Publications by authors named "Witthaya Poomipak"

10 Publications

  • Page 1 of 1

Comparative genome characterization of Leptospira interrogans from mild and severe leptospirosis patients.

Genomics Inform 2021 Sep 30;19(3):e31. Epub 2021 Sep 30.

Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.

Leptospirosis is a zoonotic disease caused by spirochetes from the genus Leptospira. In Thailand, Leptospira interrogans is a major cause of leptospirosis. Leptospirosis patients present with a wide range of clinical manifestations from asymptomatic, mild infections to severe illness involving organ failure. For better understanding the difference between Leptospira isolates causing mild and severe leptospirosis, illumina sequencing was used to sequence genomic DNA in both serotypes. DNA of Leptospira isolated from two patients, one with mild and another with severe symptoms, were included in this study. The paired-end reads were removed adapters and trimmed with Q30 score using Trimmomatic. Trimmed reads were constructed to contigs and scaffolds using SPAdes. Cross-contamination of scaffolds was evaluated by ContEst16s. Prokka tool for bacterial annotation was used to annotate sequences from both Leptospira isolates. Predicted amino acid sequences from Prokka were searched in EggNOG and David gene ontology database to characterize gene ontology. In addition, Leptospira from mild and severe patients, that passed the criteria e-value < 10e-5 from blastP against virulence factor database, were used to analyze with Venn diagram. From this study, we found 13 and 12 genes that were unique in the isolates from mild and severe patients, respectively. The 12 genes in the severe isolate might be virulence factor genes that affect disease severity. However, these genes should be validated in further study.
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http://dx.doi.org/10.5808/gi.21037DOI Listing
September 2021

Recent developments in neoantigen-based cancer vaccines.

Asian Pac J Allergy Immunol 2020 Jun;38(2):91-101

The Center of Excellence in Systems Biology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

Cynics point out that a cure for cancer has been "around the corner" for the last 50 years. Nevertheless, the recent convergence of deep DNA, RNA, and proteomic technologies with enhanced understanding of the nuances of the adaptive immune system has generated great optimism amongst researchers. The extraordinary heterogeneity of various cancers, once thought to be a major therapeutic hurdle, may now be bypassed via "personalized" vaccine treatments. Specifically, these treatments involve the identification of MHC-bound peptides that are unique to a patient's cancer (neoantigens), followed by immunization with peptides, RNA, or DNA that encodes these neoantigens via various delivery systems, thus amplifying the immune system's response to the particular cancer. Such approaches have shown dramatic results in animal studies. Not surprisingly, then, the field of neoantigen-based immunotherapy has advanced at a spectacular rate, necessitating that interested individuals stay apprised of recent developments. Following an introduction to the subject, we thus focus on aspects that are particularly fast-moving; the cellular sources of neoantigens, which are surprisingly diverse, the tools that are used for their identification, and the status of the numerous clinical trials that are now being conducted.
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http://dx.doi.org/10.12932/AP-120520-0841DOI Listing
June 2020

High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses.

Viruses 2019 10 25;11(11). Epub 2019 Oct 25.

Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.

Victoria and Yamagata lineages of influenza B viruses are globally circulating in seasonal epidemics. Madin-Darby canine kidney (MDCK) cells are permissive for viral isolation and vaccine manufacture. Nevertheless, the interplay between influenza B viruses and host microRNAs has not been investigated in this cell line. Therefore, the present study aims at high-throughput analysis of canine microRNA profile upon infection of influenza B viruses. Briefly, MDCK cells were infected with Victoria or Yamagata lineage at MOI of 0.01. After being harvested at 6, 12 and 24 h post infection, microRNAs were subjected to high-throughput sequencing based on MiSeq platform (Illumina). The results demonstrated that five microRNAs including cfa-miR-197, cfa-miR-215, cfa-miR361, cfa-miR-1841, and cfa-miR-1842 were overexpressed in both Victoria and Yamagata lineage infections. Interestingly, computational prediction showed that karyopherin alpha 6 (KPNA6) was targeted by cfa-miR-197 and cfa-miR-215. Moreover, the binding sites of both microRNAs were assessed by 3'-UTR reporter assay. The results showed that only cfa-miR-197 could bind to the target sites of KPNA6, leading to suppressing luciferase activity. Additionally, silencing of KPNA6 was confirmed by overexpression of cfa-miR-197. This study provides canine microRNA responses to seasonal influenza B viruses, suggesting that virus-mediated microRNAs might play crucial roles in host gene regulation.
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http://dx.doi.org/10.3390/v11110986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893747PMC
October 2019

Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells.

