Recovery and characterization of ß-glucosidase-producing non-Saccharomyces yeasts from the fermentation broth of Vitis labruscana Baily × Vitis vinifera L. for investigation of their fermentation characteristics.

The present study focuses on investigating 60 strains of yeast isolated from the natural fermentation broth of Vitis labruscana Baily × Vitis vinifera L. These strains underwent screening using lysine culture medium and esculin culture medium, resulting in the identification of 27 local non-Saccharomyces yeast strains exhibiting high ß-glucosidase production. Subsequent analysis of their fermentation characteristics led to the selection of four superior strains (Z-6, Z-11, Z-25, and Z-58) with excellent ß-glucosidase production and fermentation performance. Notably, these selected strains displayed a dark coloration on esculin medium and exhibited robust gas production during Duchenne tubules' fermentation test. Furthermore, all four non-Saccharomyces yeast strains demonstrated normal growth under specific conditions including SO2 mass concentration ranging from 0.1 to 0.3 g/L, temperature between 25 and 30 °C, glucose mass concentration ranging from 200 to 400 g/L, and ethanol concentration at approximately 4%. Molecular biology identification confirmed that all selected strains belonged to Pichia kudriavzevii species which holds great potential for wine production.

Isolation, identification, and tolerance analysis of yeast during the natural fermentation process of Sidamo coffee beans.

Yeast, which plays a pivotal role in the brewing, food, and medical industries, exhibits a close relationship with human beings. In this study, we isolated and purified 60 yeast strains from the natural fermentation broth of Sidamo coffee beans to screen for indigenous beneficial yeasts. Among them, 25 strains were obtained through morphological characterization on nutritional agar medium from Wallerstein Laboratory (WL), with molecular biology identifying Saccharomyces cerevisiae strain YBB-47 and the remaining 24 yeast strains identified as Pichia kudriavzevii. We investigated the fermentation performance, alcohol tolerance, SO2 tolerance, pH tolerance, sugar tolerance, temperature tolerance, ester production capacity, ethanol production capacity, H2S production capacity, and other brewing characteristics of YBB-33 and YBB-47. The results demonstrated that both strains could tolerate up to 3% alcohol by volume at a high sucrose mass concentration (400 g/L) under elevated temperature conditions (40 ℃), while also exhibiting a remarkable ability to withstand an SO2 mass concentration of 300 g/L at pH 3.2. Moreover, S. cerevisiae YBB-47 displayed a rapid gas production rate and strong ethanol productivity. whereas P. kudriavzevii YBB-33 exhibited excellent alcohol tolerance. Furthermore, this systematic classification and characterization of coffee bean yeast strains from the Sidamo region can potentially uncover additional yeasts that offer high-quality resources for industrial-scale coffee bean production.

Purification and characterization of an α-l-arabinofuranosidase, α-l-AFase, for hydrolyzed ginsenoside Rc from Bacillus subtilis.

Natural ginsenoside needs to be converted into rare ginsenoside before it can be readily absorbed into the bloodstream for action. In this study, an α-l-arabinofuranosidase (α-l-AFase) gene Bsafs2 was cloned from Bacillus subtilis (B. subtilis). Bsafs2 was ligated to the expression vector pET28a(+), and the expression vector was constructed and transformed into Escherichia coli (E. coli) BL21 heterologous recombinant expression to obtain α-l-AFase. α-l-AFase can hydrolyze at the C20 site of Ginsenoside Rc to obtain rare ginsenoside Rd. Studies on the enzymatic property showed that α-l-AFase had good tolerance to ethanol, glucose, and l-arabinose. The optimum temperature of α-l-AFase was 40 °C and pH = 5.5. Kinetic parameters Km of α-l-AFase for pNPαAraf and Ginsenoside Rc were 1.93 and 8.9 mmol/L, the Vmax were 26 and 154 µmol/min/mg, the Kcat were 24.14 and 1.48 S-1, respectively. This study provides the enzyme source for the biotransformation of Ginsenoside Rc.

Mechanism of the effect of TREM2 on cognitive function in autistic mice.

