Antibiotic-Induced Gut Microbiota Dysbiosis Modulates Host Transcriptome and m6A Epitranscriptome via Bile Acid Metabolism.

Gut microbiota can influence host gene expression and physiology through metabolites. Besides, the presence or absence of gut microbiome can reprogram host transcriptome and epitranscriptome as represented by N6-methyladenosine (m6A), the most abundant mammalian mRNA modification. However, which and how gut microbiota-derived metabolites reprogram host transcriptome and m6A epitranscriptome remain poorly understood. Here, investigation is conducted into how gut microbiota-derived metabolites impact host transcriptome and m6A epitranscriptome using multiple mouse models and multi-omics approaches. Various antibiotics-induced dysbiotic mice are established, followed by fecal microbiota transplantation (FMT) into germ-free mice, and the results show that bile acid metabolism is significantly altered along with the abundance change in bile acid-producing microbiota. Unbalanced gut microbiota and bile acids drastically change the host transcriptome and the m6A epitranscriptome in multiple tissues. Mechanistically, the expression of m6A writer proteins is regulated in animals treated with antibiotics and in cultured cells treated with bile acids, indicating a direct link between bile acid metabolism and m6A biology. Collectively, these results demonstrate that antibiotic-induced gut dysbiosis regulates the landscape of host transcriptome and m6A epitranscriptome via bile acid metabolism pathway. This work provides novel insights into the interplay between microbial metabolites and host gene expression.

Jolkinolide B synergistically potentiates the antitumor activity of GPX4 inhibitors via inhibiting TrxR1 in cisplatin-resistant bladder cancer cells.

Glutathione peroxidase 4 (GPX4) is a promising anticancer therapeutic target; however, the application of GPX4 inhibitors (GPX4i) is limited owing to intrinsic or acquired drug resistance. Hence, understanding the mechanisms underlying drug resistance and discovering molecules that can overcome drug resistance are crucial. Herein, we demonstrated that GPX4i killed bladder cancer cells by inducing lipid reactive oxygen species-mediated ferroptosis and apoptosis, and cisplatin-resistant bladder cancer cells were also resistant to GPX4i, representing a higher half-maximal inhibitory concentration value than that of parent bladder cancer cells. In addition, thioredoxin reductase 1 (TrxR1) overexpression was responsible for GPX4i resistance in cisplatin-resistant bladder cancer cells, and inhibiting TrxR1 restored the sensitivity of these cells to GPX4i. In vitro and in vivo studies revealed that Jolkinolide B (JB), a natural diterpenoid and previously identified as a TrxR1 inhibitor, potentiated the antiproliferative efficacy of GPX4i (RSL3 and ML162) against cisplatin-resistant bladder cancer cells. Furthermore, GPX4 knockdown and inhibition could augment JB-induced paraptosis and apoptosis. Our results suggest that inhibiting TrxR1 can effectively improve GPX4 inhibition-based anticancer therapy. A combination of JB and GPX4i, which is well-tolerated and has several anticancer mechanisms, may serve as a promising therapy for treating bladder cancer.

Online biomedical named entities recognition by data and knowledge-driven model.

Named entity recognition (NER) is an important task for the natural language processing of biomedical text. Currently, most NER studies standardized biomedical text, but NER for unstandardized biomedical text draws less attention from researchers. Named entities in online biomedical text exist with errors and polymorphisms, which negatively impact NER models' performance and impede support from knowledge representation methods. In this paper, we propose a neural network method that can effectively recognize entities in unstandardized online medical/health text. We introduce a new pre-training scheme that uses large-scale online question-answering pairs to enhance transformers' model capacity on online biomedical text. Moreover, we supply models with knowledge representations from a knowledge base called multi-channel knowledge labels, and this method overcomes the restriction from languages, like Chinese, that require word segmentation tools to represent knowledge. Our model outperforms other baseline methods significantly in experiments on a dataset for Chinese online medical entity recognition and achieves state-of-the-art results.

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.

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.

Co-effects of m6A and chromatin accessibility dynamics in the regulation of cardiomyocyte differentiation.

BACKGROUND: Cardiomyocyte growth and differentiation rely on precise gene expression regulation, with epigenetic modifications emerging as key players in this intricate process. Among these modifications, N6-methyladenosine (m6A) stands out as one of the most prevalent modifications on mRNA, exerting influence over mRNA metabolism and gene expression. However, the specific function of m6A in cardiomyocyte differentiation remains poorly understood. RESULTS: We investigated the relationship between m6A modification and cardiomyocyte differentiation by conducting a comprehensive profiling of m6A dynamics during the transition from pluripotent stem cells to cardiomyocytes. Our findings reveal that while the overall m6A modification level remains relatively stable, the m6A levels of individual genes undergo significant changes throughout cardiomyocyte differentiation. We discovered the correlation between alterations in chromatin accessibility and the binding capabilities of m6A writers, erasers, and readers. The changes in chromatin accessibility influence the recruitment and activity of m6A regulatory proteins, thereby impacting the levels of m6A modification on specific mRNA transcripts. CONCLUSION: Our data demonstrate that the coordinated dynamics of m6A modification and chromatin accessibility are prominent during the cardiomyocyte differentiation.

