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1.
Enhanced antibacterial activity of calcium silicate-based hybrid cements for bone repair.
Lin, MC, Chen, CC, Wu, IT, Ding, SJ
Materials science & engineering. C, Materials for biological applications. 2020;:110727
Abstract
Calcium silicate cement has attracted much attention for bone defect repair and regeneration due to its osteogenic properties. Biomaterial-associated infections and washout have become a common clinical problem. In order to enhance the antibacterial and washout performance of calcium silicate cement to meet clinical needs, different types of chitosan, including chitosan polysaccharide (CTS), quaternary ammonium chitosan (QTS), and chitosan oligosaccharide (COS), as a liquid phase were added to the calcium silicate powder. The physicochemical properties, in vitro bioactivity, antibacterial efficacy, and osteogenic effects (MG63 cells) of the cement were evaluated. Antibacterial activity was conducted with Gram-negative Escherichia coli (E. coli) and a Gram-positive Staphylococcus aureus (S. aureus) bacteria. The amount of intracellular reactive oxygen species (ROS) produced in the bacteria cultured with the chitosan solution was also detected. The experimental results showed that the chitosan additive did not affect the crystalline phase of calcium silicate cement, but increased the setting time and strength of the cement in a concentration-dependent manner. Within the scope of this study, CTS and QTS solutions with a concentration of not <1 wt% improved the washout resistance of the control cement, while the COS solutions failed to strengthen the cement. When soaked in simulated body fluid (SBF) for 1 day, all cement samples formed apatite spherules. As the soaking time increased, the diametral tensile strength of all cements decreased and the porosity increased. The assays of MG63 cell function showed lower osteogenic activity of osteoblastic cells grown on the surfaces of the chitosan-incorporated cements in comparison with the control cement without chitosan. At the same 1% concentration, compared with QTS and COS cement, CTS cement had lower cell attachment, proliferation, differentiation, and mineralization. Conversely, the CTS cement resulted in the highest bacteriostasis ratio among the three hybrid cements against two bacteria. The ROS production followed the order of CTS > QTS > COS at the same 1% concentration. In conclusion, calcium silicate cement with 1% QTS may be a viable candidate for bone defect repair in view of anti-washout performance, setting time, antibacterial activity, and osteogenic activity shown in this study.
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2.
Synthesis and characterization of cellulose/TiO2 nanocomposite: Evaluation of in vitro antibacterial and in silico molecular docking studies.
M V, A, Harb, M, Sundaram, R
Carbohydrate polymers. 2020;:116868
Abstract
Cellulose/TiO2 nanocomposite was synthesized using coagulation in sodium hydroxide-thiourea-urea aqueous solution medium by precipitation method. This method was accomplished green and cost-effective for the fabrication of composite nanomaterials. Structure, morphology and optical properties of the nanocomposite were characterized by X-ray diffraction, energy dispersive X-ray spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and ultraviolet diffuse reflectance spectra respectively. XRD results showed the anatase structure of TiO2 while FESEM micrograph showed evidence of particle size ranging from 20 to 40 nm for cellulose/TiO2 nanocomposite. The Fourier transfer infrared spectroscopy investigation reveals that the TiO2 is bound to hydroxyl groups to the cellulose by hydrogen bonding. The optical energy bandgap is found to be 2.71 eV for nanocomposite from the UV-DRS. The mechanical strength of the composites gently escalated with the addition of TiO2 nanoparticles into cellulose polymer matrix. Cellulose/TiO2 nanocomposite was screened for their in vitro antibacterial activity against Staphylococcus aureus and Escherichia coli bacteria have been investigated. Additionally, the results obtained from in silico molecular docking studies confirm the interaction of nanocomposite with proteins, were in good agreement with the experimental data. This finding provides a novel and simple method for the synthesis of cellulose/TiO2 nanocomposite as functional biomaterials.
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3.
Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis.
Smolskaya, S, Logashina, YA, Andreev, YA
International journal of molecular sciences. 2020;(3)
Abstract
Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.
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4.
Enzymatic reactions and microorganisms producing the various isomers of hydroxyproline.
