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.

The complete genome of Ziziphus jujuba cv. dongzao, an economic crop in Yellow River Delta of China.

The complete chloroplast genome of Ziziphus jujuba cv. dongzao, known as an important economic cultivar in Yellow River Delta of China was reported. It exhibits a quadripartite structure with 161,493 bp including a large single copy region (89,178 bp), a small single copy region (19,357 bp) and a pair of inverted repeats regions (26,479 bp). It has 36.79% GC content and 114 unique genes. Phylogenetic analysis showed that it was a member of Ziziphus and more closely related to Berchemiella wilsonii. Border analysis revealed that there were some differences in the borders of the four related cultivars.

Association of the FCN2 Gene Single Nucleotide Polymorphisms with Susceptibility to Pulmonary Tuberculosis.

Ficolin-2 (FCN2) is an innate immune pattern recognition molecule that can activate the complement pathway, opsonophagocytosis, and elimination of the pathogens. The present study aimed to investigate the association of the FCN2 gene single nucleotide polymorphisms (SNPs) with susceptibility to pulmonary tuberculosis (TB). A total of seven SNPs in exon 8 (+6359 C>T and +6424 G>T) and in the promoter region (-986 G>A, -602 G>A, -557 A>G, -64 A>C and -4 A>G) of the FCN2 gene were genotyped using the PCR amplification and DNA sequencing methods in the healthy controls group (n = 254) and the pulmonary TB group (n = 282). The correlation between SNPs and pulmonary TB was analyzed using the logistic regression method. The results showed that there were no significant differences in the distribution of allelic frequencies of seven SNPs between the pulmonary TB group and the healthy controls group. However, the frequency of the variant homozygous genotype (P = 0.037, -557 A>G; P = 0.038, -64 A>C; P = 0.024, +6424 G>T) in the TB group was significantly lower than the control group. After adjustment for age and gender, these variant homozygous genotypes were found to be recessive models in association with pulmonary TB. In addition, -64 A>C (P = 0.047) and +6424 G>T (P = 0.03) were found to be codominant models in association with pulmonary TB. There was strong linkage disequilibrium (r2 > 0.80, P < 0.0001) between 7 SNPs except the -602 G>A site. Therefore, -557 A>G, -64 A>C and +6424 G>T SNPs of the FCN2 gene were correlated with pulmonary TB, and may be protective factors for TB. This study provides a novel idea for the prevention and control of TB transmission from a genetics perspective.

[Expression of neuron-specific enolase and synaptophysin in esophageal development of human embryos].

OBJECTIVE: To investigate the expression of neuron-specific enolase (NSE) and synaptophysin(SYN) proteins in different developmental stages of human embryonic esophagus. METHODS: Immunohistochemistry was used to detect the expressions of NSE and SYN proteins in embryonic esophagus tissues of fetuses of 2, 3 and 4 month gestational age (n=16). One-way ANOVA and LSD-t test were employed to compare the staining intensity and number of positive expression cells in embryonic esophageal tissues of different gestational age. RESULTS: In fetuses with 2, 3 and 4 months of gestation, the number of NSE-positive nerve cells in the myenteric nerve plexus and submucosa of human embryonic esophageal tissues were 18.38 ± 8.37, 25.00 ± 11.54 and 38.00 ± 15.09, respectively; the staining intensity of NSE-positive nerve cells and nerve fibers in myenteric nerve plexus and submucosa of embryonic esophageal tissues were 74.38 ± 14.93, 62.25 ± 18.59 and 56.44 ± 14.70, respectively. NSE-positive cells were detected in the esophageal epithelium only at the third month. In the fetuses at 2, 3 and 4 months of gestation, SYN in all layers of esophageal tissue were positively or strong positively expressed, especially in the myenteric plexus and submucosal plexus. The staining intensity of SYN-positive cells in embryonic esophagus tissues of 2, 3 and 4 month gestation were 54.69 ± 9.34, 51.84 ± 6.10 and 46.41 ± 6.44, respectively. CONCLUSION: SYN and NSE may be involved in the regulation of nerve system of esophageal tissues during the human embryonic development.

[Expressions of microtubule-associated protein 2 and nestin in the development of human embryo and fetal tongue muscles].

OBJECTIVE: To explore the role of microtubule-associated protein 2 (MAP-2) and nestin in the development of tongue muscles of human embryos and fetuses. METHODS: PV immunohistochemistry was used to detect the expressions of MAP-2 and nestin proteins in the tongue tissues of human embryos and fetuses at the second, third and fourth months of gestation. RESULTS: MAP-2 and nestin positivity was detected in the tongue muscles of human embryos at 2 to 4 months of gestation. In the embryos at the second month of gestation, no obvious MAP-2 positive cells were found in the tongue muscles; at 3 and 4 months, the number of MAP-2-positive cells in the tongue muscles was 24.14∓8.28 and 15.86∓3.89, with the expression intensity of 109.42∓11.62 and 124.27∓8.73, respectively. At 2, 3 and 4 months of gestation, the number of nestin-positive cells in the tongue muscles was 12.50∓3.17, 19.00∓7.63, and 22.80∓6.91, with expression intensity of 119.99∓24.02, 102.20∓11.76, and 98.24∓10.66, respectively. As the gestational age increased, the number of MAP-2-positive cell number continued to decline following a transient increase but the expression intensity kept increasing; nestin-positive cells increased continuously but the expression intensity kept decreasing in the embryonic or fetal tongue muscles. CONCLUSION: MAP-2 and nestin proteins are involved in the regulation of the development of tongue muscles in human embryos and fetuses.

