GLORI for absolute quantification of transcriptome-wide m6A at single-base resolution.

N6-methyladenosine (m6A) is the most abundant posttranscriptional chemical modification in mRNA, involved in regulating various physiological and pathological processes throughout mRNA metabolism. Recently, we developed GLORI, a sequencing method that enables the production of a globally absolute-quantitative m6A map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving m6A intact. The RNA library can then be prepared using a modified library construction protocol from enhanced UV crosslinking and immunoprecipitation (eCLIP) or commercial kits. Here we provide a detailed protocol for proper RNA sample handling and provide further guidelines for the use of a tailored bioinformatics pipeline (GLORI-tools) for subsequent data analysis. Compared with current methods, this new method is both exceptionally sensitive and robust, capable of identifying ~80,000 m6A sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for m6A sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating m6A biology across various fields. Basic expertise in molecular biology and knowledge of bioinformatics are required for the protocol. The entire procedure can be completed within a week, with the library construction process taking ~4 d.

MicroRNA transcriptome analysis reveals the immune regulatory mechanism of Crassostrea hongkongesis against Vibrio harveyi infection.

MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate target-genes expression and play crucial roles in post-transcriptional regulation and immune system regulation. The Hong Kong oyster (Crassostrea hongkongesis), as the main marine aquaculture shellfish in the South China Sea, not only has high economic and ecological value, but also is an ideal model for conducting research on pathogen host interaction. Vibrio harveyi, a Gram negative luminescent marine bacterium, is widely distributed in coastal water environments and can cause large-scale death of C. hongkongesis. However, little in formation is available on the immune regulatory mechanisms of C. hongkongesis infected with V. harveyi. Therefore, we performed microRNA transcriptome analysis for elucidating the immunoregulation mechanism of C. hongkongesis infected with V. harveyi. The results show that a total of 308468208 clean reads and 288371159 clean tags were obtained. 222 differentially expressed miRNAs were identified. A total of 388 target genes that were differentially expressed and negatively correlated with miRNA expression were predicted by 222 DEmiRs. GO enrichment analysis of 388 DETGs showed that they were mainly enriched in the immune-related term of membrane-bounded vesicle, endocytic vesicle lumen, antigen processing and presentation of exogenous peptide antigen via MHC class I, antigen processing and presentation of peptide antigen via MHC class I, and other immune-related term. KEGG enrichment analysis showed that DETGs were mainly enriched in the Complement and coagulation cascades, Herpes simplex virus 1 infection, Bacterial invasion of epithelial cells, Antigen processing and presentation and NOD-like receptor signaling pathway. The 16 key DEmiRs and their target genes form a regulatory network for seven immune-related pathways. These results suggest that V. harveyi infection induces a complex miRNA response with wide-ranging effects on immune gene expression in the C. hongkongesis. This study explored the immune response of C. hongkongesis to V. harveyi infection at the level of miRNAs, which provides new ideas for the healthy culture and selective breeding of C. hongkongesis.

An optimized fluorescent biosensor for monitoring long-chain fatty acyl-CoAs metabolism in vivo.

Long-chain fatty acyl-CoAs (LCACoAs) are intermediates in lipid metabolism that exert a wide range of cellular functions. However, our knowledge about the subcellular distribution and regulatory impacts of LCACoAs is limited by a lack of methods for detecting LCACoAs in living cells and tissues. Here, we report our development of LACSerHR, a genetically encoded fluorescent biosensor that enables precise measurement of subtle fluctuations in the levels of endogenous LCACoAs in vivo. LACSerHR significantly improve the fluorescent brightness and analyte affinity, in vitro and in vivo testing showcased LACSerHR's large dynamic range. We demonstrate LACSerHR's capacity for real-time evaluation of LCACoA levels in specific subcellular compartments, for example in response to disruption of ACSL enzyme function in HEK293T cells. Moreover, we show the application of LACSerHR for sensitive measurement of elevated LCACoA levels in the livers of mouse models for two common metabolic diseases (NAFLD and type 2 diabetes). Thus, our LACSerHR sensor is a powerful, broadly applicable tool for studying LCACoAs metabolism and disease.

Transcriptome analysis of digestive diverticula of Hong Kong oyster (Crassostrea hongkongesis) infected with Vibrio harveyi.

