Intestinal Dysbacteriosis Contributes to Persistent Cognitive Impairment after Resolution of Acute Liver Failure.

Regulating the gut microbiota alleviates hepatic encephalopathy (HE). It remains unclear whether it is imperative to withhold treatment for microbial imbalance after liver functional recovery. The aim of this work was to elucidate the alterations in cognitive behavior, liver function, synaptic transmission, and brain metabolites in acute liver failure (ALF) mice before and after hepatic function recovery. In this study, thioacetamide was injected intraperitoneally to establish an ALF mouse model, which induced HE. By performing hierarchical clustering analysis, we showed that the liver functions normalized, but cognitive dysfunction and intestinal dysbacteriosis were found in the ALF mice 14 days after thioacetamide injection. In addition, fecal microbiota transplantation from the ALF mice with liver function recovery could induce liver injury and cognitive impairment. Moreover, alterations in synaptic transmission were found in the ALF mice with liver function improvement, and the correlations between the gut bacteria and synaptic transmission in the cortex were significant. Finally, we detected apparent alterations in the brain metabolic profiles of the ALF mice after liver function improvement by performing 1H nuclear magnetic resonance spectroscopy, suggesting a risk of HE. These results show that intestinal dysbacteriosis in ALF mice with liver function recovery is sufficient to induce liver injury and cognitive impairment. These results indicate that continuous care may be necessary for monitoring microbial imbalance even in patients with ALF-induced HE whose liver function has recovered significantly.

Lysyl Oxidase 3 Is a Dual-Specificity Enzyme Involved in STAT3 Deacetylation and Deacetylimination Modulation.

In mammalian cells, histone deacetylase (HDAC) and Sirtuin (SIRT) are two families responsible for removing acetyl groups from acetylated proteins. Here, we describe protein deacetylation coupled with deacetylimination as a function of lysyl oxidase (LOX) family members. LOX-like 3 (Loxl3) associates with Stat3 in the nucleus to deacetylate and deacetyliminate Stat3 on multiple acetyl-lysine sites. Surprisingly, Loxl3 N-terminal scavenger receptor cysteine-rich (SRCR) repeats, rather than the C-terminal oxidase catalytic domain, represent the major deacetylase/deacetyliminase activity. Loxl3-mediated deacetylation/deacetylimination disrupts Stat3 dimerization, abolishes Stat3 transcription activity, and restricts cell proliferation. In Loxl3-/- mice, Stat3 is constitutively acetylated and naive CD4+ T cells are potentiated in Th17/Treg cell differentiation. When overexpressed, the SRCR repeats from other LOX family members can catalyze protein deacetylation/deacetylimination. Thus, our findings delineate a hitherto-unknown mechanism of protein deacetylation and deacetylimination catalyzed by lysyl oxidases.

Pretransplant use of immune checkpoint inhibitors for hepatocellular carcinoma: A multicenter, retrospective cohort study.

Immune checkpoint inhibitors (ICIs) as a downstaging or bridging therapy for liver transplantation (LT) in hepatocellular carcinoma patients are rapidly increasing. However, the evidence about the feasibility and safety of pre-LT ICI therapy is limited and controversial. To this end, a multicenter, retrospective cohort study was conducted in 11 Chinese centers. The results showed that 83 recipients received pre-LT ICI therapy during the study period. The median post-LT follow-up was 8.1 (interquartile range 3.3-14.6) months. During the short follow-up, 23 (27.7%) recipients developed allograft rejection, and 7 of them (30.4%) were diagnosed by liver biopsy. Multivariate logistics regression analysis showed that the time interval between the last administration of ICI therapy and LT (TLAT) ≥ 30 days was an independent protective factor for allograft rejection (odds ratio = 0.096, 95% confidence interval 0.026-0.357; P < .001). Multivariate Cox analysis showed that allograft rejection was an independent risk factor for overall survival (hazard ratio = 9.960, 95% confidence interval 1.006-98.610; P = .043). We conclude that patients who receive a pre-LT ICI therapy with a TLAT shorter than 30 days have a much higher risk of allograft rejection than those with a TLAT longer than 30 days. The presence of rejection episodes might be associated with higher post-LT mortality.

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.

General Synthesis of Metal Indium Sulfide Atomic Layers for Photocatalysis.