[Effects of Citalopram on frontal cortical neurons' bax mRNA bcl-2 mRNA expression and cell apoptosis of rat after stress].

OBJECTIVE: To study the effects of Citalopram on the mRNA expression of bax and bel-2 in frontal cortical neurons and on cell apoptosis of rats after stress. METHODS: Twenty-four healthy male SD rats were randomly divided into three groups (n = 8). The control group did no receive any treatment, the stress group was subject to stress and given normal saline and experimental group was given Citalopram irrigation stomach after stress. Rats were forced to swim to establish chronic stress model (15 min/d, 4 weeks), bax, bcl-2 mRNA expression were tested by in situ hybridization technique (ISH), TUNEL assay was used to determine cell apoptosis, Nikon image analysis software were used to measure the number of positive cells in each index. RESULTS: Compared with the control group, the stress group showed a larger number of bax mRNA expressing cells( P < 0.01), a smaller number of bcl-2 mRNA expressing cells (P < 0.01), and the staining intensity of positive cells was significantly reduced( P < 0.01). Compared with the stress group, the experiment group showed more reduced number of bax mRNA positive cells( P < 0.01) and significantly increased bcl-2 mRNA positive cells( P < 0.05), a small amount of positive cells were found, compared with that in the stress group, nuclear condensation in the experimental group was reduced significantly and the staining was obviously weaker( P < 0.01). CONCLUSION: Citalopram significantly antagonizes bax mRNA and potentiatesbcl-2 mRNA protein expression and inhibits apoptosis of rat prefrontal cortical neurons caused by chronic stress, which might be one possible mechanism of Citalopram for prevention and treatment of psychosis caused by chronic stress.

[Effects of citalopram on the expression of PCNA and C-fos and cell apoptosis in rat frontal cortical neurons after stress].

OBJECTIVE: To study the effects of citalopram on the expression of proliferating cell nuclear antigen (PCNA) and proto-oncogene protein (C-fos) and cell apoptosis in frontal cortical neurons of rat after stress. METHODS: Twenty four healthy male SD rats were randomly divided into three groups (n = 8): control group, stress group (treated with saline, ig) , experimental group (treated with Citalopram 4 mg/kg x d for 28 days, ig). Rats were forced to swim to establish chronic stress model. The protein expression levels of PCNA and C-fos were tested by immunohistochemistry assay. TUNEL assay was used to test cell apoptosis. Nikon image analysis software was used to determine the number of positive cells in each index. RESULTS: Compared with the control group, the stress group showed a smaller amount of PCNA-positive cells, a larger number of C-fos positive cells, and the volume of positive cells was significantly reduced. Compared with the stress group, the PCNA positive cells were increased significantly, the C-fos positive cells and TUNEL positive cells were decreased significantly, nuclear condensation phenomenon in frontal cortical neurons and the staining was significantly lighter in experimental group (P < 0.05). CONCLUSION: Citalopram significantly antagonize PCNA, C-fos protein expression and cell apoptosis of rat prefrontal cortical neurons caused by chronic stress, which might be the one of mechanisms of citalopram for prevention and treatment of psychosis caused by chronic stress.

[Expression of PCNA, C-fos and Bax proteins in human embryonic tongue tissues].

OBJECTIVE: To investigate of proliferating cell nuclear antigen (PCNA), C-fos and Bax proteins in human embryonic tongue tissue of different developmental stages. METHODS: Immunohistochemistry was used to detect the expressions of PCNA, C-fos and Bax proteins in embryonic tongue tissues of fetuses with 2, 3 and 4 month gestational age (n=16). One-way ANOVA and LSD-t test were employed to compare the number of positive expression cells in tongue tissues of fetuses with different gestational age. RESULTS: In the fetuses at 2, 3 and 4 months of gestation, the numbers of PCNA-positive cells in tongue epithelial tissues were 20.20 ± 7.13, 39.10 ± 13.44 and 26.00 ± 9.02, respectively; those in tongue muscle and fiber tissues were 17.20 ± 8.99, 22.30 ± 6.57 and 32.40 ± 14.72, respectively. In fetuses at 2 month of gestation, no C-fos-positive cells were found in tongue tissues; while at 3 and 4 months of gestation, the numbers of C-fos-positive cells in the tongue epithelial layers were 25.10 ± 7.91, 17.40 ± 2.80; those in tongue muscle and fiber tissues were 24.50 ± 4.67 and 28.00 ± 7.75, respectively. Only weak positive expression of Bax protein was observed in the third month of gestation in embryonic tongue tissues. A significant difference was noted in PCNA expression in tongue epithelial layers, the muscle and fiber tissues (P<0.01 and P<0.05) among 3 embryonic periods. A significant difference was found in C-fos expression in tongue epithelial layers (P<0.01), but not in tongue muscle and fiber tissues (P>0.05) among 3 periods. CONCLUSION: Dynamic changes were seen in PCNA and C-fos expressions in embryonic tongue tissues in different gestational ages of fetus, indicating these two proteins may participate in regulation of the development and differentiation of tongue tissues in human embryos and fetuses.