Mol Cell Proteomics 2018 11 10;17(11):2197-2215. Epub 2018 Aug 10.

¶Center of Excellence in Systems Biology, Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Interferon lambda (IFN-λ) is a relatively unexplored, yet promising antiviral agent. IFN-λ has recently been tested in clinical trials of chronic hepatitis B virus infection (CHB), with the advantage that side effects may be limited compared with IFN-α, as IFN-λ receptors are found only in epithelial cells. To date, IFN-λ's downstream signaling pathway remains largely unelucidated, particularly via proteomics methods. Here, we report that IFN-λ3 inhibits HBV replication in HepG2.2.15 cells, reducing levels of both HBV transcripts and intracellular HBV DNA. Quantitative proteomic analysis of HBV-transfected cells was performed following 24-hour IFN-λ3 treatment, with parallel IFN-α2a and PBS treatments for comparison using a dimethyl labeling method. The depth of the study allowed us to map the induction of antiviral proteins to multiple points of the viral life cycle, as well as facilitating the identification of antiviral proteins not previously known to be elicited upon HBV infection ( IFITM3, XRN2, and NT5C3A). This study also shows up-regulation of many effectors involved in antigen processing/presentation indicating that this cytokine exerted immunomodulatory effects through several essential molecules for these processes. Interestingly, the 2 subunits of the immunoproteasome cap (PSME1 and PSME2) were up-regulated whereas cap components of the constitutive proteasome were down-regulated upon both IFN treatments, suggesting coordinated modulation toward the antigen processing/presentation mode. Furthermore, in addition to confirming canonical activation of interferon-stimulated gene (ISG) transcription through the JAK-STAT pathway, we reveal that IFN-λ3 restored levels of RIG-I and RIG-G, proteins known to be suppressed by HBV. Enrichment analysis demonstrated that several biological processes including RNA metabolism, translation, and ER-targeting were differentially regulated upon treatment with IFN-λ3 IFN-α2a. Our proteomic data suggests that IFN-λ3 regulates an array of cellular processes to control HBV replication.
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http://dx.doi.org/10.1074/mcp.RA118.000735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210224PMC
November 2018

Human MicroRNAs Expression Profiles in Influenza B Virus-Infected Cells based on Illumina MiSeq Platform.

Microrna 2018 ;7(3):204-214

Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

Background: Influenza B virus causes influenza-like illness in humans. MicroRNAs (miRNAs) are small non-coding RNAs regulating gene expression through mRNA degradation or translational repression. MiRNAs have evolved to regulate many cellular processes including the viral infection response.

Objective: This study aims to investigate the miRNA profiles of human cells infected with influenza B virus.

Methods: A549 cells were infected with influenza B viruses (MOI = 0.5). MiRNAs were extracted at 24 and 48 hours post-infection. MiRNAs were used to construct four DNA libraries: influenza Binfected and an uninfected control for both time points. Then high-throughput sequencing was performed using the Miseq platform (Illumina). Sequencing data were analyzed by Miseq reporter software. The miRNAs were categorized and counted based on the frequency of reads. All filtered contigs were aligned with data from miRbase. The relative expression of each miRNA between uninfected and influenza B-infected cells was calculated.

Results: There were 13 down-regulated miRNAs and 21 up-regulated miRNAs observed in influenza B infected cells at 24 hours post infection. At 48 hours post infection, 14 miRNAs were downregulated, whereas 8 miRNAs were up-regulated.

