Identification and Quantification of Locus-Specific 8-Oxo-7,8-dihydroguanine in DNA at Ultrahigh Resolution Based on G-Triplex-Assisted Rolling Circle Amplification.

Damage of reactive oxygen species to various molecules such as DNA has been related to many chronic and degenerative human diseases, aging, and even cancer. 8-Oxo-7,8-dihydroguanine (OG), the most significant oxidation product of guanine (G), has become a biomarker of oxidative stress as well as gene regulation. The positive effect of OG in activating transcription and the negative effect in inducing mutation are a double-edged sword; thus, site-specific quantification is helpful to quickly reveal the functional mechanism of OG at hotspots. Due to the possible biological effects of OG at extremely low abundance in the genome, the monitoring of OG is vulnerable to signal interference from a large amount of G. Herein, based on rolling circle amplification-induced G-triplex formation and Thioflavin T fluorescence enhancement, an ultrasensitive strategy for locus-specific OG quantification was constructed. Owing to the difference in the hydrogen-bonding pattern between OG and G, the nonspecific background signal of G sites was completely suppressed through enzymatic ligation of DNA probes and the triggered specificity of rolling circle amplification. After the signal amplification strategy was optimized, the high detection sensitivity of OG sites with an ultralow detection limit of 0.18 amol was achieved. Under the interference of G sites, as little as 0.05% of OG-containing DNA was first distinguished. This method was further used for qualitative and quantitative monitoring of locus-specific OG in genomic DNA under oxidative stress and identification of key OG sites with biological function.

New G-Triplex DNA Dramatically Activates the Fluorescence of Thioflavin T and Acts as a Turn-On Fluorescent Sensor for Uracil-DNA Glycosylase Activity Detection.

G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.

Fumarate suppresses B-cell activation and function through direct inactivation of LYN.

Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.

Qualitative and Quantitative Analytical Techniques of Nucleic Acid Modification Based on Mass Spectrometry for Biomarker Discovery.

Nucleic acid modifications play important roles in biological activities and disease occurrences, and have been considered as cancer biomarkers. Due to the relatively low amount of nucleic acid modifications in biological samples, it is necessary to develop sensitive and reliable qualitative and quantitative methods to reveal the content of any modifications. In this review, the key processes affecting the qualitative and quantitative analyses are discussed, such as sample digestion, nucleoside extraction, chemical labeling, chromatographic separation, mass spectrometry detection, and data processing. The improvement of the detection sensitivity and specificity of analytical methods based on mass spectrometry makes it possible to study low-abundance modifications and their biological functions. Some typical nucleic acid modifications and their potential as biomarkers are displayed, and efforts to improve diagnostic accuracy are discussed. Future perspectives are raised for this research field.

Ultrasensitive HPLC-MS Quantification of S-(2-Succino) Cysteine Based on Ethanol/Acetyl Chloride Derivatization in Fumarate Accumulation Cells.

Succination is a nonenzymatic and irreversible post-translational modification (PTM) with important biological significance, yielding S-(2-succino) cysteine (2SC) residue. This PTM is low in abundance and often requires a large amount of protein samples for 2SC quantification. In this work, an efficient quantification method based on ethanol/acetyl chloride chemical derivatization was developed. The three carboxyl groups of 2SC were all esterified to increase hydrophobicity, greatly improving its ionization efficiency. The sensitivity was increased by 112 times; the limit of detection was reduced to 0.885 fmol, and the protein usage was reduced by at least 10 times. The established method was used to detect the overall concentration of 2SC in fumarate accumulation cells quantitatively.

Aging-induced pseudouridine synthase 10 impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSC) exhibit compromised reconstitution capacity and differentiation-bias towards myeloid lineage, however, the molecular mechanism behind it remains not fully understood. In this study, we observed that the expression of pseudouridine (Ψ) synthase 10 is increased in aged hematopoietic stem and progenitor cells (HSPC) and enforced protein of Ψ synthase 10 (PUS10) recapitulates the phenotype of aged HSC, which is not achieved by its Ψ synthase activity. Consistently, we observed no difference of transcribed RNA pseudouridylation profile between young and aged HSPC. No significant alteration of hematopoietic homeostasis and HSC function is observed in young Pus10-/- mice, while aged Pus10-/- mice exhibit mild alteration of hematopoietic homeostasis and HSC function. Moreover, we observed that PUS10 is ubiquitinated by E3 ubiquitin ligase CRL4DCAF1 complex and the increase of PUS10 in aged HSPC is due to aging-declined CRL4DCAF1- mediated ubiquitination degradation signaling. Taken together, this study for the first time evaluated the role of PUS10 in HSC aging and function, and provided a novel insight into HSC rejuvenation and its clinical application.