Hara, R, Kino, K
Applied microbiology and biotechnology. 2020;(11):4771-4779
Abstract
Hydroxyproline is an industrially important compound with applications in the pharmaceutical, nutrition, and cosmetic industries. trans-4-Hydroxy-L-proline is recognized as the most abundant of the eight possible isomers (hydroxy group at C-3 or C-4, cis- or trans-configuration, and L- or D-form). However, little attention has been paid to the rare isomers, probably due to their limited availability. This mini-review provides an overview of recent advances in microbial and enzymatic processes to develop practical production strategies for various hydroxyprolines. Here, we introduce three screening strategies, namely, activity-, sequence-, and metabolite-based approaches, allowing identification of diverse proline-hydroxylating enzymes with different product specificities. All naturally occurring hydroxyproline isomers can be produced by using suitable hydroxylases in a highly regio- and stereo-selective manner. Furthermore, crystal structures of relevant hydroxylases provide much insight into their functional roles. Since hydroxylases acting on free L-proline belong to the 2-oxoglutarate-dependent dioxygenase superfamily, cellular metabolism of Escherichia coli coupled with a hydroxylase is a valuable source of 2-oxoglutarate, which is indispensable as a co-substrate in L-proline hydroxylation. Further, microbial hydroxyproline 2-epimerase may serve as a highly adaptable tool to convert L-hydroxyproline into D-hydroxyproline. KEY POINTS • Proline hydroxylases serve as powerful tools for selectivel-proline hydroxylation. • Engineered Escherichia coli are a robust platform for hydroxyproline production. • Hydroxyproline epimerase convertsl-hydroxyproline intod-hydroxyproline.
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5.
The biosynthesis of the molybdenum cofactors in Escherichia coli.
Leimkühler, S
Environmental microbiology. 2020;(6):2007-2026
Abstract
The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5'-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years.
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6.
Making iron-sulfur cluster: structure, regulation and evolution of the bacterial ISC system.
Baussier, C, Fakroun, S, Aubert, C, Dubrac, S, Mandin, P, Py, B, Barras, F
Advances in microbial physiology. 2020;:1-39
Abstract
Iron sulfur (Fe-S) clusters rank among the most ancient and conserved prosthetic groups. Fe-S clusters containing proteins are present in most, if not all, organisms. Fe-S clusters containing proteins are involved in a wide range of cellular processes, from gene regulation to central metabolism, via gene expression, RNA modification or bioenergetics. Fe-S clusters are built by biogenesis machineries conserved throughout both prokaryotes and eukaryotes. We focus mostly on bacterial ISC machinery, but not exclusively, as we refer to eukaryotic ISC system when it brings significant complementary information. Besides covering the structural and regulatory aspects of Fe-S biogenesis, this review aims to highlight Fe-S biogenesis facets remaining matters of discussion, such as the role of frataxin, or the link between fatty acid metabolism and Fe-S homeostasis. Last, we discuss recent advances on strategies used by different species to make and use Fe-S clusters in changing redox environmental conditions.
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7.
ZnO, AgCl and AgCl/ZnO nanocomposites incorporated chitosan in the form of hydrogel beads for photocatalytic degradation of MB, E. coli and S. aureus.
Taghizadeh, MT, Siyahi, V, Ashassi-Sorkhabi, H, Zarrini, G
International journal of biological macromolecules. 2020;:1018-1028
Abstract
Significant improvement of effective and low-cost decolorization and disinfecting technologies is required to address the problems created by dyes and dangerous microorganisms from water and wastewaters. This article expresses the degradation of methylene blue (MB), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as gram negative and positive bacteria via a chitosan/AgCl/ZnO (CS/AgCl/ZnO) nanocomposite hydrogel beads system as a photocatalyst under visible light irradiation. The techniques such as FT-IR, SEM, EDAX, TGA, and XRD were applied to recognize the synthesized beads. Decolorization and disinfection experimental results revealed that the hydrogel beads system effectively degrade MB and bacteria. Also, the effects of the initial amount of catalysts, pH, coions and initial concentration of dye on the photocatalytic decolorization were investigated. Moreover, kinetics analysis indicates that the photocatalytic degradation rate of MB and bacteria can be described by Langmuir-Hinshelwood (L-H) and Weibull inactivation models, respectively. We provide a reusable and recoverable effective organic/inorganic photocatalyst in the form of beads that could solve the disadvantages of powder photocatalytic, without reducing the efficiency.
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8.
Probiotic Lactobacillus and Bifidobacterium Strains Counteract Adherent-Invasive Escherichia coli (AIEC) Virulence and Hamper IL-23/Th17 Axis in Ulcerative Colitis, but Not in Crohn's Disease.
