB, Vardy LA, Tan MH, Nagarajan N, and Wan Y
- Subjects
- Human Embryonic Stem Cells metabolism, Humans, Isomerism, RNA genetics, Tetrahymena genetics, Transcriptome, Nanopore Sequencing methods, Nucleic Acid Conformation, RNA chemistry, Sequence Analysis, RNA methods
- Abstract
Current methods for determining RNA structure with short-read sequencing cannot capture most differences between distinct transcript isoforms. Here we present RNA structure analysis using nanopore sequencing (PORE-cupine), which combines structure probing using chemical modifications with direct long-read RNA sequencing and machine learning to detect secondary structures in cellular RNAs. PORE-cupine also captures global structural features, such as RNA-binding-protein binding sites and reactivity differences at single-nucleotide variants. We show that shared sequences in different transcript isoforms of the same gene can fold into different structures, highlighting the importance of long-read sequencing for obtaining phase information. We also demonstrate that structural differences between transcript isoforms of the same gene lead to differences in translation efficiency. By revealing isoform-specific RNA structure, PORE-cupine will deepen understanding of the role of structures in controlling gene regulation.
- Published
- 2021
- Full Text
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25. Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators.
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Li J, Han S, Li H, Udeshi ND, Svinkina T, Mani DR, Xu C, Guajardo R, Xie Q, Li T, Luginbuhl DJ, Wu B, McLaughlin CN, Xie A, Kaewsapsak P, Quake SR, Carr SA, Ting AY, and Luo L
- Subjects
- Animals, Axons metabolism, Brain metabolism, Dendrites metabolism, Drosophila Proteins metabolism, Drosophila melanogaster metabolism, Gene Expression Profiling methods, Gene Expression Regulation, Developmental genetics, Membrane Proteins metabolism, Neurogenesis physiology, Olfactory Nerve metabolism, Olfactory Pathways cytology, Olfactory Pathways physiology, Receptors, Lipoprotein metabolism, Smell physiology, Olfactory Pathways metabolism, Olfactory Receptor Neurons metabolism, Proteomics methods
- Abstract
Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
26. Atlas of Subcellular RNA Localization Revealed by APEX-Seq.
- Author
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Fazal FM, Han S, Parker KR, Kaewsapsak P, Xu J, Boettiger AN, Chang HY, and Ting AY
- Subjects
- Fluorescent Dyes chemistry, HEK293 Cells, Humans, Microscopy, Fluorescence, Mitochondria genetics, RNA chemistry, RNA, Messenger chemistry, RNA, Messenger metabolism, Transcriptome, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism, Endonucleases metabolism, Multifunctional Enzymes metabolism, RNA metabolism, Sequence Analysis, RNA methods
- Abstract
We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
27. Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking.
- Author
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Kaewsapsak P, Shechner DM, Mallard W, Rinn JL, and Ting AY
- Subjects
- Biotinylation methods, Cell Line, Humans, Proteins metabolism, Gene Expression Profiling methods, Organelles chemistry, RNA analysis, Staining and Labeling methods
- Abstract
The spatial organization of RNA within cells is a crucial factor influencing a wide range of biological functions throughout all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, cytosol, and endoplasmic reticulum (ER), with specificity and sensitivity that rival or exceed those of conventional approaches. We further identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.
- Published
- 2017
- Full Text
- View/download PDF
28. Recruiting the host's immune system to target Helicobacter pylori's surface glycans.
- Author
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Kaewsapsak P, Esonu O, and Dube DH
- Subjects
- Animals, Azides chemistry, Cell Line, Helicobacter Infections therapy, Humans, Immune System drug effects, Phosphines chemistry, Polysaccharides, Bacterial chemistry, Adjuvants, Immunologic chemistry, Adjuvants, Immunologic pharmacology, Helicobacter Infections immunology, Helicobacter pylori immunology, Polysaccharides, Bacterial immunology
- Abstract
Due to the increased prevalence of bacterial strains that are resistant to existing antibiotics, there is an urgent need for new antibacterial strategies. Bacterial glycans are an attractive target for new treatments, as they are frequently linked to pathogenesis and contain distinctive structures that are absent in humans. We set out to develop a novel targeting strategy based on surface glycans present on the gastric pathogen Helicobacter pylori (Hp). In this study, metabolic labeling of bacterial glycans with an azide-containing sugar allowed selective delivery of immune stimulants to azide-covered Hp. We established that Hp's surface glycans are labeled by treatment with the metabolic substrate peracetylated N-azidoacetylglucosamine (Ac4 GlcNAz). By contrast, mammalian cells treated with Ac4 GlcNAz exhibited no incorporation of the chemical label within extracellular glycans. We further demonstrated that the Staudinger ligation between azides and phosphines proceeds under acidic conditions with only a small loss of efficiency. We then targeted azide-covered Hp with phosphines conjugated to the immune stimulant 2,4-dinitrophenyl (DNP), a compound capable of directing a host immune response against these cells. Finally, we report that immune effector cells catalyze selective damage in vitro to DNP-covered Hp in the presence of anti-DNP antibodies. The technology reported herein represents a novel strategy to target Hp based on its glycans., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2013
- Full Text
- View/download PDF
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