N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.

N(6)-methyldeoxyadenosine (6mA or m(6)A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here, we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms.

DNA N(6)-methyladenine: a new epigenetic mark in eukaryotes?

DNA N(6)-adenine methylation (N(6)-methyladenine; 6mA) in prokaryotes functions primarily in the host defence system. The prevalence and significance of this modification in eukaryotes had been unclear until recently. Here, we discuss recent publications documenting the presence of 6mA in Chlamydomonas reinhardtii, Drosophila melanogaster and Caenorhabditis elegans; consider possible roles for this DNA modification in regulating transcription, the activity of transposable elements and transgenerational epigenetic inheritance; and propose 6mA as a new epigenetic mark in eukaryotes.

The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity.

The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes1,2. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

m6A modification plays an integral role in mRNA stability and translation during pattern-triggered immunity.

Plants employ distinct mechanisms to respond to environmental changes. Modification of mRNA by N 6-methyladenosine (m6A), known to affect the fate of mRNA, may be one such mechanism to reprogram mRNA processing and translatability upon stress. However, it is difficult to distinguish a direct role from a pleiotropic effect for this modification due to its prevalence in RNA. Through characterization of the transient knockdown-mutants of m6A writer components and mutants of specific m6A readers, we demonstrate the essential role that m6A plays in basal resistance and pattern-triggered immunity (PTI). A global m6A profiling of mock and PTI-induced Arabidopsis plants as well as formaldehyde fixation and cross-linking immunoprecipitation-sequencing of the m6A reader, EVOLUTIONARILY CONSERVED C-TERMINAL REGION2 (ECT2) showed that while dynamic changes in m6A modification and binding by ECT2 were detected upon PTI induction, most of the m6A sites and their association with ECT2 remained static. Interestingly, RNA degradation assay identified a dual role of m6A in stabilizing the overall transcriptome while facilitating rapid turnover of immune-induced mRNAs during PTI. Moreover, polysome profiling showed that m6A enhances immune-associated translation by binding to the ECT2/3/4 readers. We propose that m6A plays a positive role in plant immunity by destabilizing defense mRNAs while enhancing their translation efficiency to create a transient surge in the production of defense proteins.

A KDPG sensor RccR governs Pseudomonas aeruginosa carbon metabolism and aminoglycoside antibiotic tolerance.

Pseudomonas aeruginosa harbors sophisticated transcription factor (TF) networks to coordinately regulate cellular metabolic states for rapidly adapting to changing environments. The extraordinary capacity in fine-tuning the metabolic states enables its success in tolerance to antibiotics and evading host immune defenses. However, the linkage among transcriptional regulation, metabolic states and antibiotic tolerance in P. aeruginosa remains largely unclear. By screening the P. aeruginosa TF mutant library constructed by CRISPR/Cas12k-guided transposase, we identify that rccR (PA5438) is a major genetic determinant in aminoglycoside antibiotic tolerance, the deletion of which substantially enhances bacterial tolerance. We further reveal the inhibitory roles of RccR in pyruvate metabolism (aceE/F) and glyoxylate shunt pathway (aceA and glcB), and overexpression of aceA or glcB enhances bacterial tolerance. Moreover, we identify that 2-keto-3-deoxy-6-phosphogluconate (KDPG) is a signal molecule that directly binds to RccR. Structural analysis of the RccR/KDPG complex reveals the detailed interactions. Substitution of the key residue R152, K270 or R277 with alanine abolishes KDPG sensing by RccR and impairs bacterial growth with glycerol or glucose as the sole carbon source. Collectively, our study unveils the connection between aminoglycoside antibiotic tolerance and RccR-mediated central carbon metabolism regulation in P. aeruginosa, and elucidates the KDPG-sensing mechanism by RccR.

Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library.

Recent years have witnessed rapid progress in the field of epitranscriptomics. Functional interpretation of the epitranscriptome relies on sequencing technologies that determine the location and stoichiometry of various RNA modifications. However, contradictory results have been reported among studies, bringing the biological impacts of certain RNA modifications into doubt. Here, we develop a synthetic RNA library resembling the endogenous transcriptome but without any RNA modification. By incorporating this modification-free RNA library into established mapping techniques as a negative control, we reveal abundant false positives resulting from sequence bias or RNA structure. After calibration, precise and quantitative mapping expands the understanding of two representative modification types, N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We propose that this approach provides a systematic solution for the calibration of various RNA-modification mappings and holds great promise in epitranscriptomic studies.

