Hyper-production of porcine contagious pleuropneumonia subunit vaccine proteins in Escherichia coli by developing a bicistronic T7 expression system.

The ApxII toxin and the outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae are important vaccine antigens against porcine contagious pleuropneumonia (PCP), a prevalent infectious disease affecting the swine industry worldwide. Previous studies have reported the recombinant expression of ApxII and Oml in Escherichia coli; however, their yields were not satisfactory. Here, we aimed to enhance the production of ApxII and Oml by constructing a bicistronic expression system based on the widely used T7 promoter. To create efficient T7 bicistronic expression cassettes, 16 different fore-cistron sequences were introduced downstream of the T7 promoter. The expression of three vaccine antigens Oml1, Oml7, and ApxII in the four strongest bicistronic vectors were enhanced compared to the monocistronic control. Further optimization of the fermentation conditions in micro-well plates (MWP) led to improved production. Finally, the production yields reached unprecedented levels of 2.43 g L-1 of Oml1, 2.59 g L-1 of Oml7, and 1.21 g L-1 of ApxII, in a 5 L bioreactor. These three antigens also demonstrated well-protective immunity against A. pleuropneumoniae infection. In conclusion, this study establishes an efficient bicistronic T7 expression system that can be used to express recombinant proteins in E. coli and achieves the hyper-production of PCP vaccine proteins.

High-throughput process development from gene cloning to protein production.

In the post-genomic era, the demand for faster and more efficient protein production has increased, both in public laboratories and industry. In addition, with the expansion of protein sequences in databases, the range of possible enzymes of interest for a given application is also increasing. Faced with peer competition, budgetary, and time constraints, companies and laboratories must find ways to develop a robust manufacturing process for recombinant protein production. In this review, we explore high-throughput technologies for recombinant protein expression and present a holistic high-throughput process development strategy that spans from genes to proteins. We discuss the challenges that come with this task, the limitations of previous studies, and future research directions.

Flavonoids from Seabuckthorn (Hippophae rhamnoides L.) restore CUMS-induced depressive disorder and regulate the gut microbiota in mice.

Seabuckthorn (Hippophae rhamnoides L.), which is enriched with flavonoids, including isorhamnetin, quercetin and kaempferol, is a representative example of "medicine food homology" targeting several diseases. Major depressive disorders seriously threaten mental health worldwide and may even lead to death. Chronic unpredictable mild stress (CUMS)-induced depressive-like symptoms in mice are usually considered as the highest similarity to the situation in humans. Herein, we determined the potential functions of the flavonoid-enriched fraction from Seabuckthorn, which was named SBF, in treating major depressive disorder in mice. In the CUMS-induced mouse model, the intake of SBF reversed their depressive behaviors and relieved the CUMS-disturbed levels of neurotrophins, neurotransmitters, stress-related hormones, and inflammation-related cytokines. Additionally, the treatment of depressive mice with SBF showed ability to regulate the gut microbiota, especially in decreasing the abundance of Lactobacillaceae, while increasing the abundance of Lachnospiraceae at the family level. The results suggest the beneficial effects of Seabuckthorn flavonoids in functioning as a health food supplement to treat major depressive disorders.

Leaderless Bicistronic Design for Precise and Reliable Control of Gene Expression in Corynebacterium Glutamicum.

In synthetic biology, the precise control of gene expression is challenging due to the limited orthogonality of expression elements. Here, to address this issue and improve the reusability of genetic elements, we developed a bicistronic expression cassette in Corynebacterium glutamicum based on a leaderless promoter lacking a 5'UTR. The created leaderless bicistronic design (BCD) significantly improved the orthogonality of expression elements across different genes of interest. We also explored the importance of the fore-cistron and SD motif in maintaining the strength of leaderless BCDs. Additionally, we established a library containing 55,901 fore-cistrons and demonstrated that the regulatory range of gene expression in leaderless BCDs can be broader by modifying the fore-cistron sequence. This study provides a novel synthetic biology tool based on leaderless BCD for fine-tuning gene expression in C. glutamicum using fore-cistrons. Moreover, the strategy developed here can also be applied to improve the performance of other leaderless promoters in other bacteria.

The neurotrophic activities of brain-derived neurotrophic factor are potentiated by binding with apigenin, a common flavone in vegetables, in stimulating the receptor signaling.

