scholarly journals The Combination of Bacillus Subtilis wt55 with AiiO-AIO6 Improve The Resistance of Zebrafish To A. Veronii Infection by Simple Collocation Rather Than B. Subtilis Quorum-Quenching Recombinant Expression Strain

2021 ◽  
Author(s):  
Yuan-Yuan Yao ◽  
Rui Xia ◽  
Ya-Lin Yang ◽  
Chen-Chen Gao ◽  
Feng-Li Zhang ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Proinflammatory Cytokine ◽  
Recombinant Expression ◽  
Expression System ◽  
Quorum Quenching ◽  
Combination Group ◽  
Germ Free ◽  
Expression Strain ◽  
Simple Combination

Abstract Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. The bacteriostatic effect of B. subtilis wt55 strain and quenching enzyme AiiO-AIO6 in vitro showed wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Therefore simple combination of B. subtilis wt55 strain and AiiO-AIO6 was used to evaluate potential synergistic effect. The results showed this combination could significantly reduce the number of invasive A. veronii in gut after challenge, corresponding to the lower intestinal alkaline phosphatase activity. In vitro co-culture experiments showed this combination could inhibit the growth of A. veronii. Direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the combination of wt55 and AiiO-AIO6 could significantly reduce the expression of NF-κB and proinflammatory cytokine IL-1β, increase the expression of lysozyme gene. The gut microbiota induced by either experimental diet was transferred to germ-free zebrafish, and the results showed that intestinal microbiota also plays a regulatory role. The gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of TGF-β and lysozyme. Given the effectiveness of this combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

scholarly journals The Combination of Bacillus Subtilis wt55 with AiiO-AIO6 Improve the Resistance of Zebrafish to A. Veronii Infection by Simple Collocation Rather Than B. Subtilis Quorum-Quenching Recombinant Expression Strain

2021 ◽  
Author(s):  
Yuan-Yuan Yao ◽  
Rui Xia ◽  
Ya-Lin Yang ◽  
Chen-Chen Gao ◽  
Feng-Li Zhang ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Proinflammatory Cytokine ◽  
Recombinant Expression ◽  
Expression System ◽  
Quorum Quenching ◽  
Combination Group ◽  
Germ Free ◽  
Expression Strain ◽  
Simple Combination

Abstract Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. The bacteriostatic effect of B. subtilis wt55 strain and quenching enzyme AiiO-AIO6 in vitro showed wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Therefore simple combination of B. subtilis wt55 strain and AiiO-AIO6 was used to evaluate potential synergistic effect. The results showed this combination could significantly reduce the number of invasive A. veronii in gut after challenge, corresponding to the lower intestinal alkaline phosphatase activity. In vitro co-culture experiments showed this combination could inhibit the growth of A. veronii. Direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the combination of wt55 and AiiO-AIO6 could significantly reduce the expression of NF-κB and proinflammatory cytokine IL-1β, increase the expression of lysozyme gene. The gut microbiota induced by either experimental diet was transferred to germ-free zebrafish, and the results showed that intestinal microbiota also plays a regulatory role. The gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of TGF-β and lysozyme. Given the effectiveness of this combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

scholarly journals The Combination of Bacillus Subtilis wt55 with AiiO-AIO6 Improve the Resistance of Zebrafish to Aeromonas Veronii Infection by Simple Collocation Rather Than B. Subtilis Quorum-Quenching Recombinant Expression Strain

2021 ◽  
Author(s):  
Yuan-Yuan Yao ◽  
Rui Xia ◽  
Ya-Lin Yang ◽  
Chen-Chen Gao ◽  
Feng-Li Zhang ◽  
...  
Keyword(s):  
Survival Rate ◽  
Gut Microbiota ◽  
Proinflammatory Cytokine ◽  
Recombinant Expression ◽  
Expression System ◽  
Quorum Quenching ◽  
Germ Free ◽  
Expression Strain ◽  
Simple Combination

Abstract Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. Firstly, the bacteriostatic activity of B. subtilis wt55 strain and quenching enzyme AiiO-AIO6 on the growth of A. veronii were tested in vitro, and the results showed that this two had different effects on A. veronii: wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Then, the synergistic effects of simple combination of B. subtilis wt55 and AiiO-AIO6 was evaluated next. The results showed this combination could improve the survival rate and significantly reduce the number of invasive A. veronii in gut after challenge compared to the other groups, corresponding to the lower intestinal alkaline phosphatase activity. In vitro co-culture experiments showed this combination could inhibit the growth of A. veronii. Direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the combination of wt55 and AiiO-AIO6 could significantly reduce the expression of NF-κB and proinflammatory cytokine IL-1β, increase the expression of lysozyme gene. The gut microbiota induced by either experimental diet was transferred to germ-free zebrafish, and the results showed that intestinal microbiota also plays a regulatory role. The gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of TGF-β and lysozyme. Given the effectiveness of this combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

scholarly journals A118 GUT MICROBIOTA TRANSPLANTATION FROM A PATIENT WITH SEVERE CONSTIPATION INDUCED CHANGES IN COLONIC FUNCTION AND STRUCTUR OF GNOTOBIOTIC MICE

