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.

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.

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.

Is COPD associated with increased mortality and morbidity in hospitalized pneumonia? A systematic review and meta-analysis.

This review aimed to investigate whether chronic obstructive pulmonary disease (COPD) is associated with increased mortality and morbidity in patients hospitalized with community-acquired pneumonia (CAP). EMBASE, PubMed and Web of Science were searched for cohort studies and case-control studies investigating the impact of COPD on CAP. The primary outcome was all-cause mortality, and secondary outcomes included length of hospital stay, intensive care unit (ICU) admission and need for mechanical ventilation. Methodological quality was assessed using the Newcastle-Ottawa Scale. The Mantel-Haenszel method and inverse variance method were used to calculate pooled relative risks (RRs) and mean differences (MD), respectively. Eleven studies (nine cohort studies and two case-control studies), involving 257 958 patients, were included. The overall methodological quality was high. COPD was not associated with increased mortality in hospitalized CAP patients (RR, 1.20; 95% confidence interval (CI): 0.92-1.56; P = 0.19; I(2) = 55%) in cohort studies, and was associated with reduced mortality in case-control studies (RR, 0.82; 95% CI: 0.74-0.90; P < 0.0001; I(2) = 80%). COPD was not associated with longer hospital stay (MD, 0.11; 95% CI: -0.42 to 0.64; P = 0.68; I(2) = 21%), more frequent ICU admission (RR, 0.97; 95% CI: 0.70-1.35; P = 0.87; I(2) = 65%), and more need for mechanical ventilation (RR 0.91, 95% CI: 0.71-1.16; P = 0.44; I(2) = 4%).The current available evidence indicates that COPD may not be associated with increased mortality and morbidity in patients hospitalized with CAP. This conclusion should be re-evaluated by prospective population-based cohort studies.

Development of brucine-loaded microsphere/thermally responsive hydrogel combination system for intra-articular administration.

Intra-articular drug delivery system could directly deliver a drug to an affected joint and offer the possibility of reaching high drug concentrations at the site of action with limited systemic toxicity. However, depending on their chemical structure, some active compounds were rapidly cleared from the joint, thus requiring numerous injections, which could cause infection or joint disability. To control the release behavior for prolonged time periods, a novel biologically based drug delivery vehicle was designed for intra-articular using microsphere/thermally responsive hydrogel combination system in this paper. And brucine was the test drug. The system was constructed by dispersing the brucine microspheres which was prepared by using a spray-drying method in a thermally responsive biopolymer hydrogel contained with chitosan-glycerol-borax. The microspheres were spherical as evidenced by the scanning electron microscopy (SEM) photographs. And the entrapment rate was 98.60% w/w with an average size range of 0.9-4.5 µm. Fourier transforms infrared (FT-IR) spectroscopy and X-ray diffraction (XRD) revealed the absence of drug-polymer interaction and amorphous nature of an entrapped drug. From the in vitro drug release study we could see that there was a burst release of microsphere, which was obviously retarded when dispersed in hydrogel. And the studies of biocompatibility with synovium showed that no apparent thickening or hyperplasia of the synovium, a small quality of phlogocyte imbibitions was observed. The results of FX imaging in rats showed that by intra-articular injection the BMH could stay in articular for over 7 days were consistent with our in vitro release. And the results of pharmacodynamics revealed the BMH could benefit OA joint by suppressing the levels of TNF-α and IL-1ß, protect the damaged joint from degradation. The novel microsphere/thermoresponsive hydrogel combination system could be a promising treatment option for OA and RA. In conclusion, the system appears to be generally biocompatible with synovium and could control the drug release for several days; hence it might be suitable for the development of treatment strategies for rheumatic diseases.

[Brucine chitosan thermosensitive hydrogel for intra-articular injection].

The aim of this study was to develop a sustained release converse thermosensitive hydrogel for intra-articular injection using chitosan-glycerol-borax as matrix, its physical properties and biocompatibility were investigated. Taking gelation time and gelation condition as index, the influence of concentration of chitosan, ratio of chitosan to glycerol, pH on physical properties of hydrogel were investigated. And then the in vitro drug release, rheological properties and biocompatibility were studied. The thermosensitive hydrogel flows easily at room temperature and turns to gelation at body temperature, which can certainly prolong the release of drug and has good biocompatibility.

