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Fulltext Optimization of nucleic acid extraction and amplification of a thiamine biosynthesis gene fragment from selected Malaysian seaweeds

Md Ahaik FA, Mohd Taufik SH, Faiqah Johari NA, Zainal Abidin AA, Balia Yusof ZN

Genes Genet Syst, 2023 Feb 22;97(5):247-256.
PMID: 36631109 DOI: 10.1266/ggs.22-00088

Obtaining high-quality nucleic acid extracted from seaweeds is notoriously difficult due to contamination with polysaccharides and polyphenolic compounds after cell disruption. Specific methods need to be employed for RNA isolation in different seaweed species, and therefore studies of the thiamine biosynthesis pathway have been limited. Two selected Malaysian species which are highly abundant and underutilized, namely Gracilaria sp. and Padina sp., representing the red and brown seaweeds, respectively, were collected to develop optimized total RNA extraction methods. Prior to that, DNA was extracted, and amplification of the 18S rRNA gene and the THIC gene (encoding the first enzyme in the pyrimidine branch of the thiamine biosynthesis pathway) from the DNA template was successful in Gracilaria sp. only. RNA was then extracted from both seaweeds using three different existing methods, with some modifications, using cetyltrimethylammonium bromide, guanidine thiocyanate and sodium dodecyl sulphate. Methods I and III proved to be efficient for Padina sp. and Gracilaria sp., respectively, for the extraction of highly purified RNA, with A260/A280 values of 2.0 and 1.8. However, amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and the THIC gene was successful in only Gracilaria sp. cDNA derived from extracted RNA. Further modifications are required to improve the exploitation of nucleic acids from brown seaweeds, which has been proven to be difficult. This work should pave the way for molecular studies of seaweeds generally and for the elucidation, specifically, of the thiamine biosynthesis pathway.

Matched MeSH terms: RNA/metabolism
Berberine and RNAi-Targeting Telomerase Reverse Transcriptase (TERT) and/or Telomerase RNA Component (TERC) Caused Oxidation in Colorectal Cancer Cell Line, HCT 116: An Integrative Approach using Molecular and Metabolomic Studies

Samad MA, Saiman MZ, Abdul Majid N, Karsani SA, Yaacob JS

Cell Biochem Biophys, 2024 Mar;82(1):153-173.
PMID: 38198024 DOI: 10.1007/s12013-023-01210-8

Colorectal cancer (CRC) is the most common cancer in both men and women and is associated with increased telomerase levels and activity. The potential downstream effects of TERT and/or TERC downregulation by berberine (a telomerase inhibitor) or RNA interference (RNAi) on various target RNAs, proteins, relative telomerase activity (RTA), relative telomere length (RTL), hydrogen peroxide concentration [H2O2], percentage of cell cycle distribution, cell size and granularity as well as cellular metabolites were explored in HCT 116 cell line. Knockdown of TERT decreased TERC. The downregulation of TERT and/or TERC caused increment of [H2O2], G0/G1 phase arrest in addition to decreased S and G2/M phases, as well as diminished cell size. RTL was later reduced as a result of TERT, TERT and/or TERC downregulation which decreased RTA. It was discovered that xanthine oxidase (XO) was significantly and positively correlated at FDR-adjusted p value RNAs, proteins, metabolites, oxidative stress mechanism and subsequently phenotypic changes in HCT 116 which is valuable to understand the intricate biological interactions and mechanism of telomerase in CRC.

Matched MeSH terms: RNA/metabolism
Fulltext Identification and characterization of a novel Epstein-Barr Virus-encoded circular RNA from LMP-2 Gene

Tan KE, Ng WL, Marinov GK, Yu KH, Tan LP, Liau ES, et al.

Sci Rep, 2021 Jul 13;11(1):14392.
PMID: 34257379 DOI: 10.1038/s41598-021-93781-w

Epstein-Barr virus (EBV) has been recently found to generate novel circular RNAs (circRNAs) through backsplicing. However, comprehensive catalogs of EBV circRNAs in other cell lines and their functional characterization are still lacking. In this study, we have identified a list of putative EBV circRNAs in GM12878, an EBV-transformed lymphoblastoid cell line, with a significant majority encoded from the EBV latent genes. A novel EBV circRNA derived from the exon 5 of LMP-2 gene which exhibited highest prevalence, was further validated using RNase R assay and Sanger sequencing. This circRNA, which we term circLMP-2_e5, can be universally detected in a panel of EBV-positive cell lines modelling different latency programs. It ranges from lower expression in nasopharyngeal carcinoma (NPC) cells to higher expression in B cells, and is localized to both the cytoplasm and the nucleus. We provide evidence that circLMP-2_e5 is expressed concomitantly with its cognate linear LMP-2 RNA upon EBV lytic reactivation, and may be produced as a result of exon skipping, with its circularization possibly occurring without the involvement of cis elements in the short flanking introns. Furthermore, we show that circLMP-2_e5 is not involved in regulating cell proliferation, host innate immune response, its linear parental transcripts, or EBV lytic reactivation. Taken together, our study expands the current repertoire of putative EBV circRNAs, broadens our understanding of the biology of EBV circRNAs, and lays the foundation for further investigation of their function in the EBV life cycle and disease development.

