Plasmid-Stabilizing Strains for Antibiotic-Free Chemical Fermentation.

Plasmid-mediated antibiotic-free fermentation holds significant industrial potential. However, the requirements for host elements and energy during plasmid inheritance often cause cell burden, leading to plasmid loss and reduced production. The stable maintenance of plasmids is primarily achieved through a complex mechanism, making it challenging to rationally design plasmid-stabilizing strains and characterize the associated genetic factors. In this study, we introduced a fluorescence-based high-throughput method and successfully screened plasmid-stabilizing strains from the genomic fragment-deletion strains of Escherichia coli MG1655 and Bacillus subtilis 168. The application of EcΔ50 in antibiotic-free fermentation increased the alanine titer 2.9 times. The enhanced plasmid stability in EcΔ50 was attributed to the coordinated deletion of genes involved in plasmid segregation and replication control, leading to improved plasmid maintenance and increased plasmid copy number. The increased plasmid stability of BsΔ44 was due to the deletion of the phage SPP1 surface receptor gene yueB, resulting in minimized sporulation, improved plasmid segregational stability and host adaptation. Antibiotic-free fermentation results showed that strain BsΔyueB exhibited a 61.99% higher acetoin titer compared to strain Bs168, reaching 3.96 g/L. When used for the fermentation of the downstream product, 2,3-butanediol, strain BsΔyueB achieved an 80.63% higher titer than Bs168, reaching 14.94 g/L using rich carbon and nitrogen feedstocks. Overall, our work provided a plasmid-stabilizing chassis for E. coli and B. subtilis, highlighting their potential for antibiotic-free fermentation of valuable products and metabolic engineering applications.

PIEZO1 targeting in macrophages boosts phagocytic activity and foam cell apoptosis in atherosclerosis.

The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.

[Microbial production of food compounds with carbon dioxide and derived low-carbon molecules as substrates].

The construction and optimization of microbial cell factories are crucial steps and key technologies in achieving green biomanufacturing. As concern has been aroused regarding the excessive carbon dioxide (CO2) emissions and food security, a new and promising research field, microbial conversion of CO2 into food compounds, has emerged. The research in this field not only holds significant implications for achieving the carbon peaking and carbon neutrality goals but also plays a role in maintaining food security. This paper provides a comprehensive review and outlook of the research on utilizing CO2 and its derived low-carbon chemicals for the production of food compounds, focusing on the production of glucose, sugar derivatives, and single-cell proteins and the development of artificial CO2 fixation pathways.

Identification of functional sgRNA mutants lacking canonical secondary structure using high-throughput FACS screening.

Coexpressing multiple identical single guide RNAs (sgRNAs) in CRISPR-dependent engineering triggers genetic instability and phenotype loss. To provide sgRNA derivatives for efficient DNA digestion, we design a high-throughput digestion-activity-dependent positive screening strategy and astonishingly obtain functional nonrepetitive sgRNA mutants with up to 48 out of the 61 nucleotides mutated, and these nonrepetitive mutants completely lose canonical secondary sgRNA structure in simulation. Cas9-sgRNA complexes containing these noncanonical sgRNAs maintain wild-type level of digestion activities in vivo, indicating that the Cas9 protein is compatible with or is able to adjust the secondary structure of sgRNAs. Using these noncanonical sgRNAs, we achieve multiplex genetic engineering for gene knockout and base editing in microbial cell factories. Libraries of strains with rewired metabolism are constructed, and overproducers of isobutanol or 1,3-propanediol are identified by biosensor-based fluorescence-activated cell sorting (FACS). This work sheds light on the remarkable flexibility of the secondary structure of functional sgRNA.

Species-specific design of artificial promoters by transfer-learning based generative deep-learning model.

Native prokaryotic promoters share common sequence patterns, but are species dependent. For understudied species with limited data, it is challenging to predict the strength of existing promoters and generate novel promoters. Here, we developed PromoGen, a collection of nucleotide language models to generate species-specific functional promoters, across dozens of species in a data and parameter efficient way. Twenty-seven species-specific models in this collection were finetuned from the pretrained model which was trained on multi-species promoters. When systematically compared with native promoters, the Escherichia coli- and Bacillus subtilis-specific artificial PromoGen-generated promoters (PGPs) were demonstrated to hold all distribution patterns of native promoters. A regression model was developed to score generated either by PromoGen or by another competitive neural network, and the overall score of PGPs is higher. Encouraged by in silico analysis, we further experimentally characterized twenty-two B. subtilis PGPs, results showed that four of tested PGPs reached the strong promoter level while all were active. Furthermore, we developed a user-friendly website to generate species-specific promoters for 27 different species by PromoGen. This work presented an efficient deep-learning strategy for de novo species-specific promoter generation even with limited datasets, providing valuable promoter toolboxes especially for the metabolic engineering of understudied microorganisms.

Arterial Adventitial Vasa Vasorum Hyperplasia involved in Atherosclerotic Plaque Formation in a Rabbit Model.