The studies of anti-inflammatory and analgesic activities and pharmacokinetics of Oxytropis falcate Bunge extraction after transdermal administration in rats.

The aim of this study was to evaluate the activities of anti-inflammatory and analgesic of the total flavonoids extraction from Oxytropis falcate Bunge (FEO) after transdermal administration. The pharmacokinetics and absolute bioavailability of FEO in rat, furthermore, was studied. Firstly, the anti-inflammatory and analgesic effects of the FEO were studied by xylene-induced ear edema, adjuvant-induced joint inflammation law in rats, acetic acid-induced writhing and hot-plate tests in mice. Secondly, we developed a sensitive and specific HPLC method to analyze 2', 4'-dihydroxychalcone (TFC, the mainly ingredient of FEO) in rat plasma to study the pharmacokinetic of TEC. The results showed FEO has anti-inflammatory and analgesic property in a dose-dependent manner, and that the high dose group (90.6 mg/kg) of FEO appeared more significantly effective than the positive drug. From the pharmacokinetic studies of TFC in rats, we got the main pharmacokinetic parameters of TFC, providing a basis for the future studies in clinic.

Comparative pharmacokinetics and bioavailability studies of quercetin, kaempferol and isorhamnetin after oral administration of Ginkgo biloba extracts, Ginkgo biloba extract phospholipid complexes and Ginkgo biloba extract solid dispersions in rats.

The aim of this study was to improve the oral bioavailability of Ginkgo biloba extract (GBE) through preparing G. biloba extract phospholipid complexes (GBP) and G. biloba extract solid dispersions (GBS). Firstly we prepared the GBP and GBS and studied their physicochemical properties by differential scanning calorimetry (DSC), powder X-ray diffraction (XRD) and dissolution. Then we studied the pharmacokinetic characteristics and bioavailability in rats. The results showed that the bioavailability of quercetin, kaempferol and isorhamnetin in rats was increased remarkably after oral administration of GBP and GBS comparing with GBE. The bioavailabilities of GBP increased more than that of GBS.

Synthesis of a novel polymer bile salts-(polyethylene glycol)2000-bile salts and its application to the liver-selective targeting of liposomal DDB.

PURPOSE: The objective of this study was to achieve a sustained and targeted delivery of liposome to the liver, by modifying the phospholipid [phosphatidylcholine (PC)/cholesterol (10 : 1) liposomes with a novel polymer bile salts-(polyethylene glycol)(2000)-bile salts (BP(2)B). METHODS: First, we generated a novel BP(2)B by N,N'-dicyclohexylcarbodiimide/4-dimethylaminopyridine esterification method and confirmed by Fourier transform infraredand (1) H-NMR spectra. Second, we prepared the BP(2)B-modified liposomes (BP(2)BL) that included BP(2)B, and the effect of the weight ratios of BP(2)B/PC on entrapment efficiency was investigated and BP(2)B/PC = 3% (w/w) was determined as the optimum ratio for the 4,4'-dimethoxy-5,6,5',6'-bi (methylenedioxy)-2,2'-bicarbomethoxybiphenyl liposomes. And then, the ability of the liver target of BP(2)BL was studied by calculating the targeted parameters. RESULTS AND DISCUSSION: All the results revealed that the introduction of polyoxyethylene chains could control interactions of bile salt moieties on liposome surfaces with the receptor compared with traditional liposomes (CL), marking BP(2)BL as a suitable carrier for hepatic parenchymal cell-specific and sustained targeting. It was suggested that liposomes containing such novel BP(2)B have great potential as drug delivery carriers for the liver-selective targeting that has targeted and sustained drug delivery.

[The enhancing effect of borneol on the absorption of tetramethylpyrazine].

