Improvement of the fibrinolytic activity, acid resistance and thermostability of nattokinase by surface charge engineering.

Nattokinase is a promising thrombolytic drug due to its powerful fibrinolytic effect and few side effects. However, the low fibrinolytic activity and stability of nattokinase have limited its industrial production and oral application. In this study, the basic and neutral amino acid residues on the surface of recombinant nattokinase AprY from Bacillus mojavensis LY-06 (rAprY) were mutated to acidic amino acid residues by surface charge engineering strategy, and two variants K12D and N109D with 92.6 % and 8.4 % increased fibrinolytic activity were obtained. The R45E variant with enhanced acid stability and thermostability was also screened, its acid stability at pH 4 and t1/2 at 55 °C were 3.7-fold and 1.8-fold higher than that of wild type rAprY, respectively. Bioinformatics analysis showed that the increased activities of K12D and N109D variants were related to the increased flexibility of the region around their active centers. The increased rigidity of 97-103 amino acid residues around the active center of R45E may be the reason for its enhanced stability and reduced catalytic activity. The multipoint mutation K12D-N109D (M2)'s catalytic activity did not increase cumulatively, but its pH stability did. The nattokinase variants generated in this study have potential for industrial production and application.

Combined Computer-Aided Predictors to Improve the Thermostability of Nattokinase.

Food-derived nattokinase has strong thrombolytic activity and few side effects. In the field of medicine, nattokinase has been developed as an adjuvant drug for the treatment of thrombosis, and nattokinase-rich beverages and health foods have also shown great potential in the field of food development. At present, the poor thermostability of nattokinase limits its industrial production and application. In this study, we used several thermostability-prediction algorithms to predict nattokinase from Bacillus mojavensis LY-06 (AprY), and screened two variants S33T and T174V with increased thermostability and fibrinolytic activity. The t1/2 of S33T and T174V were 8.87-fold and 2.51-fold those of the wild type AprY, respectively, and their enzyme activities were also increased (1.17-fold and 1.28-fold, respectively). Although the thermostability of N218L was increased by 2.7 times, the fibrinolytic activity of N218L was only 73.3% of that of wild type AprY. The multiple-point mutation results showed that S33T-N218L and S33T-T174V-N218L variants lost their activity, and the T174V-N218L variant did not show any significant change in catalytic performance, while S33T-T174V increased its thermostability and activity by 21.3% and 24.8%, respectively. Although the S33T-T174V variant did not show the additive effect of thermostability, it combined the excellent transient thermostability of S33T with the better thrombolytic activity of T174V. Bioinformatics analysis showed that the overall structure of S33T and T174V variants tended to be stable, while the structure of S33T-T174V variant was more flexible. Local structure analysis showed that the increased rigidity of the active center region (positions 64-75) and the key loop region (positions 129-130, 155-163, 187-192, 237-241, and 268-270) determined the increased thermostability of all variants. In addition, the enhanced flexibility of S33T-T174V variant in the Ca1 binding region (positions 1-4, 75-82) and the peripheral region of the catalytic pocket (positions 210-216) may account for the inability to superpose its thermostability. We explored the effective strategy to enhance the thermostability of nattokinase, and the resulting variants have potential industrial production and application.

Exercise-induced endothelial Mecp2 lactylation suppresses atherosclerosis via the Ereg/MAPK signalling pathway.

BACKGROUND AND AIMS: Lactylation, a recently identified post-translational modification (PTM), plays a central role in the regulation of multiple physiological and pathological processes. Exercise is known to provide protection against cardiovascular disease. However, whether exercise-generated lactate changes lactylation and is involved in the exercise-induced attenuation of atherosclerotic cardiovascular disease (ASCVD) remains unclear. The purpose of this study was to investigate the effects and mechanisms of exercise-induced lactylation on ASCVD. METHODS AND RESULTS: Using the high-fat diet-induced apolipoprotein-deficient mouse model of ASCVD, we found that exercise training promoted Mecp2 lysine lactylation (Mecp2k271la); it also decreased the expression of vascular cell adhesion molecule 1 (Vcam-1), intercellular adhesion molecule 1 (Icam-1), monocyte chemoattractant protein 1 (Mcp-1), interleukin (IL)-1ß, IL-6, and increased the level of endothelial nitric oxide synthase (Enos) in the aortic tissue of mice. To explore the underlying mechanisms, mouse aortic endothelial cells (MAECs) were subjected to RNA-sequencing and CHIP-qPCR, which confirmed that Mecp2k271la repressed the expression of epiregulin (Ereg) by binding to its chromatin, demonstrating Ereg as a key downstream molecule for Mecp2k271la. Furthermore, Ereg altered the mitogen-activated protein kinase (MAPK) signalling pathway through regulating the phosphorylation level of epidermal growth factor receptor, thereby affecting the expression of Vcam-1, Icam-1, Mcp-1, IL-1ß, IL-6, and Enos in ECs, which in turn promoted the regression of atherosclerosis. In addition, increasing the level of Mecp2k271la by exogenous lactate administration in vivo also inhibits the expression of Ereg and the MAPK activity in ECs, resulting in repressed atherosclerotic progression. CONCLUSIONS: In summary, this study provides a mechanistic link between exercise and lactylation modification, offering new insight into the anti-atherosclerotic effects of exercise-induced PTM.

