ABSTRACT
Proper cell fate determination relies on precise spatial and temporal genome-wide cooperation between regulatory elements (REs) and their targeted genes. However, the lengths of REs defined using different methods vary, which indicates that there is sequence redundancy and that the context of the genome may be unintelligible. We developed a method called MAE-seq (Massive Active Enhancers by Sequencing) to experimentally identify functional REs at a 25-bp scale. In this study, MAE-seq was used to identify 626879, 541617 and 554826 25-bp enhancers in mouse embryonic stem cells (mESCs), C2C12 and HEK 293T, respectively. Using â¼1.6 trillion 25 bp DNA fragments and screening 12 billion cells, we identified 626879 as active enhancers in mESCs as an example. Comparative analysis revealed that most of the histone modification datasets were annotated by MAE-Seq loci. Furthermore, 33.85% (212195) of the identified enhancers were identified as de novo ones with no epigenetic modification. Intriguingly, distinct chromatin states dictate the requirement for dissimilar cofactors in governing novel and known enhancers. Validation results show that these 25-bp sequences could act as a functional unit, which shows identical or similar expression patterns as the previously defined larger elements, Enhanced resolution facilitated the identification of numerous cell-specific enhancers and their accurate annotation as super enhancers. Moreover, we characterized novel elements capable of augmenting gene activity. By integrating with high-resolution Hi-C data, over 55.64% of novel elements may have a distal association with different targeted genes. For example, we found that the Cdh1 gene interacts with one novel and two known REs in mESCs. The biological effects of these interactions were investigated using CRISPR-Cas9, revealing their role in coordinating Cdh1 gene expression and mESC proliferation. Our study presents an experimental approach to refine the REs at 25-bp resolution, advancing the precision of genome annotation and unveiling the underlying genome context. This novel approach not only advances our understanding of gene regulation but also opens avenues for comprehensive exploration of the genomic landscape.
Subject(s)
Genome , Regulatory Sequences, Nucleic Acid , Animals , Mice , Regulatory Sequences, Nucleic Acid/genetics , Chromatin , Genomics/methods , Gene Expression Regulation , Enhancer Elements, GeneticABSTRACT
A photocatalytic annulation cascade of unactivated N-alkene-linked indoles with Langlois' reagent by a radical relay is developed at room temperature under blue LED irradiation. The reaction afforded a series of tri/difluoromethylated pyrrolo[1,2-a]indoles in moderate to good yields. The DFT study suggests that the reaction is ascribed to a rhodamine 6G-induced cyclization cascade involving vinyl addition-radical relay and hydrogen-atom-abstraction (HAA) processes, and interestingly, pyrrolo[1,2-a]indoles are applied as fluorescent dyes into the fluorescence spectrum and live-cell imaging. This paper represents an initial example on photocatalytic cyclization cascades by radical relay and the HAA process.
ABSTRACT
As a representative gas of food spoilage, the development of rapid hydrogen sulfide (H2S) analysis strategies for food safety control is in great demand. Despite traditional methods for H2S detection possessing great achievements, they are still incapable of meeting the requirement of portability and quantitative detection at the same time. Herein, a nanozyme catalysis pressure-powered sensing platform that enables visual quantification with the naked eye is proposed. In this methodology, Pt nanozyme inherits the catalase-like activity to facilitate the decomposition of H2O2 to O2, which can significantly improve the pressure in the closed container, further pushing the movement of indicator dye. Furthermore, H2S was found to effectively inhibit the catalytic activity of Pt nanozyme, indicating that the catalase-like activity of PtNPs may be regulated by varying concentrations of H2S. Therefore, by utilizing a self-designed pressure-powered microchannel device, the concentration of H2S was successfully converted into a distinct signal variation in distance. The effectiveness of the as-designed sensor in assessing the spoilage of red wine by H2S determination has been demonstrated. It exhibits a strong correlation between the change in dye distance and H2S concentration within the range of 1-250 µM, with a detection limit of 0.17 µM. This method is advantageous as it enhances the quantitative detection of H2S with the naked eye based on the portable pressure-powered sensing platform, as compared to traditional H2S biosensors. Such a pressure-powered distance variation platform would greatly broaden the application of H2S-based detection in food spoilage management.
