Diabetes-induced cardiomyopathy is ameliorated by heat-killed Lactobacillus reuteri GMNL-263 in diabetic rats via the repression of the toll-like receptor 4 pathway.

PURPOSE: Diabetes mellitus (DM) leads to disorders such as cardiac hypertrophy, cardiac myocyte apoptosis, and cardiac fibrosis. Previous studies have shown that Lactobacillus reuteri GMNL-263 decreases cardiomyopathy by reducing inflammation. In this study, we investigated the potential benefit of GMNL-263 supplementation in treating diabetes-induced cardiomyocytes in rats with DM. METHODS: Five-week-old male Wistar rats were randomly divided into three groups, control, DM, and rats with DM treated with different dosages of L. reuteri GMNL-263. After undergoing treatment for 4 weeks, all rats were euthanized for further analysis. RESULTS: We observed that cardiac function and structure of rats with DM was rescued by GMNL-263. Activation of toll-like receptor 4 (TLR4)-related inflammatory, hypertrophic, and fibrotic signaling pathways in the hearts of rats with DM was reduced by treatment with GMNL-263. CONCLUSION: Our findings demonstrate that GMNL-263 inhibited diabetes-induced cardiomyocytes via the repression of the TLR4 pathway. Moreover, these findings suggest that treatment with high-dose GMNL-263 could be a precautionary therapy for reducing the diabetes-induced cardiomyopathy.

Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.

Direct in situ butanol recovery inside the packed bed during continuous acetone-butanol-ethanol (ABE) fermentation.

In this study, the integrated in situ extraction-gas stripping process was coupled with continuous ABE fermentation using immobilized Clostridium acetobutylicum. At the same time, oleyl alcohol was cocurrently flowed into the packed bed reactor with the fresh medium and then recycled back to the packed bed reactor after removing butanol in the stripper. A high glucose consumption of 52 g/L and a high butanol productivity of 11 g/L/h were achieved, resulting in a high butanol yield of 0.21 g-butanol/g-glucose. This can be attributed to both the high bacterial activity for solvent production as well as a threefold increase in the bacterial density inside the packed bed reactor. Also reported is that 64 % of the butanol produced can be recovered by the integrated in situ extraction-gas stripping process. A high butanol productivity and a high glucose consumption were simultaneously achieved.

Potential production platform of n-butanol in Escherichia coli.

We proposed a potential production platform of n-butanol in Escherichia coli. First, a butyrate-conversion strain was developed by removal of undesired genes and recruiting endogenous atoDA and Clostridium adhE2. Consequently, this E. coli strain grown on the M9 mineral salt with yeast extract (M9Y) was shown to produce 6.2g/L n-butanol from supplemented butyrate at 36h. The molar conversion yield of n-butanol on butyrate reaches 92%. Moreover, the production platform was advanced by additional inclusion of a butyrate-producing strain. This strain was equipped with a pathway comprising atoDA and heterologous genes for the synthesis of butyrate. Without butyrate, the butyrate-conversion and the butyrate-producing strains were co-cultured in M9Y medium and produced 5.5g/L n-butanol from glucose at 24h. The production yield on glucose accounts for 69% of the theoretical yield. Overall, it indicates a promise of the developed platform for n-butanol production in E. coli.

The effect of serine protease inhibitors on airway inflammation in a chronic allergen-induced asthma mouse model.

Serine protease inhibitors reportedly attenuated airway inflammation and had antioxidant in multiorgan. However, the effects of the serine protease inhibitors nafamostat mesilate (FUT), gabexate mesilate (FOY), and ulinastatin (UTI) on a long-term challenged mouse model of chronic asthma are unclear. BALB/c mice (6 mice/group) were intratracheally inoculated with five doses of Dermatophagoides pteronyssinus (Der p; 50 µL, 1 mg/mL) at one-week intervals. Therapeutic doses of FUT (0.0625 mg/kg), FOY (20 mg/kg), or UTI (10,000 U/kg) were, respectively, injected intraperitoneally into these mice. Control mice received sterile PBS. At 3 days after the last challenge, mice were sacrificed to assess airway hyperresponsiveness (AHR), remodeling, and inflammation; lung histological features; and cytokine expression profiles. Compared with untreated controls, mice treated with FUT, FOY, and UTI had decreased AHR and goblet cell hyperplasia, decreased eosinophil and neutrophil infiltration, decreased Der p-induced IL-4 levels in serum and IL-5, IL-6, IL-13, and IL-17 levels in bronchoalveolar lavage fluid, and inhibited nuclear factor (NF)-κB activity in lung tissues. The serine protease inhibitors FUT, FOY, and UTI have potential therapeutic benefits for treating asthma by downregulating Th2 cytokines and Th17 cell function and inhibiting NF-κB activation in lung tissue.