OBJECTIVE: It was previously believed that atherosclerotic (AS) plaque starts to develop from the intima and that intraplaque vasa vasorum (VV) hyperplasia promotes adventitial VV (AVV) hyperplasia. However, recent studies have shown that arterial AVV hyperplasia precedes early intimal thickening, suggesting its possible role as an initiating factor of AS. To provide further insight into this process, in this study, we examine the evolution of AAV and VV development in a preclinical model of early AS with longitudinal ultrasound imaging. METHODS: Models of early AS were established. Duplex ultrasound scanning and contrast-enhanced ultrasound were performed for diagnosis. Pearson correlation tests were used to analyze the relationships between AVV hyperplasia and VV hyperplasia, or between AVV hyperplasia and intima-media thickness (IMT). RESULTS: During 0-12 wk of high-fat feeding, AVV gradually increased and intima-media thickened gradually in the observation area; in the 2nd wk of high-fat feeding, the observation area showed obvious AVV proliferation; at the 4th wk, the intima-media membrane became thicker; at the 12th wk, early plaque formation and intraplaque VV proliferation were observed. There was a strong positive correlation between AVV proliferation and IMT thickening and a strong negative correlation between AVV proliferation and the change rate of vessel diameter. CONCLUSION: This study demonstrated that AVV proliferation in the arteries occurred earlier than IMT thickening and was positively correlated with IMT. At present, the indicators of ultrasonic diagnosis of AS, such as IMT, Intraplaque VV, Echo property, all appear in the advanced stage of AS. The AVV may be an innovative diagnostic target for the early stage of AS plaque.

Chondroitin sulphate modified MoS2 nanoenzyme with multifunctional activities for treatment of Alzheimer's disease.

Nano-MoS2 exhibit oxidoreductase-like activities, and has been shown to effectively eliminate excessive intracellular ROS and inhibit Aß aggregation, thus demonstrating promising potential for anti-Alzheimer's disease (anti-AD) intervention. However, the low water dispersibility and high toxicity of nano-MoS2 limits its further application. In this study, we developed a chondroitin sulphate (CS)-modified MoS2 nanoenzyme (CS@MoS2) by harnessing the excellent biocompatibility of CS and the exceptional activities of nano-MoS2 to explore its potential in anti-AD research. Promisingly, CS@MoS2 significantly inhibited Aß1-40 aggregation and prevented toxic injury in SH-SY5Y cells caused by Aß1-40. In addition, CS@MoS2 protected these cells from oxidative stress damage by regulating ROS production, as well as promoting the activities of SOD and GSH-Px. CS@MoS2 also modulated the intracellular Ca2+ imbalance and downregulated Tau hyperphosphorylation by activating GSK-3ß. CS@MoS2 suppressed p-NF-κB (p65) translocation to the nucleus by inhibiting MAPK phosphorylation, and modulated the expression of downstream anti- and proinflammatory cytokines. Owing to its multifunctional activities, CS@MoS2 effectively improved spatial learning, memory, and anxiety in D-gal/AlCl3-induced AD mice. Taken together, these results indicate that CS@MoS2 has significant potential for improving the therapeutic efficacy of the prevention and treatment of AD, while also presenting a novel framework for the application of nanoenzymes.

Arterial Adventitial Vasa Vasorum Density Reflects The Progression Of Unstable Plaques: A Retrospective Clinical Study.

OBJECTIVE: Arterial adventitial vasa vasorum (AVV) plays an important role in the occurrence and development of atherosclerotic (AS) disease. AS is a systemic disease, and plaque is not only a local vascular event, but also occurs at multiple sites throughout the vascular bed. Currently, effective anti-AVV therapies are lacking. Therefore, we posed the following scientific questions: "does human carotid adventitial vasa vasorum density reflect plaque neovascularization and intimal-media hyperplasia in carotid?"; and "is it possible to reduce human AVV density by sonodynamic therapy (SDT)?" METHODS: A retrospective study was conducted on 160 patients with carotid atherosclerosis. Duplex ultrasound scanning (DUS), contrast-enhanced ultrasound (CEUS), coronary angiography, and coronary CT angiography (CTA) were used for diagnosis and screening. Pearson correlation tests and Receiver operating characteristic (ROC) curve were used to analyze the relationships between AVV hyperplasia, vasa vasorum (VV) hyperplasia and the intima-media thickness (IMT). SDT was developed for the treatment of arterial AVV hyperplasia and AS plaques. RESULTS: The presence of local AVV in carotid unstable plaques correlated with the echogenic properties of the carotid plaque and the extent of plaque progression; Furthermore local AVV hyperplasia in patients with carotid atherosclerotic plaques was associated with acute coronary syndrome (ACS) events; Local AVV hyperplasia in patients with carotid atherosclerotic plaques was associated with coronary artery stenosis. Notably, SDT reduced local AVV hyperplasia and shrank the plaques in human femoral and carotid atherosclerotic lesions. CONCLUSIONS: The presence of AVV in human carotid arteries reflects the severity of carotid and coronary artery AS. Further, SDT can reduce the hyperplasia of local AVV in human femoral and carotid plaques.