In-source fragmentation of nucleosides in electrospray ionization towards more sensitive and accurate nucleoside analysis.

Nucleosides have been found to suffer in-source fragmentation (ISF) in electrospray ionization mass spectrometry, which leads to reduced sensitivity and ambiguous identification. In this work, a combination of theoretical calculations and nuclear magnetic resonance analysis revealed the key role of protonation at N3 near the glycosidic bond during ISF. Therefore, an ultrasensitive liquid chromatography-tandem mass spectrometry system for 5-formylcytosine detection was developed with 300 fold signal enhancement. Also, we established a MS1-only platform for nucleoside profiling and successfully identified sixteen nucleosides in the total RNA of MCF-7 cells. Taking ISF into account, we can realize analysis with higher sensitivity and less ambiguity, not only for nucleosides, but for other molecules with similar protonation and fragmentation behaviors.

Exploring acoustic characteristics of different aircraft types by fusing with aircraft tracking data.

In this study, we develop a method that assigns acoustic signals with Automatic Dependent Surveillance-Broadcast (ADS-B) data to build a labeled dataset of acoustic signals from aircraft without expensive ground-truth experiments. An exploration of the resultant labeled dataset enables an assessment of the acoustic characteristics from three types of aircraft. The fusion framework is evaluated using data from an acoustic sensor and collocated ADS-B receiver in the middle of a large urban area at Southern Methodist University in Dallas, Texas. Our results demonstrate the benefit of combining multiple types of data to generate a labeled dataset leveraging open-source aircraft surveillance data. By studying three classes of aircraft, we find that the smaller fixed wing single engine (FWSE) class is mostly detected within approximately 5000 m, while the larger fixed wing multi-engine (FWME) class is commonly detected out to greater distances above 7500 m. The FWSE class has a median source frequency at 100 Hz, compared to FWME class with median source frequency at 80 Hz, while rotorcraft has a source frequency falling into a lower range of 30-100 Hz.

Probabilistic Modeling of Multicamera Interference for Time-of-Flight Sensors.

The behavior of multicamera interference in 3D images (e.g., depth maps), which is based on infrared (IR) light, is not well understood. In 3D images, when multicamera interference is present, there is an increase in the amount of zero-value pixels, resulting in a loss of depth information. In this work, we demonstrate a framework for synthetically generating direct and indirect multicamera interference using a combination of a probabilistic model and ray tracing. Our mathematical model predicts the locations and probabilities of zero-value pixels in depth maps that contain multicamera interference. Our model accurately predicts where depth information may be lost in a depth map when multicamera interference is present. We compare the proposed synthetic 3D interference images with controlled 3D interference images captured in our laboratory. The proposed framework achieves an average root mean square error (RMSE) of 0.0625, an average peak signal-to-noise ratio (PSNR) of 24.1277 dB, and an average structural similarity index measure (SSIM) of 0.9007 for predicting direct multicamera interference, and an average RMSE of 0.0312, an average PSNR of 26.2280 dB, and an average SSIM of 0.9064 for predicting indirect multicamera interference. The proposed framework can be used to develop and test interference mitigation techniques that will be crucial for the successful proliferation of these devices.

Dimensioning Cuboid and Cylindrical Objects Using Only Noisy and Partially Observed Time-of-Flight Data.

One of the challenges of using Time-of-Flight (ToF) sensors for dimensioning objects is that the depth information suffers from issues such as low resolution, self-occlusions, noise, and multipath interference, which distort the shape and size of objects. In this work, we successfully apply a superquadric fitting framework for dimensioning cuboid and cylindrical objects from point cloud data generated using a ToF sensor. Our work demonstrates that an average error of less than 1 cm is possible for a box with the largest dimension of about 30 cm and a cylinder with the largest dimension of about 20 cm that are each placed 1.5 m from a ToF sensor. We also quantify the performance of dimensioning objects using various object orientations, ground plane surfaces, and model fitting methods. For cuboid objects, our results show that the proposed superquadric fitting framework is able to achieve absolute dimensioning errors between 4% and 9% using the bounding technique and between 8% and 15% using the mirroring technique across all tested surfaces. For cylindrical objects, our results show that the proposed superquadric fitting framework is able to achieve absolute dimensioning errors between 2.97% and 6.61% when the object is in a horizontal orientation and between 8.01% and 13.13% when the object is in a vertical orientation using the bounding technique across all tested surfaces.