The Hong Kong oyster (Crassostrea hongkongesis), as the main marine aquaculture shellfish in the South China Sea, not only has high economic and ecological value, but also is an ideal model for conducting research on pathogen host interaction. However, diseases caused by Vibrio pose a serious impediment to the culture of C. hongkongesis. In this study, we performed transcriptome analysis of digestive diverticula of C. hongkongesis infected with V. harveyi. A total of 977, 689, 912 high quality reads and 955, 208, 562 valid reads were obtained. At 12, 24, 48 and 72 h post-infection, 1402, 2168, 2727 and 1398 differentially expressed genes (DEGs) were captured, respectively. GO enrichment analysis showed that DEGs were significantly enriched in cellular processes, catalytic activity, cell part and other terms. KEGG enrichment analysis revealed that these DEGs were mainly closely related to Necroptosis, RIG-I-like receptor signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway and other pathways are related. The results of WGCNA analysis indicated that THBS1, CA10, Trpm2, THAP12, PTPRT, HSPA12A, and ADAM10 were the hub genes in the gene co-expression network. This study will provide new ideas at the transcriptome level for the immune regulatory mechanisms and adaptability of the C. hongkongesis to V. infection, as well as for achieving selective breeding for Vibrio resistance in the C. hongkongesis.

"Help Us!": a content analysis of COVID-19 help-seeking posts on Weibo during the first lockdown.

BACKGROUND: Social media is playing an increasingly important role in public emergencies for help-seekers, especially during the global COVID-19 pandemic. Wuhan, China, firstly official reported COVID-19 cases and implemented lockdown measures to prevent the spread of the virus. People during the first lockdown were restricted from seeking help face-to-face. Social media is more prominent as an online tool for people seeking help, especially for patients, than in other stages of the COVID-19 pandemic. OBJECTIVE: This study aimed to explore the urgent needs presented in help-seeking posts in Wuhan during the first COVID-19 lockdown, the content features of these posts, and how they influenced online user engagement. METHODS: This study collected posts from Weibo posted with specific help tags during the first COVID-19 lockdown in Wuhan: from 23 January 2020 to 24 March 2020, and eventually received 2055 data, including textual content, comments, retweets, and publishing location. Content analysis was conducted, and manual coding was performed on help-seeking typology, narrative mode, narrative subject, and emotional valence. RESULTS: The result showed that help-seeking posts primarily were seeking medical (97.7%). Features of these posts were mainly adopting a mixed narrative mode (46.4%), released by relatives of patients (61.7%), and expressing negative emotions (93.2%). Chi-square tests suggested that help-seeking posts with mixed narrative modes released by relatives express more frequent negative emotions. Results of negative binomial regression indicated posts of seeking information (B = 0.52, p < .001, e0.52 = 1.68), with mixed narrative mode (B = 0.63, p < .001, e0.63 = 1.86), released by themselves (as referential groups) and with neutral emotions increased comments. Posts of seeking medical (B = 0.57, p < .01, e0.57 = 1.77), with mixed narrative mode (B = 1.88, p < .001, e1.88 = 6.53), released by people of unrelated patients (B = 0.47, p < .001, e0.47 = 1.60) and with neutral emotions increased retweets. CONCLUSIONS: This study provides evidence of what actual public demands are to be considered and addressed by governments and public administrators before implementing closure and lockdown policies to limit the spread of the virus. Meanwhile, our findings offer strategies for people help-seeking on social media in similar public health emergencies.

Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI.

N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian cells and the best-studied epitranscriptomic mark. Despite the development of various tools to map m6A, a transcriptome-wide method that enables absolute quantification of m6A at single-base resolution is lacking. Here we use glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to develop an absolute m6A quantification method that is conceptually similar to bisulfite-sequencing-based quantification of DNA 5-methylcytosine. We apply GLORI to quantify the m6A methylomes of mouse and human cells and reveal clustered m6A modifications with differential distribution and stoichiometry. In addition, we characterize m6A dynamics under stress and examine the quantitative landscape of m6A modification in gene expression regulation. GLORI is an unbiased, convenient method for the absolute quantification of the m6A methylome.

Ultrafine Co6W6C as an efficient anode catalyst for direct hydrazine fuel cells.

Ultrafine ternary carbide Co6W6C@C nanoparticles (NPs) were successfully synthesized and these NPs exhibited high catalytic activities for the hydrazine oxidation reaction (HzOR) under alkaline conditions. In a practical O2-hydrazine fuel cell test, its peak power density reached 203 mW cm-2, a value superior to that of the state-of-the-art commercial Pt/C catalyst.