Nucleic-acid detection via isothermal amplification and collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative polymerase chain reaction (qPCR). Here, we report the clinical validation of the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) -the virus that causes COVID-19 (coronavirus disease 2019) -in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per
Global DNA hypomethylation promoting genomic instability leads to cancer and deterioration of human health with age. Aim: To invent a biotechnology that can reprogram this process. Methods: We used Alu siRNA to direct Alu interspersed repetitive sequences methylation in human cells. We evaluated the correlation between DNA damage and Alu methylation levels. Results: We observed an inverse correlation between Alu element methylation and endogenous DNA damage in white blood cells. Cells transfected with Alu siRNA exhibited high Alu methylation levels, increased proliferation, reduced endogenous DNA damage and improved resistance to DNA damaging agents. Conclusion: Alu methylation stabilizes the genome by preventing accumulation of DNA damage. Alu siRNA could be useful for evaluating reprograming of the global hypomethylation phenotype in cancer and aging cells. DNA methylation at interspersed repetitive sequences (IRSs) plays an important role in maintaining genome stability. Cells with IRS hypomethylation exhibit increased mutation rates [1,2]. Here, we tested whether DNA damage, an alteration in the chemical structure of DNA and a precursor to mutation [3], plays a role in mediating how global hypomethylation promotes genomic instability. Our recent study found that global hypomethylation is associated with plasma 8-hydroxy-2 -deoxyguanosine (8-OHdG) in biliary atresia patients [4]. Moreover, urinary 8-OHdG, DNA strand breaks and global DNA hypomethylation are associated with low serum folate [5] and oxidative stress [6]. Therefore, we hypothesized that the human genome may use DNA methylation in IRSs to prevent DNA damage.This study developed a technology to add DNA methylation at Alu elements. The human genome contains greater than one million Alu elements [7]. Several reports demonstrated de novo methylation by siRNA in plants [8][9][10]. In human cells, small RNA was used to promote DNA methylation, shRNA for long interspersed element-1 (LINE-1) and hepatitis B virus, and siRNA for HIV-1 promoter region [11][12][13]. There are many types of IRS such as Alu elements, LINE-1, and human endogenous retrovirus [14][15][16][17]. To increase DNA methylation, we tested siRNA to unique sequences and several IRS sequences. Our preliminary trial demonstrated that only Alu siRNA could increase methylation of the target sequences. Here, we evaluated whether Alu siRNA is a useful tool to explore the role of global hypomethylation in genomic instability [18].Alu hypomethylation may play a role in causing genomic instability in cancer and aging cells. Genomic instability, the main enabling characteristic of cancer and aging processes [18,19], may mainly be promoted by IRS hypomethylation. IRS hypomethylation is commonly observed both in aging [14,20] and cancer cells [21]. IRS
Although, increased oxidative stress and hypomethylation of long interspersed nuclear element-1 (LINE-1) associate with bladder cancer (BCa) development, the relationship between these alterations is unknown. We evaluated the oxidative stress and hypomethylation of the LINE-1 in 61 BCa patients and 45 normal individuals. To measure the methylation levels and to differentiate the LINE-1 loci into hypermethylated, partially methylated and hypomethylated, peripheral blood cells, urinary exfoliated cells and cancerous tissues were evaluated by combined bisulfite restriction analysis PCR. The urinary total antioxidant status (TAS) and plasma protein carbonyl content were determined. The LINE-1 methylation levels and patterns, especially hypomethylated loci, in the blood and urine cells of the BCa patients were different from the levels and patterns in the healthy controls. The urinary TAS was decreased, whereas the plasma protein carbonyl content was increased in the BCa patients relative to the controls. A positive correlation between the methylation of LINE-1 in the blood-derived DNA and urinary TAS was found in both the BCa and control groups. The urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content provided the best diagnostic potential for BCa prediction. Based on post-diagnostic samples, the combination test improved the diagnostic power to a sensitivity of 96% and a specificity of 96%. In conclusion, decreased LINE-1 methylation is associated with increased oxidative stress both in healthy and BCa subjects across the various tissue types, implying a dose-response association. Increases in the LINE-1 hypomethylation levels and the number of hypomethylated loci in both the blood- and urine-derived cells and increase in the oxidative stress were found in the BCa patients. The combination test of the urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content may be useful for BCa screening and monitoring of treatment.
The mechanism that causes genomic instability in nondividing aging cells is unknown. Our previous study of mutant yeast suggested that 2 types of replication-independent endogenous DNA double-strand breaks (RIND-EDSBs) exist and that they play opposing roles. The first type, known as physiologic RIND-EDSBs, were ubiquitous in the G phase of both yeast and human cells in certain genomic locations and may act as epigenetic markers. Low RIND-EDSB levels were found in mutants that lacked chromatin-condensing proteins, such as the high-mobility group box (HMGB) proteins and Sir2. The second type is referred to as pathologic RIND-EDSBs. High pathological RIND-EDSB levels were found in DSB repair mutants. Under normal physiologic conditions, these excess RIND-EDSBs are repaired in much the same way as DNA lesions. Here, chronological aging in yeast reduced physiological RIND-EDSBs and cell viability. A strong correlation was observed between the reduction in RIND-EDSBs and viability in aging yeast cells ( r = 0.94, P < 0.0001). We used galactose-inducible HO endonuclease (HO) and nhp6a∆, an HMGB protein mutant, to evaluate the consequences of reduced physiological RIND-EDSB levels. The HO-induced cells exhibited a sustained reduction in RIND-EDSBs at various levels for several days. Interestingly, we found that lower physiologic RIND-EDSB levels resulted in decreased cell viability ( r = 0.69, P < 0.0001). Treatment with caffeine, a DSB repair inhibitor, increased pathological RIND-EDSBs, which were distinguished from physiologic RIND-EDSBs by their lack of sequences prior to DSB in untreated cells [odds ratio (OR) ≤1]. Caffeine treatment in both the HO-induced and nhp6a∆ cells markedly increased OR ≤1 breaks. Therefore, physiological RIND-EDSBs play an epigenetic role in preventing pathological RIND-EDSBs, a type of DNA damage. In summary, the reduction of physiological RIND-EDSB level is a genomic instability mechanism in chronologically aging cells.-Thongsroy, J., Patchsung, M., Pongpanich, M., Settayanon, S., Mutirangura, A. Reduction in replication-independent endogenous DNA double-strand breaks promotes genomic instability during chronological aging in yeast.
BackgroundCellular senescence due to genomic instability is believed to be one of the mechanisms causing health problems in diabetes mellitus (DM). Low methylation levels of Alu elements or Alu hypomethylation, an epigenomic event causing genomic instability, were commonly found in aging people and patients with aging phenotypes, such as osteoporosis.ResultsWe investigate Alu methylation levels of white blood cells of type 2 DM, pre-DM, and control. The DM group possess the lowest Alu methylation (P < 0.001, P < 0.0001 adjusted age). In the DM group, Alu hypomethylation is directly correlated with high fasting blood sugar, HbA1C, and blood pressure.ConclusionGenome-wide hypomethylation may be one of the underlining mechanisms causing genomic instability in type 2 DM. Moreover, Alu methylation levels may be a useful biomarker for monitoring cellular senescence in type 2 DM patients.Electronic supplementary materialThe online version of this article (10.1186/s13148-017-0395-6) contains supplementary material, which is available to authorized users.
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