AIMS: We aimed to identify the neurotrophic activities of apigenin (4',5,7-trihydroxyflavone) via its coordination with brain-derived neurotrophic factor (BNDF) and an elevated signaling of tyrosine kinase receptor B (Trk B receptor). METHODS: The direct binding of apigenin to BDNF was validated by ultrafiltration and biacore assay. Neurogenesis, triggered by apigenin and/or BDNF, was determined in cultured SH-SY5Y cells and rat cortical neurons. The amyloid-beta (Aß)25-35 -induced cellular stress was revealed by propidium iodide staining, mitochondrial membrane potential, bioenergetic analysis, and formation of reactive oxygen species levels. Activation of Trk B signaling was tested by western blotting. RESULTS: Apigenin and BDNF synergistically maintained the cell viability and promoted neurite outgrowth of cultured neurons. In addition, the BDNF-induced neurogenesis of cultured neurons was markedly potentiated by applied apigenin, including the induced expressions of neurofilaments, PSD-95 and synaptotagmin. Moreover, the synergy of apigenin and BDNF alleviated the (Aß)25-35 -induced cytotoxicity and mitochondrial dysfunction. The synergy could be accounted by phosphorylation of Trk B receptor, and which was fully blocked by a Trk inhibitor K252a. CONCLUSION: Apigenin potentiates the neurotrophic activities of BDNF through direct binding, which may serve as a possible treatment for its curative efficiency in neurodegenerative diseases and depression.

Flavonoids from Seabuckthorn (Hippophae rhamnoides L.) mimic neurotrophic functions in inducing neurite outgrowth in cultured neurons: Signaling via PI3K/Akt and ERK pathways.

BACKGROUND: Various brain disorders, including neurodegenerative diseases and major depressive disorders, threaten an increasing number of patients. Seabuckthorn, a fruit from Hippophae rhamnoides L., is an example of "medicine food homology". The fruit has enriched flavonoids that reported to have benefits in treating cognitive disorders. However, the studies on potential functions of Seabuckthorn and/or its flavonoid-enriched fraction in treating neurodegenerative disorders are limited. PURPOSE: This study aimed to determine the ability and mechanism of the flavonoid-enriched fraction of Seabuckthorn (named as SBF) in mimicking the neurotrophic functions in inducing neurite outgrowth of cultured neurons. METHODS: Cultured PC12 cell line, SH-SY5Y cell line and primary neurons (cortical and hippocampal neurons isolated from E17-19 SD rat embryos) were the employed models to evaluate SBF in inducing neurite outgrowth by comparing to the effects of NGF and BDNF. Immuno-fluorescence staining was applied to identify the morphological change during the neuronal differentiation. Luciferase assay was utilized for analyzing the transcriptional regulation of neurofilaments and cAMP/CREB-mediated gene. Western blot assay was conducted to demonstrate the expressions of neurofilaments and phosphorylated proteins. RESULTS: The application of SBF induced neuronal cell differentiation, and this differentiating activation was blocked by the inhibitors of PI3K/Akt and ERK pathways. Additionally, SBF showed synergy with neurotrophic factors in stimulating the neurite outgrowth of cultured neurons. Moreover, the major flavonoids within SBF, i.e., isorhamnetin, quercetin and kaempferol, could account for the neurotrophic activities of SBF. CONCLUSION: Seabuckthorn flavonoids mimicked neurotrophic functions in inducing neuronal cell differentiation via activating PI3K/Akt and ERK pathways. The results suggest the beneficial functions of Seabuckthorn as a potential health food supplement in treating various brain disorders, e.g., neurodegenerative diseases.

The ethanolic extract of peanut shell attenuates the depressive-like behaviors of mice through modulation of inflammation and gut microbiota.

Peanut shell is an agricultural byproduct being wasted on a large scale, which is in urgent need to be recycled. To fully utilize its pharmacological ingredients, e.g. luteolin, eriodyctiol, and 5,7-dihydroxychromone, we evaluated the curative effect of ethanol extract deriving from peanut shell (PSE) in treating chronic unpredictable mild stress (CUMS)-induced depressive mice. The chronic stress lasted for 10 weeks, and PSE at 100-900 mg/kg/day was gavaged to mice in the last 2 weeks of modeling. The depressive behaviors were assessed by analyses of sucrose preference, tail suspension, and forced swimming. The brain injury was demonstrated by Hematoxylin and Eosin (H&E), Nissl body, and TdT-mediated dUTP nick end labeling (TUNEL) stainings in the mouse hippocampus. Biochemical indicators were analyzed, including levels of neurotrophic factors, neurotransmitters, stress hormones, and inflammatory mediators. The feces were collected for the 16S rDNA sequencing of gut microbiome. Administration of PSE improved the sucrose water consumption of depressive mice, while it decreased the immobile time in tail suspension and forced swimming tests. Meanwhile, the anti-depressive effect of PSE was supported by ameliorated histochemical staining, increased levels of neurotrophic factors and neurotransmitters, as well as down-regulated stress hormones. Furthermore, the treatment of PSE was able to mitigate the levels of inflammatory cytokines in brain, serum, and small intestine. Besides, the tight junction proteins, e.g., occludin and ZO-1, of gut showed elevated expressions, which coincided with the elevated abundance and diversity of gut microbiota upon PSE treatment. This study validated the therapeutic efficacy of PSE in fighting against depression, as well as its modulatory action on inflammation and gut microbiota, which promoted the recycling of this agricultural waste to be health supplements of added value.

