Browsing by Author "Kurilung A."
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Item Metadata only Accurate Prediction of Ion Mobility Collision Cross-Section Using Ion’s Polarizability and Molecular Mass with Limited Data(2023-01-01) Wisanpitayakorn P.; Sartyoungkul S.; Kurilung A.; Sirivatanauksorn Y.; Visessanguan W.; Sathirapongsasuti N.; Khoomrung S.; Wisanpitayakorn P.; Mahidol UniversityThe rotationally averaged collision cross-section (CCS) determined by ion mobility-mass spectrometry (IM-MS) facilitates the identification of various biomolecules. Although machine learning (ML) models have recently emerged as a highly accurate approach for predicting CCS values, they rely on large data sets from various instruments, calibrants, and setups, which can introduce additional errors. In this study, we identified and validated that ion’s polarizability and mass-to-charge ratio (m/z) have the most significant predictive power for traveling-wave IM CCS values in relation to other physicochemical properties of ions. Constructed solely based on these two physicochemical properties, our CCS prediction approach demonstrated high accuracy (mean relative error of <3.0%) even when trained with limited data (15 CCS values). Given its ability to excel with limited data, our approach harbors immense potential for constructing a precisely predicted CCS database tailored to each distinct experimental setup. A Python script for CCS prediction using our approach is freely available at https://github.com/MSBSiriraj/SVR_CCSPrediction under the GNU General Public License (GPL) version 3.Item Metadata only An enhancement of extrachromosomal circular DNA enrichment and amplification to address the extremely low overlap between replicates(2026-04-01) Burnham C.M.; Kurilung A.; Wanchai V.; Regenberg B.; Delgado-Calle J.; Basnakian A.G.; Nookaew I.; Burnham C.M.; Mahidol UniversityExtrachromosomal circular DNA (eccDNA) of chromosomal origin is present in all eukaryotic organisms and tissues that have been tested. Populations of eccDNA exhibit immense diversity and a characteristically low degree of overlap between samples, suggesting low inheritance of eccDNA between cells or a deficiency in the methods by which eccDNA is detected. This study revisits the Circle-Seq approach for enrichment of eccDNA to address these limitations, hypothesizing that experimental procedures significantly contribute to the observed low eccDNA overlap. We optimized the protocol by reducing the time needed to complete the procedure. Linear DNA is digested by increasing Exonuclease V activity. We employed CRISPR-Cas9 for mitochondrial linearization, which proved superior to using restriction enzymes. A key finding is the critical role of random hexamer primer concentration and genomic DNA input in rolling circle amplification (RCA) for generating high-quality long concatemeric tandem copy amplicons from eccDNA, essential for confident de novo eccDNA construction from long-read sequencing data. Lower primer concentrations substantially increased the percentage of concatemer-derived eccDNA and improved the overlap of identified eccDNAs in technical replicates. Applying this revised approach to human myeloma and breast cancer cell lines, as well as xenograft models, demonstrated >50% overlap in detected eccDNA, a substantial improvement over the <1% overlap observed in previous studies. Additionally, the oncogenic signature of eccDNAs can be identified across all replicates. These findings provide guidelines for developing standardized procedures for eccDNA profiling, advancing our understanding of eccDNA biology, and its potential clinical applications.Item Metadata only CAIM: coverage-based analysis for identification of microbiome(2024-07-25) Acheampong D.A.; Jenjaroenpun P.; Wongsurawat T.; Kurilung A.; Pomyen Y.; Kandel S.; Kunadirek P.; Chuaypen N.; Kusonmano K.; Nookaew I.; Acheampong D.A.; Mahidol UniversityAccurate taxonomic profiling of microbial taxa in a metagenomic sample is vital to gain insights into microbial ecology. Recent advancements in sequencing technologies have contributed tremendously toward understanding these microbes