ABSTRACT
BACKGROUND: Current solid-phase reversible immobilization (SPRI) beads technology is widely used in molecular biology due to its convenience for DNA manipulation. However, the high performance commercial SPRI beads have no price advantage over our method. Furthermore, the use of commercially available SPRI beads standards does not provide the flexibility required for a number of specific nucleic acid handling scenarios. RESULTS: We report an efficient DNA purification strategy by combining home-made beads-suspension buffer with SPRI beads. The method tests the critical concentrations of polyethylene glycol (PEG) 8000 and beads to maximise recovery. And the composition of the SPRI beads DNA purification system (SDPS) was determined at 20% PEG 8000, 2 M NaCl and 16.3 mM MgCl2, and 1.25 mg/ml beads (1/8th original concentration). Then, we tested the DNA recovery of the SDPS, and the result showed that it was comparable to the control (AMPure XP beads). In the study, we have also developed an adjustment SPRI beads DNA purification system (ASDPS), the volume of ASDPS per reaction is 0.6× reaction volume (beads/samples). The performance of ASDPS is similar to SDPS and the control. But the cost of our methods is only about 1/24th of the control. To further assess its performance, we prepare the DNA-seq libraries to evaluate the yield, library quality, capture efficiency and consistency. We have compared all these results with the performance of the control and confirmed its efficiency. CONCLUSION: We have proposed an alternative DNA purification approach with great flexibility, allowing researchers to manipulate DNA in different conditions. And ultimately, its application will benefit molecular biology research in the future.
Subject(s)
DNA , Cost-Benefit AnalysisABSTRACT
It is largely unknown how mammalian genomes evolve under rapid speciation and environmental adaptation. An excellent model for understanding fast evolution is provided by the genus Sus, which diverged relatively recently and lacks postzygotic isolation. Here, we present a high-quality reference genome of the Visayan warty pig, which is specialized to a tropical island environment. Comparing the genome sequences and chromatin contact maps of the Visayan warty pig (Sus cebifrons) and domestic pig (Sus scrofa), we characterized the dynamics of chromosomal structure evolution during Sus speciation, revealing the similar chromosome conformation as the potential biological mechanism of frequent postdivergence hybridization among Suidae. We further investigated the different signatures of adaptive selection and domestication in Visayan warty pig and domestic pig with specific emphasize on the evolution of olfactory and gustatory genes, elucidating higher olfactory diversity in Visayan warty pig and positive and relaxed evolution of bitter and fat taste receptors, respectively, in domestic pig. Our comprehensive evolutionary and comparative genome analyses provide insight into the dynamics of genomes and how these change over relative short evolutionary times, as well as how these genomic differences encode for differences in the phenotypes.
Subject(s)
Chromosomes , Genome , Animals , Genomics , Sus scrofa/genetics , Swine/geneticsABSTRACT
Chromosome conformation-capture technologies are widely used in 3D genomics; however, experimentally, such methods have high-noise limitations and, therefore, require significant bioinformatics efforts to extract reliable distal interactions. Miscellaneous undesired linear DNAs, present during proximity-ligation, represent a main noise source, which needs to be minimized or eliminated. In this study, different exonuclease combinations were tested to remove linear DNA fragments from a circularized DNA preparation. This method efficiently removed linear DNAs, raised the proportion of annulation and increased the valid-pairs ratio from â¼40% to â¼80% for enhanced interaction detection in standard Hi-C. This strategy is applicable for development of various 3D genomics technologies, or optimization of Hi-C sequencing efficiency.
