scholarly journals Design and heterologous expression of a novel dimeric LL37 variant in Pichia pastoris

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Na Zhan ◽  
Licong Zhang ◽  
Hong Yang ◽  
Yalan Zheng ◽  
Xinke Wei ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Pichia Pastoris ◽  
Immune Cells ◽  
Selection Index ◽  
White Blood Cells ◽  
Substitution Method ◽  
Salt Ions ◽  
New Ideas ◽  
Microbial Systems ◽  
Strong Antimicrobial Activity

Abstract Background The antimicrobial peptide LL37 is produced by white blood cells (mainly neutrophils) and various epithelial cells, and has the outstanding advantages of participating in immune regulation, causing chemotaxis of immune cells and promoting wound healing. However, the central domain of LL37 needs to be improved in terms of antimicrobial activity. Results In this study, the amino acid substitution method was used to improve the antimicrobial activity of the LL37 active center, and a dimeric design with a better selection index was selected. A flexible linker was selected and combined with the 6 × His-SUMO tag and LG was successfully expressed using Pichia pastoris as a host. Recombinant LG displayed strong antimicrobial activity by destroying the cell membrane of bacteria but had low hemolytic activity. In addition, compared with monomeric peptide FR, rLG had improved ability to tolerate salt ions. Conclusion This research provides new ideas for the production of modified AMPs in microbial systems and their application in industrial production.

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

scholarly journals IMMU-45. CUSTOMIZED LIPID NANOPARTICLES DELIVER NUCLEIC ACIDS TO IMMUNE CELLS IN BRAIN TUMORS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii114-ii114
Author(s):  
Adam Grippin ◽  
Brandon Wummer ◽  
Hector Mendez-Gomez ◽  
Tyler Wildes ◽  
Kyle Dyson ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Nucleic Acids ◽  
Immune Cells ◽  
White Blood Cells ◽  
Lipid Nanoparticles ◽  
Intracranial Tumor ◽  
Innate Immune ◽  
Checkpoint Blockade ◽  
Normal Brain ◽  
Innate Immune Cells

Abstract BACKGROUND Brain tumors are notoriously difficult to treat in part due to their isolation behind the blood brain barrier and their power to suppress antitumor immune responses. We have previously reported cationic liposome formulations capable of delivering immune modulatory nucleic acids to immune cells in various peripheral organs, but there is currently no reliable method to deliver nucleic acids into brain tumors without direct injection into the tumor site. OBJECTIVE Here, we report the development of a customized lipid nanoparticle to deliver immune modulatory nucleic acids to immune cells in brain tumors. APPROACH Cationic liposomes composed of varying lipid combinations were evaluated for their ability to deliver functional mRNA and siRNA to innate immune cells in vitro and in intracranial tumor models. Nucleic acids were labelled with Cy3 to monitor particle distribution in vivo. RESULTS Lipids composed of DOTAP and cholesterol selectively delivered mRNA and siRNA to intracranial GL261 and KR158b tumors. Interestingly, these particles selectively delivered these nucleic acids to CD45+ white blood cells with minimal delivery to CD45- tumor cells or normal brain tissue. Encapsulation of siRNA blocking programmed death ligand 1 (PDL1) significantly reduced PDL1 expression on innate immune cells in brain tumors, with the greatest effects on tumor-associated macrophages. Co-administration of systemic checkpoint blockade with intravenous administration of these lipid nanoparticles bearing PDL1 siRNA enabled systemic and intratumoral checkpoint blockade, leading to 37% long term survivorship in an otherwise fatal intracranial tumor model. CONCLUSIONS Our customized lipid nanoparticles enable potent intratumoral immune modulation via delivery of nucleic acids to immune cells in brain tumors.

High-titer production and strong antimicrobial activity of sophorolipids fromRhodotorula bogoriensis

2015 ◽  
Vol 31 (4) ◽  
pp. 867-874 ◽  
Author(s):  
Daniel K.Y. Solaiman ◽  
Richard D. Ashby ◽  
Nicole V. Crocker
Keyword(s):  
Antimicrobial Activity ◽  
High Titer ◽  
Strong Antimicrobial Activity

scholarly journals A Review of Antimicrobial Activity of Dental Mesenchymal Stromal Cells: Is There Any Potential?