at species resolution through a whole shotgun metagenomic approach. In this study, we developed a new bioinformatics tool, coverage-based analysis for identification of microbiome (CAIM), for accurate taxonomic classification and quantification within both long- and short-read metagenomic samples using an alignment-based method. CAIM depends on two different containment techniques to identify species in metagenomic samples using their genome coverage information to filter out false positives rather than the traditional approach of relative abundance. In addition, we propose a nucleotide-count-based abundance estimation, which yield lesser root mean square error than the traditional read-count approach. We evaluated the performance of CAIM on 28 metagenomic mock communities and 2 synthetic datasets by comparing it with other top-performing tools. CAIM maintained a consistently good performance across datasets in identifying microbial taxa and in estimating relative abundances than other tools. CAIM was then applied to a real dataset sequenced on both Nanopore (with and without amplification) and Illumina sequencing platforms and found high similarity of taxonomic profiles between the sequencing platforms. Lastly, CAIM was applied to fecal shotgun metagenomic datasets of 232 colorectal cancer patients and 229 controls obtained from 4 different countries and 44 primary liver cancer patients and 76 controls. The predictive performance of models using the genome-coverage cutoff was better than those using the relative-abundance cutoffs in discriminating colorectal cancer and primary liver cancer patients from healthy controls with a highly confident species markers.Item Metadata only Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs(2024-03-01) Boonciew P.; Saisongkorh W.; Brameld S.; Thongpin M.; Kurilung A.; Krangvichian P.; Niyomtham W.; Patarakul K.; Phichitraslip T.; Hampson D.J.; Prapasarakul N.; Boonciew P.; Mahidol UniversityLeptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.Item Metadata only LC-MS/MS identifies elevated imidazole propionate and gut-derived metabolite alterations in peritoneal dialysis patients(2025-01-01) Manokasemsan W.; Jariyasopit N.; Wanichthanarak K.; Poungsombat P.; Kurilung A.; Limjiasahapong S.; Thapa K.; Sirivatanauksorn Y.; Raksasuk S.; Srithongkul T.; Kitiyakara C.; Khoomrung S.; Manokasemsan W.; Mahidol UniversityWe developed a robust LC–MS/MS method for the simultaneous quantification of 16 uremic toxins (UTs) and 14 bile acids (BAs) in plasma and fecal samples within a single method. The method demonstrated high sensitivity, broad metabolite coverage, and excellent accuracy, precision, and throughput. Using this platform, targeted metabolites were quantified in peritoneal dialysis (PD) patients (n = 31) and healthy controls (HC; n = 60). Of the 30 targeted metabolites included in the validation method, 20 were detected in fecal samples and 12 in plasma in this study. Fecal samples exhibited greater BA diversity, whereas UTs were evenly distributed across both matrices. Fecal profiles showed minimal differences between PD and HC, suggesting limited gut-level alteration. In contrast, plasma analysis revealed nine metabolites significantly elevated in PD, including indoxyl sulfate, phenyl sulfate, hippuric acid, and imidazole propionate (ImP), lithocholic acid, cinnamoylglycine, m-hydroxyhippuric acid, phenylacetylglutamine, and phenylacetylglycine. Notably, plasma ImP—an underexplored metabolite—was elevated independently of diabetes or cardiovascular disease, implicating impaired renal clearance as its primary driver. These results highlight the systemic impact of gut-derived metabolites in kidney failure and position targeted UT–BA profiling as a powerful complementary tool for clinical metabolomics in chronic kidney disease and PD.Item Metadata only LC-QTOF-MSE with MS1-based precursor ion quantification and SiMD-assisted identification enhances human urine metabolite analysis(2025-01-01) Kurilung A.; Limjiasahapong S.; Wanichthanarak K.; Manokasemsan W.; Kaewnarin K.; Duangkumpha K.; Manocheewa S.; Tansawat R.; Chaiteerakij R.; Nookaew I.; Sirivatanauksorn