Matched MeSH terms: RNA/metabolism
High-performance interactive analysis of split aptamer and HIV-1 Tat on multiwall carbon nanotube-modified field-effect transistor

Fatin MF, Rahim Ruslinda A, Gopinath SCB, Arshad MKM

Int J Biol Macromol, 2019 Mar 15;125:414-422.
PMID: 30529550 DOI: 10.1016/j.ijbiomac.2018.12.066

Interaction between split RNA aptamer and the clinically important target, HIV-1 Tat was investigated on a biosensing surface transduced by functionally choreographed multiwall carbon nanotubes (MWCNTs). Acid oxidation was performed to functionalize MWCNTs with carboxyl functional groups. X-ray photoelectron spectroscopy analysis had profound ~2.91% increment in overall oxygen group and ~1% increment was noticed with a specific carboxyl content owing to CO and OCO bonding. The interaction between split RNA aptamer and HIV-1 Tat protein was quantified by electrical measurements with the current signal (Ids) over a gate voltage (Vgs). Initially, 34.4 mV gate voltage shift was observed by the immobilization of aptamer on MWCNT. With aptamer and HIV-1 Tat interaction, the current flow was decreased with the concomitant gate voltage shift of 23.5 mV. The attainment of sensitivity with split aptamer and HIV-1 Tat interaction on the fabricated device was 600 pM. To ensure the genuine interaction of aptamer with HIV-1 Tat, other HIV-1 proteins, Nef and p24 were interacted with aptamer and they displayed the negligible interferences with gate voltage shift of 3.5 mV and 5.7 mV, which shows 4 and 2.5 folds lesser than HIV-1 Tat interaction, respectively.

Matched MeSH terms: RNA/metabolism
Fulltext Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation

Srinivasan S, Yeri A, Cheah PS, Chung A, Danielson K, De Hoff P, et al.

Cell, 2019 04 04;177(2):446-462.e16.
PMID: 30951671 DOI: 10.1016/j.cell.2019.03.024

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.

Matched MeSH terms: RNA/metabolism
Fulltext Transcriptomic analysis of the role of RasGEF1B circular RNA in the TLR4/LPS pathway

Ng WL, Marinov GK, Chin YM, Lim YY, Ea CK

Sci Rep, 2017 09 25;7(1):12227.
PMID: 28947785 DOI: 10.1038/s41598-017-12550-w

Circular RNAs (circRNAs) have recently emerged as a large class of novel non-coding RNA species. However, the detailed functional significance of the vast majority of them remains to be elucidated. Most functional characterization studies targeting circRNAs have been limited to resting cells, leaving their role in dynamic cellular responses to stimuli largely unexplored. In this study, we focus on the LPS-induced cytoplasmic circRNA, mcircRasGEF1B, and combine targeted mcircRasGEF1B depletion with high-throughput transcriptomic analysis to gain insight into its function during the cellular response to LPS stimulation. We show that knockdown of mcircRasGEF1B results in altered expression of a wide array of genes. Pathway analysis revealed an overall enrichment of genes involved in cell cycle progression, mitotic division, active metabolism, and of particular interest, NF-κB, LPS signaling pathways, and macrophage activation. These findings expand the set of functionally characterized circRNAs and support the regulatory role of mcircRasGEF1B in immune response during macrophage activation and protection against microbial infections.

Matched MeSH terms: RNA/metabolism*
Massive Proteogenomic Reanalysis of Publicly Available Proteomic Datasets of Human Tissues in Search for Protein Recoding via Adenosine-to-Inosine RNA Editing

Levitsky LI, Ivanov MV, Goncharov AO, Kliuchnikova AA, Bubis JA, Lobas AA, et al.

J Proteome Res, 2023 Jun 02;22(6):1695-1711.
PMID: 37158322 DOI: 10.1021/acs.jproteome.2c00740

The proteogenomic search pipeline developed in this work has been applied for reanalysis of 40 publicly available shotgun proteomic datasets from various human tissues comprising more than 8000 individual LC-MS/MS runs, of which 5442 .raw data files were processed in total. This reanalysis was focused on searching for ADAR-mediated RNA editing events, their clustering across samples of different origins, and classification. In total, 33 recoded protein sites were identified in 21 datasets. Of those, 18 sites were detected in at least two datasets, representing the core human protein editome. In agreement with prior artworks, neural and cancer tissues were found to be enriched with recoded proteins. Quantitative analysis indicated that recoding the rate of specific sites did not directly depend on the levels of ADAR enzymes or targeted proteins themselves, rather it was governed by differential and yet undescribed regulation of interaction of enzymes with mRNA. Nine recoding sites conservative between humans and rodents were validated by targeted proteomics using stable isotope standards in the murine brain cortex and cerebellum, and an additional one was validated in human cerebrospinal fluid. In addition to previous data of the same type from cancer proteomes, we provide a comprehensive catalog of recoding events caused by ADAR RNA editing in the human proteome.