The metal indium sulfides have attracted extensive research interest in photocatalysis due to regulable atomic configuration and excellent optoelectronic properties. However, the synthesis of metal indium sulfide atomic layers is still challenging since intrinsic non-van-der-Waals layered structures of some components. Here, a surfactant self-assembly growth mechanism is proposed to controllably synthesize metal indium sulfide atomic layers. Eleven types of atomic layers with tunable compositions, thickness, and defect concentrations are successfully achieved namely In2S3, MgIn2S4, CaIn2S4, MnIn2S4, FeIn2S4, ZnIn2S4, Zn2In2S5, Zn4In16S33, CuInS2, CuIn5S8, and CdIn2S4. The typical CaIn2S4 shows a defect-dependence activity for CO2 photoreduction. The designed S vacancies in CaIn2S4 can serve as catalytic centers to activate CO2 molecules via localized electrons for π-back-donation. The engineered S vacancies tune the non-covalent interaction with CO2 and intermediates, manages to tune the free energy, and lower the reaction energy barrier. As a result, the defect-rich CaIn2S4 displays 2.82× improved reduction rate than defect-poor CaIn2S4. Meantime, other components also display promising photocatalytic performance, such as Zn2In2S5 with a H2O2 photosynthesis rate of 292 µmol g-1 h-1 and CuInS2 with N2-NH4 + conversion rate of 54 µmol g-1 h-1. This work paves the way for the multidisciplinary exploration of metal indium sulfide atomic layers with unique photocatalysis properties.

Magnified x ray ghost imaging with tapered polycapillary optics free of the penumbra effect.

X ray ghost imaging (XGI) offers both radiation dose-reduction potential and cost-effective benefits owing to the utilization of a single-pixel detector. Most XGI schemes with laboratory x ray sources require a mechanically moving mask for either structured illumination or structured detection. In either configuration, however, its resolution remains limited by the source size and the unit size of the mask. Upon propagation, the details of the object can actually be magnified by the divergence of x rays, but at the same time, the penumbra effect produced by the finite source size is dramatically intensified, which ultimately leads to a degradation of image quality in XGI. To address these limitations, this work proposes a magnified XGI scheme using structured detection equipped with tapered polycapillary optics, which can efficiently suppress the object's penumbra as well as resolve the magnified details of the object. In general, the resolution of this scheme is no longer affected by the source size but by the microcapillary size of polycapillary. Our work fundamentally achieves cancellation of penumbra effect-induced aberration, thus paving the way for high-resolution magnified XGI.

Reduction of Backward Scatterings at the Low-Coherence Kunwu Laser Facility.

We report the first experimental observation on the reduction of backward scatterings by an instantaneous broadband laser with 0.6% bandwidth in conditions of interest for inertial confinement fusion at the low-coherence Kunwu laser facility. The backscatter of stimulated Brillouin scattering (SBS) was robustly reduced by half at intensities of 1-5×10^{14} W/cm^{2} with the 0.53-µm broadband laser in comparison with the monochromatic laser. As SBS dominates energy loss of laser-plasma interactions, the reduction of that demonstrates the enhancement of laser-target coupling by the use of broadband laser. The mitigation of filamentation leads to the reduction of stimulated Raman backscattering at low intensities. In addition, the three-halves harmonic emission was reduced with the broadband laser as well.

Realization of Gapped and Ungapped Photonic Topological Anderson Insulators.

It is commonly believed that topologically nontrivial one-dimensional systems support edge states rather than bulk states at zero energy. In this work, we find an unanticipated case of topological Anderson insulator (TAI) phase where two bulk modes are degenerate at zero energy, in addition to degenerate edge modes. We term this "ungapped TAI" to distinguish it from the previously known gapped TAIs. Our experimental realization of both gapped and ungapped TAIs relies on coupled photonic resonators, in which the disorder in coupling is judiciously engineered by adjusting the spacing between the resonators. By measuring the local density of states both in the bulk and at the edges, we demonstrate the existence of these two types of TAIs, together forming a TAI plateau in the phase diagram. Our experimental findings are well supported by theoretical analysis. In the ungapped TAI phase, we observe stable coexistence of topological edge states and localized bulk states at zero energy, highlighting the distinction between TAIs and traditional topological insulators.

Site-specific quantification of Adenosine-to-Inosine RNA editing by Endonuclease-Mediated qPCR.