A useful method integrating production and immobilization of recombinant cellulase.

The development of cellulase-based bioprocess is afflicted by the processing efficiency of enzymes. To address this issue, a method based on artificial oil bodies (AOBs) was proposed to integrate production and immobilization of recombinant cellulase. First, the heterologous endoglucanase (celA), cellobiohydrolase (celK), and ß-glucosidase (gls) genes were individually fused with oleosin, a structural protein of plant seed oils. After expression in Escherichia coli, each fusion protein of insolubility was mixed together with plant oils. AOBs were assembled by subjecting the mixture to sonication. Consequently, active CelA, CelK, and Gls were resumed and co-immobilized on AOBs surface. Finally, the assembly condition (including the protein ratio) and the reaction condition were further optimized by response surface methodology. The resulting AOBs-bound cellulase remained stable for 4 cycles of cellulose-hydrolyzed reactions. Overall, the result shows a promise of this proposed approach for processing recombinant cellulase, which may provide a facile method to investigate optimum combination of cellulase components towards various cellulosic materials.

High production of poly(3-hydroxybutyrate) in Escherichia coli using crude glycerol.

Poly(3-hydroxybutyrate) (PHB) is a prominent bio-plastic and recognized as the potential replacement of petroleum-derived plastics. To make PHB cost-effective, the production scheme based on crude glycerol was developed using Escherichia coli. The heterogeneous synthesis pathway of PHB was introduced into the E. coli strain capable of efficiently utilizing glycerol. The central metabolism that links to the synthesis of acetyl-CoA and NADPH was further reprogrammed to improve the PHB production. Key genes were targeted for manipulation, involving those in glycolysis, the pentose phosphate pathway, and the tricarboxylic cycle. As a result, the engineered strain gained a 22-fold increase in the PHB titer. Finally, the fed-batch fermentation was conducted with the producer strain to give the PHB titer, content, and productivity reaching 36.3 ± 3.0 g/L, 66.5 ± 2.8%, and 1.2 ± 0.1 g/L/h, respectively. The PHB yield on crude glycerol accounts for 0.3 g/g. The result indicates that the technology platform as developed is promising for the production of bio-plastics.

Caleosin-assembled oil bodies as a potential delivery nanocarrier.

Encapsulation of hydrophobic agents with nanocarriers is challenging. Therefore, we have sought to use nanoscale artificial oil bodies (NOBs) as an alternative delivery carrier. To constitute NOBs, caleosin (Cal), a structural protein of plant seed oil bodies, was first fused with ZH2 (Cal-ZH2). ZH2 is a bivalent anti-HER2/neu affibody with a high affinity towards the HER2/neu receptor. After overproduction in Escherichia coli, insoluble Cal-ZH2 was isolated and used to assemble NOBs in one step. Consequently, resulting NOBs had a zeta potential around -49 mV and ranged in size from 150 to 200 nm. Upon loading with a hydrophobic fluorescence dye, NOBs were found to be selectively internalized into HER2/neu-positive tumor cells. Further analyses showed that more than 90% cells were invaded by dye-loaded NOBs and the cargo dye was released in cells with time. In addition, the in vitro assay revealed the release of the dye from NOBs in a slow and prolong manner. Overall, these results indicate the potential of Cal-based NOBs as a delivery vehicle.

The Immunomodulatory Effect of You-Gui-Wan on Dermatogoides-pteronyssinus-Induced Asthma.