How immunogenetically different are domestic pigs from wild boars: a perspective from single-nucleotide polymorphisms of 19 immunity-related candidate genes.

The coexistence of wild boars and domestic pigs across Eurasia makes it feasible to conduct comparative genetic or genomic analyses for addressing how genetically different a domestic species is from its wild ancestor. To test whether there are differences in patterns of genetic variability between wild and domestic pigs at immunity-related genes and to detect outlier loci putatively under selection that may underlie differences in immune responses, here we analyzed 54 single-nucleotide polymorphisms (SNPs) of 19 immunity-related candidate genes on 11 autosomes in three pairs of wild boar and domestic pig populations from China, Iberian Peninsula, and Hungary. Our results showed no statistically significant differences in allele frequency and heterozygosity across SNPs between three pairs of wild and domestic populations. This observation was more likely due to the widespread and long-lasting gene flow between wild boars and domestic pigs across Eurasia. In addition, we detected eight coding SNPs from six genes as outliers being under selection consistently by three outlier tests (BayeScan2.1, FDIST2, and Arlequin3.5). Among four non-synonymous outlier SNPs, one from TLR4 gene was identified as being subject to positive (diversifying) selection and three each from CD36, IFNW1, and IL1B genes were suggested as under balancing selection. All of these four non-synonymous variants were predicted as being benign by PolyPhen-2. Our results were supported by other independent lines of evidence for positive selection or balancing selection acting on these four immune genes (CD36, IFNW1, IL1B, and TLR4). Our study showed an example applying a candidate gene approach to identify functionally important mutations (i.e., outlier loci) in wild and domestic pigs for subsequent functional experiments.

Dichlorido{2-[(2,6-dimethyl-phen-yl)imino-meth-yl]pyridine-κN,N'}zinc.

In the asymmetric unit of the title compound, [ZnCl(2)(C(14)H(14)N(2))], the central Zn(II) ion is four-coordinated in a distorted tetra-hedral environment by two N atoms of the ligand 2-[(2,6-dimethyl-phen-yl)imino-meth-yl]pyridine and two chloride anions. In the crystal, adjacent mol-ecules are connected through C-H⋯Cl hydrogen bonds between a C-H group of the ligand and a Cl(-) anion, leading to a chain-like structure along the b direction.

[Expression of nNOS, Pax3 and Cx43 proteins in early developing posterior horn of embryonic and fetal human spinal cord].

OBJECTIVE: To investigate the distribution pattern of the expressions neuronal nitric oxide synthase (nNOS), Pax3 and connexin 43 (Cx43) proteins in the early developing posterior horn of embryonic and fetal human spinal cord. METHODS: Immunohistochemistry was used to detect the expressions of nNOS, Pax3 and Cx43 proteins in the posterior horn of the spinal cord during the second, third and fourth month of human embryonic and fetal development. RESULTS: In the second to fourth month of gestation, the expressions of nNOS and Pax3 proteins increased gradually from weak expression to strong expression in the posterior horn of the spinal cord. In the second to third month of development, Cx43 protein expression was negative in the posterior horn of the spinal cord, but positive in the myelin sheath. In the fourth month, positive Cx43 expression was detected in some of the cells in the posterior horn of the spinal cord. CONCLUSION: nNOS, Pax3 and Cx43 proteins are closely related to the growth and development of the spinal cord in human embryos and fetuses.

[Expression of Cx43 and Pax3 in the small intestinal muscular layers of early human embryos].

OBJECTIVE: To explore the patterns of Cx43 and Pax3 protein expressions in the small intestinal muscular layers of human embryo during early development. METHODS: Immunohistochemistry with SABC method was employed to examine the expression of Cx43 and Pax3 proteins in the muscular layers of the small intestine in early human embryos in the second to fourth months of gestation. RESULTS: In the second month of gestation, the muscle layer of the small intestine was negative for Cx43 and Pax3 protein expressions. In the third month, Cx43 and Pax3 expressions were negative in the inner circular muscle layer, but some positive cells were found in the longitudinal muscle layer and the myenteric plexus. In the fourth month, positive expression of Cx43 and Pax3 proteins were seen in the entire muscle layer. CONCLUSION: Cx43 and Pax3 proteins are closely related to the growth and development of the cells and tissues in the small intestinal muscle layer in human embryos.