Exercise Mitigates Endothelial Pyroptosis and Atherosclerosis by Downregulating NEAT1 Through N6-Methyladenosine Modifications.

BACKGROUND: The benefits of exercise on the cardiovascular system are widely recognized; however, the underlying mechanisms are unknown. Here, we report the effect of the long noncoding RNA NEAT1 (nuclear paraspeckle assembly transcript 1), which is regulated by exercise, on atherosclerosis development after N6-methyladenosine (m6A) modifications. METHODS: Using clinical cohorts and NEAT1-/- mice, we determined the exercise-mediated expression and role of NEAT1 in atherosclerosis. To investigate the mechanism of epigenetic modification of NEAT1 regulated by exercise, we identified METTL14 (methyltransferase-like 14)-a key m6A modification enzyme under exercise-and found that METTL14 alters the expression and role of NEAT1 through m6A modification and elucidated the specific mechanism of METTL14 in vitro and in vivo. Finally, the NEAT1 downstream regulatory network was investigated. RESULTS: We found that NEAT1 expression was downregulated with exercise and that downregulation of NEAT1 was an important factor in the improvement of atherosclerosis with exercise. Exercise-mediated loss of function of NEAT1 can delay atherosclerosis. Mechanistically, we showed that exercise induced a significant downregulation of m6A modification and METTL14, which binds to the m6A sites of NEAT1 and promotes NEAT1 expression through subsequent YTHDC1 (YTH domain-containing 1) recognition to promote endothelial pyroptosis. Furthermore, NEAT1 induces endothelial pyroptosis by binding KLF4 (Kruppel-like factor 4) to promote the transcriptional activation of the key pyroptotic protein NLRP3 (NOD-like receptor thermal protein domain-associated protein 3), whereas exercise can attenuate NEAT1-mediated endothelial pyroptosis to improve atherosclerosis. CONCLUSIONS: Our study of NEAT1 provides new insights into the improvement of atherosclerosis by exercise. This finding demonstrates the role of exercise-mediated NEAT1 downregulation in atherosclerosis while expanding our understanding of the mechanisms by which exercise regulates long noncoding RNA function through epigenetic modifications.

Histone Lactylation Boosts Reparative Gene Activation Post-Myocardial Infarction.

BACKGROUND: Inflammation resolution and cardiac repair initiation after myocardial infarction (MI) require timely activation of reparative signals. Histone lactylation confers macrophage homeostatic gene expression signatures via transcriptional regulation. However, the role of histone lactylation in the repair response post-MI remains unclear. We aimed to investigate whether histone lactylation induces reparative gene expression in monocytes early and remotely post-MI. METHODS: Single-cell transcriptome data indicated that reparative genes were activated early and remotely in bone marrow and circulating monocytes before cardiac recruitment. Western blotting and immunofluorescence staining revealed increases in histone lactylation levels, including the previously identified histone H3K18 lactylation in monocyte-macrophages early post-MI. Through joint CUT&Tag and RNA-sequencing analyses, we identified Lrg1, Vegf-a, and IL-10 as histone H3K18 lactylation target genes. The increased modification and expression levels of these target genes post-MI were verified by chromatin immunoprecipitation-qPCR and reverse transcription-qPCR. RESULTS: We demonstrated that histone lactylation regulates the anti-inflammatory and pro-angiogenic dual activities of monocyte-macrophages by facilitating reparative gene transcription and confirmed that histone lactylation favors a reparative environment and improves cardiac function post-MI. Furthermore, we explored the potential positive role of monocyte histone lactylation in reperfused MI. Mechanistically, we provided new evidence that monocytes undergo metabolic reprogramming in the early stage of MI and demonstrated that dysregulated glycolysis and MCT1 (monocarboxylate transporter 1)-mediated lactate transport promote histone lactylation. Finally, we revealed the catalytic effect of IL (interleukin)-1ß-dependent GCN5 (general control non-depressible 5) recruitment on histone H3K18 lactylation and elucidated its potential role as an upstream regulatory element in the regulation of monocyte histone lactylation and downstream reparative gene expression post-MI. CONCLUSIONS: Histone lactylation promotes early remote activation of the reparative transcriptional response in monocytes, which is essential for the establishment of immune homeostasis and timely activation of the cardiac repair process post-MI.