Y.; Khoomrung S.; Kurilung A.; Mahidol UniversityThis study presents the development and validation of a liquid chromatography–quadrupole-time-of-flight mass spectrometry method with data-independent acquisition (LC-QTOF-MSE) for targeted quantification, post-targeted screening, and untargeted metabolite profiling. Using MS1-based precursor ion quantification, the method demonstrated excellent analytical performance with linearity (R² > 0.99), accuracy (84 %–131 %), and precision (1 %–17 % relative standard deviation (RSD)). Although LC-QTOF‑MSE sensitivity is at least nine-fold lower than LC-triple quadrupole MS with multiple reaction monitoring, it remains adequate for quantifying urinary metabolites, particularly those that fragment poorly or yield low‑intensity product ions. For post‑targeted screening and untargeted profiling, an in‑house reference library (the Siriraj Metabolomics Data Warehouse, SiMD), comprising 174 curated metabolite standards, was integrated into the workflow to enhance metabolite identification confidence. The official website for SiMD can be accessed at https://si-simd.com/. To demonstrate the method's utility, 11 amino and organic acids were quantified in urine samples from 100 healthy individuals. Four compounds—L-methionine, L-histidine, L-tryptophan, and trans-ferulic acid—were significantly higher levels in females (P < 0.05), likely reflecting sex-specific physiological or dietary intake differences. Post‑targeted screening identified 29 additional metabolites and assigned them to level 1 (m/z, RT, isotope pattern, and MS/MS spectra matched to reference standards) based on the Metabolomics Standards Initiative guidelines. Untargeted retrospective profiling revealed level 1 seven metabolites, including ribitol, creatine, glucuronic acid, trans-ferulic acid, succinic acid, dimethylglycine, and 3-hydroxyphenylacetic acid related to sex variation (VIP > 1.5). In summary, the LC-QTOF-MSE method coupled with SiMD provides a robust and comprehensive workflow for metabolomics analysis. It enables reliable target quantification and enhances confidence in metabolite identification while also reducing sample and instrumental demands. These features make it particularly well-suited for clinical metabolomics studies.Item Metadata only Measurement of very low-molecular weight metabolites by traveling wave ion mobility and its use in human urine samples(2024-05-01) Kurilung A.; Limjiasahapong S.; Kaewnarin K.; Wisanpitayakorn P.; Jariyasopit N.; Wanichthanarak K.; Sartyoungkul S.; Wong S.C.C.; Sathirapongsasuti N.; Kitiyakara C.; Sirivatanauksorn Y.; Khoomrung S.; Kurilung A.; Mahidol UniversityThe collision cross-sections (CCS) measurement using ion mobility spectrometry (IMS) in combination with mass spectrometry (MS) offers a great opportunity to increase confidence in metabolite identification. However, owing to the lack of sensitivity and resolution, IMS has an analytical challenge in studying the CCS values of very low-molecular-weight metabolites (VLMs ≤ 250 Da). Here, we describe an analytical method using ultrahigh-performance liquid chromatography (UPLC) coupled to a traveling wave ion mobility-quadrupole-time-of-flight mass spectrometer optimized for the measurement of VLMs in human urine samples. The experimental CCS values, along with mass spectral properties, were reported for the 174 metabolites. The experimental data included the mass-to-charge ratio (m/z), retention time (RT), tandem MS (MS/MS) spectra, and CCS values. Among the studied metabolites, 263 traveling wave ion mobility spectrometry (TWIMS)-derived CCS values (TWCCSN2) were reported for the first time, and more than 70% of these were CCS values of VLMs. The TWCCSN2 values were highly repeatable, with inter-day variations of <1% relative standard deviation (RSD). The developed method revealed excellent TWCCSN2 accuracy with a CCS difference (ΔCCS) within ±2% of the reported drift tube IMS (DTIMS) and TWIMS CCS values. The complexity of the urine matrix did not affect the precision of the method, as evidenced by ΔCCS within ±1.92%. According to the Metabolomics Standards Initiative, 55 urinary metabolites