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 137-138
Author(s):  
X Bai ◽  
G De Palma ◽  
J Lu ◽  
S M Collins ◽  
P Bercik
Keyword(s):  
Gut Microbiota ◽  
Fecal Microbiota Transplantation ◽  
Fecal Microbiota ◽  
Colonic Transit ◽  
Slow Transit Constipation ◽  
Antibiotic Exposure ◽  
Ach Release ◽  
Germ Free ◽  
Gi Transit

Abstract Background Increasing evidence suggests that gut microbiota play a key role in gastrointestinal (GI) tract function. We have previously shown that fecal microbiota transplantation diarrhea predominant IBS patients into germ-free mice induces faster GI transit, increased permeability and innate immune activation. However, it is unknown whether gut dysfunction is induced by microbiota from patients with chronic constipation. Aims Here, we investigated the role of the intestinal microbiota in the expression of severe slow transit constipation in a patient with previous C difficile infection and extensive antibiotic exposure. Methods Germ-free (GF) mice (14 weeks old) were gavaged with diluted fecal content from the patient with constipation (PA) or a sex and age-matched healthy control (HC). 12 weeks later, we assessed gut motility and GI transit using videofluoroscopy and a bead expulsion test.. We then investigated intestinal and colonic smooth muscle isometric contraction in vitro using electric field stimulation (EFS), and acetylcholine (Ach) release was assessed by superfusion using [3H] choline. Histological changes were evaluated by H&E and immunohistochemistry. Results Mice with PA microbiota had faster whole GI transit (score 18.9 ± 0.9 (N=9) than mice with HC microbiota (15.4 ± 1.0, N=10, p=0.032), with markers located mainly in the distal small bowel and cecum. However, bead expulsion from the colon was significantly longer in PA mice (420.8 s ± 124.6 s, N=9) than in HC mice (82.6 s ± 20.0 s, N=10, p=0.026). This delayed colonic transit was likely due to colonic retroperistalsis visualized videofluoroscopically by retrograde flow of barium in the right colon of PA mice. There was no difference between the two groups in small intestinal or colonic tissues in Ach release or contractility induced by carbachol or KCl,. EFS caused transient biphasic relaxation and contraction in small intestine and colon, with the colonic contraction being stronger in the PA group. Microscopic tissue analysis showed disruption of the interstitial cells of Cajal (ICC) network and increased lymphocyte infiltration in colonic mucosa and submucosa in PA mice. Conclusions These results indicate that the microbiota is a driver of delayed colonic transit in a patient whose constipation started following extensive antibiotic exposure for C. difficile infection. The observed dysmotility pattern was not due to lower muscle contractility but likely caused by immune mediated changes in the ICC network. Funding Agencies CIHR

scholarly journals DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein–dynactin–cargo adaptor complexes

2017 ◽  
Vol 114 (9) ◽  
pp. E1597-E1606 ◽  
Author(s):  
Ha Thi Hoang ◽  
Max A. Schlager ◽  
Andrew P. Carter ◽  
Simon L. Bullock
Keyword(s):  
Single Molecule ◽  
Heavy Chain ◽  
Muscular Atrophy ◽  
Recombinant Expression ◽  
Neurological Diseases ◽  
Expression System ◽  
Cytoplasmic Dynein ◽  
In Vitro Motility

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein–dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo–motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

scholarly journals Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

1999 ◽  
Vol 22 (3) ◽  
pp. 273-283 ◽  
Author(s):  
C Sen Gupta ◽  
RR Dighe
Keyword(s):  
Pichia Pastoris ◽  
Structure Function ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Recombinant Expression ◽  
Expression System ◽  
Biologically Active ◽  
Maximal Response ◽  
Glycoprotein Hormone

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.

scholarly journals DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes

2016 ◽  
Author(s):  
Ha Thi Hoang ◽  
Max A. Schlager ◽  
Andrew P. Carter ◽  
Simon L Bullock
Keyword(s):  
Single Molecule ◽  
Heavy Chain ◽  
Muscular Atrophy ◽  
Recombinant Expression ◽  
Neurological Diseases ◽  
Expression System ◽  
Cytoplasmic Dynein ◽  
Recombinant Expression System

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4 MDa motor complex that traffics organelles, vesicles and macromolecules towards microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterise 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor BICD2. Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2, and are therefore expected to result in linkage of cargoes to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

scholarly journals The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update

2021 ◽  
Author(s):  
Nazaret Peña-Gil ◽  
Cristina Santiso-Bellón ◽  
Roberto Gozalbo-Rovira ◽  
Javier Buesa ◽  
Vicente Monedero ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Animal Studies ◽  
Viral Infections ◽  
Host Cells ◽  
Blood Group Antigens ◽  
Virus Attachment ◽  
Germ Free ◽  
Complex Relationships