Subject(s)
Chromosomes, Mammalian , Exodeoxyribonucleases , Genomics/methods , Animals , Cell Line , Cells, Cultured , Chromatin , MiceABSTRACT
BACKGROUND: As a powerful tool, RNA-Seq has been widely used in various studies. Usually, unmapped RNA-seq reads have been considered as useless and been trashed or ignored. RESULTS: We develop a strategy to mining the full length sequence by unmapped reads combining with specific reverse transcription primers design and high throughput sequencing. In this study, we salvage 36 unmapped reads from standard RNA-Seq data and randomly select one 149 bp read as a model. Specific reverse transcription primers are designed to amplify its both ends, followed by next generation sequencing. Then we design a statistical model based on power law distribution to estimate its integrality and significance. Further, we validate it by Sanger sequencing. The result shows that the full length is 1556 bp, with insertion mutations in microsatellite structure. CONCLUSION: We believe this method would be a useful strategy to extract the sequences information from the unmapped RNA-seq data. Further, it is an alternative way to get the full length sequence of unknown cDNA.
Subject(s)
High-Throughput Nucleotide Sequencing , DNA, Complementary , RNA-Seq , Sequence Analysis, RNA , Exome SequencingABSTRACT
An alternative process of Pd-catalyzed C-4 selective coupling of 2,4-dichloropyridines with boronic esters was developed, which afforded 24 examples of C-4 coupled pyridines in moderate to good yields. After further arylation, 21 examples of C-2, C-4 diarylated pyridines with a significant photophysical property were obtained, which were applied as pyridine-based dyes into live-cell imaging with good biocompatibility and low toxicity.
Subject(s)
Coloring Agents , Palladium , Catalysis , Cells , Molecular Structure , PyridinesABSTRACT
Procaterol hydrochloride (Prh) can inhibit KClO3 oxidation of fluorescein isothiocyanate (FITC) to form a non-phosphorescent compound, which causes room temperature phosphorescence (RTP) of FITC in the system to enhance sharply the linear relationship between ∆Ip and the Prh content. Thus, a rapid response and highly sensitive phosphorescence sensor for the determination of Prh has been developed based on the inhibiting effect of Prh on KClO3 oxidation of FITC. This simple, high sensitivity (detection limit (LD) calculated by 3Sb /k was 0.019 fg/spot, sample volume 0.40 µl, corresponding concentration 4.8 × 10(-14) g ml(-1) ) and selective sensor with a wide linear range (0.080-11.20 g/spot) has been applied to detect Prh in blood samples, and the results were consistent with those obtained by high-performance liquid chromatography (HPLC). Simultaneously, the mechanism of the phosphorescence sensor for the detection of Prh was also investigated using infrared spectroscopy.
Subject(s)
Fluorescein-5-isothiocyanate/chemistry , Luminescent Measurements/methods , Procaterol/analysis , Procaterol/pharmacology , Animals , Chlorates/chemistry , Chromatography, High Pressure Liquid , Fluorescent Dyes/chemistry , Humans , Limit of Detection , Oxidation-Reduction , Procaterol/blood , Procaterol/urine , Sensitivity and Specificity , Spectrophotometry, Infrared , Sus scrofaABSTRACT
A highly sensitive fluorescent probe for clenbuterol hydrochloride (CLB) detection has been first designed based on its catalytic effect on NaIO4 oxidating eosine Y (R). And this environment-friendly, simple, rapid, selective and sensitive fluorescent probe has been utilized to detect CLB in the practical samples with the results consisting with those obtained by GC/MS. The structures of R and CLB were characterized by infrared spectra. The mechanism of the proposed assay for the detection of CLB was also discussed.
Subject(s)
Clenbuterol/analysis , Eosine Yellowish-(YS)/chemistry , Fluorescent Dyes/chemistry , Periodic Acid/chemistry , Catalysis , Luminescent Measurements , Molecular Structure , Oxidation-ReductionABSTRACT
Fluorescein (HFin) could emit strong and stable room temperature phosphorescence (RTP) signal on polyamide membrane (PAM) using Pb(2+) as the ion perturber. Carbaryl could activate effect on NaIO4 oxidating HFin, which caused the RTP signal of the system to quench sharply. The phosphorescence intensity (ΔI p) of activating system higher 3.3 times (119.4/36.0) than that of non-activating system, and is directly proportional to the content of carbaryl. Thus, an activating solid substrate room temperature phosphorimetry (SSRTP) for carbaryl detection has been established. This sensitive (the limit of quantification (LOQ) was 2.0 × 10(-13) g mL(-1)), selective, simple and rapid method has been applied to determine trace carbaryl in water samples with the results consisting with those obtained by fluorimetry, showing its high accuracy. The apparent activation energy (E) and rate constant (k) of this activating reaction were 20.77 kJ mol(-1) and 1.85 × 10(-4) s(-1), respectively. Meanwhile, the mechanism of activating SSRTP for carbaryl detection was also discussed using infrared spectra (IR).