This study aimed to investigate the mechanism of the effect of TREM2 on cognitive function in autistic mice. TREM2 overexpression and knockdown viruses were given to autism spectrum disorder (ASD) mice and BV2 microglia cell line. To assess cognitive performance, all groups of mice took part in the open field, new object recognition, Morris water maze, and three-box social experiments. Double immunofluorescence labeling demonstrated co-localization of LC3II and NeuN. Proteins from the PI3K/Akt/mTOR pathway were identified. In vivo, behavior studies revealed that TREM2 could successfully improve ASD mice's social interaction and cognitive performance. Besides, we discovered that TREM2 could increase autophagy in ASD mice. In vitro, overexpressing TREM2 reduced the expression of PI3K/AKT/mTOR pathway proteins, whereas knocking down TREM2 increased the expression of PI3K/AKT/mTOR pathway proteins. In conclusion, TREM2 could inhibit PI3K/Akt/mTOR signaling pathway, enhance autophagy, and improve the social communication ability and cognitive function of ASD mice.

Micro-scale urbanization-based risk factors for dengue epidemics.

Dengue is one of the world's most rapidly spreading mosquito-borne viral diseases. As it is found mostly in urban and semi-urban areas, urbanization and associated human activities that affect the environment and larval habitats could become risk factors (e.g., lane width, conditions of street ditches) for the spread of dengue. However, there are currently no systematic studies of micro-scale urbanization-based risk factors for the spread of dengue epidemics. We describe the study area, two micro-scale environmental risk factors associated with urbanization, and meteorological data. Since the observations involve spatial and temporal correlations, we also use some statistical methods for the analysis of spatial and spatial-temporal data for the relationship between urbanization and dengue. In this study, we analyzed data from Kaohsiung, a densely populated city in southern Taiwan, and found a positive correlation between environmental risk factors associated with urbanization (ditches positive for mosquito larvae and closely packed streets termed "dengue lanes") and clustering effects in dengue cases. The statistical analysis also revealed that the occurrence of positive ditches was significantly associated with that of dengue lanes in the study area. The relationship between climate variables and positive ditches was also analyzed in this paper, indicating a relationship between dengue and both rainfall and temperature, with temperature having a greater effect. Overall, this work is immediately relevant and applicable for policymakers in government, who will need to reduce these favorable habitats for vector-born disease spreaders and implement regulations for new urban constructions to thus reduce dengue spread in future outbreaks.

CRISPR-Cas-Driven Single Micromotor (Cas-DSM) Enables Direct Detection of Nucleic Acid Biomarkers at the Single-Molecule Level.

The target-dependent endonuclease activity (also known as the trans-cleavage activity) of CRISPR-Cas systems has stimulated great interest in the development of nascent sensing strategies for nucleic acid diagnostics. Despite many attempts, the majority of the sensitive CRISPR-Cas diagnostics strategies mainly rely on nucleic acid preamplification, which generally needs complex probes/primers designs, multiple experimental steps, and a longer testing time, as well as introducing the risk of false-positive results. In this work, we propose the CRISPR-Cas-Driven Single Micromotor (Cas-DSM), which can directly detect the nucleic acid targets at a single-molecule level with high specificity. We have demonstrated that the Cas-DSM is a reliable and practical method for the quantitative detection of DNA/RNA in various complex clinical samples as well as in individual cells without any preamplification processes. Due to the excellent features of the CRISPR/Cas system, including constant temperature, simple design, high specificity, and flexible programmability, the Cas-DSM could serve as a simple and universal platform for nucleic acid detection. More importantly, this work will provide a breakthrough for the development of next-generation amplification-free CRISPR/Cas sensing toolboxes.

CLEC5A mediates Zika virus-induced testicular damage.

BACKGROUND: Zika virus (ZIKV) infection is clinically known to induce testicular swelling, termed orchitis, and potentially impact male sterility, but the underlying mechanisms remain unclear. Previous reports suggested that C-type lectins play important roles in mediating virus-induced inflammatory reactions and pathogenesis. We thus investigated whether C-type lectins modulate ZIKV-induced testicular damage. METHODS: C-type lectin domain family 5 member A (CLEC5A) knockout mice were generated in a STAT1-deficient immunocompromised background (denoted clec5a-/-stat1-/-) to enable testing of the role played by CLEC5A after ZIKV infection in a mosquito-to-mouse disease model. Following ZIKV infection, mice were subjected to an array of analyses to evaluate testicular damage, including ZIKV infectivity and neutrophil infiltration estimation via quantitative RT-PCR or histology and immunohistochemistry, inflammatory cytokine and testosterone detection, and spermatozoon counting. Furthermore, DNAX-activating proteins for 12 kDa (DAP12) knockout mice (dap12-/-stat1-/-) were generated and used to evaluate ZIKV infectivity, inflammation, and spermatozoa function in order to investigate the potential mechanisms engaged by CLEC5A. RESULTS: Compared to experiments conducted in ZIKV-infected stat1-/- mice, infected clec5a-/-stat1-/- mice showed reductions in testicular ZIKV titer, local inflammation and apoptosis in testis and epididymis, neutrophil invasion, and sperm count and motility. CLEC5A, a myeloid pattern recognition receptor, therefore appears involved in the pathogenesis of ZIKV-induced orchitis and oligospermia. Furthermore, DAP12 expression was found to be decreased in the testis and epididymis tissues of clec5a-/-stat1-/- mice. As for CLEC5A deficient mice, ZIKV-infected DAP12-deficient mice also showed reductions in testicular ZIKV titer and local inflammation, as well as improved spermatozoa function, as compared to controls. CLEC5A-associated DAP12 signaling appears to in part regulate ZIKV-induced testicular damage. CONCLUSIONS: Our analyses reveal a critical role for CLEC5A in ZIKV-induced proinflammatory responses, as CLEC5A enables leukocytes to infiltrate past the blood-testis barrier and induce testicular and epididymal tissue damage. CLEC5A is thus a potential therapeutic target for the prevention of injuries to male reproductive organs in ZIKV patients.