The traditional Chinese medicine You-Gui-Wan (YGW) contains ten species of medicinal plants and has been used to improve health in remissive states of asthma for hundreds of years in Asia. However, little is known about the immunomodulatory mechanisms in vivo. Therefore, this study investigated the pathologic and immunologic responses to YGW in mice that had been repeatedly exposed to Dermatogoides-pteronyssinus (Der p). YGW reduced Der-p-induced airway hyperresponsiveness and total IgE in serum. It also inhibited eosinophil infiltration by downregulating the protein expression of IL-5 in serum and changed the Th2-bios in BALF by upregulating IL-12. Results of the collagen assay and histopathologic examination showed that YGW reduced airway remodeling in the lung. In addition, after YGW treatment there was a relative decrease in mRNA expression of TGF-ß1, IL-13, eotaxin, RANTES, and MCP-1 in lung in the YGW group. The results of EMSA and immunohistochemistry revealed that YGW inhibited NF-κB expression in epithelial lung cells. YGW exerts its regulative effects in chronic allergic asthmatic mice via its anti-inflammatory activity and by inhibiting the progression of airway remodeling.

Glutamate as a non-conventional substrate for high production of the recombinant protein in Escherichia coli.

The economic viability of the biomass-based biorefinery is readily acknowledged by implementation of a cascade process that produces value-added products such as enzymes prior to biofuels. Proteins from the waste stream of biorefinery processes generally contain glutamate (Glu) in abundance. Accordingly, this study was initiated to explore the potential of Glu for production of recombinant proteins in Escherichia coli. The approach was first adopted by expression of D-hydantoinase (HDT) in commercially-available BL21(DE3) strain. Equipped with the mutant gltS (gltS*), the strain grown on Glu produced the maximum HDT as compared to the counterpart on glucose, glycerol, or acetate. The Glu-based production scheme was subsequently reprogrammed based on the L-arabinose-regulated T7 expression system. The strain with gltS* was further engineered by rewiring metabolic pathways. With low ammonium, the resulting strain produced 1.63-fold more HDT. The result indicates that Glu can serve as a carbon and nitrogen source. Overall, our proposed approach may open up a new avenue for the enzyme biorefinery platform based on Glu.

Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu-positive tumor cells.

Targeting of non-phagocytic tumor cells and prompt release of gene cargos upon entry into tumors are two limiting steps in the bacterial gene delivery path. To tackle these problems, the non-pathogenic Escherichia coli strain BL21(DE3) was engineered to display the anti-HER2/neu affibody on the surface. After co-incubation with tumor cells for 3 h, the anti-HER2/neu affibody-presenting E. coli strain was selectively internalized into HER2/neu-positive SKBR-3 cells. The invasion efficiency reached as high as 30%. Furthermore, the bacteria were equipped with the phage ϕX174 lysin gene E-mediated autolysis system. Carrying the transgene (e.g., eukaryotic green fluorescent protein, GFP), the tumor-targeting bacteria were subjected to the thermal shock to trigger the autolysis system upon entry into HER2/neu-positive cells. Flow cytometric analysis revealed that 3% of infected cells expressed GFP 24 h post thermal induction. Overall, the results show a promise of the proposed approach for developing bacteria as a delivery carrier.

Selective internalization of self-assembled artificial oil bodies by HER2/neu-positive cells.

A novel delivery carrier was developed using artificial oil bodies (AOBs). Plant seed oil bodies (OBs) consist of a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with the storage protein oleosin (Ole). Ole consists of a central hydrophobic domain with two amphiphatic arms that extrude from the surface of OBs. In this study, a bivalent anti-HER2/neu affibody domain (ZH2) was fused with Ole at the C terminus. After overproduction in Escherichia coli, the fusion protein (Ole-ZH2) was recovered to assemble AOBs. The size of self-assembled AOBs was tailored by varying the oil/Ole-ZH2 ratio and pH to reach a nanoscale. Upon co-incubation with tumor cells, the nanoscale AOBs encapsulated with a hydrophobic fluorescence dye were selectively internalized by HER2/neu-overexpressing cells and displayed biocompatibility with the cells. In addition, the ZH2-mediated endosomal entry of AOBs occurred in a time- and AOB dose-dependent manner. The internalization efficiency was as high as 90%. The internalized AOBs disintegrated at the non-permissive pH (e.g. in acidic endosomes) and the cargo dye was released. Results of in vitro study revealed a sustained and prolonged release profile. Taken together, our findings indicate the potential of AOBs as a delivery carrier.