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing.

N6-methyladenosine (m6A) has been increasingly recognized as a new and important regulator of gene expression. To date, transcriptome-wide m6A detection primarily relies on well-established methods using next-generation sequencing (NGS) platform. However, direct RNA sequencing (DRS) using the Oxford Nanopore Technologies (ONT) platform has recently emerged as a promising alternative method to study m6A. While multiple computational tools are being developed to facilitate the direct detection of nucleotide modifications, little is known about the capabilities and limitations of these tools. Here, we systematically compare ten tools used for mapping m6A from ONT DRS data. We find that most tools present a trade-off between precision and recall, and integrating results from multiple tools greatly improve performance. Using a negative control could improve precision by subtracting certain intrinsic bias. We also observed variation in detection capabilities and quantitative information among motifs, and identified sequencing depth and m6A stoichiometry as potential factors affecting performance. Our study provides insight into the computational tools currently used for mapping m6A based on ONT DRS data and highlights the potential for further improving these tools, which may serve as the basis for future research.

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.

Sebaceous Carcinoma of the Submandibular Gland a Case Report and Review of the Literature.

Objective: Sebaceous carcinoma (SC) of the submandibular gland is extremely rare. Owing to the low morbidity and nonspecific clinical manifestations, diagnosis is commonly delayed, which increases metastasis and mortality. To date, there have been five reported cases of SC of the submandibular gland. Here, we present a new case and review the relevant literature. Methods and Results: A 36-year-old woman presented with an enlarged left submandibular gland. Clinical features included a non-tender solitary nodular mass with normal overlying skin. There were no special findings on computed tomography or ultrasound examination except for a swollen mass in the left submandibular gland. The patient underwent surgical resection. Pathological examination confirmed the diagnosis of SC with nerve infiltration. Immunohistochemical examination of this case showed positive staining for P63, P40, CK7, CK8/18, MLH1, MSH2, MSH6, and PMS2. The specimen was negative for androgen receptor, CEA, S-100, CK5/6, SOX-10, SOX-11, SMA, and GCDFP-15. The KI-67 labeling index was determined to be 15%. PAS and anti-epithelial membrane antigen were positive in partial area. The patient is still undergoing follow-up, and no metastasis or recurrence has been observed for 2 months. Conclusion: This case highlighted the fact that despite its rarity, SC should be considered as a differential diagnosis for masses located in the head and face. Early and accurate diagnosis, followed by wide surgical excision, has a favorable prognosis. Therefore, clinicians should be familiar with the clinical and pathological features of this disease.

High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome.

N6-deoxyadenosine methylation (6mA) is the most widespread type of DNA modification in prokaryotes and is also abundantly distributed in some unicellular eukaryotes. However, 6mA levels are remarkably low in mammals. The lack of a precise and comprehensive mapping method has hindered more advanced investigations of 6mA. Here, we report a new method MM-seq (modification-induced mismatch sequencing) for genome-wide 6mA mapping based on a novel detection principle. We found that modified DNA bases are prone to form a local open region that allows capture by antibody, for example, via a DNA breathing or base-flipping mechanism. Specified endonuclease or exonuclease can recognize the antibody-stabilized mismatch-like structure and mark the exact modified sites for sequencing readout. Using this method, we examined the genomic positions of 6mA in bacteria (E. coli), green algae (C. reinhardtii), and mammalian cells (HEK239T, Huh7, and HeLa cells). In contrast to bacteria and green algae, human cells possess a very limited number of 6mA sites which are sporadically distributed across the genome of different cell types. After knocking out the RNA m6A methyltransferase METTL3 in mouse ES cells, 6mA becomes mostly diminished. Our results imply that rare 6mA in the mammalian genome is introduced by RNA m6A machinery via a non-targeted mechanism.

Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via. CaM/Src/Pitx2 Activation in Atrial Myocytes.

Hypertension is a major cardiovascular risk factor for atrial fibrillation (AF) worldwide. However, the role of mechanical stress caused by hypertension on downregulating the L-type calcium current (ICa,L), which is vital for AF occurrence, remains unclear. Therefore, the aim of the present study was to investigate the role of Piezo1, a mechanically activated ion channel, in the decrease of ICa,L in response to high hydrostatic pressure (HHP, one of the principal mechanical stresses) at 40 mmHg, and to elucidate the underlying pathways. Experiments were conducted using left atrial appendages from patients with AF, spontaneously hypertensive rats (SHRs) treated with valsartan (Val) at 30 mg/kg/day and atrium-derived HL-1 cells exposed to HHP. The protein expression levels of Piezo1, Calmodulin (CaM), and Src increased, while that of the L-type calcium channel a1c subunit protein (Cav1.2) decreased in the left atrial tissue of AF patients and SHRs. SHRs were more vulnerable to AF, with decreased ICa,L and shortened action potential duration, which were ameliorated by Val treatment. Validation of these results in HL-1 cells in the context of HHP also demonstrated that Piezo1 is required for the decrease of ICa,L by regulating Ca2+ transient and activating CaM/Src pathway to increase the expression of paired like homeodomain-2 (Pitx2) in atrial myocytes. Together, these data demonstrate that HHP stimulation increases AF susceptibility through Piezo1 activation, which is required for the decrease of ICa,L via. the CaM/Src/Pitx2 pathway in atrial myocytes.