Facial Anthropometric Analysis of Gender-Related Characteristics in Computed Tomography.

BACKGROUND: In recent years, facial feminization surgery (FFS) has gained increasing popularity because of increases in transgender individuals and the acceptance of diversity in gender identity. However, there is still a scarcity of anthropometric research to guide evidence-based practices for FFS in Taiwan. AIM AND OBJECTIVES: The purpose of this study was to provide a reference for surgeons to achieve optimal outcomes for patients undergoing FFS. The anthropometric analysis could help surgeons meet patients' specific requirements and improve patients' alignment with their gender identity. MATERIALS AND METHODS: The study group consisted of 100 patients (50 males and 50 females) who had undergone cranial computed tomography at Chang Gung Memorial Hospital in Taiwan because of the indication of blunt injuries to the head and face with suspected skull and facial fractures. The computed tomography images were imported into the OsiriX image software to conduct an anthropometric evaluation. The parameters used in the measurements included 2 aspects: bone and soft tissue anthropometric analysis. RESULTS: Anthropometric data were obtained from 50 males (age 32.6 ± 11.4 years) and 50 females (age 33.7 ± 10.3 years). The results for bone measurements showed that both the forehead bossing length and nasal bone width in the male group were significantly greater. The frontal angle in both bone and soft tissue in the male group was significantly smaller. The chin height and bigonial width in both bone and soft tissue in the male group were significantly greater. Although the average gonial angle was greater in the female group, the difference was not significant. For the measurements of lip projection, the results showed that there was no significant difference. Although this group of Asian males had more acute nasolabial angles, the difference was not statistically significant. However, the average nasofrontal angle among females was significantly more obtuse than among males. CONCLUSION: The results revealed that Asian males tend to have more prominent superior orbital rims, wider nasal bones, and wider and taller mandibles compared with Asian females. Despite showing some trends, the gonial angle and lip projections did not reveal any significant differences, which is likely because of a large amount of variation.

Analysis of mandibular asymmetry in adolescent and adult patients with unilateral posterior crossbite on cone-beam computed tomography.

INTRODUCTION: This study aimed to evaluate mandibular asymmetry in unilateral posterior crossbite (UPXB) patients and compare the asymmetry between adolescents and adults with UPXB. METHODS: This study included and analyzed cone-beam computed tomography scans of 125 subjects. The subjects were divided into a UPXB group and a control group according to the presence or absence of UPXB, and each group included adolescent patients (aged 10-15 years) and adult patients (aged 20-40 years). Linear, angular, and volumetric measurements were obtained to evaluate the asymmetries of the mandibles. RESULTS: Both adolescent and adult patients in the UPXB group presented asymmetries in condylar unit length, ramal height, body length, and mediolateral ramal inclination (P <0.05). Adult patients with UPXB showed greater asymmetries than adolescents. Differences with condylar unit length, condylar unit width, ramal height, condylar unit volume, and hemimandibular volume were significantly greater in adult UPXB patients than adolescent UPXB patients (P <0.05). CONCLUSIONS: The worsening of mandibular asymmetries in UPXB adults suggests that asymmetry in UPXB patients may progress over time; therefore, early treatment should be considered for UPXB adolescent patients. Further studies are still needed to evaluate the effectiveness of early treatment.

Mapping single-nucleotide m6A by m6A-REF-seq.

The past few years have witnessed rapid progress in the field of RNA modifications. As the most prevailing modification on eukaryotic mRNA, m6A is characterized to play a vital role in various cellular activities. However, limitations of the detection method impede functional studies of m6A. Here we introduce m6A-REF-seq, a powerful and straightforward method to identify m6A at single-nucleotide resolution. m6A-REF-seq relies on the recognition of RNA endonuclease MazF towards m6A at the ACA motif, providing an orthogonal method independent of the m6A antibody being adopted by most of current methods. We describe a detailed protocol to perform m6A-REF-seq, including NGS library construction and sequencing data analysis. In particular, we describe an optimized assay to validate individual m6A sites identified by m6A-REF-seq, which can also be applied to detect any candidate m6A sites.

