Syringaldehyde Exhibits Antibacterial and Antioxidant Activities against Mycobacterium marinum Infection.

Tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis (Mtb), which has a unique resistance to many antimicrobial agents. TB has emerged as a significant worldwide health issue because of the rise of multidrug-resistant strains causing drug-resistant TB (DR-TB). As a result, the development of new drugs or effective strategies is crucial for patients with TB. Mycobacterium marinum (Mm) and Mtb are both species of mycobacteria. In zebrafish, Mm proliferates and forms chronic granulomatous infections, which are similar to Mtb infections in lung tissue. Syringaldehyde (SA) is a member of the phenolic aldehyde family found in various plants. Here, we investigated its antioxidative and antibacterial properties in Mm-infected cells and zebrafish. Our results demonstrated that SA inhibits Mm-infected pulmonary epithelial cells and inhibits the proliferation of Mm in Mm-infected zebrafish, suggesting that SA provides an antibacterial effect during Mm infection. Further study demonstrated that supplementation with SA inhibits the production of malondialdehyde (MDA) and reactive oxygen species (ROS) and increases the levels of reduced glutathione (GSH) in Mm-infection-induced macrophages. SA inhibits the levels of MDA in Mm-infected zebrafish, suggesting that SA exerts antioxidative effects in vivo. Additionally, we found that SA promotes the expression of NRF2/HO-1/NQO-1 and the activation of the AMPK-α1/AKT/GSK-3ß signaling pathway. In summary, our data demonstrated that SA exerts antioxidative and antibacterial effects during Mm infection both in vivo and in vitro and that the antioxidative effects of SA may be due to the regulation of NRF2/HO-1/NQO-1 and the AMPK-α1/AKT/GSK-3ß signaling pathway.

miR-4687-5p Affects Intracellular Survival of Mycobacterium tuberculosis through Its Regulation of NRAMP1 Expression in A549 Cells.

Tuberculosis (TB), as one of the leading causes of death, poses a serious predicament to the world. MicroRNAs (miRNAs) play a role in the post-transcriptional regulation of gene expression. It has been reported that the expression of miRNAs changes upon mycobacterial infection; the screening and identification of miRNAs regulating the expression of genes could benefit our understanding of TB pathogenesis and generate effective strategies for its control and prevention. In this study, luciferase assays showed that miR-4687-5p is bound to the 3'-untranslated region of natural resistance-associated macrophage protein 1 (NRAMP1). Additionally, we found a significant increase in miR-4687-5p expression in Mycobacterium tuberculosis (Mtb)-infected A549 cells. Concomitantly, we detected a reduced level of NRAMP1 expression, suggesting that NRAMP1 is one of the targets of miR-4687-5p. Infection experiments evidenced that the transfection of miR-4687-5p induced a decrease in NRAMP1 expression and increased intracellular Mtb loads post-infection, indicating that miR-4687-5p promotes the intracellular survival of Mtb through its downregulation of the NRAMP1 protein level. We also found that the transfection of miR-4687-5p induced increased apoptosis and decreased cell proliferation post-infection with Mtb. The results presented in our study suggest that miR-4687-5p may be indicative of the susceptibility of Mtb infection to humans and could act as a potential therapeutic target for tuberculosis treatment.

Alternative Polyadenylation of Malic Enzyme 1 Is Essential for Accelerated Adipogenesis.

Understanding the mechanism of adipogenesis is an important basis for improving meat quality traits of livestock. Alternative polyadenylation (APA) is a vital mechanism to regulate the expression of eukaryotic genes. However, how the individual APA functions in adipogenesis remains elusive. This study was intended to investigate the effect of malic enzyme 1 (ME1) APA on adipogenesis. Here, intracellular lipid droplets were stained using Oil red O. 3' RACE was used to verify APA events of the ME1 gene. Interactions between ME1 3' untranslated region (3' UTR)-APA isoforms and miRNAs, as well as differential expression of isoforms, were examined using dual-luciferase reporter and molecular experiments. The mechanism of ME1 APA on adipogenesis was explored by gain and loss of function assays. In this study, two ME1 isoforms with different 3' UTR lengths were detected during adipogenesis. Moreover, the ME1 isoform with a short 3' UTR was significantly upregulated compared with the one with a long 3' UTR. Mechanistically, only the long ME1 isoform was targeted by miR-153-3p to attenuate adipogenesis, while the short one escaped the regulation of miR-153-3p to accelerate adipogenesis. Our results reveal a novel mechanism of ME1 APA in regulating adipogenesis.