were identified with a confidence level of 1. Among these 55 metabolites, 53 (96%) were VLMs. The larger number of confirmed compounds found in this study was a result of the addition of TWCCSN2 values, which clearly increased metabolite identification confidence.Item Metadata only Novel Organization of the Staphylococcal Cassette Chromosome mec Composite Island in Clinical Staphylococcus haemolyticus and Staphylococcus hominis Subspecies hominis Isolates from Dogs(2022-08-01) Phumthanakorn N.; Wongsurawat T.; Jenjaroenpun P.; Kurilung A.; Prapasarakul N.; Mahidol UniversityStaphylococcus haemolyticus and Staphylococcus hominis subsp. hominis are common coagulase-negative staphylococcus opportunistic pathogens. In Thailand, the clinical strains S. haemolyticus 1864 and 48 and S. hominis subsp. hominis 384 and 371 have been recovered from sick dogs. These strains were methicillin resistant with the nontypeable staphylococcal cassette chromosome mec (NT-SCCmec). The SCCmec element distribution in the clinical isolates from dogs was analyzed using whole-genome sequencing, which revealed the presence of different SCCmec composite islands (CIs) and gene structure. The SCCmec-CIs of c SCCmec1864 (13 kb) and c SCC1864 (11 kb) with a class C1 mec complex but no ccr gene were discovered in S. haemolyticus 1864. The CIs of c SCCmec48 with a C1 mec complex (28 kb), SCC48 with ccrA4B4 (23 kb), and c SCC48 (2.6 kb) were discovered in S. haemolyticus 48. In SCC48, insertion sequence IS256 contained an aminoglycoside-resistant gene [aph(20)-Ia]. Two copies of IS431 containing the tetracycline-resistant gene tet(K) were found downstream of c SCC48. In S. hominis subsp. hominis, the SCCmec-CI in strain 384 had two separate sections: c SCCmec384 (20 kb) and SCCars (23 kb). c SCCmec384 lacked the ccr gene complex but carried the class A mec complex. Trimethoprim-resistant dihydrofolate reductase (dfrC) was discovered on c SCCmec384 between two copies of IS257. In strain 371, SCCmec VIII (4A) (37 kb) lacking a direct repeat at the chromosomal end was identified. This study found SCCmec elements in clinical isolates from dogs that were structurally complex and varied in their genetic content, with novel organization.Item Metadata only Quantifying fecal and plasma short-chain fatty acids in healthy Thai individuals(2024-12-01) Manokasemsan W.; Jariyasopit N.; Poungsombat P.; Kaewnarin K.; Wanichthanarak K.; Kurilung A.; Duangkumpha K.; Limjiasahapong S.; Pomyen Y.; Chaiteerakij R.; Tansawat R.; Srisawat C.; Sirivatanauksorn Y.; Sirivatanauksorn V.; Khoomrung S.; Manokasemsan W.; Mahidol UniversityShort-chain fatty acids (SCFAs) are involved in important physiological processes such as gut health and immune response, and changes in SCFA levels can be indicative of disease. Despite the importance of SCFAs in human health and disease, reference values for fecal and plasma SCFA concentrations in healthy individuals are scarce. To address this gap in current knowledge, we developed a simple and reliable derivatization-free GC-TOFMS method for quantifying fecal and plasma SCFAs in healthy individuals. We targeted six linear- and seven branched-SCFAs, obtaining method recoveries of 73–88% and 83–134% in fecal and plasma matrices, respectively. The developed methods are simpler, faster, and more sensitive than previously published methods and are well suited for large-scale studies. Analysis of samples from 157 medically confirmed healthy individuals showed that the total SCFAs in the feces and plasma were 34.1 ± 15.3 µmol/g and 60.0 ± 45.9 µM, respectively. In fecal samples, acetic acid (Ace), propionic acid (Pro), and butanoic acid (But) were all significant, collectively accounting for 89% of the total SCFAs, whereas the only major SCFA in plasma samples was Ace, constituting of 93% of the total plasma SCFAs. There were no statistically significant differences in the total fecal and plasma SCFA concentrations between sexes or among age groups. The data revealed, however, a positive correlation for several nutrients, such as carbohydrate, fat, iron from vegetables, and water, to most of the targeted SCFAs. This is the first large-scale