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PloS One 2024A differentially methylated region (DMR) is a genomic region that has significantly different methylation patterns between biological conditions. Identifying DMRs...
A differentially methylated region (DMR) is a genomic region that has significantly different methylation patterns between biological conditions. Identifying DMRs between different biological conditions is critical for developing disease biomarkers. Although methods for detecting DMRs in microarray data have been introduced, developing methods with high precision, recall, and accuracy in determining the true length of DMRs remains a challenge. In this study, we propose a normalized kernel-weighted model to account for similar methylation profiles using the relative probe distance from "nearby" CpG sites. We also extend this model by proposing an array-adaptive version in attempt to account for the differences in probe spacing between Illumina's Infinium 450K and EPIC bead array respectively. We also study the asymptotic results of our proposed statistic. We compare our approach with a popular DMR detection method via simulation studies under large and small treatment effect settings. We also discuss the susceptibility of our method in detecting the true length of the DMRs under these two settings. Lastly, we demonstrate the biological usefulness of our method when combined with pathway analysis methods on oral cancer data. We have created an R package called idDMR, downloadable from GitHub repository with link: https://github.com/DanielAlhassan/idDMR, that allows for the convenient implementation of our array-adaptive DMR method.
Topics: DNA Methylation; Humans; CpG Islands; Oligonucleotide Array Sequence Analysis; Mouth Neoplasms; Algorithms; Software; Computer Simulation
PubMed: 38941289
DOI: 10.1371/journal.pone.0306036 -
ChemMedChem Jun 2024Sirtuin 6 (Sirt6), an NAD+-dependent deacylase, has emerged as a promising target for aging-related diseases and cancer. Advancing the medicinal chemistry of Sirt6...
Sirtuin 6 (Sirt6), an NAD+-dependent deacylase, has emerged as a promising target for aging-related diseases and cancer. Advancing the medicinal chemistry of Sirt6 modulators is crucial for the development of chemical probes aimed at unraveling the intricate biological functions of Sirt6 and unlocking its therapeutic potential. A proprietary DNA-encoded library yielded Sirt6 inhibitor 2-Pr, displaying remarkable inhibitory activity and isoform-selectivity, and featuring a chemical structure distinct from reported Sirt6 modulators. In this study, we explore the inhibitory mechanism of 2-Pr, evaluating the impact of chemical modifications and presenting a crystal structure of the Sirt6/ADP-ribose/2-Pr complex. Notably, co-crystal structure analysis reveals an unexpected and unprecedented binding mode of Sirt6, with 2-Pr spanning the acyl channel of the enzyme, extending into the acetyl-lysine binding pocket, and reaching toward the C-site. This unique binding mode guides potential avenues for developing potent and selective Sirt6 inhibitors.
PubMed: 38940296
DOI: 10.1002/cmdc.202400273 -
Frontiers in Cellular and Infection... 2024The rapid detection of Mycobacterium tuberculosis (MTB) is essential for controlling tuberculosis. We designed a portable thermocycler-based real-time fluorescence...
BACKGROUND
The rapid detection of Mycobacterium tuberculosis (MTB) is essential for controlling tuberculosis. We designed a portable thermocycler-based real-time fluorescence loop-mediated isothermal amplification assay (cyp141-RealAmp) using six oligonucleotide primers derived from cyp141 to detect MTB. A combined number of 213 sputum samples (169 obtained from clinically diagnosed cases of pulmonary TB and 44 from a control group without tuberculosis) underwent Acid-fast bacillus (AFB) smear, culture, Xpert MTB/RIF assays, and cyp141-RealAmp assay.
RESULTS
By targeting MTB cyp141, this technique could detect as low as 10 copies/reaction within 30 min, and it was successfully rejected by other mycobacteria and other bacterial species tested. Of the 169 patients, there was no statistical difference between the detection rate of cyp141-RealAmp (92.90%, 95% CI: 89.03-96.07) and that of Xpert MTB/RIF (94.67%, 95% CI: 91.28-98.06) ( > 0.05), but both were statistically higher than that of culture (65.68%, 95% CI: 58.52-72.84) (< 0.05) and AFB (57.40%, 95% CI: 49.94-64.86) (< 0.05). Both cyp141-RealAmp and Xpert MTB/RIF had a specificity of 100%. Furthermore, a high concordance between cyp141-RealAmp and Xpert MTB/RIF was found ( = 0.89).
CONCLUSION
The cyp141-RealAmp assay was shown to be effective, responsive, and accurate in this study. This method offers a prospective strategy for the speedy and precise detection of MTB.
Topics: Mycobacterium tuberculosis; Humans; Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Molecular Diagnostic Techniques; Sputum; Tuberculosis, Pulmonary; DNA Primers; Female; Fluorescence; Adult; Male; Tuberculosis; Middle Aged
PubMed: 38938885
DOI: 10.3389/fcimb.2024.1349063 -
Biology Methods & Protocols 2024Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the...
Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single-point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wild-type KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wild-type counterpart.