2022 ◽  
Vol 2 ◽  
Author(s):  
Oleh Andrukhov ◽  
Alice Blufstein ◽  
Christian Behm
Keyword(s):  
Vitamin D ◽  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Mesenchymal Stromal Cells ◽  
Immune Cells ◽  
Stromal Cells ◽  
Multilineage Differentiation ◽  
Differentiation Potential ◽  
Health Maintenance ◽  
Immunomodulatory Properties

Antimicrobial defense is an essential component of host-microbial homeostasis and contributes substantially to oral health maintenance. Dental mesenchymal stromal cells (MSCs) possess multilineage differentiation potential, immunomodulatory properties and play an important role in various processes like regeneration and disease progression. Recent studies show that dental MSCs might also be involved in antibacterial defense. This occurs by producing antimicrobial peptides or attracting professional phagocytic immune cells and modulating their activity. The production of antimicrobial peptides and immunomodulatory abilities of dental MSCs are enhanced by an inflammatory environment and influenced by vitamin D3. Antimicrobial peptides also have anti-inflammatory effects in dental MSCs and improve their differentiation potential. Augmentation of antibacterial efficiency of dental MSCs could broaden their clinical application in dentistry.

scholarly journals PSVII-32 In vitro evaluation of antimicrobial activity of short- and medium-chain fatty acid salts and their combinations against Streptococcus suis

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 465-466
Author(s):  
Cinta Sol ◽  
Mónica Puyalto ◽  
Bernat Canal ◽  
Ana Maria Carvajal ◽  
Manuel Gómez ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Antimicrobial Activity ◽  
Fatty Acid ◽  
Sodium Butyrate ◽  
Chain Fatty Acid ◽  
Medium Chain Fatty Acid ◽  
Gram Positive ◽  
Medium Chain ◽  
Gram Positive Bacteria ◽  
Strong Antimicrobial Activity

Abstract The aim of this study was to investigate the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of organic acid salts against six field isolates of Streptococcus suis. The three products evaluated were sodium salt of coconut fatty acids distillate (DIC) alone and two combinations with sodium butyrate (NaBut): DIC70:30, being 70% of NaBut protected with 30% of DIC; and DIC50:50, being 50% of NaBut protected with 50% of DIC. Antimicrobial susceptibility testing was performed to estimate the MIC values for each product and strain by the broth microdilution method at pH 6.0. MBC values were also determined by sub-culturing supernatant from wells without evident bacterial growth. The values of MIC50/MBC50 were calculated as the concentration which inhibited/killed 50% of the isolates tested. The MIC50 showed DIC as the most effective (8 ppm) against S. suis followed by DIC50:50 (32 ppm) and DIC70:30 (64 ppm). The MBC50 demonstrated a similar trend, DIC being the most effective (16 ppm) followed by DIC50:50 (64 ppm) and DIC70:30 (64 ppm). It is well known that butyric acid is a short-chain fatty acid which has strong antimicrobial activity against Gram-negative bacteria. In contrast, coconut fatty acids distillate is a medium-chain fatty acid source (MCFA) rich in lauric acid which has strong antimicrobial activity against Gram-positive bacteria. Both products are generally available as salts to facilitate their application in feed. In this study, the results showed that DIC was the most effective against the Gram-positive bacteria tested, followed by DIC50:50 and DIC70:30, the sodium butyrate-based products. As expected, a higher concentration of MCFA in the tested product was associated with a higher inhibitory and bactericidal activity. Further studies would be required to better understand these interactions as well as in vivo studies to demonstrate the effects on microbial populations.

