Clostridium butyricum Protects IPEC-J2 Cells from ETEC K88-Induced Oxidative Damage by Activating the Nrf2/ARE Signaling Pathway.

Clostridium butyricum (CB) is a naturally occurring probiotic compound that can alleviate the oxidative damage induced by enterotoxigenic Escherichia coli K88 (ETEC K88) in porcine intestinal epithelial (IPEC-J2) cells. In this study, we investigate the molecular mechanism underlying this effect. Based on cell viability, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX) assessments, the optimal concentration of ETEC K88 was determined to be 1 × 103 cfu/mL. Viable bacteria counts in cells pretreated with CB and then infected with ETEC K88 show that CB can adhere to IPEC-J2 cells and that optimal adhesion is achieved at the multiple infection index (MOI) of 50 at 3 h of pretreatment. The results of qPCR indicate that although ETEC significantly decreases the expression levels of antioxidant enzymes regulated by NF-E2-related factor 2 (Nrf2) compared to the control group, CB reverses this effect. To confirm that Nrf2 is directly involved in the mechanism by which CB alleviates oxidative stress, siRNA was used to silence the expression of Nrf2 gene in IPEC-J2 cells. Compared to the NC+ETEC and siRNA+ETEC groups, the expressions of SOD1, SOD2, GPX1, and GPX2 in the NC+CB+ETEC and siRNA+CB+ETEC groups are significantly increased at 12 h and 24 h. This shows that CB can reduce ETEC K88-induced oxidative damage in IPEC-J2 cells by activating the expression of antioxidant enzymes implicated in the Kelch-like ECH-associated protein-1- (Keap1-) Nrf2/antioxidant response element (ARE) signaling pathway.

Clostridium Butyricum ZJU-F1 Benefits the Intestinal Barrier Function and Immune Response Associated with Its Modulation of Gut Microbiota in Weaned Piglets.

This study investigated the effects of dietary C. butyricum ZJU-F1 on the apparent digestibility of nutrients, intestinal barrier function, immune response, and microflora of weaned piglets, with the aim of providing a theoretical basis for the application of Clostridium butyricum as an alternative to antibiotics in weaned piglets. A total of 120 weanling piglets were randomly divided into four treatment groups, in which piglets were fed a basal diet supplemented with antibiotics (CON), Bacillus licheniformis (BL), Clostridium butyricum ZJU-F1 (CB), or Clostridium butyricum and Bacillus licheniformis (CB-BL), respectively. The results showed that CB and CB-BL treatment increased the intestinal digestibility of nutrients, decreased intestinal permeability, and increased intestinal tight junction protein and mucin expression, thus maintaining the integrity of the intestinal epithelial barrier. CB and CB-BL, as exogenous probiotics, were also found to stimulate the immune response of weaned piglets and improve the expression of antimicrobial peptides in the ileum. In addition, dietary CB and CB-BL increased the proportion of Lactobacillus. The levels of butyric acid, propionic acid, acetic acid, and total acid were significantly increased in the ceca of piglets fed CB and CB-BL. Furthermore, we validated the effects of C. butyricum ZJU-F1 on the intestinal barrier function and immune response in vitro and found C. butyricum ZJU-F1 improved intestinal function and enhanced the TLR-2-MyD88-NF-κB signaling.

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT.

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.

Co-production of 1,3 propanediol and long-chain alkyl esters from crude glycerol.

Crude glycerol is an excellent carbon source for bacterial production systems. Bacterial fermentation often generates by-products that can offer an additional carbon pool to improve the product profile for optimal valorization. In this study, the properties of two phylogenetically distinct bacteria, Acinetobacter baylyi ADP1 and Clostridium butyricum, were coupled in a one-pot batch process to co-produce 1,3 propanediol (PDO) and long-chain alkyl esters (wax esters, WEs) from crude glycerol. In the process, A. baylyi deoxidized the growth medium allowing glycerol fermentation and PDO production by C. butyricum. Reaeration of the co-cultivations enabled A. baylyi to metabolize the fermentation by-products, acetate and butyrate, and synthesize intracellular WEs. To improve PDO production and A. baylyi growth, carbon and macronutrients in the growth medium were screened and optimized using Plackett-Burman and Box-Behnken models. The validation experiment revealed a good correlation between the observed and predicted values. The salting-out method recovered 89.5% PDO from the fermentation broth and in vacuo extraction resulted in a PDO content of 5.3 g L-1. Nuclear magnetic resonance revealed a WE content and yield of 34.4 ±â€¯1.4 mg L-1 and 34.2 ±â€¯3.2 mg WE g-1 dry cell weight, respectively. A molar yield of 0.65 mol PDO mol-1 and 0.62 µmol WE mol-1 crude glycerol was achieved with the synthetic consortium. This work emphasizes the strength of response surface methodology in improving production processes from the mutualistic association of divergent bacterial species in consortium. The co-production of PDO and WEs from crude glycerol is demonstrated for the first time in this study.

