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Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA.Genome Research Jul 2021Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased...
Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.
PubMed: 34140313
DOI: 10.1101/gr.266551.120 -
Nature Biotechnology Apr 2024Current N-methyladenosine (mA) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio,...
Current N-methyladenosine (mA) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale mA RNA immunoprecipitation and sequencing (picoMeRIP-seq) for studying mA in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark mA mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.
Topics: Animals; Mice; Zebrafish; RNA; RNA, Messenger; Embryonic Stem Cells; Cells, Cultured
PubMed: 37349523
DOI: 10.1038/s41587-023-01831-7 -
Molecules (Basel, Switzerland) Jan 2021Melatonin is a hormone secreted in the pineal gland with several functions, especially regulation of circadian sleep cycle and the biological processes related to it.... (Review)
Review
Melatonin is a hormone secreted in the pineal gland with several functions, especially regulation of circadian sleep cycle and the biological processes related to it. This review evaluates the bioavailability of melatonin and resulting metabolites, the presence of melatonin in wine and beer and factors that influence it, and finally the different benefits related to treatment with melatonin. When administered orally, melatonin is mainly absorbed in the rectum and the ileum; it has a half-life of about 0.45-1 h and is extensively inactivated in the liver by phase 2 enzymes. Melatonin (MEL) concentration varies from picograms to ng/mL in fermented beverages such as wine and beer, depending on the fermentation process. These low quantities, within a dietary intake, are enough to reach significant plasma concentrations of melatonin, and are thus able to exert beneficial effects. Melatonin has demonstrated antioxidant, anticarcinogenic, immunomodulatory and neuroprotective actions. These benefits are related to its free radical scavenging properties as well and the direct interaction with melatonin receptors, which are involved in complex intracellular signaling pathways, including inhibition of angiogenesis and cell proliferation, among others. In the present review, the current evidence on the effects of melatonin on different pathophysiological conditions is also discussed.
Topics: Animals; Anticarcinogenic Agents; Antioxidants; Beer; Circadian Rhythm; Fermentation; Humans; Melatonin; Neuroprotective Agents; Wine
PubMed: 33440795
DOI: 10.3390/molecules26020343 -
Astrobiology Dec 2022For exploring nearby stars, let us consider the challenges of a picogram- to nanogram-scale probe to land, replicate, and produce a communications module based on...
For exploring nearby stars, let us consider the challenges of a picogram- to nanogram-scale probe to land, replicate, and produce a communications module based on biominerals at the destination. A billion such probes could be launched for similar cost as a single gram-scale probe. One design is a highly reflective light sail, traveling a long straight line toward the gravitational well of a destination star, and then photo-deflected to the closest nonluminous mass-ideally a planet or moon with exposed liquid water.
PubMed: 36475966
DOI: 10.1089/ast.2022.0008 -
Tissue Engineering. Part B, Reviews Aug 2022The recent discovery of mesenchymal stem cells within periapical lesions (PL-MSC) has presented novel opportunities for managing periradicular diseases in adult teeth by... (Review)
Review
The recent discovery of mesenchymal stem cells within periapical lesions (PL-MSC) has presented novel opportunities for managing periradicular diseases in adult teeth by way of enhancing tissue regeneration. This discovery coincides with the current paradigm shift toward biologically driven treatment strategies in endodontics, which have typically been reserved for non-vital immature permanent teeth. One such approach that shows promise is utilizing local endogenous non-collagenous dentine extracellular matrix components (dECM) to recruit and upregulate the intrinsic regenerative capacity of PL-MSCs . At picogram levels, these morphogens have demonstrated tremendous ability to enhance the cellular activities in and animal studies that would otherwise be necessary for periradicular regeneration. Briefly, these include proliferation, viability, migration, differentiation, and mineralization. Therefore, topical application of dECMs during ortho- or retrograde root canal treatment could potentially enhance and sustain the regenerative mechanisms within diseased periapical tissues that are responsible for attaining favorable clinical and radiographic outcomes. This would provide many advantages when compared with conventional antimicrobial-only therapies for apical periodontitis (AP), which do not directly stimulate healing and have had stagnant success rates over the past five decades despite significant advances in operative techniques. The aim of this narrative review was to present the novel concept of exploiting endogenous dECMs as clinical tools for treating AP in mature permanent teeth. A large scope of literature was summarized to discuss the issues associated with conventional treatment modalities; current knowledge surrounding PL-MSCs; composition of the dECM; inductive potentials of dECM morphogens in other odontogenic stem cell niches; how treatment protocols can be adapted to take advantage of dECMs and PL-MSCs; and finally, the challenges currently impeding successful clinical translation alongside directions for future research. Impact statement Apical periodontitis (AP) is an inflammatory condition that is associated with a great degree of morbidity and ultimately leads to tooth loss. The purpose of this review was to summarize the current evidence pertaining to stem cell therapy in endodontics and present a novel clinical methodology through which they may be utilized to address AP. A comprehensive overview of the basic science, clinical translation, and potential challenges are presented in this review.
Topics: Animals; Cell Differentiation; Dentin; Periapical Periodontitis; Root Canal Therapy; Tissue Engineering
PubMed: 34309453
DOI: 10.1089/ten.TEB.2021.0074 -
Molecules (Basel, Switzerland) Mar 2021Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities,...
Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities, a simple and sensitive approach for picogram detection of lipolytic enzyme quantity is still highly desirable. Here we report a lipase detection assay which is 1000-fold more sensitive than previously reported methods. Our assay enables the detection of as low as 5 pg and 180 pg of lipolytic activity by direct spotting and zymography, respectively. Furthermore, we demonstrated that the detection sensitivity was adjustable by varying the buffering capacity, which allows for screening of both high and low abundance lipolytic enzymes. Coupled with liquid chromatography-mass spectrometry, our method provides a useful tool for sensitive detection and identification of lipolytic enzymes.
Topics: Chromatography, Liquid; Chromogenic Compounds; Electrophoresis, Polyacrylamide Gel; Enzyme Assays; Esterases; Lipase; Lipolysis; Mass Spectrometry; Substrate Specificity
PubMed: 33799781
DOI: 10.3390/molecules26061542 -
Bulletin of the National Research Centre 2022COVID-19 outbreak has engulfed different parts of the world, affecting more than 163 million people and causing more than 3 million deaths worldwide due to human... (Review)
Review
BACKGROUND
COVID-19 outbreak has engulfed different parts of the world, affecting more than 163 million people and causing more than 3 million deaths worldwide due to human transmission. Thus, it has become critical to identify the risk factors and laboratory parameters to identify patients who have high chances of worsening clinical symptoms or poor clinical outcomes. Therefore, the study aims to identify inflammatory markers that can help identify patients at increased risk for progression to critical illness, thus decreasing the risk of any mortality. Our study focussed on the predictive utility of C-reactive protein, Interleukin-6, D-dimer and Procalcitonin in assisting the management of COVID-19 patients with adverse clinical effects. Through literature search in electronic databases, we included the retrospective studies that evaluated the biomarkers among confirmed COVID-19 patients before initiation of treatment and who had a definite outcome (dead or discharged). Biomarkers were expressed in standardized difference in mean value, calculated based on study sizes and mean values between survivors and non-survivors considered the effect size. We carried out a meta-regression analysis to identify the causes of the heterogeneity between the studies.
RESULTS
Number of studies eligible for C-reactive protein, D-dimer and Interleukin-6 markers were eight, seven and four, respectively. Using random effect model revealed that the overall effect size with 95% confidence interval (CI) for C-reactive protein, D-dimer and Interleukin-6 were 1.45 (0.79-2.12) milligrams/litre, 1.12 (0.64-1.59) micrograms/millilitre Fibrinogen Equivalent Units and 1.34 (0.43-2.24) picograms/millilitre respectively was statistically significant ( < 0.05) inferring that the mean scores of these marker were significantly higher among the non-survivors compared to the survivors. Two studies were eligible for Procalcitonin marker and there was no heterogeniety ( -statistics = 0) between these studies. Therefore, fixed-effect model revealed that the overall effect size (95% CI) for Procalcitonin was 0.75 (0.30-1.21) Nanograms/millilitre was also high among non-survivors.
CONCLUSIONS
The study found that serum levels of C-reactive protein, Interleukin-6 and D-dimer showed significant elevation in non-survivors compared to survivors. Raised inflammatory markers aid in the risk stratification of COVID-19 patients and their proper management.
PubMed: 35261542
DOI: 10.1186/s42269-022-00733-z -
Biosensors Dec 2022As an antibody-free sensing membrane for the detection of the antibiotic tetracycline (TC), a liquid PVC membrane doped with the ion-pair tetracycline/θ-shaped anion...
As an antibody-free sensing membrane for the detection of the antibiotic tetracycline (TC), a liquid PVC membrane doped with the ion-pair tetracycline/θ-shaped anion [3,3'-Co(1,2-CBH)] ([-COSAN]) was formulated and deposited on a SWCNT modified gold microelectrode. The chosen transduction technique was electrochemical impedance spectroscopy (EIS). The PVC membrane was composed of: the tetracycline/[-COSAN] ion-pair, a plasticizer. A detection limit of 0.3 pg/L was obtained with this membrane, using bis(2-ethylhexyl) sebacate as a plasticizer. The sensitivity of detection of tetracycline was five times higher than that of oxytetracycline and of terramycin, and 22 times higher than that of demeclocycline. A shelf-life of the prepared sensor was more than six months and was used for detection in spiked honey samples. These results open the way to having continuous monitoring sensors with a high detection capacity, are easy to clean, avoid the use of antibodies, and produce a direct measurement.
Topics: Plasticizers; Tetracycline; Anti-Bacterial Agents; Microelectrodes; Antibodies; Oxytetracycline
PubMed: 36671906
DOI: 10.3390/bios13010071 -
Beilstein Journal of Nanotechnology 2022Quartz crystal microbalance (QCM) has been widely used for various sensing applications, including chirality detection due to the high sensitivity to nanogram or... (Review)
Review
Quartz crystal microbalance (QCM) has been widely used for various sensing applications, including chirality detection due to the high sensitivity to nanogram or picogram mass changes, fast response, real-time detection, easy operation, suitability in different media, and low experimental cost. The sensing performance of QCM is dependent on the surface design of the recognition layers. Various strategies have been employed for studying the relationship between the structural features and the specific detection of chiral isomers. This review provides an overview of the construction of chiral sensing layers by various nanostructures and materials in the QCM system, which include organic molecules, supermolecular assemblies, inorganic nanostructures, and metal surfaces. The sensing mechanisms based on these surface nanostructures and the related potentials for chiral detection by the QCM system are also summarized.
PubMed: 36348938
DOI: 10.3762/bjnano.13.100