"Liu-Liang-Chung" syndrome with multiple congenital anomalies and the distinctive craniofacial features caused by dominant ZEB2 gene gain mutation.

BACKGROUND: Contiguous gene gain syndrome including entire ZEB2 may be a novel syndrome. In the past, there were no easily distinct and recognizable features as a guide for precise clinical and genetic diagnosis of the syndrome. CASE PRESENTATION: We report a novel case with the syndrome with a novel de novo 22.16 Mb duplication at 2q21.2-q24.1. The syndrome is characterized by multiple anomalies including the same typical craniofacial phenotype that is entirely different from Mowat-Wilson syndrome (MWS), and other quite similar features of MWS consisting of development delay, congenital heart disease, abdominal abnormalities, urogenital abnormalities, behavioral problems and so on, in which the distinctive craniofacial features can be more easily recognized. CONCLUSIONS: Contiguous gene gain syndrome including entire ZEB2 characterized with similar multiple congenital anomalies of MWS and the distinctive craniofacial features is mainly caused by large 2q22 repeats including ZEB2 leading to dominant singe ZEB2 gene gain mutation, which is recommended to be named "Liu-Liang-Chung" syndrome. We diagnose this novel syndrome to distinguish it from MWS. Some variable additional features in the syndrome including remarkable growth and development retardation and protruding ears were recognized for the first time.

[Analysis of a patient with tuberous sclerosis complex due to mosaicism TSC2 mutation].

OBJECTIVE: To analyze variants of TSC1 and TSC2 genes in a Chinese patient with tuberous sclerosis complex (TSC). METHODS: Peripheral blood samples were collected from the patient and her parents with informed consent. Following extraction of genomic DNA, potential variants of the TSC1 and TSC2 genes was detected by using targeted capture next-generation sequencing (NGS) and Sanger sequencing. RESULTS: The patient was found to harbor a de novo mosaicism variant c.3295_3298delG (Val1100CysfsTer3) of the TSC2 gene, with the proportion of the mutant allele determined as 13.4%, which was confirmed by Sanger sequencing. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.3295_3298delG (Val1100CysfsTer3) variant was predicted to be pathogenic (PVS1+PS2+PM2). CONCLUSION: The mosaicism heterozygous variant of c.3295_3298delG of the TSC2 gene, as detected by both NGS and Sanger sequencing, probably underlay the TSC2 in this patient.

Moringa oleifera seed ethanol extract and its active component kaempferol potentiate pentobarbital-induced sleeping behaviours in mice via a GABAergic mechanism.