Rotavirus (RV) and norovirus (NoV) are the leading cause of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections, since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive and Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections there are too many discrepancies that prevent drawing conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances, enteric viral infection. The ways microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restricts infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responders and non-responders individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and the intestinal viruses, new avenues will be open for the development of novel anti-NoV and anti-RV therapies.

scholarly journals The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update

2021 ◽  
Vol 22 (24) ◽  
pp. 13473
Author(s):  
Nazaret Peña-Gil ◽  
Cristina Santiso-Bellón ◽  
Roberto Gozalbo-Rovira ◽  
Javier Buesa ◽  
Vicente Monedero ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Animal Studies ◽  
Viral Infections ◽  
Host Cells ◽  
Blood Group Antigens ◽  
Virus Attachment ◽  
Germ Free ◽  
Complex Relationships

Rotavirus (RV) and norovirus (NoV) are the leading causes of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive or Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections, there are too many discrepancies that prevent the ability to draw conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances enteric viral infection. The ways that microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restrict infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responder and non-responder individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and intestinal viruses, new avenues will open for the development of novel anti-NoV and anti-RV therapies.

scholarly journals Establishment of a recombinant expression system for connective tissue growth factor (CTGF) that models CTGF processing in utero

Reproduction ◽  
2003 ◽  
pp. 271-284 ◽  
Author(s):  
DK Ball ◽  
EE Moussad ◽  
MA Rageh ◽  
SA Kemper ◽  
DR Brigstock
Keyword(s):  
Growth Factor ◽  
Connective Tissue ◽  
Connective Tissue Growth Factor ◽  
Recombinant Expression ◽  
Expression System ◽  
Limited Proteolysis ◽  
In Utero ◽  
Tissue Growth ◽  
Recombinant Expression System

Connective tissue growth factor (CTGF) stimulates cell proliferation, migration, adhesion and extracellular matrix production, and functions in processes such as development, differentiation, angiogenesis, implantation, wound healing and fibrosis. CTGF is a 38 kDa protein that comprises four discrete structural modules (modules 1-4) but is susceptible to limited proteolysis in utero yielding bioactive isoforms that comprise either modules 3 and 4 (16-20 kDa) or module 4 (10 kDa). Here we report the development of a stable cell line, termed DB1, that was generated by transfecting cDNA encoding full-length human CTGF into Chinese hamster ovary cells that were mutant for heparin sulphate and chondroitin sulphate. DB1 cells produced 38 kDa CTGF and low molecular mass CTGFs that had N-termini between modules 2 and 3 at Ala(181) (20 kDa), Leu(184) (18 kDa) or Ala(197) (16 kDa) or between modules 3 and 4 at Gly(253) (10 kDa). CTGF was exported from DB1 cells as early as 5 min after synthesis and all isoforms were readily purified from conditioned medium by sequential steps of heparin affinity, cation exchange, and reverse-phase chromatography. The 38 kDa CTGF was faithfully glycosylated and underwent limited proteolysis in the presence of thrombin, kallikrein or uterine fluids, the last of which was antagonized by anti-thrombin III. All CTGF isoforms promoted cell adhesion, mitosis and epithelial transdifferentiation in vitro as well as subcutaneous fibrosis in vivo. The establishment of this recombinant expression system allows for mass-scale production of all previously reported uterine CTGF isoforms, demonstrates that module 4 contains functional domains involved in a broad range of biological activities, and will facilitate studies of CTGF processing in vitro.

Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system

2008 ◽  
Vol 389 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Stephan Frey ◽  
Martin Haslbeck ◽  
Otmar Hainzl ◽  
Johannes Buchner
Keyword(s):  
Mammalian Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Basic Research ◽  
Medical Diagnostics ◽  
Free Expression ◽  
Translation System ◽  
Functional Antibodies ◽  
The Stability

Abstract Antibodies are an important component of the immune system of higher eukaryotes. Furthermore, they are effective tools in basic research, medical diagnostics and therapy. Recombinant expression of these heterotetrameric, disulfide-bridged proteins is usually performed in mammalian cells. Here, we describe the cell-free expression of a mouse monoclonal antibody, MAK33, in a coupled transcription/translation system, based on an Escherichia coli lysate. Both the heavy and the light chain can be produced efficiently in this setup. However, they fail to form functional antibodies. With a view to overcome folding and oxidation defects, we supplemented the system with the oxidoreductases PDI (protein disulfide isomerase) and DsbC and the ER-specific chaperones Grp94 and BiP; furthermore, we optimized the redox conditions. We found that functional antibodies can only be obtained in the presence of an oxidoreductase. In contrast, the addition of Grp94 and/or BiP had no influence on the productive folding reaction. The comparison of the antibody expressed in vitro with MAK33 expressed in cell culture showed that the in vitro expressed antibody is correctly assembled, disulfide-bridged and shows identical antigen affinity. The stability of the in vitro expressed non-glycosylated IgG is comparable to that of the authentic antibody.

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