Leccese, G, Bibi, A, Mazza, S, Facciotti, F, Caprioli, F, Landini, P, Paroni, M
Cells. 2020;(8)
Abstract
Hypersecretion of proinflammatory cytokines and dysregulated activation of the IL-23/Th17 axis in response to intestinal microbiota dysbiosis are key factors in the pathogenesis of inflammatory bowel diseases (IBD). In this work, we studied how Lactobacillus and Bifidobacterium strains affect AIEC-LF82 virulence mechanisms and the consequent inflammatory response linked to the CCR6-CCL20 and IL-23/Th17 axes in Crohn's disease (CD) and ulcerative colitis (UC) patients. All Lactobacillus and Bifidobacterium strains significantly reduced the LF82 adhesion and persistence within HT29 intestinal epithelial cells, inhibiting IL-8 secretion while not affecting the CCR6-CCL20 axis. Moreover, they significantly reduced LF82 survival within macrophages and dendritic cells, reducing the secretion of polarizing cytokines related to the IL-23/Th17 axis, both in healthy donors (HD) and UC patients. In CD patients, however, only B. breve Bbr8 strain was able to slightly reduce the LF82 persistence within dendritic cells, thus hampering the IL-23/Th17 axis. In addition, probiotic strains were able to modulate the AIEC-induced inflammation in HD, reducing TNF-α and increasing IL-10 secretion by macrophages, but failed to do so in IBD patients. Interestingly, the probiotic strains studied in this work were all able to interfere with the IL-23/Th17 axis in UC patients, but not in CD patients. The different interaction mechanisms of probiotic strains with innate immune cells from UC and CD patients compared to HD suggest that testing on CD-derived immune cells may be pivotal for the identification of novel probiotic strains that could be effective also for CD patients.
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9.
Optimization of enhanced microbial production of zinc bacitracin by submerged fermentation technology.
Hassan, A, Ali, S, Farooq, MA, Tahir, HM, Awan, MU, Mughal, TA
Journal of basic microbiology. 2020;(7):585-599
Abstract
Bacitracin is one of the most important antibiotics used in different biomedical fields. It helps to achieve sizeable amount of foreign exchange due to its use in the poultry feed. The cheap agricultural wastes are readily available for the preparation of fermentation media used for bacitracin production. The microorganisms could be mutated with different chemicals and UV radiation to improve bacitracin production. Thus, the current study was focused on the synthesis of low-cost and effective bacitracin by mutant strains of Bacillus licheniformis, employing the submerged fermentation technique. The bacteria were exposed to the UV irradiation for various time periods ranging from 5 to 40 min. These mutants were named as BLAA-5-BLAA-40. Mutant strain BLAA-25 produced maximum bacitracin, with significantly high activity (142.81 IU/mg) against Klebsiella pneumoniae but less activity against Escherichia coli (115.19 IU/mg). Several fermentation conditions were investigated to optimize bacitracin production. The highest bacitracin yield was obtained by an inoculum size of 10%, fermentation period 48 hr, pH 7, T = 37°C, using soybean meal as a substrate. Among all substrates, cucumber peel was the substrate showing the highest minimum inhibitory concentration (2.3 mg/ml and 2.7 mg/ml against K. pneumoniae and E. coli respectively). A comparison between commercial and experimentally produced Zn bacitracin showed that commercial bacitracin has a low activity (63.2 IU/mg) as compared with experimental bacitracin. Hence, the agro wastes and mutation could be used to increase the synthesis of Zn bacitracin in B. licheniformis.
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10.
Efficient refolding of recombinant reteplase expressed in Escherichia coli strains using response surface methodology.
Fathi-Roudsari, M, Maghsoudi, A, Maghsoudi, N, Niazi, S, Soleiman, M
International journal of biological macromolecules. 2020;:1321-1327
Abstract
Reteplase is a deleted variant of human tissue plasminogen activator with a complex structure containing nine disulfide bonds. Reteplase is expressed as inclusion bodies in Escherichia coli and needs the additional step of refolding for activation. In this study an experimental design was performed to find the optimal refolding condition for reteplase. The influence of 14 chemical additives was assessed by one factor at a time method and then Taguchi design followed by response surface methodology was employed to find compounds with most significant effects on reteplase refolding and their optimum concentration. We found that 0.13 M histidine, 1.64 M methionine, 0.33 M cysteine, and 0.34 M arginine in addition to the GSH/GSSG is the optimal condition for refolding of reteplase. We also investigated the refolding yield for inclusion bodies obtained from different E. coli strains and found that BL21 (DE3) has the best recovery yield in comparison to Rosetta-gami and Shuffle T7.