Management Strategies for Geriatric Maxillofacial Fractures: An Anthropometric and Clinical Study in Taiwan.

BACKGROUND: Geriatric maxillofacial trauma has become an increasingly pressing clinical issue in Taiwan because of increased life expectancy. AIM AND OBJECTIVES: The purposes of this study were to investigate the anthropometric changes and the posttrauma outcomes in the aging population and to optimize the management strategies for geriatric facial fractures. MATERIALS AND METHODS: From 2015 to 2020, a total of 30 patients 65 years or older were identified to have suffered from maxillofacial fractures and presented at the emergency department of the Chang Gung Memorial Hospital (CGMH). These patients were categorized into group III, representing the elderly group. Two other groups (group I, age 18-40 years; group II, age 41-64 years) of patients were categorized based on their age. After applying propensity score matching to reduce bias caused by a large case number difference, patient demographics, anthropometric data, and management methods were compared and analyzed. RESULTS: Among 30 patients 65 years or older who met the inclusion criteria, the mean age of the matched group III was 77.31 ± 14.87 years, and the mean number of retained teeth was 11.77 (range, 3-20 teeth). The elderly patients had a significantly lower number of retained teeth (group I vs group II vs group III, 27.3 vs 25.23 vs 11.77; P < 0.001). Anthropometric data showed that facial bone structure degenerated significantly with advancing age. Outcome analysis demonstrated that falls accounted for 43.3% of injury mechanisms in the elderly group, followed by motorcycle accidents (30%) and car accidents (23.3%). Nineteen elderly patients (63%) received nonsurgical management. On the other hand, 86.7% of cases in the other 2 age groups underwent surgery. The average numbers of total hospital and intensive care unit stays in group III patients were 16.9 (range, 3-49 days) and 4.57 (range, 0-47 days), which was significantly longer than the other 2 age groups. CONCLUSIONS: Our results suggested that not only surgery is feasible for elderly patients with facial fractures, but an acceptable result is often obtainable. However, an eventful course, including extended hospital/intensive care unit stays and an increased risk of associated injuries and complications, may be expected.

Crystal structure of the yeast heterodimeric ADAT2/3 deaminase.

BACKGROUND: The adenosine-to-inosine (A-to-I) editing in anticodons of tRNAs is critical for wobble base-pairing during translation. This modification is produced via deamination on A34 and catalyzed by the adenosine deaminase acting on tRNA (ADAT) enzyme. Eukaryotic ADATs are heterodimers composed of the catalytic subunit ADAT2 and the structural subunit ADAT3, but their molecular assemblies and catalytic mechanisms are largely unclear. RESULTS: Here, we report a 2.8-Å crystal structure of Saccharomyces cerevisiae ADAT2/3 (ScADAT2/3), revealing its heterodimeric assembly and substrate recognition mechanism. While each subunit clearly contains a domain resembling their prokaryotic homolog TadA, suggesting an evolutionary gene duplication event, they also display accessory domains for additional structural or functional purposes. The N-lobe of ScADAT3 exhibits a positively charged region with a potential role in the recognition and binding of tRNA, supported by our biochemical analysis. Interestingly, ScADAT3 employs its C-terminus to block tRNA's entry into its pseudo-active site and thus inactivates itself for deamination despite the preservation of a zinc-binding site, a mechanism possibly shared only among yeasts. CONCLUSIONS: Combining the structural with biochemical, bioinformatic, and in vivo functional studies, we propose a stepwise model for the pathway of deamination by ADAT2/3. Our work provides insight into the molecular mechanism of the A-to-I editing by the eukaryotic ADAT heterodimer, especially the role of ADAT3 in catalysis.

Cu2+-modified hollow carbon nanospheres: an unusual nanozyme with enhanced peroxidase-like activity.