CONTEXT: Moringa oleifera Lam. (Moringaceae) (MO) is an important food plant that has high nutritional and medical value. However, there is limited information on whether its seeds can improve sleep. OBJECTIVE: This study investigated the effects of MO seed ethanol extracts (EEMOS) on sleep activity improvement and examined the underlying mechanisms. MATERIALS AND METHODS: Male ICR mice were placed into six groups (n = 12) and treated as follows: Control (sodium carboxymethyl cellulose, 20 mL/kg), estazolam tablets (2 mg/kg), EEMOS (1, 2 g/kg) and kaempferol (1, 2 mg/kg). These samples were successively given intragastric for 14 d. Locomotor activity assay, pentobarbital-induced sleeping and pentetrazol-induced seizures tests were utilized to examine the sedative-hypnotic effects (SHE) of EEMOS. RESULTS: Compared with the control group, the results revealed that EEMOS (2 g/kg) and KA (2 mg/kg) possessed good SHE and could significantly elevate the levels of γ-aminobutyric acid and reduce the levels of glutamic acid in the mouse hypothalamus (p < 0.05). Moreover, SHE was blocked by picrotoxin, flumazenil and bicuculline (p < 0.05). EEMOS (2 g/kg) and KA (2 mg/kg) significantly upregulated the protein expression levels of glutamic acid decarboxylase-65 (GAD65) and α1-subunit of GABAA receptors in the hypothalamus of mice (p < 0.05), not affecting glutamic acid decarboxylase-67 (GAD67) and γ2-subunit expression levels (p > 0.05). Additionally, they cause a significant increase in Cl- influx in human cerebellar granule cells at a concentration of 8 µg/mL (p < 0.05). DISCUSSION AND CONCLUSIONS: These findings demonstrated that EEMOS could improve sleep by regulating GABAA-ergic systems, and encourage further clinical trials to treat insomnia.

Epidemiological analysis of the Kaohsiung city strategy for dengue fever quarantine and epidemic prevention.

BACKGROUND: Dengue is endemic in over 100 countries and is an important public health problem worldwide. Dengue fever is not endemic in Taiwan; the importation of dengue viruses from neighboring countries via close commercial links and air travel is considered to be the cause of local outbreaks. Therefore, efforts toward disease control have focused on preventing the importation of dengue into Taiwan. In this study, we investigated the relationships between the numbers of imported and indigenous dengue cases to test the validity of this strategy. METHODS: Data on cases of dengue fever that occurred between 2013 and 2018 were obtained from the surveillance systems of the Taiwan Center for Disease Control and Kaohsiung City Health Department. Standard epidemiological data, including the monthly numbers of indigenous and imported cases of dengue, were calculated. Potential associations between the numbers of indigenous and imported cases were investigated using correlation analyses. RESULTS: We identified a possible relationship between the period of disease concealment and the number of imported dengue cases, which resulted in epidemics of indigenous dengue fever within local communities. Further analysis of confirmed cases during previous epidemics in Kaohsiung City found that the risk of indigenous dengue fever may be related to the likelihood that patients with imported dengue fever will stay within local communities. CONCLUSION: Given the correlations found between imported and indigenous cases of dengue fever, as well as the relationship between the disease concealment period and the risk of indigenous dengue fever, prevention of disease importation and efficient identification of dengue cases within high-risk communities remain the major priorities for disease control.

Fe3O4/PVDF catalytic membrane treatment organic wastewater with simultaneously improved permeability, catalytic property and anti-fouling.

Fe3O4/Polyvinylidene fluoride (PVDF) three-channel hollow fiber catalytic membrane was successfully fabricated via non-solvent induced phase inversion and used for organic wastewater degradation in this work. The effects of Fe3O4 nanoparticles addition on the surface and cross-section morphologies, hydrophilicity and thermal properties of the catalytic membrane were characterized by the field emission scanning electron microscopy (SEM), water contact angle and thermogravimetric analysis (TGA), respectively. The obtained catalytic membrane exhibited good hydrophilicity, a high pure water flux of 175.8 L m-2 h-1 and a high removal of methylene blue (up to 97.6%) with Fenton catalytic reaction. Meanwhile, the catalytic membrane shows excellent anti-fouling property due to the presence of Fenton reaction. Our results show that Fe3O4/PVDF three-channel hollow fiber catalytic membrane was a promising alternative for the degradation of organic contaminants.

Hepatitis C virus genotypes 1-3 infections regulate lipogenic signaling and suppress cholesterol biosynthesis in hepatocytes.