ABSTRACT
Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-ß stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-ß stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.
Subject(s)
Fibroblasts , Genome-Wide Association Study , Animals , Mice , Promoter Regions, Genetic , Genome , Genomics , Mammals/geneticsABSTRACT
As one of the major public health problems, cancers seriously threaten the human health. Among them, lung cancer is considered to be one of the most life-threatening malignancies. Therefore, developing early diagnosis technology and timely treatment for lung cancer is urgent. Recent research has witnessed that measuring changes of biomarkers expressed in lung cancer has practical significance. Meanwhile, we note that bioimaging with organic fluorescent probes plays an important role for its high sensitivity, real-time analysis and simplicity of operation. In the past years, kinds of organic fluorescent probes targeting lung cancer related biomarker have been developed. Herein, we summarize the research progress of organic fluorescent probes for the detection of lung cancer related biomarkers in this review, along with their design principle, luminescence mechanism and bioimaging application. Additionally, we put forward some challenges and future prospects from our perspective.
Subject(s)
Fluorescent Dyes , Lung Neoplasms , Humans , Lung Neoplasms/diagnostic imaging , Biomarkers, Tumor , LuminescenceABSTRACT
A simple, sensitive and reliable dopamine (DA) biosensor was developed based on a carbon dots (CDs) and chitosan (CS) composite film modified glassy carbon electrode (CDs-CS/GCE). Under optimal conditions, the CDs-CS/GCE showed a better electrochemical response for the detection of DA than that of the glassy carbon electrode (GCE). The oxidation peak current (Ipa) of DA was linear with the concentration of DA in the range from 0.1 µM to 30.0 µM with the limit of detection as 11.2 nM (3S/N). The CDs-CS/GCE was applied to the detection of DA content in an injection solution of DA with satisfactory results.
Subject(s)
Biosensing Techniques/methods , Carbon/chemistry , Chitosan/chemistry , Dopamine/analysis , Nanocomposites/chemistry , Buffers , Diffusion , Dopamine/chemistry , Electrochemistry , Electrodes , Hydrogen-Ion Concentration , Limit of Detection , Oxidation-ReductionABSTRACT
Efficient, stable and low-cost catalysts are essential for improving the efficiency of hydrogen evolution reactions. Herein, the MoO2@Ni2P heterostructure electrocatalyst was synthesized in a self-supported system on a carbon paper (CP) by two-step deposition and phosphorization at low temperature. The self-supported nanoarray structure makes the catalyst to effectively and efficiently transfer electrons and exposes more of its active sites. Moreover, the strong interaction between Ni2P and MoO2 helps to effectively optimize the electronic structure. The density of states calculations demonstrate that there is an increase in the density of electronic states near the MoO2/Ni2P Fermi level. This shows that MoO2/Ni2P has fast charge transfer kinetics. MoO2 modulates the d-band center of nickel, resulting in moderate adsorption/desorption of hydrogen. The above results show that MoO2@Ni2P has good hydrogen evolution activity. The experimental results show that the overpotential (η10) of MoO2@Ni2P/CP in the alkaline environment is only 57 mV with a Tafel slope of 61 mV dec-1. It is similar to the commercial noble-metal catalysts and outperforms most reported catalysts.
