Effect and potential mechanism of nitrite reductase B on nitrite degradation by Limosilactobacillus fermentum RC4.

Nitrite has the potential risk of hypoxic poisoning or cancer in pickled food. In our previous study, Limosilactobacillus fermentum (L. fermentum) RC4 is effective in nitrite degradation by producing nitrite reductase B (NirB). To investigate the detailed mechanism from the genome, response, and regulation of NirB, the whole-genome sequence of L. fermentum RC4 was analyzed, the L. fermentum-EGFP-nirB with enhanced green fluorescent protein (EGFP) labeled the nitrite reductase large subunit nirB, and the recombined L. fermentum-NirB with overexpression NirB strain was conducted. The key genes within the dominant metabolism pathways may be involved in stress tolerance to regulate the degrading process. The green fluorescence density of EGFP indicated that NirB activity has a threshold and peaked under 300 mg/L nitrite concentration. NirB overexpressed in L. fermentum RC4 boosted the enzyme activity by 39.6% and the degradation rate by 10.5%, when fermented in 300 mg/L for 40 h, compared to the control group. RNA-seq detected 248 differential genes mainly enriched in carbohydrate, amino acid, and energy metabolism. The ackA gene for pyruvate metabolism and the mtnN gene for cysteine metabolism were up-regulated. NirB regulates these genes to produce acid and improve stress resistance for L. fermentum RC4 to accelerate nitrite degradation.

Activation of p62-Keap1-Nrf2 Pathway Protects 6-Hydroxydopamine-Induced Ferroptosis in Dopaminergic Cells.

Parkinson's disease (PD) is a common neurodegenerative disorder primarily caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc). However, the manner of death of dopaminergic neurons remains indistinct. Ferroptosis is a form of cell death involving in the iron-dependent accumulation of glutathione depletion and lipid peroxide. Besides, previous studies indicated that ferroptosis might be involved in the death of dopaminergic neurons. In this study, we aim to explore the protective effect of the p62-Keap1-Nrf2 pathway against 6-hydroxydopamine (6-OHDA)-induced ferroptosis in dopaminergic cells. Firstly, our results demonstrated that 6-OHDA-induced ferroptosis could be observed in vivo zebrafish and in vitro human dopaminergic cell line (SH-SY5Y cells) model. Moreover, ferroptosis induced by 6-OHDA mitigates in SH-SY5Y cells upon ferrostatin-1 (Fer, an inhibitor of ferroptosis) treatment via upregulating the protein expression of glutathione peroxidase 4 (GPX4). Then, we found that high p62/SQSTM1 (p62) expression could protect SH-SY5Y cells against ferroptosis through promoting Nrf2 nuclear transfer and upregulating the expression of the antioxidant protein heme oxygenase-1 (HO-1). Ultimately, high p62 expression activates the Nrf2/HO-1 signaling pathway through binding to Kelch-like ECH-associated protein 1 (Keap1). Collectively, the activation of the p62-Keap1-Nrf2 pathway prevents 6-OHDA-induced ferroptosis in SH-SY5Y cells, targeting this pathway in combination with a pharmacological inhibitor of ferroptosis can be a potential approach for PD therapy.

Enhancement of Tobacco (Nicotiana tabacum L.) Seed Lipid Content for Biodiesel Production by CRISPR-Cas9-Mediated Knockout of NtAn1.

Tobacco (Nicotiana tabacum L.) seed lipid is a promising non-edible feedstock for biodiesel production. In order to meet the increasing demand, achieving high seed lipid content is one of the major goals in tobacco seed production. The TT8 gene and its homologs negatively regulate seed lipid accumulation in Arabidopsis and Brassica species. We speculated that manipulating the homolog genes of TT8 in tobacco could enhance the accumulation of seed lipid. In this present study, we found that the TT8 homolog genes in tobacco, NtAn1a and NtAn1b, were highly expressed in developing seed. Targeted mutagenesis of NtAn1 genes was created by the CRISPR-Cas9-based gene editing technology. Due to the defect of proanthocyanidin (PA) biosynthesis, mutant seeds showed the phenotype of a yellow seed coat. Seed lipid accumulation was enhanced by about 18 and 15% in two targeted mutant lines. Protein content was also significantly increased in mutant seeds. In addition, the seed yield-related traits were not affected by the targeted mutagenesis of NtAn1 genes. Thus, the overall lipid productivity of the NtAn1 knockout mutants was dramatically enhanced. The results in this present paper indicated that tobacco NtAn1 genes regulate both PAs and lipid accumulation in the process of seed development and that targeted mutagenesis of NtAn1 genes could generate a yellow-seeded tobacco variety with high lipid and protein content. Furthermore, the present results revealed that the CRISPR-Cas9 system could be employed in tobacco seed de novo domestication for biodiesel feedstock production.