A Cu2+-modified carboxylated hollow carbon nanospheres (Cu2+-HCNSs-COOH) was designed with enhanced peroxidase-like activity for the detection of hydrogen peroxide (H2O2) and degradation of methylene blue (MB). Hollow polymer nanospheres were fabricated from aniline, pyrrole, Triton-100, and ammonium persulfate via confined interfacial copolymerization reaction, which can be pyrolyzed to create HCNSs with the hollow gap diameter of about 20 nm under high temperature. Combining the synergistic effect of coordination and electrostatic interaction, Cu2+-HCNSs-COOH was constructed by anchoring Cu2+ on the surface of HCNSs-COOH. Furthermore, Cu2+-HCNSs-COOH has higher affinity for 3,3',5,5'-tetramethylbenzidine and H2O2 of 0.20 mM and 0.88 mM, respectively. Based on the rapid response of Cu2+-HCNSs-COOH to H2O2, we constructed a colorimetric sensing platform by detecting the absorbance of the 3,3',5,5'-tetramethylbenzidine-H2O2 system at 652 nm for quantifying H2O2, which holds good linear relationship between 1 and 150 µM and has a detection limit of 0.61 µM. We also investigated the degradation of MB in the presence of Cu2+-HCNSs-COOH and H2O2, which can degrade 80.7% pollutants within 30 min. This research developed an unusual nanozyme for bioassays and water pollution treatment, which broadened the way for the rapid development of clinical diagnostics and water pollution treatment.

An attention-based multi-task model for named entity recognition and intent analysis of Chinese online medical questions.

In this paper, we propose an attention-based multi-task neural network model for text classification and sequence tagging and then apply it to the named entity recognition and the intent analysis of Chinese online medical questions. We found that the use of both attention and multi-task learning improved the performance of these tasks. Our method achieved superior performance in named entity recognition and intent analysis compared with other baseline methods; the method is a light-weight solution that is suitable for deployment on small servers. Furthermore, we took advantage of the model's capabilities for these two tasks and built a simple question-answering system for cardiovascular issues. Users and service providers can monitor the logic of the answers generated by this system.

Ubiquitously expressed genes participate in cell-specific functions via alternative promoter usage.

How do different cell types acquire their specific identities and functions is a fundamental question of biology. Previously significant efforts have been devoted to search for cell-type-specifically expressed genes, especially transcription factors, yet how do ubiquitously expressed genes participate in the formation or maintenance of cell-type-specific features remains largely unknown. Here, we have identified 110 mouse embryonic stem cell (mESC) specifically expressed transcripts with cell-stage-specific alternative transcription start sites (SATS isoforms) from 104 ubiquitously expressed genes, majority of which have active epigenetic modification- or stem cell-related functions. These SATS isoforms are specifically expressed in mESCs, and tend to be transcriptionally regulated by key pluripotency factors through direct promoter binding. Knocking down the SATS isoforms of Nmnat2 or Usp7 leads to differentiation-related phenotype in mESCs. These results demonstrate that cell-type-specific transcription factors are capable to produce cell-type-specific transcripts with alternative transcription start sites from ubiquitously expressed genes, which confer ubiquitously expressed genes novel functions involved in the establishment or maintenance of cell-type-specific features.

Androgenetic haploid embryonic stem cells produce live transgenic mice.

Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals. Mammalian haploids are restricted to germline cells and are occasionally found in tumours with massive chromosome loss. Recent success in establishing haploid embryonic stem (ES) cells in medaka fish and mice raised the possibility of using engineered mammalian haploid cells in genetic studies. However, the availability and functional characterization of mammalian haploid ES cells are still limited. Here we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into an enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to germlines of chimaeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte-injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. Our findings show the developmental pluripotency of androgenentic haploids and provide a new tool to quickly produce genetic models for recessive traits. They may also shed new light on assisted reproduction.

Widespread occurrence of N6-methyladenosine in bacterial mRNA.

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m(6)A as well as m(6)A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m(6)A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m(6)A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m(6)A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m(6)A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m(6)A pattern that is distinct from those in eukaryotes. Most m(6)A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m(6)A peaks indicates that the majority of m(6)A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m(6)A in these pathways.

[GC-MS combined with AMDIS and Kováts retention index to investigate dynamic change rules of volatile components from Atractylodis Macrocephalae Rhizoma with different stir-baking degrees].