Production of Succinic Acid from Amino Acids in Escherichia coli.

Glutamate (Glu) and aspartate (Asp) are the most abundant amino acids in various sources of protein waste, recognized as a sustainable resource. In this study, Escherichia coli was engineered to produce succinic acid (SA) from Glu and Asp. Succinate dehydrogenase involved in the tricarboxylic acid was inactivated in the Glu-utilizing strain. To grow on Asp, this mutant strain was subjected to metabolic evolution. One resulting strain capable of metabolizing Asp was further evolved to improve the growth of Glu and Asp. After the deletion of arcA, the resulting strain was employed for the aerobic production of SA. The shake-flask culture was conducted with the minimal medium containing 10 g/L Glu and 10 g/L Asp. Finally, it resulted in the SA production, with a titer, the molar yield, and productivity reaching 72.8 mM (i.e., 8.6 g/L), 0.54 (ca. 75.4% of the theoretical yield), and 0.66 g/L/h, respectively. Overall, this study opens up a new avenue of the biorefinery platform based on renewable amino acids.

A Strategy to Improve Production of Recombinant Proteins in Escherichia coli Based on a Glucose-Glycerol Mixture and Glutamate.

Enzymes have a wide range of applications in many sectors of the industry, and the market value has skyrocketed in recent years. Glucose and glycerol are two renewable carbon sources of importance. Therefore, it is appealing to produce recombinant enzymes with these carbon substrates on the basis of economic viability. In this study, glycerol metabolism and glucose metabolism in Escherichia coli (E. coli) were manipulated in a systematic way. In addition, glutamate (Glu) was used for replacement of yeast extract to reduce the cost and the quality-variation problem. A strategy was further developed to incorporate Glu into the central metabolism. The engineered E. coli strain finally enabled efficient co-utilization of glucose and glycerol and improved biomass and protein production by 4.3 and 8.2-folds, respectively. The result illustrates that this proposed approach is promising for effective production of recombinant proteins.

Rewiring of glycerol metabolism in Escherichia coli for effective production of recombinant proteins.

BACKGROUND: The economic viability of a protein-production process relies highly on the production titer and the price of raw materials. Crude glycerol coming from the production of biodiesel is a renewable and cost-effective resource. However, glycerol is inefficiently utilized by Escherichia coli. RESULTS: This issue was addressed by rewiring glycerol metabolism for redistribution of the metabolic flux. Key steps in central metabolism involving the glycerol dissimilation pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle were pinpointed and manipulated to provide precursor metabolites and energy. As a result, the engineered E. coli strain displayed a 9- and 30-fold increase in utilization of crude glycerol and production of the target protein, respectively. CONCLUSIONS: The result indicates that the present method of metabolic engineering is useful and straightforward for efficient adjustment of the flux distribution in glycerol metabolism. The practical application of this methodology in biorefinery and the related field would be acknowledged.

Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads.

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.

A simple strategy to effectively produce d-lactate in crude glycerol-utilizing Escherichia coli.

BACKGROUND: Fed-batch fermentation has been conventionally implemented for the production of lactic acid with a high titer and high productivity. However, its operation needs a complicated control which increases the production cost. RESULTS: This issue was addressed by simplifying the production scheme. Escherichia coli was manipulated for its glycerol dissimilation and d-lactate synthesis pathways and then subjected to adaptive evolution under high crude glycerol. Batch fermentation in the two-stage mode was performed by controlling the dissolved oxygen (DO), and the evolved strain deprived of poxB enabled production of 100 g/L d-lactate with productivity of 1.85 g/L/h. To increase productivity, the producer strain was further evolved to improve its growth rate on crude glycerol. The fermentation was performed to undergo the aerobic growth with low substrate, followed by the anaerobic production with high substrate. Moreover, the intracellular redox of the strain was balanced by fulfillment of the anaerobic respiratory chain with nitrate reduction. Without controlling the DO, the microbial fermentation resulted in the homofermentative production of d-lactate (ca. 0.97 g/g) with a titer of 115 g/L and productivity of 3.29 g/L/h. CONCLUSIONS: The proposed fermentation strategy achieves the highest yield based on crude glycerol and a comparable titer and productivity as compared to the approach by fed-batch fermentation. It holds a promise to sustain the continued development of the crude glycerol-based biorefinery.