Effects of dietary supplementation with Clostridium butyricum on laying performance, egg quality, serum parameters, and cecal microflora of laying hens in the late phase of production.

This experiment was conducted to evaluate the effects of dietary supplementation with Clostridium butyricum on laying performance, egg quality, serum parameters, and cecal microflora of laying hens in the late phase of production. Jinghong-1 strain laying hens (n = 960; 48 wk of age) were randomly allocated to 5 treatment groups with 6 replicates of 32 hens. Hens were fed with basal diet (control) and basal diet supplemented with 2.5 × 104 (CB1), 5 × 104 (CB2), 1 × 105 (CB3), and 2 × 105 (CB4) cfu/g C. butyricum for 10 wk. The results showed that egg production, egg mass, and eggshell strength increased quadratically as supplemental C. butyricum increased, and these responses were maximized in the CB2 group (P < 0.05). Compared with the control group, the addition of C. butyricum resulted in quadratic effects on serum total protein, uric acid, calcium, complement component C3 and catalase concentrations, and these responses were maximized or minimized in the CB2 group (P < 0.05). Linear and quadratic increases were observed in serum IgM, total superoxide dismutase, and glutathione peroxidase concentrations, and these responses were maximized in CB2 or CB3 group (P < 0.05). The addition of C. butyricum in the CB2 group resulted in linearly increasing levels of serum IgG concentration as compared with the control group (P < 0.05). Spleen index increased (P < 0.05) in the CB2 group. Hens fed with C. butyricum reduced (P > 0.05) the population of E. coli, while Bifidobacterium counts increased quadratically and maximized in the CB2 group (P < 0.05). In conclusion, the results indicated that dietary supplementation with C. butyricum (5 × 104 or 1 × 105 cfu/g) could improve laying performance and egg quality by promoting immune function, enhancing antioxidative capacity, and benefiting the cecal microflora of laying hens in the late phase of production.

Computational Tale of Two Enzymes: Glycerol Dehydration With or Without B12.

We present a series of QM/MM calculations aimed at understanding the mechanism of the biological dehydration of glycerol. Strikingly and unusually, this process is catalyzed by two different radical enzymes, one of which is a coenzyme-B12-dependent enzyme and the other which is a coenzyme-B12-independent enzyme. We show that glycerol dehydration in the presence of the coenzyme-B12-dependent enzyme proceeds via a 1,2-OH shift, which benefits from a significant catalytic reduction in the barrier. In contrast, the same reaction in the presence of the coenzyme-B12-independent enzyme is unlikely to involve the 1,2-OH shift; instead, a strong preference for direct loss of water from a radical intermediate is indicated. We show that this preference, and ultimately the evolution of such enzymes, is strongly linked with the reactivities of the species responsible for abstracting a hydrogen atom from the substrate. It appears that the hydrogen-reabstraction step involving the product-related radical is fundamental to the mechanistic preference. The unconventional 1,2-OH shift seems to be required to generate a product-related radical of sufficient reactivity to cleave the relatively inactive C-H bond arising from the B12 cofactor. In the absence of B12, it is the relatively weak S-H bond of a cysteine residue that must be homolyzed. Such a transformation is much less demanding, and its inclusion apparently enables a simpler overall dehydration mechanism.

Dietary supplementation with Clostridium butyricum modulates serum lipid metabolism, meat quality, and the amino acid and fatty acid composition of Peking ducks.

The aim of this study was to investigate the effects of Clostridium butyricum (C. butyricum) on the performance, serum lipid metabolism, muscle morphology, meat quality, and fatty acid profiles of Peking ducks. A total of 1,500 Peking ducks were randomly divided into five groups with five replicates and were fed a non-antibiotic basal diet (Control) or a basal diet supplemented with either 200, 400, or 600 mg/kg of C. butyricum (2.0 × 109 CFU/g) or 150 mg of aureomycin/kg for 42 d. Compared with the control group, supplementation with C. butyricum increased the average daily weight gain but reduced the feed/gain ratio from 1 to 42 d of age. Similarly, dietary C. butyricum increased the activities of antioxidant enzymes but decreased the malondialdehyde (MDA) and lipid metabolites concentration. C. butyricum supplementation increased the muscle pH value at 45 min postmortem, the redness of the meat, and the contents of inosine acid (IMP) and intramuscular fat (IMF) in Peking ducks. By contrast, C. butyricum supplementation lowered the lightness, drip loss, and the shear force of breast meat. Supplementation with C. butyricum increased the concentrations of essential amino acids and flavor amino acids, as well as arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and total polyunsaturated fatty acids (PUFA) in breast muscle. Dietary C. butyricum could positively improve performance, lipid metabolism, meat quality, and the amino acid and fatty acid composition in a dose-dependent manner. Therefore, C. butyricum is proposed as a feasible alternative feed additive for the production of healthier Peking duck meat with favorable properties.