To investigate the dynamic change rules of volatile components from Atractylodis Macrocephalae Rhizoma with different stir-baking degrees (from slight stir-baking, stir-baking to yellow, stir-baking to brown, to stir-baking to scorch). In the present experiment, the Atractylodis Macrocephalae Rhizoma samples with different stir-baking degrees were collected at different processing time points. The contents of volatile oil in various samples were determined by steam distillation method, and the volatile compounds were extracted by using static headspace sampling method. Gas chromatography-mass spectrography (GC-MS) and automated mass spectral deconrolution and identification system (AMDIS) were combined with Kováts retention index to analyze the chemical constituents of the volatile compounds. The results showed that with the deepening of the stir-baking degree, the content of volatile oil was decreased step by step in 4 phases, and both the compositions and contents of volatile components from Atractylodis Macrocephalae Rhizoma showed significant changes. The results showed that the dynamic change rules of volatile components from Atractylodis Macrocephalae Rhizoma in the process of stir-baking were closely related to the processing degree; in addition, Atractylodis Macrocephalae Rhizoma and honey bran had adsorption on each other. These results can provide a scientific basis for elucidating the stir-baking (with bran) mechanism of Atractylodis Macrocephalae Rhizoma.

ISRNA: an integrative online toolkit for short reads from high-throughput sequencing data.

UNLABELLED: Integrative Short Reads NAvigator (ISRNA) is an online toolkit for analyzing high-throughput small RNA sequencing data. Besides the high-speed genome mapping function, ISRNA provides statistics for genomic location, length distribution and nucleotide composition bias analysis of sequence reads. Number of reads mapped to known microRNAs and other classes of short non-coding RNAs, coverage of short reads on genes, expression abundance of sequence reads as well as some other analysis functions are also supported. The versatile search functions enable users to select sequence reads according to their sub-sequences, expression abundance, genomic location, relationship to genes, etc. A specialized genome browser is integrated to visualize the genomic distribution of short reads. ISRNA also supports management and comparison among multiple datasets. AVAILABILITY: ISRNA is implemented in Java/C++/Perl/MySQL and can be freely accessed at http://omicslab.genetics.ac.cn/ISRNA/.

In vivo suppression of microRNA-24 prevents the transition toward decompensated hypertrophy in aortic-constricted mice.

RATIONALE: During the transition from compensated hypertrophy to heart failure, the signaling between L-type Ca(2+) channels in the cell membrane/T-tubules and ryanodine receptors in the sarcoplasmic reticulum becomes defective, partially because of the decreased expression of a T-tubule-sarcoplasmic reticulum anchoring protein, junctophilin-2. MicroRNA (miR)-24, a junctophilin-2 suppressing miR, is upregulated in hypertrophied and failing cardiomyocytes. OBJECTIVE: To test whether miR-24 suppression can protect the structural and functional integrity of L-type Ca(2+) channel-ryanodine receptor signaling in hypertrophied cardiomyocytes. METHODS AND RESULTS: In vivo silencing of miR-24 by a specific antagomir in an aorta-constricted mouse model effectively prevented the degradation of heart contraction, but not ventricular hypertrophy. Electrophysiology and confocal imaging studies showed that antagomir treatment prevented the decreases in L-type Ca(2+) channel-ryanodine receptor signaling fidelity/efficiency and whole-cell Ca(2+) transients. Further studies showed that antagomir treatment stabilized junctophilin-2 expression and protected the ultrastructure of T-tubule-sarcoplasmic reticulum junctions from disruption. CONCLUSIONS: MiR-24 suppression prevented the transition from compensated hypertrophy to decompensated hypertrophy, providing a potential strategy for early treatment against heart failure.

High-resolution N(6) -methyladenosine (m(6) A) map using photo-crosslinking-assisted m(6) A sequencing.

N(6) -methyladenosine (m(6) A) is an abundant internal modification in eukaryotic mRNA and plays regulatory roles in mRNA metabolism. However, methods to precisely locate the m(6) A modification remain limited. We present here a photo-crosslinking-assisted m(6) A sequencing strategy (PA-m(6) A-seq) to more accurately define sites with m(6) A modification. Using this strategy, we obtained a high-resolution map of m(6) A in a human transcriptome. The map resembles the general distribution pattern observed previously, and reveals new m(6) A sites at base resolution. Our results provide insight into the relationship between the methylation regions and the binding sites of RNA-binding proteins.