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An Integrative Approach Using Molecular and Metabolomic Studies Reveals the Connection of Glutamic Acid with Telomerase and Oxidative Stress in Berberine-Treated Colorectal Cancer Cell Line HCT 116

Samad MA, Zamani AI, Abdul Majid N, Karsani SA, Baharum SN, Yaacob JS, et al.

Appl Biochem Biotechnol, 2025 Feb 26.
PMID: 40009339 DOI: 10.1007/s12010-025-05200-9

Colorectal cancer (CRC) is one of the common deadliest cancers worldwide. In Malaysia, the numbers of new CRC cases were horrific and worrisome. Telomerase is both prognostic indicator and predictor of carcinogenesis in CRC patients. Berberine, a telomerase inhibitor, was used in clinical trials and metabolomic studies; however, the association of telomerase with metabolites and metabolic pathways was not fully understood. Colorectal cancer cell line HCT 116 was cultured and treated with 10.54 µg/mL berberine. The cells were harvested at different time points to conduct subsequent analyses. The methods used in this research were real time-polymerase chain reaction (RT-PCR) to assess RNA expressions; Western blot to determine protein levels; TELOTAGGG Telomerase PCR ELISA to determine relative telomerase activity (RTA); 4',6-diamidino-2-phenylindole (DAPI) staining to determine percentage of nuclei damage; fluorescence microscopy for cell area; spectrophotometric potassium iodide assay for intracellular hydrogen peroxide concentration [H2O2]; as well as liquid chromatography mass spectrometry (LCMS) and tandem mass spectrometry (MS/MS) to investigate the intracellular metabolites. Partial least square-discriminant analysis (PLS-DA) score plot exhibited an improved separation compared to principal component analysis (PCA) when metabolomic data analysis of HCT 116 at various berberine treatment durations was conducted. Time and berberine treatment had an impact on RTA in HCT 116. RTA was discovered to be positively and negatively correlated to 14 and 2 metabolites, respectively. Glutamic acid was consistently found correlated to RTA. Other four metabolites, i.e., MG(14:0), [3-[hydroxy(phosphonooxy)phosphoryl]oxyphenyl] phosphono hydrogen phosphate), (3S,6S)-6-[[(3S,6R)-6-[(2S,3S,5S)-2,5-diiodo-4-methoxy-6-methyloxan-3-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid, and 1-[5-O-(5'-adenylyloxyphosphonyl)-beta-D-ribofuranosyl]-5-amino-1H-imidazole-4-carboxamide, were newly discovered to be connected to RTA in HCT 116. Four metabolic pathways that majorly affected shared glutamic acid and glutamine. Nitrogen metabolism, D-glutamine and D-glutamate metabolism, glyoxylate and dicarboxylate metabolism, and aminoacyl-tRNA biosynthesis have been identified to be associated with RTA. Network analyses hinted that glutamic acid was also associated with oxidative stress mechanism. The multiple roles glutamic acid acted in diverse metabolic pathways and interaction networks emphasized the importance of glutamic acid in HCT 116 regarding RTA. This research establishes the association between RTA and several chosen RNAs, proteins, metabolites, and oxidative stress mechanisms, consequential in morphological alteration in HCT 116, to expand the knowledge of the intricate biological relationships and telomerase mechanism in CRC.
Fulltext Aspergillus fumigatus AR04 obeys Arrhenius' rule in cultivation temperature shifts from 30 to 40°C

Nieland S, Barig S, Salzmann J, Gehrau F, Zamani AI, Richter A, et al.

Microb Biotechnol, 2021 Jul;14(4):1422-1432.
PMID: 33421319 DOI: 10.1111/1751-7915.13739

To set a benchmark in fungal growth rate, a differential analysis of prototrophic Aspergillus fumigatus AR04 with three ascomycetes applied in > 103 t year-1 scale was performed, i.e. Ashbya gosspyii (riboflavin), Aspergillus niger (citric acid) and Aspergillus oryzae (food-processing). While radial colony growth decreased 0.5-fold when A. gossypii was cultivated at 40°C instead of 28°C, A. fumigatus AR04 responded with 1.7-fold faster hyphal growth. A. niger and A. oryzae formed colonies at 40°C, but not at 43°C. Moreover, all A. fumigatus strains tested grew even at 49°C. In chemostat experiments, A. fumigatus AR04 reached steady state at a dilution rate of 0.7 h-1 at 40°C, 120% more than reported for A. gossypii at 28°C. To study mycelial growth rates under unlimited conditions, carbon dioxide increase rates were calculated from concentrations detected online in the exhaust of batch fermentations for 3 h only. All rates calculated suggest that A. fumigatus AR04 approximates Arrhenius' rule when comparing short cultivations at 30°C with those at 40°C. Linearization of the exponential phase and comparison of the slopes revealed an increase to 192% by the 10°C up-shift.
Fulltext Comparative metabolomics of Phialemonium curvatum as an omnipotent fungus cultivated on crude palm oil versus glucose

Zamani AI, Barig S, Ibrahim S, Mohd Yusof H, Ibrahim J, Low JYS, et al.