To explore the mechanism of the absorption enhancement of borneol, the effect of borneol on the intestinal absorption and the pharmacokinetics of tetramethylpyrazine phosphate after oral administration were investigated. In situ intestinal recirculation was performed to study the effect of various concentrations of borneol on the absorption of tetramethylpyrazine phosphate at duodenum, jejunum, ileum and colon. After oral administration of tetramethylpyrazine phosphate, the mixture of tetramethylpyrazine phosphate and borneol and the mixture of tetramethylpyrazine phosphate and verapamil in rats, the concentrations of tetramethylpyrazine phosphate in plasma were determined by RP-HPLC at predesigned time. The pharmacokinetic parameters were compared based on the results of the three animal experiments, and analyzed with software program 3p97. The result showed that tetramethylpyrazine phosphate could be absorbed at all of the four intestinal segments with increasing absorption amount per unit as follows: colon > duodenum > jejunum > ileum, but without saturation, which demonstrated that tetramethylpyrazine phosphate was absorbed via simple diffusion. Borneol could enhance the intestinal absorption of tetramethylpyrazine phosphate, however, not in proportion. There was no obvious difference between the test group and the control group when 10 microg x mL(-1) borneol was added (P > 0.05), while when the concentration comes to 25 microg x mL(-1) and 50 microg x mL(-1), significant differences were observed (P < 0.05). Borneol and verapamil did enhance the bioavailability of tetramethylpyrazine phosphate after oral administration in rats. The enhancing effect of borneol showed only when the concentration came to a certain level but with no specific sites existed in the intestine. One of the mechanisms of borneol on the enhancing effect on absorption of tetramethylpyrazine phosphate might be the inhibition of the metabolism of CYP 3A and exocytosis of P-gp.

A simple HPLC method for the determination of bifendate: application to a pharmacokinetic study of bifendate liposome.

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method with ultraviolet detector (UV) has been developed for the determination of bifendate in 100 microl plasma of rats. Sample preparation was carried out by deproteinization with 100 microl of acetonitrile. A 20 microl of supernatant was directly injected into the HPLC system with methanol-double distilled water (65/35, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Separation was performed with a microBondapak C(18) column at 30 degrees C. The peak was detected at 278 nm. The calibration curve was linear (r(2)=0.9989) in the concentration range of 0.028-2.80 microg/ml in plasma. The intra- and inter-day variation coefficients were not more than 6.55% and 6.07%, respectively. The limit of detection was 5 ng/ml. The mean recoveries of bifendate were ranged from 94.53% to 99.36% in plasma. The present method has been successfully applied to the pharmacokinetic study of bifendate liposome in rats.

[Preparation of verapamil hydrochloride controlled-onset extended-release pellets and its pharmacokinetics in dogs].

AIM: To prepare verapamil hydrochloride controlled-onset extended-release pellets (VH-COERP) and study its release behavior in vitro. To compare the pharmacokinetic characteristics and bioavailability in six Beagle dogs after oral administration of VH-COERP and verapamil hydrochloride delayed-release pellets (VH-DRP) as reference. METHODS: The core of VH-COERP were prepared in the fluidized bed (mini-glatt) by spraying water solution containing drugs onto sucrose-starch pellets with hydroroxy propyl methyl cellulose (HPMC) as the inner coating swelling layer and ethylcellulouse aqueous dispersion as the outer coating controlled layer. Through modifying the coating level of inner and outer layer, the VH-COERP with the optimized cumulative release profile was obtained. The concentration of VH in plasma of six dogs and its pharmacokinetic behaviors after oral administration of VH-COERP and VH-DRP at different times were studied by RP-HPLC. The pharmacokinetic parameters were computed by software program 3P97. RESULTS: The lag time, the release behavior and the amount of VH from VH-COERP within 24 hours were not influenced by the pH of dissolution medium and post-process, but obviously influenced by the different kinds of added material in swelling layer and the coating level of the inner swelling layer and the outer controlled layer. In vitro the lag time of release profile of VH from VH-COERP was 5 h and then VH was extended release from VH-COERP in the following time. Compared with the VH-DRP, VH-COERP in vivo has an obviously lag time (4 h) , Tmax was also delayed (8 h) and the relative bioavailability was (94.56 +/- 7.64)%. CONCLUSION: The release profile of VH from VH-COERP was shown to be extended-release after an conspicuous lag time in vitro and in vivo. So the drug can be taken by the patient before bed time and begin to work at the morning.