Conclusion: This study suggested that miRNAs may play important roles in host gene regulation in response to viral infection.
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http://dx.doi.org/10.2174/2211536607666180515111048DOI Listing
December 2018

Comparison of Four Human Papillomavirus Genotyping Methods: Next-generation Sequencing, INNO-LiPA, Electrochemical DNA Chip, and Nested-PCR.

Ann Lab Med 2018 Mar;38(2):139-146

Center of Excellence in Clinical Virology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok.

Background: Human papillomavirus (HPV) infection causes cervical cancer, thus necessitating early detection by screening. Rapid and accurate HPV genotyping is crucial both for the assessment of patients with HPV infection and for surveillance studies.

Methods: Fifty-eight cervicovaginal samples were tested for HPV genotypes using four methods in parallel: nested-PCR followed by conventional sequencing, INNO-LiPA, electrochemical DNA chip, and next-generation sequencing (NGS).

Results: Seven HPV genotypes (16, 18, 31, 33, 45, 56, and 58) were identified by all four methods. Nineteen HPV genotypes were detected by NGS, but not by nested-PCR, INNO-LiPA, or electrochemical DNA chip.

Conclusions: Although NGS is relatively expensive and complex, it may serve as a sensitive HPV genotyping method. Because of its highly sensitive detection of multiple HPV genotypes, NGS may serve as an alternative for diagnostic HPV genotyping in certain situations.
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http://dx.doi.org/10.3343/alm.2018.38.2.139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736673PMC
March 2018

Original Research: Analysis of hepatic microRNA alterations in response to hepatitis B virus infection and pegylated interferon alpha-2a treatment.

Exp Biol Med (Maywood) 2016 10 4;241(16):1803-10. Epub 2016 May 4.

Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Research Unit of Hepatitis and Liver Cancer, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand

Interferons play important roles in defense mechanisms against viral infection, and thus interferon therapy has been a standard treatment in chronic hepatitis B patients. Interferons signaling pathways promote interferon-inducible genes including microRNAs. In this research, we aimed to determine microRNAs expression profiles in vitro and in vivo For in vitro model, Huh7 cells were transfected with or without hepatitis B virus plasmid for 6 h, and then treated with 100 ng of pegylated-interferon alpha-2a for 24 h. In vivo, we defined microRNAs expression profiles in pair-liver tissues of chronic hepatitis B patients in comparison between before and after treatment of pegylated-interferon alpha-2a for 48 weeks. Cellular small RNAs were extracted followed by library preparation. To determine microRNAs expression profiles, the next-generation sequencing was carried out on MiSeq platform (Illumina®). In vitro analysis demonstrated that microRNAs can be classified into up-regulated and down-regulated microRNAs in response to hepatitis B virus, interferon, and combination of hepatitis B virus and interferon. Moreover, in vivo analysis revealed microRNAs profiles in non-responders, responders without hepatitis B surface antigen clearance, and responders with hepatitis B surface antigen clearance. The target genes of the candidate microRNAs were determined in terms of roles in cellular pathways and immune response, which might be related to treatment in chronic hepatitis B patients. Results revealed that two down-regulated microRNAs including miR-185-5p and miR-186-5p were correlated in both in vitro and in vivo studies. These two microRNAs might be represented as specific hepatic microRNAs responding to hepatitis B virus and pegylated-interferon alpha-2a treatment, which may remarkable and attractive for further study involving in the association of their target genes and prediction of pegylated-interferon alpha-2a response. Interestingly, microRNAs expression patterns might be useful for understanding the response mechanism and serve as biomarkers for prediction of pegylated-interferon alpha-2a treatment response in patients with chronic hepatitis B.
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http://dx.doi.org/10.1177/1535370216647184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027934PMC
October 2016

DETECTION BY DUPLEX RT-COUPLED NESTED PCR OF HEPATITIS A AND ROTAVIRUS IN OYSTERS FROM THAILAND EAST COAST.