The expression of Nramp1 modulates the uptake of Mycobacterium tuberculosis by macrophages through alternating inflammatory responses.

Natural-resistance-associated macrophage protein-1 (NRAMP1) is a transmembrane protein of the mammalian SLC11 gene family. Previously, genome-wide association study (GWAS) have shown that the single nucleotide polymorphisms (SNPs) of NRAMP1 are associated with human susceptibility to tuberculosis (TB), and the detection of clinical samples have demonstrated that the expression levels of NRAMP1 are concomitant with the susceptibility to TB in humans and cows, but underlying mechanism is unknown. In this study, we completed a series of experiments to investigate how the expression of Nramp1 affects the infection of macrophages with Mycobacterium tuberculosis (Mtb). We found that the increase of Nramp1 expression induced the decrease of Mtb infection efficiency and the higher-level expression of pro-inflammatory cytokines and chemokines, However, the knockdown of Nramp1 promoted the efficiency of bacilli infection to macrophages and induced lower-levels of expression of pro-inflammatory cytokines and chemokines. Collectively, the results in this study demonstrated that the levels of Nramp1 expression affect Mtb infection of macrophage and regulate pro-inflammatory responses of macrophages to Mtb infection, indicating the population with the low-expression level of NRAMP1 predispose to Mtb infection and TB development, and suggesting SNPs in NRAMP1 modulate the host susceptibility to TB through its regulation of NRAMP1 expression.

CRISPR/Cas9-induced asap1a and asap1b co-knockout mutant zebrafish displayed abnormal embryonic development and impaired neutrophil migration.

ASAP1 (Arf-GAP with SH3 domain, the ankyrin repeat and the PH domain) is the GTPase activating protein of the small G protein Arf. To understand more about the physiological functions of ASAP1 in vivo, we chose to use the zebrafish as an animal model, and analyzed the characterization of asap1 using loss-of-function studies. Here, two isoforms in zebrafish, asap1a and asap1b, were found to be homologous to human ASAP1, and the gene knockout zebrafish lines for asap1a and asap1b were established using the CRISPR/Cas9 technique with different insertions and deletions of bases. Zebrafish with asap1a and asap1b co-knockout showed a significant reduction in survival and hatching rates, as well as an increase in malformation rates during the early stages of development, while the asap1a or asap1b single knockout mutants did not affect the growth and development of individual zebrafish. Exploring the gene expression compensation between asap1a and asap1b using qRT-PCR, we found that asap1b had increased expression when asap1a was knocked out, showing a clear compensatory effect against asap1a knockout; In turn, asap1a did not have detectable compensating expression after asap1b knockout. Furthermore, the co-knockout homozygous mutants displayed impaired neutrophil migration to Mycobacterium marinum infection, and showed an increased bacterial load. Together, these are the first inherited asap1a and/or asap1b mutant zebrafish lines by the CRISPR/Cas9 gene editing approach, and by serving as useful models, they can significantly contribute to better annotation and follow-up physiological studies of human ASAP1.

Shortening of HO1 3'UTRs by Alternative Polyadenylation Suppresses Adipogenesis in 3T3-L1.

Appropriately increasing intramuscular fat content can help improve meat quality, so it is necessary to explore the internal molecular mechanism of preadipocyte differentiation. The role of heme oxygenase 1 (HO1) in cell oxidative stress, energy metabolism, cell proliferation, and differentiation has gradually been revealed. Here, we used 3'RACE to identify the full-length 3' untranslated region (3'UTR) of HO1 and found that a very short 3'UTR variant was produced by alternative polyadenylation (APA). HO1 with a long 3'UTR variant was identified as a direct target of miR155-5P and miR377-3P. Our experimental results verified the inhibitory effect of HO1 on preadipocyte differentiation. In addition, our research confirms that by escaping microRNA inhibitory effects, the HO1 3'UTR short variant produced by APA has a higher level of expression. Thus, the HO1 3'UTR short variant has a stronger inhibitory effect on the preadipocyte differentiation than the HO1 3'UTR long variants in 3T3-L1.