study to report SCFA reference intervals in the plasma and feces of healthy individuals, and thereby delivers valuable data for microbiome, metabolomics, and biomarker research.Item Metadata only The Emergence of blaNDM-Encoding Plasmids in Enterobacteriaceae Isolated from Shared Water Resources for Livestock and Human Utilization in Central Thailand(2026-01-01) Songsaeng W.; Kurilung A.; Prapasarakul N.; Wongsurawat T.; Am-In N.; Lugsomya K.; Lohwacharin J.; Damrongsiri S.; Shein H.Z.; Sirichokchatchawan W.; Songsaeng W.; Mahidol UniversityBackground/Objectives: The environmental dissemination of antimicrobial-resistant Enterobacteriaceae poses a remarkable threat to public health. This study investigates the environmental presence and dissemination of carbapenemase-producing Enterobacteriaceae (CPE) in 30 important water bodies selected according to their interconnection with and utilization by livestock and community people in central Thailand. Methods: Water samples were collected from 30 selected water bodies. Enterobacteriaceae were isolated and screened for CPE and multidrug resistance. Carbapenemase genes (blaNDM-5, blaNDM-1 and blaIMI-1) were detected and their locations (plasmid and chromosome) determined. Plasmid types were further characterized, and conjugation experiments were performed to assess transferability among bacterial species. Results: From all selected samples, six isolates (20%) were identified as multidrug-resistant CPE including one Escherichia coli, one Klebsiella pneumoniae and four Enterobacter roggenkampii carrying blaNDM-5, blaNDM-1 and blaIMI-1 genes, respectively. The blaNDM-5 and blaNDM-1 genes were located on phage-like pO111 type plasmid and IncC plasmid, while blaIMI-1 was located on chromosomes. The plasmids also consisted of components that closely resembled those found in resistance plasmids obtained from clinical and environmental isolates worldwide. Additionally, through plasmid conjugation experiment, carbapenemase genes were transferable with a high rate among bacterial species. Conclusions: These findings indicated that water bodies are polluted and there is an urgent need for integrated strategies to monitor and mitigate the spread of antibiotic resistance across human, animal and environmental health domains in aquatic environments.Item Metadata only Traveling Wave Ion Mobility-Derived Collision Cross Section Database for Plant Specialized Metabolites: An Application to Ventilago harmandiana Pierre(2022-10-07) Jariyasopit N.; Limjiasahapong S.; Kurilung A.; Sartyoungkul S.; Wisanpitayakorn P.; Nuntasaen N.; Kuhakarn C.; Reutrakul V.; Kittakoop P.; Sirivatanauksorn Y.; Khoomrung S.; Mahidol UniversityThe combination of ion mobility mass spectrometry (IM-MS) and chromatography is a valuable tool for identifying compounds in natural products. In this study, using an ultra-performance liquid chromatography system coupled to a high-resolution quadrupole/traveling wave ion mobility spectrometry/time-of-flight MS (UPLC-TWIMS-QTOF), we have established and validated a comprehensive TWCCSN2and MS database for 112 plant specialized metabolites. The database included 15 compounds that were isolated and purified in-house and are not commercially available. We obtained accurate m/z, retention times, fragment ions, and TWIMS-derived CCS (TWCCSN2) values for 207 adducts (ESI+and ESI-). The database included novel 158 TWCCSN2values from 79 specialized metabolites. In the presence of plant matrix, the CCS measurement was reproducible and robust. Finally, we demonstrated the application of the database to extend the metabolite coverage of Ventilago harmandiana Pierre. In addition to pyranonaphthoquinones, a group of known specialized metabolites in V. harmandiana, we identified flavonoids, xanthone, naphthofuran, and protocatechuic acid for the first time through targeted analysis. Interestingly, further investigation using IM-MS of unknown features suggested the presence of organonitrogen compounds and lipid and lipid-like molecules, which is also reported for the first time. Data are available on the MassIVE (https://massive.ucsd.edu, data set identifier MSV000090213).