Ultrasensitive and Single-Base Resolution Quantification of 8-Oxo-7,8-dihydroguanine in DNA by Extension and Ligation-Based qPCR.

Oxidative DNA damage is tightly linked to the development of multiple age-related diseases. The prominent oxidation product is 8-oxo-7,8-dihydroguanine (OG), which has been proved to be an important epigenetic-like biomarker. Quantification of the locus-specific OG frequency includes quantitative and locating information, which is of great significance for exploring the functional roles of OG in disease induction and gene regulation. Herein, an ultrasensitive quantification of OG at single-base resolution was established using real-time fluorescence quantitative polymerase chain reaction as an amplification tool. Based on the coding property of Bsu DNA polymerase that incorporates adenine on the opposite site of OG and the selectivity of the ligase for perfectly matched sequences, the difference between OG and G on the sequence could be enlarged. Well-performed Taq DNA ligase was selected out, and as low as 46.2 zmol of target DNA with an OG site and an OG frequency of 5% could be detected. G contents on a specific site were also detectable based on the similar principle, thus the OG frequency of this locus could be accurately determined by a standard addition method. This strategy was successfully applied to the evaluation of locus-specific OG in both model DNA and genomic DNA from human cervical carcinoma cell lines under multiple oxidative stress, showing the potential for functional research and dynamic monitoring of critical OG sites.

Probabilistic Modeling of Motion Blur for Time-of-Flight Sensors.

Synthetically creating motion blur in two-dimensional (2D) images is a well-understood process and has been used in image processing for developing deblurring systems. There are no well-established techniques for synthetically generating arbitrary motion blur within three-dimensional (3D) images, such as depth maps and point clouds since their behavior is not as well understood. As a prerequisite, we have previously developed a method for generating synthetic motion blur in a plane that is parallel to the sensor detector plane. In this work, as a major extension, we generalize our previously developed framework for synthetically generating linear and radial motion blur along planes that are at arbitrary angles with respect to the sensor detector plane. Our framework accurately captures the behavior of the real motion blur that is encountered using a Time-of-Flight (ToF) sensor. This work uses a probabilistic model that predicts the location of invalid pixels that are typically present within depth maps that contain real motion blur. More specifically, the probabilistic model considers different angles of motion paths and the velocity of an object with respect to the image plane of a ToF sensor. Extensive experimental results are shown that demonstrate how our framework can be applied to synthetically create radial, linear, and combined radial-linear motion blur. We quantify the accuracy of the synthetic generation method by comparing the resulting synthetic depth map to the experimentally captured depth map with motion. Our results indicate that our framework achieves an average Boundary F1 (BF) score of 0.7192 for invalid pixels for synthetic radial motion blur, an average BF score of 0.8778 for synthetic linear motion blur, and an average BF score of 0.62 for synthetic combined radial-linear motion blur.

Ultrasensitive Simultaneous Detection of Multiple Rare Modified Nucleosides as Promising Biomarkers in Low-Put Breast Cancer DNA Samples for Clinical Multi-Dimensional Diagnosis.

Early cancer diagnosis is essential for successful treatment and prognosis, and modified nucleosides have attracted widespread attention as a promising group of cancer biomarkers. However, analyzing these modified nucleosides with an extremely low abundance is a great challenge, especially analyzing multiple modified nucleosides with a different abundance simultaneously. In this work, an ultrasensitive quantification method based on chemical labeling, coupled with LC-MS/MS analysis, was established for the simultaneous quantification of 5hmdC, 5fdC, 5hmdU and 5fdU. Additionally, the contents of 5mdC and canonical nucleosides could be obtained at the same time. Upon derivatization, the detection sensitivities of 5hmdC, 5fdC, 5hmdU and 5fdU were dramatically enhanced by several hundred times. The established method was further applied to the simultaneous detection of nine nucleosides with different abundances in about 2 µg genomic DNA of breast tissues from 20 breast cancer patients. The DNA consumption was less than other overall reported quantification methods, thereby providing an opportunity to monitor rare, modified nucleosides in precious samples and biology processes that could not be investigated before. The contents of 5hmdC, 5hmdU and 5fdU in tumor tissues and normal tissues adjacent to the tumor were significantly changed, indicating that these three modified nucleosides may play certain roles in the formation and development of tumors and be potential cancer biomarkers. While the detection rates of 5hmdC, 5hmdU and 5fdU alone as a biomarker for breast cancer samples were 95%, 75% and 85%, respectively, by detecting these three cancer biomarkers simultaneously, two of the three were 100% consistent with the overall trend. Therefore, simultaneous detection of multiple cancer biomarkers in clinical samples greatly improved the accuracy of cancer diagnosis, indicating that our method has great application potential in clinical multidimensional diagnosis.