ABSTRACT
Gene regulatory networks are now at the forefront of precision biology, which can help researchers better understand how genes and regulatory elements interact to control cellular gene expression, offering a more promising molecular mechanism in biological research. Interactions between the genes and regulatory elements involve different promoters, enhancers, transcription factors, silencers, insulators, and long-range regulatory elements, which occur at a â¼10 µm nucleus in a spatiotemporal manner. In this way, three-dimensional chromatin conformation and structural biology are critical for interpreting the biological effects and the gene regulatory networks. In the review, we have briefly summarized the latest processes in three-dimensional chromatin conformation, microscopic imaging, and bioinformatics, and we have presented the outlook and future directions for these three aspects.
ABSTRACT
Developing an effective method for the detection of aflatoxin B1 (AFB1) remains an arduous task due to the high toxicity of AFB1 to a health concern. In this study, a sensitive and reliable electrochemical aptasensor based on carbon dots/α-Fe2O3-Fe3O4 nanocomposite (CDs/α-Fe2O3-Fe3O4) is constructed for the determination of AFB1. The CDs have good electrical conductivity and large specific surface areas to improve the aptasensor's sensitivity. The α-Fe2O3-Fe3O4 can not only improve the catalytic performance of the aptasensor but also have magnetism, which can realize the recovery of CDs/α-Fe2O3-Fe3O4 to avoid material waste and environmental pollution. This electrochemical aptasensor can achieve a good linear (0.001-100.0 nM) and excellent detection limit (0.5 pM) for the determination of AFB1. In addition, the aptasensor was also applied to determine AFB1 in beer, rice, and peanuts, all results were in good agreement with HPLC, indicating that the electrochemical aptasensor has a broad application prospect.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Nanocomposites , Aflatoxin B1/analysis , Carbon , Electrochemical Techniques , Gold , Limit of DetectionABSTRACT
Sitodiplosis mosellana, a notorious pest of wheat worldwide, copes with temperature extremes during harsh summers and winters by entering obligatory diapause as larvae. However, the metabolic adaptive mechanism underlying this process is largely unknown. In this study, we performed a comparative metabolomics analysis on S. mosellana larvae at four programmed developmental stages, i.e., pre-diapause, diapause, low temperature quiescence and post-diapause development. In total, we identified 54 differential metabolites based on pairwise comparisons of the four groups. Of these metabolites, 37 decreased in response to diapause, including 4 TCA cycle intermediates (malic acid, citric acid, fumaric acid, α-ketoglutaric acid), 2 saturated fatty acids (palmitic acid, stearic acid) and most amino acids. In contrast, nine metabolites, including trehalose, glycerol, mannitol, proline, alanine, oleic acid and linoleic acid were significantly higher in both the diapause and quiescent stages than the other two stages. In addition to two of them (trehalose, proline), glutamine was also significantly highest in the cold quiescence stage. These elevated metabolites could function as cryoprotectants and/or energy reserves. These findings suggest that the reduced TCA cycle activity and elevated biosynthesis of functional metabolites are most likely responsible for maintaining low metabolic activity and cold tolerance during diapause, which is crucial for the survival and post-diapause development of this pest.
ABSTRACT
Sitodiplosis mosellana is a major wheat pest that oviposits on spikes, and resistant wheat varieties have been released. However, wheat spike volatiles mediating S. mosellana host selection or resistance are largely unknown. In this study, we found that the highly susceptible wheat varieties Xinong 822, Xinong 88, and Xiaoyan 22 were preferred for S. mosellana oviposition, and their spike volatiles were more attractive to females compared to the resistant varieties Kenong 1006, Shanmai 139, and Jinmai 47. Importantly, we found five odor components evoking obvious concentration-dependent electroantennogram (EAG) and behavioral response. Notably, 3-hexanol, cis-3-hexenylacetate, and hexyl acetate strongly attracted females, whereas ocimene, a dominant component of three resistant varieties, and α-farnesene, absent in Xinong 88, repelled females. Significant attraction was also observed in a synthetic blend mimicking Xinong 822 volatiles. These results suggest that these wheat volatiles are involved in host selection of S. mosellana and provide a basis for development of semichemical-based pest management.