PubMed: 38938409
DOI: 10.1093/biomethods/bpae041 -
International Journal of Biological... Jun 2024Food allergens elicit abnormal immune system responses among allergic individuals and sensitive detection for allergenic ingredient is greatly significant. To address...
Food allergens elicit abnormal immune system responses among allergic individuals and sensitive detection for allergenic ingredient is greatly significant. To address this need, a novel fluorescent aptasensor, assisted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), have been developed for food allergens. In this study, aptamer offers distinctive recognition capabilities in binding specific targets, while CRISPR-associated-12a protein (Cas12a) holds precise cis-cleavage for cutting fluorescent signal probes. Notably, the utilization of Cas12a cis-cleavage activity, rather than trans-cleavage, eliminates the necessity for additional fluorescent probes, thus reducing interference between substances and enhancing sensitivity. Throughout the process, complementary DNA (cDNA) plays a crucial dual role in target recognition conversion and signal presentation, representing a key challenge and innovative aspect of this study. To evaluate the performance of the aptasensor, lysozyme (LYS) is employed as a representative model target of food allergens. Under optimal conditions, the developed aptasensor could achieve an exceptional low limit of detection (LOD) of 6.10 pM with a dynamic detection range of 10 pM-320 pM. The aptasensor demonstrates high selectivity and great recovery rates. This strategy yields promising outcomes, holding the potential to serve as a valuable reference for various food allergens detection.
PubMed: 38936584
DOI: 10.1016/j.ijbiomac.2024.133444 -
Spectrochimica Acta. Part A, Molecular... Jun 2024The accurate and sensitive detection of metallothionein (MT) is of great significance in the fields of biomedical, toxicological and environmental sciences. In this...
The accurate and sensitive detection of metallothionein (MT) is of great significance in the fields of biomedical, toxicological and environmental sciences. In this work, based on the high affinity interaction between MT and the heavy metal ions of Hg and the significant signal amplification capability of Cas12a/crRNA enzyme as well, we report a simple and highly sensitive method for visual detection of MT, a biomarker in fish for heavy metal ion-induced water bio-pollution. The target MT molecules bind Hg in the Hg- complexed hairpin DNA probes to unfold the hairpin structure into ssDNAs, which hybridize with the partial dsDNA duplexes via strand displacement to yield specific sequence-containing dsDNAs. Cas12a/crRNA recognizes these specific sequences to activate its enzyme activity to cyclically cleave the ssDNA linkers in the blue colored gold nanoparticle aggregates to transit their color into red to realize visual detection of MT. Owing to the signal amplification by Cas12a/crRNA, as low as 25 nM of MT can be visually detected with naked eye. In addition, our colorimetric detection method has high selectivity for MT against other interference proteins and can detect MT in the livers and kidneys of crucian carps bought from a local supermarket. Moreover, the developed assay overcomes the limitations of conventional MT detection methods in terms of complexity, high cost and low sensitivity and can therefore offer new methods for monitoring water bio-pollutions.
PubMed: 38936209
DOI: 10.1016/j.saa.2024.124682 -
PloS One 2024Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS...
Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS events, such as expressed sequence tags (EST), microarrays and RNA-seq. However, EST has limitations in identifying low-abundance genes, while microarray and RNA-seq are high-throughput technologies, and PCR-based technology is needed for validation. To overcome the limitations of EST and shortcomings of high-throughput technologies, we established a method to identify AS events, especially for low-abundance genes, by reverse transcription (RT) PCR with gene-specific primers (GSPs) followed by nested PCR. This process includes two major steps: 1) the use of GSPs to amplify as long as the specific gene segment and 2) multiple rounds of nested PCR to screen the AS and confirm the unknown splicing variants. With this method, we successfully identified three new splicing variants, namely, GenBank Accession No. HM623886 for the bdnf gene (GenBank GeneID: 12064), GenBank Accession No. JF417977 for the trkc gene (GenBank GeneID: 18213) and GenBank Accession No. HM623888 for the glb-18 gene (GenBank GeneID: 172485). In addition to its reliability and simplicity, the method is also cost-effective and labor-intensive. In conclusion, we developed an RT-nested PCR method using gene-specific primers to efficiently identify known and novel AS variants. This approach overcomes the limitations of existing methods for detecting rare transcripts. By enabling the discovery of new isoforms, especially for low-abundance genes, this technique can aid research into aberrant splicing in disease. Future studies can apply this method to uncover AS variants involved in cancer, neurodegeneration, and other splicing-related disorders.
Topics: Alternative Splicing; Humans; Brain-Derived Neurotrophic Factor; Reverse Transcriptase Polymerase Chain Reaction; DNA Primers
PubMed: 38935635
DOI: 10.1371/journal.pone.0305201 -
Analytical Chemistry Jun 2024Bacterial viability assessment plays an important role in food-borne pathogen detection and antimicrobial drug development. Here, we first used GelRed as a DNA-binding...