C. butyricum lipoteichoic acid inhibits the inflammatory response and apoptosis in HT-29 cells induced by S. aureus lipoteichoic acid.

Lipoteichoic acid (LTA) is one of microbe-associated molecular pattern (MAMP) molecules of gram-positive bacteria. In this study, we demonstrated that Clostridium butyricum LTA (bLTA) significantly inhibited the inflammatory response and apoptosis induced by Staphylococcus aureus LTA (aLTA) in HT-29 cells. aLTA stimulated the inflammatory responses by activating a strong signal transduction cascade through NF-κB and ERK, but bLTA did not activate the signaling pathway. bLTA pretreatment inhibited the activation of the NF-κB and ERK signaling pathway induced by aLTA. The expression and release of cytokines such as IL-8 and TNF-α were also suppressed by bLTA pretreatment. aLTA treatment induced apoptosis in HT-29 cells, but bLTA did not affect the viability of the cells. Further study indicated that bLTA inhibited apoptosis in HT-29 cells induced by aLTA. These results suggest that bLTA may act as an aLTA antagonist and that an antagonistic bLTA may be a useful agent for suppressing the pro-inflammatory activities of gram-positive pathogenic bacteria.

Different effects of lipoteichoic acid from C. butyricum and S. aureus on inflammatory responses of HT-29 cells.

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and represents one of the most critical microbe-associated molecular pattern (MAMP) molecules. In this study, we isolated and purified LTA from Clostridium butyricum (bLTA) and compared its effects on the inflammatory responses of HT-29 cells with those of LTA from Staphylococcus aureus (aLTA). We also compared the effects of bLTA and aLTA on cell growth, proliferation, and apoptosis. The results showed that the length and saturation degree of the acyl chains in the two LTA molecules were obviously different. aLTA stimulated the phosphorylation of p65 and activated the NF-κB signaling pathway, inducing the expression and secretion of cytokines. Moreover, aLTA also inhibited the growth and proliferation of HT-29 cells and induced cell apoptosis. However, bLTA had no significant effects on the NF-κB signaling pathway in HT-29 cells and did not stimulate cellular inflammatory responses or induce apoptosis. These differences in activity may result from the different lengths and saturation degrees of the acyl fatty acid chains of the two LTA molecules. These differences may also account for the distinct effects elicited by probiotic bacteria and pathogenic bacteria on host cells.

Effects of Clostridium butyricum on growth performance, antioxidation, and immune function of broilers.

To investigate the effects of Clostridium butyricum on growth performance, antioxidation, and immune function of broilers, 320 one-day-old Arbor Acres commercial male chicks were assigned to one of 5 treatments with 8 replicates in a completely randomized design for 42 d. The 5 treatments were basal diet (control), basal diet supplemented with 2.5×10(8) cfu C. butyricum/kg (CB1), basal diet supplemented with 5×10(8) cfu C. butyricum/kg (CB2), basal diet supplemented with 1×10(9) cfu C. butyricum/kg (CB3), and basal diet supplemented with 150 mg aureomycin/kg (antibiotic). The results showed that all C. butyricum-supplemented groups during d 1 to 21 and the CB2 group during d 22 to 42 had higher ADG compared with the control (P<0.05). Chicks fed the CB3 diet had higher glutathione S-transferase (GST) activity (P<0.05), and chicks fed the CB2 diet had a higher glutathione (GSH) concentration in duodenal and ileal mucosa at 21 d of age than those in the control group (P<0.05). Chicks fed the CB3 diet had a lower malondialdehyde (MDA) concentration in duodenal mucosa than those in the control and CB1 groups (P<0.05). Chicks fed the CB2, CB3, and antibiotic diets had a lower MDA concentration in ileal mucosa than those in the control and CB1 groups (P<0.05). Broilers fed the CB3 diet had greater superoxide dismutase (SOD) activity in the ileal mucosa on d 21 and in jejunal mucosa on d 42 than those in the other groups (P<0.05). Chicks fed the CB2, CB3, and antibiotic diets had a higher GSH concentration in duodenal and jejunal mucosa on d 42 than those in the control group (P<0.05). Broilers fed the CB2 and CB3 diets had a lower MDA concentration in the jejunal mucosa on d 42 than those in the control and CB1 groups. Chicks fed diets supplemented with C. butyricum had a higher IgM concentration than those in the control group at 21 and 42 d of age (P<0.05). The results indicate that C. butyricum improves broilers' growth performance, antioxidation, and immune function.