A Herbal Mixture of Sesami Semen Nigrum and Longan Arillus Induces Neurite Outgrowth in Cultured Neurons and Shows Anti-Depression in Chronic Mild Stress-Induced Rats.

Medicinal food homology is referring to a group of food itself being considered as herbal medicine without a boundary of usage. Under the guidance of this food/medicine principle, the current study aims to develop anti-depressant from this food/medicine catalog. The herbal mixture of Sesami Semen Nigrum and Longan Arillus was evaluated in cultured PC12 rat pheochromocytoma cells, rat primary cortical neurons, and in chronic mild stress (CMS)-induced depressive rat model. The combination of two ethanolic extracts of Sesami Semen Nigrum and Longan Arillus in 1 : 1 ratio mimicked the function of nerve growth factor (NGF) and synergistically induced neurite outgrowth of PC12 cells. Besides, the expression and phosphorylation of tropomyosin receptor kinase A (TrkA) of the cultured cells were also elevated. This neurotrophic activity of herbal mixture was further supported by the increased expressions of biomarkers for neurogenesis and synaptogenesis in cortical neurons. Moreover, the depressed rats were soothed by the intake of herbal mixture, showing improved performance in behavior tests, as well as reversed levels of neurotransmitters and neurotrophic factors. Our results provide a new way to make full use of the current food/medicine resources, as to accelerate the development of therapeutics for depression.

Hypersecretion of OmlA antigen in Corynebacterium glutamicum through high-throughput based development process.

Outer membrane lipoprotein A (OmlA) is a vaccine antigen against porcine contagious pleuropneumonia (PCP), a disease severely affecting the swine industry. Here, we aimed to systematically potentiate the secretory production of OmlA in Corynebacterium glutamicum (C. glutamicum), a widely used microorganism in the food industry, by establishing a holistic development process based on our high-throughput culture platform. The expression patterns, expression element combinations, medium composition, and induction conditions were comprehensively screened or optimized in microwell plates (MWPs), followed by fermentation parameter optimization in a 4 × 1 L parallel fermentation system (CUBER4). An unprecedented yield of 1.01 g/L OmlA was ultimately achieved in a 5-L bioreactor following the scaling-up strategy of fixed oxygen mass transfer coefficient (kLa), and the produced OmlA antigen showed well-protective immunity against Actinobacillus pleuropneumoniae challenge. This result provides a rapid and reliable pipeline to achieve the hyper-production of OmlA, and possibly other recombinant vaccines, in C. glutamicum. KEY POINTS: • Established a holistic development process and applied it to potentiate the secretion of OmlA. • The secretion of OmlA reached an unprecedented yield of 1.01 g/L. • The recombinant OmlA antigen induced efficient protective immunity.

Bicistronic design as recombinant expression enhancer: characteristics, applications, and structural optimization.

The bicistronic design (BCD) is characterized by a short fore-cistron sequence and a second Shine-Dalgarno (SD2) sequence upstream of the target gene. The outstanding performance of this expression cassette in promoting recombinant protein production has attracted attention. Recently, the application of the BCD has been further extended to gene expression control, protein translation monitoring, and membrane protein production. In this review, we summarize the characteristics, molecular mechanisms, applications, and structural optimization of the BCD expression cassette. We also specifically discuss the challenges that the BCD system still faces. This is the first review of the BCD expression strategy, and it is believed that an in-depth understanding of the BCD will help researchers to better utilize and develop it. KEY POINTS: • Summary of the characteristics and molecular mechanisms of the BCD system. • Review of the actual applications of the BCD expression cassette. • Summary of the structural optimization of the BCD system.