Bacterial viability assessment plays an important role in food-borne pathogen detection and antimicrobial drug development. Here, we first used GelRed as a DNA-binding stain for a bacterial viability assessment. It was found that live bacteria were able to exclude GelRed, which however could easily penetrate dead ones and be absorbed nonspecifically on the bacterial periplasm. Cations were used to reduce the nonspecific adsorption and greatly increase the red fluorescence ratio of dead to live bacteria. Combined with SYTO 9 (a membrane-permeable dye) for double-staining, a ratiometric fluorescent method was established. Using O157:H7 as a bacteria model, the ratiometric fluorescent method can probe dead bacteria as low as 0.1%. A linear correlation between the ratiometric fluorescence and the theoretical ratio of dead bacteria was acquired, with a correlation coefficient of 0.97. Advantages in sensitivity, accuracy, and safety of the GelRed/SYTO9-based ratiometric fluorescent method against traditional methods were demonstrated. The established method was successfully applied to the assessment of germicidal efficacy of different heat treatments. It was found that even 50 °C treatment could lead to the death of minor bacteria. The as-developed method has many potential applications in microbial researches, and we believe it could be expanded to the viability assessment of mammalian cells.
PubMed: 38934709
DOI: 10.1021/acs.analchem.4c01737 -
Viruses May 2024Hepatitis E virus (HEV) can cause self-limiting acute and chronic hepatitis infections, particularly in immunocompromised individuals. In developing countries, HEV is...
The Full-Genome Analysis and Generation of an Infectious cDNA Clone of a Genotype 6 Hepatitis E Virus Variant Obtained from a Japanese Wild Boar: In Vitro Cultivation in Human Cell Lines.
Hepatitis E virus (HEV) can cause self-limiting acute and chronic hepatitis infections, particularly in immunocompromised individuals. In developing countries, HEV is mainly transmitted via drinking contaminated water, whereas zoonotic transmission dominates the route of infection in developed countries, including Japan. Pigs are an important reservoir for HEV infection. Wild boars, which share the same genus and species as domestic pigs, are also an HEV reservoir. During our nationwide study of HEV infection in wild boar populations in Japan, a genotype 6 (HEV-6) strain, wbJHG_23, was isolated in Hyogo Prefecture in 2023. The genomic length was 7244 nucleotides, excluding the poly(A) tract. The wbJHG_23 strain exhibited the highest nucleotide identity throughout its genome with two previously reported HEV-6 strains (80.3-80.9%). Conversely, it displayed lower similarity (73.3-78.1%) with the HEV-1-5, HEV-7, and HEV-8 strains, indicating that, although closely related, the wbJHG_23 strain differs significantly from the reported HEV-6 strains and might represent a novel subtype. The wbJHG_23 strain successfully infected the human-derived cancer cell lines, PLC/PRF/5 and A549 1-1H8 cells, suggesting that HEV-6 has the potential for zoonotic infection. An infectious cDNA clone was constructed using a reverse genetics system, and a cell culture system supporting the efficient propagation of the HEV-6 strain was established, providing important tools for further studies on this genotype. Using this cell culture system, we evaluated the sensitivity of the wbJHG_23 strain to ribavirin treatment. Its good response to this treatment suggested that it could be used to treat human infections caused by HEV-6.
Topics: Animals; Cell Line; DNA, Complementary; Genome, Viral; Genotype; Hepatitis E; Hepatitis E virus; Japan; Phylogeny; RNA, Viral; Sus scrofa; Swine; Swine Diseases
PubMed: 38932135
DOI: 10.3390/v16060842 -
Microorganisms Jun 2024Prosthetic joint infection (PJI) is one of the most serious complications of joint replacement surgery among orthopedic surgeries and occurs in 1 to 2% of primary...
Prosthetic joint infection (PJI) is one of the most serious complications of joint replacement surgery among orthopedic surgeries and occurs in 1 to 2% of primary surgeries. Additionally, the cause of PJIs is mostly bacteria from the species, accounting for more than 98%, while fungi cause PJIs in only 1 to 2% of cases and can be difficult to manage. The current gold-standard microbiological method of culturing synovial fluid is time-consuming and produces false-negative and -positive results. This study aimed to identify a novel, accurate, and convenient molecular diagnostic method. The DreamDX primer-hydrolysis probe set was designed for the pan-bacterial and pan-fungal detection of DNA from pathogens that cause PJIs. The sensitivity and specificity of DreamDX primer-hydrolysis probes were 88.89% (95% CI, 56.50-99.43%) and 97.62% (95% CI, 87.68-99.88%), respectively, compared with the microbiological method of culturing synovial fluid, and receiver operating characteristic (ROC) area under the curve (AUC) was 0.9974 (*** < 0.0001). It could be concluded that the DreamDX primer-hydrolysis probes have outstanding potential as a molecular diagnostic method for identifying the causative agents of PJIs, and that host inflammatory markers are useful as adjuvants in the diagnosis of PJIs.
PubMed: 38930616
DOI: 10.3390/microorganisms12061234