RNA molecules contain diverse modified nucleobases that play pivotal roles in numerous biological processes. Adenosine-to-inosine (A-to-I) RNA editing, one of the most prevalent RNA modifications in mammalian cells, is linked to a multitude of human diseases. To unveil the functions of A-to-I RNA editing, accurate quantification of inosine at specific sites is essential. In this study, we developed an endonuclease-mediated cleavage and real-time fluorescence quantitative PCR method for A-to-I RNA editing (EM-qPCR) to quantitatively analyze A-to-I RNA editing at a single site. By employing this method, we successfully quantified the levels of A-to-I RNA editing on various transfer RNA (tRNA) molecules at position 34 (I34) in mammalian cells with precision. Subsequently, this method was applied to tissues from sleep-deprived mice, revealing a notable alteration in the levels of I34 between sleep-deprived and control mice. The proposed method sets a precedent for the quantitative analysis of A-to-I RNA editing at specific sites, facilitating a deeper understanding of the biological implications of A-to-I RNA editing.

Balloon angioplasty as first-choice recanalization strategy for intracranial atherosclerosis-related emergent large vessel occlusion with small clot burden.

PURPOSE: The optimal primary recanalization strategy for intracranial atherosclerosis-related emergent large vessel occlusion (ICAS-ELVO) remains controversial. We aimed to explore the safety and efficacy of balloon angioplasty as the first-choice recanalization strategy for ICAS-ELVO with small clot burden. METHODS: Consecutive ICAS-ELVO patients presenting with microcatheter "first-pass effect" during endovascular treatment (EVT) were retrospectively analyzed. Patients were divided into preferred balloon angioplasty (PBA) and preferred mechanical thrombectomy (PMT) groups based on the first-choice recanalization strategy. The reperfusion and clinical outcomes between the two groups were compared. RESULTS: Seventy-six patients with ICAS-ELVO involving the microcatheter "first-pass effect" during EVT were enrolled. Compared with patients in the PMT group, those in the PBA group were associated with (i) a higher rate of first-pass recanalization (54.0% vs. 28.9%, p = .010) and complete reperfusion (expanded thrombolysis in cerebral ischemia ≥ 2c; 76.0% vs. 53.8%, p = .049), (ii) shorter puncture-to-recanalization time (49.5 min vs. 89.0 min, p < .001), (iii) lower operation costs (¥48,499.5 vs. ¥ 99,086.0, p < .001), and (iv) better 90-day functional outcomes (modified Rankin scale:0-1; 44.0% vs. 19.2%, p = .032). Logistic regression analysis revealed that balloon angioplasty as the first-choice recanalization strategy was an independent predictor of 90-day excellent functional outcomes for ICAS-ELVO patients with microcatheter "first-pass effect" (adjusted odds ratio = 6.01, 95% confidence interval: 1.15-31.51, p = .034). CONCLUSION: Direct balloon angioplasty potentially improves 90-day functional outcomes for ICAS-ELVO patients with small clot burden, and may be a more appropriate first-choice recanalization strategy than mechanical thrombectomy for these patients.

Cooperative Action between SALL4A and TET Proteins in Stepwise Oxidation of 5-Methylcytosine.

TET family enzymes successively oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, leading to eventual demethylation. 5hmC and TET enzymes occupy distinct chromatin regions, suggesting unknown mechanisms controlling the fate of 5hmC within diverse chromatin environments. Here, we report that SALL4A preferentially associates with 5hmC in vitro and occupies enhancers in mouse embryonic stem cells in a largely TET1-dependent manner. Although most 5hmC at SALL4A peaks undergoes further oxidation, this process is abrogated upon deletion of Sall4 gene, with a concomitant reduction of TET2 at these regions. Thus, SALL4A facilitates further oxidation of 5hmC at its binding sites, which requires its 5hmC-binding activity and TET2, supporting a collaborative action between SALL4A and TET proteins in regulating stepwise oxidation of 5mC at enhancers. Our study identifies SALL4A as a 5hmC binder, which facilitates 5hmC oxidation by stabilizing TET2 association, thereby fine-tuning expression profiles of developmental genes in mouse embryonic stem cells.

Topology Optimization Design Method for Acoustic Imaging Array of Power Equipment.