Southeast Asian J Trop Med Public Health 2015 Jul;46(4):624-39

Abstract. An efficient and rapid virus detection method is required for routine monitoring and risk assessment in food products. A duplex RT-coupled nested PCR method was developed to detect the simultaneous presence of hepatitis A virus (HAV) and rotavirus in commercial oysters from the eastern coast of Thailand. Primers were designed to amplify HAV VP4 and rotavirus VP7 genes. Although excess amounts of target template of one virus type interfered with RT-PCR am- plification of the other, this was overcome by including a nested duplex PCR step. Detection limit for both types of virus of this technique in oyster samples was more than 1,000-fold lower than that of the equivalent monoplex method. Out of 41 oyster samples 63% were positive for either one or both viruses. All rotaviruses belonged to group A G1P[8]. The use of multiplex RT-coupled nested PCR technique provides a cost-effective, rapid, sensitive and efficient tool to detect a wide diversity of viral pathogens and to improve control of virus infection in oysters.
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July 2015

Human microRNAs profiling in response to influenza A viruses (subtypes pH1N1, H3N2, and H5N1).

Exp Biol Med (Maywood) 2016 Feb 29;241(4):409-20. Epub 2015 Oct 29.

Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330 Thailand Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330 Thailand

MicroRNAs (miRNAs) play an important role in regulation of gene silencing and are involved in many cellular processes including inhibition of infected viral replication. This study investigated cellular miRNA expression profiles operating in response to influenza virus in early stage of infection which might be useful for understanding and control of viral infection. A549 cells were infected with different subtypes of influenza virus (pH1N1, H3N2 and H5N1). After 24 h post-infection, miRNAs were extracted and then used for DNA library construction. All DNA libraries with different indexes were pooled together with equal concentration, followed by high-throughput sequencing based on MiSeq platform. The miRNAs were identified and counted from sequencing data by using MiSeq reporter software. The miRNAs expressions were classified into up and downregulated miRNAs compared to those found in non-infected cells. Mostly, each subtype of influenza A virus triggered the upregulated responses in miRNA expression profiles. Hsa-miR-101, hsa-miR-193b, hsa-miR-23b, and hsa-miR-30e* were upregulated when infected with all three subtypes of influenza A virus. Target prediction results showed that virus infection can trigger genes in cellular process, metabolic process, developmental process and biological regulation. This study provided some insights into the cellular miRNA profiling in response to various subtypes of influenza A viruses in circulation and which have caused outbreaks in human population. The regulated miRNAs might be involved in virus-host interaction or host defense mechanism, which should be investigated for effective antiviral therapeutic interventions.
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http://dx.doi.org/10.1177/1535370215611764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935422PMC
February 2016

Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene.

Exp Biol Med (Maywood) 2015 Dec 15;240(12):1630-9. Epub 2015 Jun 15.

Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand

MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are involved in many cellular processes including inhibition of viral replication in infected cells. In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) were analyzed to identify candidate human miRNAs targeting and silencing viral genes expression. Candidate human miRNAs were predicted by miRBase and RNAhybrid based on minimum free energy (MFE) and hybridization patterns between human miRNAs and viral target genes. In silico analysis presented 76 miRNAs targeting influenza A viruses, including 70 miRNAs that targeted specific subtypes (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A viruses. Interestingly, miR-3145 is the only candidate miRNA targeting all three subtypes of influenza A viruses. The miR-3145 targets to PB1 encoding polymerase basic protein 1, which is the main component of the viral polymerase complex. The silencing effect of miR-3145 was validated by 3'-UTR reporter assay and inhibition of influenza viral replication in A549 cells. In 3'-UTR reporter assay, results revealed that miR-3145 triggered significant reduction of the luciferase activity. Moreover, expression of viral PB1 genes was also inhibited considerably (P value < 0.05) in viral infected cells expressing mimic miR-3145. In conclusion, this study demonstrated that human miR-3145 triggered silencing of viral PB1 genes and lead to inhibition of multiple subtypes of influenza viral replication. Therefore, hsa-miR-3145 might be useful for alternative treatment of influenza A viruses in the future.
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http://dx.doi.org/10.1177/1535370215589051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935342PMC
December 2015
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