Microb Cell Fact, 2020 Sep 09;19(1):179.
PMID: 32907579 DOI: 10.1186/s12934-020-01434-w

BACKGROUND: Sugars and triglycerides are common carbon sources for microorganisms. Nonetheless, a systematic comparative interpretation of metabolic changes upon vegetable oil or glucose as sole carbon source is still lacking. Selected fungi that can grow in acidic mineral salt media (MSM) with vegetable oil had been identified recently. Hence, this study aimed to investigate the overall metabolite changes of an omnipotent fungus and to reveal changes at central carbon metabolism corresponding to both carbon sources.
RESULTS: Targeted and non-targeted metabolomics for both polar and semi-polar metabolites of Phialemonium curvatum AWO2 (DSM 23903) cultivated in MSM with palm oil (MSM-P) or glucose (MSM-G) as carbon sources were obtained. Targeted metabolomics on central carbon metabolism of tricarboxylic acid (TCA) cycle and glyoxylate cycle were analysed using LC-MS/MS-TripleQ and GC-MS, while untargeted metabolite profiling was performed using LC-MS/MS-QTOF followed by multivariate analysis. Targeted metabolomics analysis showed that glyoxylate pathway and TCA cycle were recruited at central carbon metabolism for triglyceride and glucose catabolism, respectively. Significant differences in organic acids concentration of about 4- to 8-fold were observed for citric acid, succinic acid, malic acid, and oxaloacetic acid. Correlation of organic acids concentration and key enzymes involved in the central carbon metabolism was further determined by enzymatic assays. On the other hand, the untargeted profiling revealed seven metabolites undergoing significant changes between MSM-P and MSM-G cultures.
CONCLUSIONS: Overall, this study has provided insights on the understanding on the effect of triglycerides and sugar as carbon source in fungi global metabolic pathway, which might become important for future optimization of carbon flux engineering in fungi to improve organic acids production when vegetable oil is applied as the sole carbon source.
Fulltext Comparative metabolomics analysis reveals alkaloid repertoires in young and mature Mitragyna speciosa (Korth.) Havil. Leaves

Veeramohan R, Zamani AI, Azizan KA, Goh HH, Aizat WM, Razak MFA, et al.

PLoS One, 2023;18(3):e0283147.
PMID: 36943850 DOI: 10.1371/journal.pone.0283147

The fresh leaves of Mitragyna speciosa (Korth.) Havil. have been traditionally consumed for centuries in Southeast Asia for its healing properties. Although the alkaloids of M. speciosa have been studied since the 1920s, comparative and systematic studies of metabolite composition based on different leaf maturity levels are still lacking. This study assessed the secondary metabolite composition in two different leaf stages (young and mature) of M. speciosa, using an untargeted liquid chromatography-electrospray ionisation-time-of-flight-mass spectrometry (LC-ESI-TOF-MS) metabolite profiling. The results revealed 86 putatively annotated metabolite features (RT:m/z value) comprising 63 alkaloids, 10 flavonoids, 6 terpenoids, 3 phenylpropanoids, and 1 of each carboxylic acid, glucoside, phenol, and phenolic aldehyde. The alkaloid features were further categorised into 14 subclasses, i.e., the most abundant class of secondary metabolites identified. As per previous reports, indole alkaloids are the most abundant alkaloid subclass in M. speciosa. The result of multivariate analysis (MVA) using principal component analysis (PCA) showed a clear separation of 92.8% between the young and mature leaf samples, indicating a high variance in metabolite levels between them. Akuammidine, alstonine, tryptamine, and yohimbine were tentatively identified among the many new alkaloids reported in this study, depicting the diverse biological activities of M. speciosa. Besides delving into the knowledge of metabolite distribution in different leaf stages, these findings have extended the current alkaloid repository of M. speciosa for a better understanding of its pharmaceutical potential.