Acoustic imaging technology has the advantages of non-contact and intuitive positioning. It is suitable for the rapid positioning of defects such as the mechanical loosening, discharge, and DC bias of power equipment. However, the existing research lacks the optimization design of microphone array topology. The acoustic frequency domain characteristics of typical power equipment are elaborately sorted out. After that, the cut-off frequencies of acoustic imaging instruments are determined, to meet the needs of the full bandwidth test requirements. Through a simulation calculation, the circular array is demonstrated to be the optimal shape. And the design parameters affect the imaging performance of the array to varying degrees, indicating that it is difficult to obtain the optimal array topology by an exhaustive method. Aimed at the complex working conditions of power equipment, a topology optimization design method of an acoustic imaging array for power equipment is proposed, and the global optimal solution of microphone array topology is obtained. Compared with the original array, the imaging performance of the improved LF and HF array is promoted by 54% and 49%, respectively. Combined with the simulation analysis and laboratory test, it is verified that the improved array can not only accurately locate the single sound source but also accurately identify the main sound source from the interference of the contiguous sound source.

Adaptive UAV Navigation Method Based on AHRS.

To address the inaccuracy of the Constant Acceleration/Constant Velocity (CA/CV) model as the state equation in describing the relative motion state in UAV relative navigation, an adaptive UAV relative navigation method is proposed, which is based on the UAV attitude information provided by Attitude and Heading Reference System (AHRS). The proposed method utilizes the AHRS output attitude parameters as the benchmark for dead reckoning and derives a relative navigation state equation with attitude error as process noise. By integrating the extended Kalman filter output for relative state estimation and employing an adaptive decision rule designed using the innovation of the filter update phase, the proposed method recalculates motion states deviating from the actual motion using the Tasmanian Devil Optimization (TDO) algorithm. The simulation results show that, compared with the CA/CV model, the proposed method reduces the relative position errors by 12%, 23%, and 32% in the X, Y, and Z directions, respectively, and that it reduces the relative velocity errors by 350%, 330%, and 300%, respectively. There is a significant improvement in the relative navigation accuracy.

The mechanism of action and chemical synthesis of FDA newly approved drug molecules.

In 2023, the U.S. Food and Drug Administration has approved 29 small molecule drugs. These newly approved small molecule drugs possess the distinct scaffolds, thereby exhibiting diverse mechanisms of action and binding modalities. Moreover, the marketed drugs have always been an important source of new drug development and creative inspiration, thereby fostering analogous endeavors in drug discovery that potentially extend to the diverse clinical indications. Therefore, conducting a comprehensive evaluation of drug approval experience and associated information will facilitate the expedited identification of highly potent drug molecules. In this review, we comprehensively summarized the relevant information regarding the clinical applications, mechanisms of action and chemical synthesis of 29 small molecule drugs, with the aim of providing a promising structural basis and design inspiration for pharmaceutical chemists.

Comparative analysis of different treatment strategies for septic spondylitis: a retrospective study of one hundred and twelve patients.

PURPOSE: To compare the clinical efficacy and prognosis differences between conservative treatment and surgical treatment in patients with non-serious neurologically intact pyogenic spondylitis (Nsi-Nsni-PS), and to provide theoretical reference for the clinical treatment of Nsi-Nsni-PS patients. METHODS: A retrospective analysis was conducted on 112 cases of Nsi-Nsni-PS patients treated in our hospital from June 2016 to June 2021. According to different treatment methods, they were divided into conservative treatment group (53 cases) and surgical treatment group (59 cases). The general data, laboratory tests, imaging examinations, length of hospital stay, duration of antibiotic use, VAS for pain before and after treatment, ODI, local kyphotic angle correction of diseased vertebrae, and recurrence rate were collected and analyzed in both groups. SPSS 26.0 statistical software was used for analysis. Measurement data were expressed as mean ± standard deviation, and independent sample t-test or rank sum test was used for comparison between groups, while variance analysis was used for intra-group comparison. Count data were expressed as number (%) and compared between groups using chi-square test or Fisher's exact test. Mann-Whitney U test was used to evaluate the changes in local kyphotic angle between the two groups. A p value < 0.05 was considered statistically significant. RESULTS: There were no significant differences in general data and imaging characteristics between the two groups (P > 0.05); there were no statistically significant differences in the positive culture rate of pathogens, length of hospital stay, duration of antibiotic use, treatment complications, WBC, CRP, ESR levels at admission and discharge, VAS and ODI at admission and last follow-up between the two groups (P > 0.05). The WBC and CRP levels of patients in the conservative group at discharge were lower than those in the surgical group (P < 0.05), and there was no significant difference in the decrease in inflammatory indicators (WBC, CRP, ESR) between the two groups (P > 0.05). By the last follow-up, the neurological function of patients in both groups had significantly improved compared to admission (P < 0.05), with 12 out of 15 ASIA grade D patients in the conservative group recovering to grade E, and 21 out of 25 grade D patients in the surgical group recovering to grade E, with no worsening of neurological function in either group. The differences in VAS and ODI scores at the last follow-up compared to before treatment were statistically significant in both groups (P < 0.05), and all patients regained normal activity. Compared with before treatment, the correction degree of local kyphotic angle in the surgical group at the last follow-up was 0.93 ± 4.94°, slightly higher than that in the conservative group (-0.83 ± 3.37°), and the difference was statistically significant(P < 0.05). CONCLUSIONS: During our follow-up, we found that both conservative and surgical treatments achieved satisfactory clinical outcomes in patients with Nsi-Nsni-PS. Compared to conservative treatment, surgical intervention did not demonstrate significant advantages in reducing hospitalization time and antibiotic usage duration, increasing pathogen culture positivity rate, lowering treatment complications, or controlling recurrence. However, surgical intervention showed superiority in correcting the local kyphotic angle of spinal lesions, albeit with relatively increased surgical trauma, risks, and treatment costs. At the last follow-up, the surgical group did not exhibit better long-term efficacy. Therefore, when formulating clinical treatment strategies for patients with Nsi-Nsni-PS, it may be preferable to prioritize conservative treatment, supplemented by the use of sensitive or empiric antibiotics for infection management, to improve patient prognosis.