BACKGROUND: Patients with different hepatitis C virus (HCV) genotype infections are associated with varying metabolic disorders. Although alteration of lipid metabolism has been confirmed as a virus-induced metabolic derangement in chronic hepatitis C patients, the impact of various HCV genotypes on hepatic cholesterol metabolism remains elusive. In this study, we thus investigated the HCV genotype-specific lipogenic and cholesterol metabolism profiles in an in vitro cell culture system. METHODS: We first conducted HCV cell culture system (HCVcc) assays by infecting Huh7.5.1 cells with multiple infection-competent HCV strains, including the genotype 2a JFH1 and JFH1-based intergenotypic recombinants 1b and 3a. We then examined the expression levels of various lipid and cholesterol-related genes. RESULTS: The data showed that infection with individual HCV genotypes exerted unique gene expression regulatory effects on lipoproteins and cholesterol metabolism genes. Of note, all HCV strains suppressed cholesterol biosynthesis in hepatocytes through downregulating the expression of HMG-CoA reductase (HMGCR) and farnesyl-diphosphate farnesyltransferase 1 (FDFT1) - two essential enzymes for cholesterol biosynthesis. These HCV-mediated inhibitory effects could be reversed by treatment with sofosbuvir, a pangenotypic NS5B inhibitor. In addition, overexpression of HCV genotype 1b, 2a or 3a core protein significantly suppressed HMGCR mRNA transcription and translation, thus diminished cellular cholesterol biosynthesis. Nonetheless, the core protein had no effect on FDFT1 expression. CONCLUSION: Although HCV infection regulates host lipid metabolism in a genotype-specific manner, its inhibition on hepatocellular cholesterogenic gene expression and total cholesterol biosynthesis is a common effect among HCV genotype 1b, 2a and 3a.

Highly sensitive quantification of site-specific 5-hydroxymethylcytosine at single-base resolution by HpaII-mediated ligation PCR.

A highly sensitive HpaII-mediated ligation PCR assay that can precisely discriminate 5hmC from 5mC and C is developed for the quantitative determination of site-specific 5hmC in genomic DNA samples.

Pegylated IFN-α suppresses hepatitis C virus by promoting the DAPK-mTOR pathway.

Death-associated protein kinase (DAPK) has been found to be induced by IFN, but its antiviral activity remains elusive. Therefore, we investigated whether DAPK plays a role in the pegylated IFN-α (peg-IFN-α)-induced antiviral activity against hepatitis C virus (HCV) replication. Primary human hepatocytes, Huh-7, and infectious HCV cell culture were used to study the relationship between peg-IFN-α and the DAPK-mammalian target of rapamycin (mTOR) pathways. The activation of DAPK and signaling pathways were determined using immunoblotting. By silencing DAPK and mTOR, we further assessed the role of DAPK and mTOR in the peg-IFN-α-induced suppression of HCV replication. Peg-IFN-α up-regulated the expression of DAPK and mTOR, which was associated with the suppression of HCV replication. Overexpression of DAPK enhanced mTOR expression and then inhibited HCV replication. In addition, knockdown of DAPK reduced the expression of mTOR in peg-IFN-α-treated cells, whereas silencing of mTOR had no effect on DAPK expression, suggesting mTOR may be a downstream effector of DAPK. More importantly, knockdown of DAPK or mTOR significantly mitigated the inhibitory effects of peg-IFN-α on HCV replication. In conclusion, our data suggest that the DAPK-mTOR pathway is critical for anti-HCV effects of peg-IFN-α.

Lentzea cavernae sp. nov., an actinobacterium isolated from a karst cave sample, and emended description of the genus Lentzea.

A novel actinobacterial strain, designated SYSU K10001T, was isolated from a limestone sample collected from a karst cave in Xingyi county, Guizhou province, south-western China. The taxonomic position of the strain was investigated using a polyphasic approach. Cells of the strain were aerobic and Gram-stain-positive. On the basis of 16S rRNA gene sequence analysis, strain SYSU K10001T was most closely related to the type strains of the genus Lentzea, Lentzea albida NBRC 16102T (98.8 % similarity) and Lentzea waywayandensis NRRL B-16159T (98.6 %), and is therefore considered to represent a member of the genus Lentzea. DNA-DNA hybridization values between strain SYSU K10001T and related type strains of the genus Lentzea were less than 70 %. In addition, meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan. The whole-cell sugars were arabinose, fructose, mannose and xylose. The major isoprenoid quinone was MK-9(H4), while the major fatty acids (>10 %) were iso-C16 : 0 and C14 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified phospholipid and one unidentified lipid. The genomic DNA G+C content of strain SYSU K10001T was 69.4 mol%. On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU K10001T represents a novel species of the genus Lentzea, for which the name Lentzea cavernae sp. nov. is proposed. The type strain is SYSU K10001T (=KCTC 39804T=CGMCC 4.7367T=NBRC 112394T).

Feedback regulation of IFN-α/ß signaling by Axl receptor tyrosine kinase modulates HBV immunity.