Characterization of a Nattokinase from the Newly Isolated Bile Salt-Resistant Bacillus mojavensis LY-06.

Nattokinase is a potential new thrombolytic drug because of its strong thrombolytic effect, high safety, and low cost. However, there is no research reporting on bile salt-tolerant nattokinase-producing probiotics. In this study, the bile salt-tolerant nattokinase-producing strain Bacillus mojavensis LY-06 was isolated from local Xinjiang douchi, and the fermentation yield of nattokinase of 1434.64 U/mL was obtained by both a single factor experiment and an orthogonal experiment. A gene responsible for fibrinolysis (aprY) was cloned from the genome of strain Bacillus mojavensis LY-06, and the soluble expression of this gene in Escherichia coli (rAprY, fused with His-tag at C-terminus) was achieved; molecular docking elucidates the cause of insoluble expression of rAprY. The optimal pH and temperature for the fibrinolysis activity of nattokinase AprY fermented by Bacillus mojavensis LY-06 were determined to be pH 6.0 and 50 °C, respectively. However, the optimal pH of rAprY expressed in Escherichia coli was 8, and its acid stability, thermal stability, and fibrinolytic activity were lower than those of AprY. Bioinformatics analysis found that the His-tag carried at the C-terminus of rAprY could affect its acidic stability by changing the isoelectric point and surface charge of the enzyme; in contrast to AprY, changes in the number of internal hydrogen bonds and the flexibility of the loop region in the structure of rAprY resulted in lower fibrinolytic activity and poorer thermal stability.

Efficient Etherification of 2,5-Bis(hydroxymethyl)furan to 2,5-Bis(propoxymethyl)furan by an Amorphous Silica-Alumina Catalyst in a Fixed-Bed Reactor.

The efficient etherification of 2,5-bis(hydroxymethyl)furan (BHMF) to 2,5-bis(propoxymethyl)furan (BPMF) was achieved by using low-cost amorphous silica-aluminas (ASA) catalysts in a fixed-bed reactor. A considerable yield of BPMF up to 85.1 % was obtained over ASA-30 catalyst under the reaction conditions of 140 °C, 2.0 MPa of N2 , and 0.015 h-1 of WHSV. The excellent performance of ASA-30 catalyst could be attributed to the relatively stronger acidity (>375 °C) and larger mesoporous size (6 nm), thereby facilitating the conversion of BHMF to BPMF. In addition, the lower ratio of Brønsted/Lewis acid sites for ASA catalyst was found to efficiently suppress the occurrence of side reactions.

Quality indicators for cardiac rehabilitation after myocardial infarction in China: a consensus panel and practice test.

OBJECTIVES: Cardiac rehabilitation (CR) improves outcomes after myocardial infarction (MI), but it is underused in China. The purpose of this study was to develop a set of quality indicators (QIs) to improve clinical practices and to confirm the measurability and performance of the developed QIs for CR in Chinese patients after MI. DESIGN AND SETTING: The QIs were developed by a Chinese expert consensus panel during in-person meetings. The five QIs most in need of improvement were selected using a national questionnaire. Finally, the completion rate and feasibility of the QIs were verified in a group of MI survivors at university hospitals in China. PARTICIPANTS: Seventeen professionals participated in the consensus panel, 89 personnel in the field of CR participated in the national questionnaire and 165 MI survivors participated in the practice test. RESULTS: A review of 17 eligible articles generated 26 potential QIs, among which 17 were selected by the consensus panel after careful evaluation. The 17 QIs were divided into two domains: (1) improving participation and adherence and (2) CR process standardisation. Nationwide telephone and WeChat surveys identified the five QIs most in need of improvement. A multicenter practice test (n=165) revealed that the mean performance value of the proposed QIs was 43.9% (9.9%-86.1%) according to patients with post-MI. CONCLUSIONS: The consensus panel identified a comprehensive set of QIs for CR in patients with post-MI. A nationwide questionnaire survey was used to identify the QIs that need immediate attention to improve the quality of CR. Although practice tests confirmed the measurability of the proposed QIs in clinical practice, the implementation of the QIs needs to be improved. TRIAL REGISTRATION NUMBER: This study is part of a study registered in ClinicalTrials.gov (NCT03528382).