Systematic Profiling of Exosomal Small RNA Epigenetic Modifications by High-Performance Liquid Chromatography-Mass Spectrometry.

Exosomes are nanosized extracellular vesicles that have a critical role in intercellular communication and tumor microenvironment regulation. Extensive research has shown that exosomal small RNAs contribute to metastasis in multiple tumor types and that abnormal epigenetic modifications in nucleic acids also have an association with diverse diseases. However, the content of modified nucleosides on exosomal small RNAs has not been quantitatively reported. Because of the trace amounts of exosomes and matrix complexity, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a powerful tool for label-free sensitive and simultaneous determinations of six important modified nucleosides on small RNAs inside exosomes. This system performed well using only approximately 107-108 particles of exosomes to obtain modified nucleoside levels between 0.001 and 0.03, and the most striking result was that the content of m6A in exosomal small RNAs was continuously higher than that in the cells being analyzed. We hope that this conclusion helps establish a greater degree of deciphering accuracy on exosomes, which has considerable application potential in the diagnosis and prognosis of diseases.

Effect of TDI-Assisted Hydrophobic Surface Modification of Microcrystalline Cellulose on the Tensile Fracture of MCC/PLA Composite, and Estimation of the Degree of Substitution by Linear Regression.

Microcrystalline cellulose (MCC) was modified using toluene-2,4-diisocyanate (TDI) in tetrahydrofuran (THF). The reaction was set up for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 24 h at 75 °C. The study was aimed at hydrophobic modification of microcrystalline cellulose (MCC) to improve its dispersion in PLA matrix. Data from the elemental analysis were used to develop a statistical model to predict the degree of substitution (DS) of the OH on the surface of the MCC using both the water contact angle (WCA) and the time of carbamation as the independent variables. Composite was fabricated at 1%, 2%, 3%, 4%, and 5% fiber loading. Fourier transformed infrared spectroscopy was used to characterize the MCC and to confirm the successful graft of TDI to the MCC surface. The morphology and elemental analysis of the modified samples were examined with SEM-EDX. The samples' wettability was analyzed with a contact angle meter to measure the water contact angle (WCA). The tensile properties of composites were analyzed on a universal testing machine. The result showed that, after 1 h of carbamation, the minimum DS recorded was 0.11, and the maximum DS after 24 h was 0.16. The SEM revealed that the modified MCC had homogeneous dispersion in the polymer matrix. At 3% fiber loading, the tensile strength (TS) and elongation were at a maximum and had improvements of 80.67% and 79.44% as compared to neat PLA. The fractured tensile surface from SEM analysis showed that surface modification enhanced fiber-matrix adhesion and significantly improved the composite's strength and toughness. The proposed model that was developed in this study had a coefficient of determination (R2) of 93% to show that the model has a near-perfect goodness of fit and can well be an effective approach to predict the DS of OH from WCA and the time of reaction at similar or the same reaction conditions.

Rapid determination and continuous monitoring of propofol in microliter whole blood sample during anesthesia by paper spray ionization-mass spectrometry.

Propofol is a widely used intravenous anesthetic agent in sedation and general anesthesia. To improve the safety and maintain the depth of anesthesia, it is important to develop a rapid, sensitive, and reliable method to monitor the concentration of propofol in blood during anesthesia continuously. Here, we present a novel strategy based on paper spray ionization-mass spectrometry (PSI-MS) to detect propofol. Samples (in 10 µL) were mixed with methanol as protein precipitation solvent and 2,6-dimethylphenol as internal standard. Protein micro-precipitation was achieved with methanol by vortexing and centrifuging for 5 s each, and propofol was extracted to the supernatant. PSI-MS was performed in negative ionization mode, and MS signal lasted for 1 min. The analysis of a single sample was completed within 2 min. The area ratios of propofol to internal standard were calculated for quantification. Limit of detection of 5.5 ng mL-1 and limit of quantification of 18.2 ng mL-1 were achieved for propofol in whole blood. Calibration curve was linear in the range of 0.02-10 µg mL-1. The developed method was used successfully in monitoring the propofol concentration in 3 patients' whole blood during anesthesia, showing its further application in controlling and feeding-back target concentration infusion. Graphical abstract.

5hmC-MIQuant: Ultrasensitive Quantitative Detection of 5-Hydroxymethylcytosine in Low-Input Cell-Free DNA Samples.