Taiwanin E Induces Cell Cycle Arrest and Apoptosis in Arecoline/4-NQO-Induced Oral Cancer Cells Through Modulation of the ERK Signaling Pathway.

Taiwanin E is a bioactive compound extracted from Taiwania cryptomerioides Hayata. In this research endeavor, we studied the anti-cancer effect of Taiwanin E against arecoline and 4-nitroquinoline-1-oxide-induced oral squamous cancer cells (OSCC), and elucidated the underlying intricacies. OSCC were treated with Taiwanin E and analyzed through MTT assay, Flow cytometry, TUNEL assay, and Western blotting for their efficacy against OSCC. Interestingly, it was found that Taiwanin E significantly attenuated the cell viability of oral cancer cells (T28); however, no significant cytotoxic effects were found for normal oral cells (N28). Further, Flow cytometry analysis showed that Taiwanin E induced G1cell cycle arrest in T28 oral cancer cells and Western blot analysis suggested that Taiwanin E considerably downregulated cell cycle regulatory proteins and activated p53, p21, and p27 proteins. Further, TUNEL and Western blot studies instigated that it induced cellular apoptosis and attenuated the p-PI3K/p-Akt survival mechanism in T28 oral cancer cells seemingly through modulation of the ERK signaling cascade. Collectively, the present study highlights the prospective therapeutic efficacy of Taiwanin E against arecoline and 4-nitroquinoline-1-oxide-induced oral cancer.

Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli.

Genetic manipulation of cells for desired traits is the most appreciable strategy implemented in the field of bioengineering. However, this approach closely relies on the use of plasmids and is commonly afflicted by the potential problem of plasmid instability and safety caution. Meanwhile, it may also lead to the spread of antibiotic-resistant markers with replicons of plasmids to the environment. However, this issue has long been neglected. In this study, we have addressed these subjects by developing replicon-free and markerless methods for chromosomal insertion of genes and controlled expression of genomic genes in Escherichia coli. For the former application, the integration vectors of conditional replication were incorporated with the prophage attachment site and duplicated FRT sites. Their utility was illustrated by site-specific insertion of target genes, the endogenous dxs gene and three heterologous genes consisting of gps, crtI, and crtB, fused to T7 promoter into E. coli genome. For the latter application, the template vectors for promoter replacement were constructed to carry a DNA cassette containing the T7 promoter linked to a selective marker flanked with the FRT site. Subsequently, it was illustrated by replacement of the native promoter of chromosomal pckA by the T7 promoter. Finally, with the aid of FLP recombinase supplied from a helper plasmid, the regions containing replicon and/or selective markers in inserted DNAs were eliminated from integrants for both approaches. As a consequence, the expression of these five genes was subject to control by one response regulator, T7 RNA polymerase, in a regulon way, resulting in a high and stable production of lycopene in the cell. This result indicates the promise of developed methods for genome engineering in E. coli.

Development of Nanoscale Oil Bodies for Targeted Treatment of Lung Cancer.

Lung cancer is the most widespread disease and is frequently associated with a high level of epidermal growth factor receptor (EGFR). This study was thus conducted to provide a proof-of-concept approach for targeted therapy of lung cancer by development of nanoscale oil bodies (NOBs). This was carried out by fusion of anti-EGFR affibody (ZEGFR2) with oleosin (Ole), a structure protein of plant seed oils. The fusion protein (Ole-ZEGFR2) was produced in Escherichia coli. NOBs were spontaneously assembled from plant oil, phospholipids, and Ole-ZEGFR2. Consequently, Ole-ZEGFR2-based NOBs were selectively internalized by EGFR-positive lung cancer cells with an efficiency exceeding 90%. Furthermore, the hydrophobic anticancer drug, camptothecin (CPT), was encapsulated into Ole-ZEGFR2-based NOBs. The administration of the CPT formulation based on NOBs resulted in a strong antitumor activity both in vitro and in vivo.