MicroRNA-223-3p modulates dendritic cell function and ameliorates experimental autoimmune myocarditis by targeting the NLRP3 inflammasome.

Autoimmune myocarditis is a cause of dilated cardiomyopathy and heart failure. MicroRNAs regulate many immune processes, but their role in aberrant inflammation during autoimmune myocarditis remains unclear. In this study, we investigated the role of miR-223-3p in experimental autoimmune myocarditis (EAM). We found that miR-223-3p expression was significantly lower in EAM mice than that in normal mice. miR-223-3p inhibited NLRP3 inflammasome expression, promoting the polarization of dendritic cells (DCs) towards a tolerogenic DC phenotype. miR-223-3p effectively induced regulatory T cell (Treg) generation by inhibiting the function of antigen-presenting DCs. Transfer of miR-223-3p-overexpressing DCs protected mice against the development of EAM. Our findings suggest that miR-223-3p is involved in the induction of the tolerogenic DC phenotype and regulates tolerance in autoimmune myocarditis.

Knockdown of NEAT1 induces tolerogenic phenotype in dendritic cells by inhibiting activation of NLRP3 inflammasome.

Rationale: Tolerogenic dendritic cells (tol-DCs) play essential roles in immune-related diseases and induce immune tolerance by shaping T-cell responses. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) play important regulatory roles in the immune system. However, the potential roles and underlying mechanisms of lncRNAs in tol-DCs remain unclear. Methods: RNA in-situ hybridization, histochemistry, and qRT-PCR were performed to determine the distribution and expression of NEAT1 in DCs. Flow cytometry was used to analyze the tolerogenic function of DCs. Small sequencing, followed by bioinformatic analysis, was performed to determine the target genes of NEAT1. The mechanism of NEAT1 was explored using a luciferase reporter, chromatin immunoprecipitation assays, and Immunofluorescence. In-vivo experiments were used to investigate the induction of immune tolerance via NEAT1-knockdown DCs. Results: Our results show that lncRNA NEAT1 can induce tolerogenic phenotype in DCs. Mechanistically, small RNA-seq analysis revealed that NEAT1 knockdown preferentially affected the expression of miR-3076-3p. Furthermore, NEAT1 used the NLRP3 inflammasome as a molecular decoy for miR-3076-3p, thus facilitating the expression of tolerogenic phenotype in DCs. Moreover, the transcription factor E2F1 acted as a repressor of NEAT1 transcription via activity of H3K27ac. Our results also indicate that NEAT1 knockdown in DCs can induce immune tolerance in models of experimental autoimmune myocarditis and heart transplantation. Conclusions: Taken together, our study shows the mechanism used by NEAT1 in inducing tol-DCs and highlights the therapeutic potential of targeting NEAT1 for the treatment of immune-related diseases.

Effect of exercise-based cardiac rehabilitation on anxiety and depression in patients with myocardial infarction: A systematic review and meta-analysis.

BACKGROUND: Cardiac rehabilitation (CR) has been shown to provide the best social, psychological and physical conditions for patient recovery after myocardial infarction (MI). OBJECTIVES: The aim of present study was to quantify the efficacy of exercise-based CR treatments in terms of relief from symptoms of anxiety and depression symptoms among patients with MI. METHODS: Literature published up to August 2017 was reviewed systematically using relevant keywords, MeSH terms, and Emtree headings to search PubMed, Embase, CINAHL (Ebsco), Cochrane Central Register of Controlled Trials (CENTRAL) and Web of Science. The results of included studies were compared meta-analytically. RESULTS: We found that exercise-based CR had a significant effect on decreasing anxiety and depression scores. Furthermore, exercise-based CR may alleviate anxiety and depressive symptoms at different time periods. CONCLUSIONS: For patients with MI, exercise-based CR has been demonstrated to alleviate anxiety and depressive symptoms. These findings highlight CR as essential and beneficial for minimizing MI patient anxiety and depression during recovery.