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.

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.

In adjuvant-induced arthritic rats, acupuncture analgesic effects are histamine dependent: potential reasons for acupoint preference in clinical practice.

This study investigated whether immediate acupuncture effects in the acupoint are histamine dependent. Both histamine injection and manual acupuncture stimulation increased the pain threshold (PT) after treatment compared with the model group (P < 0.01), producing an analgesic effect. After pretreatment with clemastine, an H1 receptor antagonist and an antipruritic, the increase in the animals' pain threshold after acupuncture was suppressed compared with the Acu group (P < 0.01); however, there was no interference with the acupuncture-induced degranulation of mast cells. Pretreatment with disodium cromolyn did not suppress the increase in PT induced by the histamine injection at Zusanli (ST-36). We conclude that in adjuvant-induced arthritic rats, acupuncture analgesic effects are histamine dependent, and this histamine dependence determines the acupoint preference of acupoints away from the target site in acupuncture practice.

[Acupoint-injection of histamine induced analgesic effect in acute adjuvant-induced-arthritis rats].

OBJECTIVE: To observe the activation effect of histamine on mast cells in the acupoint area and its analgesic effects similar to acupuncture stimulation, so as to reveal the underlying mechanism of acupoint injection therapy. METHODS: A total of 60 SD rats were divided into control, model, normal saline (NS), histamine-injection and acupuncture groups (12 rats/group). Acute adjuvant-induced-arthritis (AIA) model was duplicated by injection of complete Freunds' Adjuvant (0.05 mL) into the left ankle articular cavity. For rats of saline and histamine groups, normal saline (50 microL) and histamine (50 miroL, 100 microg/mL) were injected into the left "Zusanli" (ST 36) acupoint area. For rats of acupuncture group, manual acupuncture (lifting-thrusting and twisting needle intermittently) was applied to ST 36 for 20 min. Paw withdrawal (pain threshold, PT) was detected by radiant heat irradiation of the hindpaw. Skin and muscle tissues of ST 36 area were sampled to be fixed in formalin, then, sliced (5 microm), and stained with Toluidine Blue, separately, followed by counting the degranulation rate of mast cells under microscope. RESULTS: Compared with normal control group and pre-modeling, PT of model, NS, acupuncture and histamine groups decreased significantly (P < 0.01), while in comparison with model group, PT of acupuncture and histamine groups increased considerably (P < 0.01), suggesting an induction of analgesic effect after acupuncture and histamine injection. The degranulation rates of acupuncture and histamine groups were remarkably higher than those of model and NS groups (P < 0.01), suggesting an activation of mast cells following regional acupuncture stimulation and histamine injection. CONCLUSION: Both manual acupuncture and histamine injection of ST 36 can relieve pain in AIA rats, which may be closely related to its effect in activating mast cells in acupoint area. It suggests that the mast cells are a new target for acupoint injection therapy.