Regulation of Trek1 expression in nasal mucosa with allergic rhinitis by specific immunotherapy.

Epithelial barrier dysfunction is involved in the pathogenesis of allergic disorders, such as nasal allergy. TWIK-related K(+) 1 (Trek1) potassium channels are required in the maintenance of the epithelial barrier function. This study aims to investigate the role of antigen-specific immunotherapy (SIT) in the regulation of Trek1 expression in the nasal mucosa. In this study, patients with nasal allergy were treated with SIT and/or Clostridium butyricum. The expression of Trek1 and histone demethylase 1 (HDAC1) in the nasal epithelia was assessed by real-time reverse transcription polymerase chain reaction and Western blotting. Serum cytokines were assessed by enzyme-linked immunosorbent assay. The results showed that Trek1 and HDAC1 were detected in the nasal epithelia. Trek1 was lower, whereas HDAC1 was higher in patients with allergic rhinitis as compared with healthy controls. Trek1-null RPMI2650 monolayers showed a markedly compromised epithelial barrier function. Treatment with SIT significantly increased the Trek1 levels in the nasal epithelia of allergic rhinitis patients that were further improved in conjunction of SIT and administration of probiotic C. butyricum. In conclusion, nasal epithelia express Trek1 that can be suppressed by allergic response. SIT can restore the expression of Trek1 in the nasal epithelia and can be further improved by conjunction with administration of C. butyricum.

Effects of multistrain probiotics on growth performance, apparent ileal nutrient digestibility, blood characteristics, cecal microbial shedding, and excreta odor contents in broilers.

This experiment was conducted to investigate the efficacy of Lactobacillus acidophilus, Bacillus subtilis, and Clostridium butyricum supplementation in broilers. A total of 400 one-day-old mixed sex Ross 308 broilers with an initial average BW of 46 ± 0.5 g were randomly allotted into 4 treatments with 5 replicate pens per treatment and 20 broilers in each pen for 35 d. Dietary treatments were (1) an antibiotic-free diet (CON), (2) CON + 5 mg/kg of avilamycin, (3) CON + 1 × 10(5) cfu of multistrain probiotics/kg of diet (P1), and (4) CON + 2 × 10(5) cfu of multistrain probiotics/kg of diet (P2). Broilers fed the P1 and P2 diets had greater BW gain than broilers fed the CON diet during d 22 to 35 (P = 0.01) and overall (P = 0.02). Feed conversion ratios in P1 and P2 were decreased (P = 0.03) compared with that in CON from d 22 to 35. Ileal digestibility of most essential amino acids, with the exception of His and Phe, were increased (P < 0.05) in P1 and P2 compared with CON. Serum IgA and IgM concentrations in P2 were higher (P < 0.05) than those in CON. The cecal Lactobacillus numbers were increased (P = 0.02), and the counts of Escherichia coli were decreased (P = 0.03) in P1 and P2 compared with CON. Dietary supplementation with multistrain probiotics decreased (P < 0.05) the excreta NH3 content compared with the CON. In conclusion, dietary supplementation with multistrain probiotics improved broiler growth performance, ileal amino acids digestibility, and humoral immunity. Furthermore, the probiotics decreased the cecal numbers of E. coli and decreased the NH3 content of excreta.

Destructive effects of butyrate on the cell envelope of Helicobacter pylori.

Helicobacter pylori can be found in the oral cavity and is mostly detected by the use of PCR techniques. Growth of H. pylori is influenced by various factors in the mouth, such as the oral microflora, saliva and other antimicrobial substances, all of which make colonization of the oral cavity by H. pylori difficult. In the present study, we analysed the effect of the cell supernatant of a representative periodontal bacterium Porphyromonas gingivalis on H. pylori and found that the cell supernatant destroyed the H. pylori cell envelope. As P. gingivalis produces butyric acid, we focused our research on the effects of butyrate and found that it significantly inhibited the growth of H. pylori. H. pylori cytoplasmic proteins and DNA were detected in the extracellular environment after treatment with butyrate, suggesting that the integrity of the cell envelope was compromised and indicating that butyrate has a bactericidal effect on H. pylori. In addition, levels of extracellular H. pylori DNA increased following treatment with the cell supernatant of butyric acid-producing bacteria, indicating that the cell supernatant also has a bactericidal effect and that this may be due to its butyric acid content. In conclusion, butyric acid-producing bacteria may play a role in affecting H. pylori colonization of the oral cavity.