One-Step Construction of 1,3,4-Oxadiazoles with Anticancer Activity from Tertiary Amines via a Sequential Copper(I)-Catalyzed Oxidative Ugi/aza-Wittig Reaction.

An unparalleled copper(I)-catalyzed synthesis of 1,3,4-oxadiazoles from tertiary amines in one step has been described. The one-pot reactions involving (N-isocyanimine)triphenylphosphorane, tertiary amines, and carboxylic acids resulted in the formation of 1,3,4-oxadiazoles in moderate to good yields through a consecutive oxidative Ugi/aza-Wittig reaction, enabling the direct functionalization of sp3 C-H bonds adjacent to the nitrogen atom. This method offered several notable advantages, including ligands-free, exceptional productivity and a high functional group tolerance. The preliminary biological evaluation demonstrated that compound 4f inhibited hepatoma cells efficiently, suggesting potentially broad applications of the approach for synthesis and medicinal chemistry.

Heterogeneous Nucleation Regulation Amends Unfavorable Crystallization Orientation and Defect Features of Antimony Selenosulfide Film for High-Efficient Planar Solar Cells.

Antimony selenosulfide (Sb2(S,Se)3) has obtained widespread concern for photovoltaic applications as a light absorber due to superior photoelectric features. Accordingly, various deposition technologies have been developed in recent years, especially hydrothermal deposition method, which has achieved a great success. However, device performances are limited with severe carrier recombination, relating to the quality of absorber and interfaces. Herein, bulk and interface defects are simultaneously suppressed by regulating heterogeneous nucleation kinetics with barium dibromide (BaBr2) introduction. In details, the Br adsorbs and dopes on the polar planes of cadmium sulfide (CdS) buffer layer, promoting the exposure of nonpolar planes of CdS, which facilitates the favorable growth of [hk1]-Sb2(S,Se)3 films possessing superior crystallinity and small interface defects. Additionally, the Se/S ratio is increased due to the replacement of S/Se by Br, causing a downshift of the Fermi levels with a benign band alignment and a shallow-level defect. Moreover, Ba2+ is located at grain boundaries by coordination with S and Se ions, passivating grain boundary defects. Consequently, the efficiency is increased from 7.70% to 10.12%. This work opens an avenue towards regulating the heterogeneous nucleation kinetics of Sb2(S,Se)3 film deposited via hydrothermal deposition approach to optimize its crystalline orientation and defect features.

Single-Base Resolution Detection of N6-Methyladenosine in RNA by Adenosine Deamination Sequencing.

N6-Methyladenosine (m6A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m6A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m6A relies on the faithful detection of individual m6A sites in RNA. However, developing a simple method for the single-base resolution detection of m6A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m6A in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not m6A, by the evolved tRNA adenosine deaminase (TadA) variant of TadA8e or the dimer protein of TadA-TadA8e. In AD-seq, adenosine is deaminated by TadA8e or TadA-TadA8e to form inosine, which pairs with cytidine and is read as guanosine in sequencing. m6A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m6A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m6A in sequencing can achieve the single-base resolution detection of m6A in RNA. Application of the proposed AD-seq successfully identified individual m6A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m6A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m6A in RNA.

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution.

DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfite-free and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.