Cell-free DNA (cfDNA)-based biomarkers such as mutation and methylation offer promising noninvasive strategies for disease diagnosis and prognosis. However, besides high-throughput sequencing, there has been no alternative approach to date to detect the epigenetic marks, such as 5-hydroxymethylcytosine (5hmC), in cfDNA. Here, we described a MnO2 oxidation and hydrazine-s-triazine reagent (i-Pr2N) labeling based method named 5hmC-MIQuant that achieved ultrasensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification of 5hmC in low-input DNA samples. This strategy improved the detection sensitivity of 5hmC by 178 times, and the limit of detection was as low as 14 amol. With simple preparation steps, 5hmC-MIQuant could quantify the 5hmC level in as little as 340 pg genomic DNA (equivalent to 57 copies of diploid genome). cfDNA samples from human plasma were successfully analyzed using 5hmC-MIQuant. This method is promising for the identification of 5hmC function in precious samples and the 5hmC-based noninvasive disease diagnosis.

Synthesis of a pH-Responsive Functional Covalent Organic Framework via Facile and Rapid One-Step Postsynthetic Modification and Its Application in Highly Efficient N1-Methyladenosine Extraction.

A facile and rapid postsynthetic modification strategy for functionalization of covalent organic framework (COF) was developed to synthesize a tailor-made pH-responsive COF called TpPa-1@Au@GSH for highly efficient extraction of N1-methyladenosine (m1A). Glutathione (GSH) was judiciously designed as the functional group for extracting and releasing m1A by pH variations. With the aid of gold nanoparticles (Au NPs) as linkers, GSH was successfully introduced to the robust substrate TpPa-1 in only one step spending only 1 h. Owing to the several-to-one immobilization of GSH on Au NPs and the large surface area of TpPa-1, this functional COF was constructed with abundant m1A binding sites. TpPa-1@Au@GSH showed excellent selectivity for m1A extraction by capturing m1A from a mixture of 14 nucleoside analogues followed by mass spectrometry analysis. It was proved to have ultrafast adsorption ability (only 1 min incubation time), high binding capacity (5 mg g-1, m1A/TpPa-1@Au@GSH), good reusability (at least 5 times), and good storage stability (at least 8 months at room temperature). Great performance was also achieved in extracting m1A from both animal and plant biological samples. The adsorption mechanism was demonstrated to be based on the electrostatic interaction. This work proposed a new approach for m1A extraction, demonstrated the high potential of COFs in biological sample pretreatment, and offered an effective and versatile route for functionalization of COFs.

Ultrasensitive Multiplex Detection of Single Nucleotide Polymorphisms Based on Short-Chain Hybridization Combined with Online Preconcentration of Capillary Electrophoresis.

Reliable multiple single nucleotide polymorphisms (SNPs) detection at low abundance is of great significance for disease diagnosis and biomedical research. Herein, we have developed a novel and simple method for multiple SNPs detection combining solid-phase capture by specific hybridization with online preconcentration of capillary gel electrophoresis-laser-induced fluorescence (CGE-LIF). The method presents an excellent performance due to its favorable traits: the solid-phase short-chain hybridization ensures the high specificity of SNP detection; the effective separation ability of CGE can easily achieve multiplex detection; the simple online preconcentration significantly improves the detection sensitivity of fluorescent probe by nearly 100-fold. For a single SNP target, the assay achieves a limit of detection as low as 0.01-0.02% for three different NRAS mutations in the same codon. For multiple SNP targets, as low as 0.05% abundance can be easily realized. Our method is simple, efficient, ultrasensitive, and universal for multiple SNPs detection without complex enzymatic or chemical ligation reaction, which shows great potential in early clinical diagnosis.

Snake venom characteristic peptides: novel fingerprints for species identification by sheathless capillary electrophoresis-electrospray ionization-mass spectrometry.

Snake venom is a complex mixture mainly consisting of proteins and peptides which varies with different species. These variations lead to different toxic mechanisms and different anti-venom serums for treatment and the determination of their use as drugs. Hence, it is important to develop a sensitive and reliable method to identify the species of snakes from venoms. Herein, we present a novel strategy based on the sheathless capillary electrophoresis-electrospray ionization-mass spectrometry (CESI-MS) system to characterize snake venom proteins. Through the determination of peptides, we found the characteristic peptides of 8 different snakes with high sensitivity (1 µg mL-1) and high selectivity, which provided a reliable method for the species identification and purity detection of snake venom samples.