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.

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.

Targeted genetic screening in bacteria with a Cas12k-guided transposase.

Microbes employ sophisticated cellular networks encoded by complex genomes to rapidly adapt to changing environments. High-throughput genome engineering methods are valuable tools for functionally profiling genotype-phenotype relationships and understanding the complexity of cellular networks. However, current methods either rely on special homologous recombination systems and are thus applicable in only limited bacterial species or can generate only nonspecific mutations and thus require extensive subsequent screening. Here, we report a site-specific transposon-assisted genome engineering (STAGE) method that allows high-throughput Cas12k-guided mutagenesis in various microorganisms, such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Exploiting the powerful STAGE technique, we construct a site-specific transposon mutant library that focuses on all possible transcription factors (TFs) in P. aeruginosa, enabling the comprehensive identification of essential genes and antibiotic-resistance-related factors. Given its broad host range activity and easy programmability, this method can be widely adapted to diverse microbial species for rapid genome engineering and strain evolution.

Analysis and Construction of a Competitive Endogenous RNA Regulatory Network of Baicalin-Induced Apoptosis in Human Osteosarcoma Cells.

BACKGROUND: Baicalin is an extract from the traditional Chinese herb Scutellaria baicalensis and has the potential to treat osteosarcoma (OS). However, the transcriptome-level mechanism of baicalin-mediated antitumor effects in OS has not yet been investigated. The aim of this study was to analyze the competitive endogenous RNA (ceRNA) regulatory network involved in baicalin-induced apoptosis of OS cells. METHODS: In this study, CCK-8 and flow cytometry assays were used to detect the antitumor effects of baicalin on human OS MG63 cells. Furthermore, transcriptome sequencing was employed to establish the long noncoding RNA (lncRNA), microRNA (miRNA), and mRNA profiles. RESULTS: Baicalin inhibited MG63 cell proliferation and induced apoptosis. Totals of 58 lncRNAs, 31 miRNAs, and 2136 mRNAs in the baicalin-treated MG63 cells were identified as differentially expressed RNAs compared to those in control cells. Of these, 2 lncRNAs, 3 miRNAs, and 18 mRNAs were included in the ceRNA regulatory network. The differentially expressed RNAs were confirmed by quantitative real-time PCR (qRT-PCR). CONCLUSIONS: By identifying the ceRNA network, our results provide new information about the possible molecular basis of baicalin, which has potential applications in OS treatment.

Targeted RNA N6 -Methyladenosine Demethylation Controls Cell Fate Transition in Human Pluripotent Stem Cells.

Deficiency of the N6 -methyladenosine (m6 A) methyltransferase complex results in global reduction of m6 A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m6 A methylation affects cell fate decisions due to the inability to modulate individual m6 A modification in ESCs with precise temporal control. Here, a targeted RNA m6 A erasure (TRME) system is developed to achieve site-specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline-inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6 A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m6 A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.

Psychological State Among the General Chinese Population Before and During the COVID-19 Epidemic: A Network Analysis.

Background: The infectious disease Coronavirus Disease 2019 (COVID-19) outbroke in 2019 spread to multiple countries. The quick spread of the virus and isolation strategies may trigger psychological problems. Our aim was to explore the dynamic network structure of the psychological state before and during the epidemic. Methods: A web-based survey was conducted in two stages: the T1 stage (1 January 2019 to 31 December 2019) and the T2 stage (1 February 2020 to 8 March 2020). In both stages, the Patient Health Questionnaire-9, General Anxiety Disorder-7, and Pittsburgh Sleep Quality Index were used to assess depression, anxiety, and sleep, respectively. Results: We matched the data based on IP addresses. We included 1,978, 1,547, and 2,061 individuals who completed the depression, anxiety, and sleep assessments, respectively, at both stages. During epidemics, psychomotor agitation/retardation, inability to relax, restless behavior, and the frequency of using medicine had high centrality. Meanwhile, the network structure of psychological symptoms becomes stronger than before the epidemic. Conclusion: Symptoms of psychomotor agitation/retardation, inability to relax, and restless behavior should be treated preferentially. It is necessary to provide mental health services, including timely and effective early psychological intervention. In addition, we should also pay attention to the way patients use medicines to promote sleep quality.