Hepatitis B virus (HBV) is known to cause age-dependent infection outcomes wherein most infections during young age result in chronicity. The mechanism underlying the differential outcome remains elusive. By using hydrodynamic injection of the replication-competent pAAV-HBV, we established a mouse model in which HBV persistence was generated in 4-5 w/o C57BL/6 young mice, but not in adult mice over 10 w/o. HBV-tolerant young mice expressed higher interferon (IFN)-α/ß levels in hepatocytes and intrahepatic plasmacytoid DCs (pDCs) than adult mice after pAAV-HBV injection. Excessive IFN-α/ß expression in young mice was associated with induction of the Axl regulatory pathway and expansion of intrahepatic Treg cells. In line with these findings, augmented IFN-ß expression increased Axl expression in the liver and HBV persistence in adult mice, whereas IFN-α/ß signaling blockage decreased Axl expression and HBV persistence in young mice. Accordingly, Axl overexpression decreased HBV clearance of adult mice whereas Axl silencing enhanced HBV clearance of young mice. In vitro, IFN-ß priming of pDCs and Axl-overexpressing macrophages enhanced Treg-cell differentiation. These findings suggest that age-dependent HBV chronicity is attributed to IFN-ß-Axl immune regulation, which is selectively induced in young mice by excessive IFN-α/ß production at early stage of HBV infection.

Special clinical entity with 15q26 deletion: a novel case report.

In the past, there were no easily distinct and recognizable features as a guide for precise clinical and genetic diagnosis of cases with chromosome microdeletions involving 15q26 including CHD2,. The present study analysed the clinical data and collected venous blood samples from a pediatric patient and his healthy family members for DNA testing. The whole-exome sequencing was performed by the next-generation sequencing (NGS). Chromosomal copy-number variations were tested based on NGS. We present a review of all cases with chromosome microdeletions affecting CHD2. A novel de novo 5.82-Mb deletion at 15q25.3-15q26.1 including CHD2 was identified in our patient who is an 11.6-year-old boy. We first found surprising efficacy of lamotrigine in controlling intractable drop seizures in the individual. These cases have development delay, behavioural problems, epilepsy, variable multiple anomalies, etc. Phenotypes of individuals with deletions involving 15q26 including CHD2 are highly variable with regard to facial features and multiple developmental anomalies. We first found the special clinical entity of development delay, behavioural problems, epilepsy, variable skeletal and muscular anomalies, abnormalities of variable multiple systems and characteristic craniofacial phenotypes in patients with chromosome microdeletions involving CHD2. The larger deletions involving 15q26 including CHD2 tend to cause the classical phenotype. A distinctive craniofacial appearance of the classical phenotype is midface hypoplasia and perifacial protrusion.

Specific quantitative detection of N6-methyladenosine at single-base resolution by extension-based isothermal exponential amplification reaction (E-IEXPAR).

BACKGROUND: N6-methyladenosine (m6A) is a common modification in RNA, crucial for various cellular functions and associated with human diseases. Quantification of m6A at single-base resolution is of great significance for exploring its biological roles and related disease research. However, existing analysis techniques, such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), face challenges like the requirement for thermal cycling or intricate primer design. Therefore, it is urgent to establish a simple, non-thermal cycling and highly sensitive assay for m6A. RESULTS: Leveraging the inhibitory effect of m6A on primer elongation and uncomplicated feature of the isothermal exponential amplification reaction (IEXPAR), we have developed an extension-based IEXPAR (E-IEXPAR). This approach requires just a single extension primer and one template, simplifying the design process in comparison to the more complex primer requirements of the LAMP methods. The reactions are conducted at constant temperatures, therby elimiating the use of thermal cycling that needed in PCR methods. By combining IEXPAR with an extension reaction, E-IEXPAR can identify m6A in RNA concentrations as low as 4 fM. We have also introduced a new analytical model to process E-IEXPAR results, which can aid to minimize the impact of unmodified adenine (A) on m6A measurements, enabling accurate m6A quantification in small mixed samples and cellular RNA specimens. SIGNIFICANCE AND NOVELTY: E-IEXPAR streamlines m6A detection by eliminating the need for intricate primer design and thermal cycling, which are common in current analytical methods. Its utilization of an extension reaction for the initial identification of m6A, coupled with a novel calculation model tailored to E-IEXPAR outcomes, ensures accurate m6A selectivity in mixed samples. As a result, E-IEXPAR offers a reliable, straightforward, and potentially economical approach for specifically assaying m6A in both biological function studies and clinical research.

Direct quantification of N6-methyladenosine fractions at specific site in RNA based on deoxyribozyme mediated CRISPR-Cas12a platform.

As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N6-methyladenosine (m6A) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the m6A modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify m6A fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only m6A-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of m6A at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of m6A-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2-3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine m6A fractions in real biological samples, even in low abundance RNA biomarkers.