Matched MeSH terms: RNA/metabolism
Fulltext Antiproliferative effects of dried Moringa oleifera leaf extract on human Wharton's Jelly mesenchymal stem cells

Ramarao KDR, Somasundram C, Razali Z, Kunasekaran W, Jin TL, Musa S, et al.

PLoS One, 2022;17(10):e0274814.
PMID: 36197921 DOI: 10.1371/journal.pone.0274814

Mesenchymal stem cells (MSCs) have seen an elevated use in clinical works like regenerative medicine. Its potential therapeutic properties increases when used in tandem with complementary agents like bio-based materials. Therefore, the present study is the first to investigate the cytotoxicity of a highly valued medicinal plant, Moringa oleifera, on human Wharton's Jelly mesenchymal stem cells (hWJMSCs) and its effects on the cells' gene expression when used as a pre-treatment agent in vitro. M. oleifera leaves (MOL) were dried and subjected to UHPLC-QTOF/MS analysis, revealing several major compounds like apigenin, kaempferol, and quercetin in the MOL, with various biological activities like antioxidant and anti-cancer properties. We then treated the hWJMSCs with MOL and noticed a dose-dependant inhibition on the cells' proliferation. RNA-sequencing was performed to explain the possible mechanism of action and revealed genes like PPP1R1C, SULT2B1, CDKN1A, mir-154 and CCNB1, whose expression patterns were closely associated with the negative cell cycle regulation and cell cycle arrest process. This is also evident from gene set enrichment analysis where the GO and KEGG terms for down-regulated pathways were closely related to the cell cycle regulation. The Ingenuity pathway analysis (IPA) software further predicted the significant activation of (p < 0.05, z-score > 2) of the G2/M DNA damage checkpoint regulation pathway. The present study suggests that MOL exhibits an antiproliferative effect on hWJMSCs via cell cycle arrest and apoptotic pathways. We believe that this study provides an important baseline reference for future works involving MOL's potential to accompany MSCs for clinical works. Future works can take advantage of the cell's strong anti-cancer gene expression found in this study, and evaluate our MOL treatment on various cancer cell lines.

Matched MeSH terms: RNA/metabolism
Lutein improves cell viability and reduces Alu RNA accumulation in hydrogen peroxide challenged retinal pigment epithelial cells

Chong YS, Mai CW, Leong CO, Wong LC

Cutan Ocul Toxicol, 2018 Mar;37(1):52-60.
PMID: 28554225 DOI: 10.1080/15569527.2017.1335748

PURPOSE: Dysfunction of the microRNA (miRNA)-processing enzyme DICER1 and Alu RNA accumulation are linked to the pathogenesis of age-related macular degeneration (AMD). This study determined the optimal dose of lutein (LUT) and zeaxanthin (ZEA) to protect human retinal pigment epithelium (RPE) cells against hydrogen peroxide (H2O2). The effect of the optimal dose of LUT and ZEA as DICER1 and Alu RNA modulators in cultured human RPE cells challenged with H2O2 was investigated.
MATERIALS AND METHODS: ARPE-19 cells were pre-treated with LUT, ZEA, or both for 24 h before 200 μM H2O2 challenge. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. DICER1 and Alu RNA were quantified by western blotting and real-time polymerase chain reaction, respectively.
RESULTS: H2O2 increased cell Alu RNA expression and decreased cell viability of ARPE-19, but had no significant impact on the DICER1 protein level. LUT, alone and in combination with ZEA pre-treatment, prior to H2O2 challenge significantly improved cell viability of ARPE-19 and reduced the level of Alu RNA compared to the negative control.
CONCLUSIONS: These results support the use of LUT alone, and in combination with ZEA, in AMD prevention and treatment. This study is also the first to report LUT modulating effects on Alu RNA.

Matched MeSH terms: RNA/metabolism*
In vitro genotoxicity tests for polyhydroxybutyrate--a synthetic biomaterial

Ali AQ, Kannan TP, Ahmad A, Samsudin AR

Toxicol In Vitro, 2008 Feb;22(1):57-67.
PMID: 17892925

The aims of this study are to determine the mutagenicity of a locally produced polyhydroxybutyrate (PHB) using Salmonella mutagenicity test and to find out if PHB altered the expression of p53 and c-myc proto-oncogenes and bcl-xl and bcl-xs anti-apoptotic genes in the human fibroblast cell line, MRC-5. Different concentrations of PHB were incubated with special genotypic variants of Salmonella strains (TA1535, TA1537, TA1538, TA98 and TA100) carrying mutations in several genes both with and without metabolic activation (S9) and the test was assessed based on the number of revertant colonies. The average number of revertant colonies per plate treated with PHB was less than double as compared to that of negative control. For the gene expression analyses, fibroblast cell lines were treated with PHB at different concentrations and incubated for 1, 12, 24 and 48 h separately. The total RNA was isolated and analysed for the expression of p53, c-myc, bcl-xl and bcl-xs genes. The PHB did not show over or under expression of the genes studied. The above tests indicate that the locally produced PHB is non-genotoxic and does not alter the expression of the proto-oncogenes and anti-apoptotic genes considered in this study.

Matched MeSH terms: RNA/metabolism

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