Combined Effect of Hop Resins and Sodium Hexametaphosphate against Certain Strains of Escherichia coli

2000 ◽  
Vol 63 (6) ◽  
pp. 735-740 ◽  
Author(s):  
TADASHI FUKAO ◽  
HARUMICHI SAWADA ◽  
YOSHIYUKI OHTA
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Food Systems ◽  
Combined Effect ◽  
Sodium Hexametaphosphate ◽  
Lag Phase ◽  
Phase Growth ◽  
E Coli ◽  
Cell Components ◽  
Strong Antimicrobial Activity

The combined antimicrobial effects of hop resins with sodium hexametaphosphate, glycerol monocaprate, and lysozyme were investigated aiming to make an effective agent against Escherichia coli. When they are used separately, the antimicrobial activity against E. coli was minimal. However, the combination of hop resins with sodium hexametaphosphate exhibited strong antimicrobial activity against E. coli, but no effect was found in combinations of hop resins with the other agents. The activity was strongest when the combination was added at the beginning of growth of the bacteria, resulting in a prolonged lag phase. However, when the antimicrobials were added during the log phase, growth was depressed considerably. By addition of these materials, cell components with absorbance near 260 nm were leaked out. This possibly may have resulted from damage to the cell membranes of the bacteria. The combined effect was also detected in model food systems such as mashed potatos. The use of hop resins and sodium hexametaphosphate in combination may thus be useful for controlling E. coli.

scholarly journals Antimicrobial Activity and HPLC Fingerprinting of CrudeOcimumExtracts

2011 ◽  
Vol 8 (3) ◽  
pp. 1430-1437 ◽  
Author(s):  
S. S. Deo ◽  
F. Inam ◽  
R. P. Mahashabde
Keyword(s):  
Antimicrobial Activity ◽  
Methanolic Extract ◽  
Ocimum Sanctum ◽  
Moderate Activity ◽  
Aqueous Extracts ◽  
Gram Positive ◽  
Strong Activity ◽  
Gram Negative ◽  
E Coli ◽  
Strong Antimicrobial Activity

The antimicrobial activity of crude methanolic and aqueous extracts ofOcimum sanctumandOcimum kilimandsacharicumagainst gram positive, gram negative and antifungal activity was evaluated to find the zone of inhibition and to set a HPLC profile or fingerprint of these extracts. The crude methanolic extract ofOcimum sanctumshowed strong antimicrobial activity againstS.aureusandC. albicansand moderate activity againstE. coliandB. subtilis. The crude methanolic extract ofOcimum kilimandsacharicumshowed strong antimicrobial activity againstS. aureus, E. coliandC. albicansat higher concentration, same as that shown by the standard forC. albicans. It showed moderate activity againstB. subtilis. The crude aqueous extracts of Ocimum sanctum showed strong antimicrobial activity againstS.aureusand moderate against others. Whereas the crude aqueous extracts ofOcimum kilimandsacharicumshowed moderate activity against the gram positive and gram negative organisms and strong activity againstC. albicansat higher concentration, same as that shown by the standard forC. albicans.

scholarly journals Characterization and Antimicrobial Activities of a Metabolite from a New Streptomyces Species from Bangladeshi Soil

2009 ◽  
Vol 2 (1) ◽  
pp. 178-185
Author(s):  
M. Akhand ◽  
M. A. A. Al-Bari ◽  
M. A. Islam ◽  
Proma Khondkar
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Activities ◽  
Assay Method ◽  
Streptomyces Species ◽  
Isolation And Characterization ◽  
Test Organisms ◽  
Actinomycete Strain ◽  
Diffusion Assay ◽  
Strong Antimicrobial Activity

A new actinomycete strain was isolated from Western part of Bangladesh and identified as a new Streptomyces species on the basis of its morphological, biochemical, cultural characteristics and 16S rRNA data. The present paper describes the isolation and characterization of compound 1 from this new Streptomyces species with the help of various chemical and spectroscopic methods. Antimicrobial activity of compound 1 was tested by disc diffusion assay method and compared with that of standard antibiotics (Kanamycin for antibacterial activity and Nystatin for antifungal activity). The compound has been found to exhibit moderate to strong antimicrobial activity against the test organisms. Cytotoxicity of the compound 1 and the pet. ether extract of Czapek Dox (alkaline) broth of Streptomyces species was evaluated in brine shrimp bioassay with LC50 values of 23.85 µg/ml (ppm) and 19.95 µg/ml (ppm), respectively. Keywords: Streptomyces; Antimicrobial activity; Cytotoxicity. © 2010 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. DOI: 10.3329/jsr.v2i1.3079                  J. Sci. Res. 2 (1), 178-185 (2010)  

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