In silico Determination of Some Conditions Leading to Glycolytic Oscillations and Their Interference With Some Other Processes in E. coli Cells

The technique of Z-contrast in STEM offers the possibility to determine the local concentration of macromolecules like lipids, proteins or DNA. Contrast formation depends on the atomic composition of the particular structure. In the case of DNA, its phosphorous content discriminates it from other biological macromolecules. In our studies, sections of E. coli, the dinoflagellate Amphidinium carterae and Euglena spec. cells were used which were obtained by cryofixation followed by freeze-substitution into acetone with 3% glutaraldehyde. The samples were then embedded either in Lowicryl HM20 at low temperature or in Epon at high temperature. Sections were coated on both sides with 30Å carbon.The DF- and the inelastic image have been recorded simultaneously with a Cryo-STEM. This Cryo-STEM is equipped with a highly dispersive Electron Energy Loss Spectrometer. With this instrument pure Z-contrast can be achieved either with a Filtered DF-image divided by the inelastic image or, as is used in this paper, by dividing the conventional DF-image by an inelastic image which has been recorded with an inelastic detector whose response is dependent on the total energy loss of the inelastically scattered electrons.

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Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

ACADEMIA JOURNAL OF BIOLOGY ◽

10.15625/2615-9023/v40n4.11689 ◽

2018 ◽

Vol 40 (4) ◽

Author(s):

Dang Thi Ngoc Ha ◽

Le Thi Thu Hong ◽

Truong Nam Hai

Keyword(s):

Recombinant Antibody ◽

Blood Type ◽

Soluble Form ◽

Supernatant Fraction ◽

Blood Group Antigens ◽

Single Chain ◽

E Coli ◽

Recombinant Scfv ◽

Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

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Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207322666190412165316 ◽

2019 ◽

Vol 22 (5) ◽

pp. 346-354

Author(s):

Yan A. Ivanenkov ◽

Renat S. Yamidanov ◽

Ilya A. Osterman ◽

Petr V. Sergiev ◽

Vladimir A. Aladinskiy ◽

...

Keyword(s):

Antibacterial Activity ◽

Large Scale ◽

Underlying Mechanism ◽

Luciferase Assay ◽

Reporter System ◽

E Coli ◽

Starting Point ◽

High Concentrations ◽

Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

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Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666191114104850 ◽

2020 ◽

Vol 21 (4) ◽

pp. 316-324

Author(s):

Manica Negahdaripour ◽

Navid Nezafat ◽

Reza Heidari ◽

Nasrollah Erfani ◽

Nasim Hajighahramani ◽

...

Keyword(s):

In Silico ◽

Group Versus ◽

Tlr Agonists ◽

E Coli ◽

Metal Affinity ◽

Th1 And Th2 Cytokines ◽

Ifn Γ ◽

Humoral Responses ◽

Future Work

Background: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. Methods: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. Results: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund’s adjuvant group. Conclusion: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

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Acetate Kinase (AcK) is Essential for Microbial Growth and Betel-derived Compounds Potentially Target AcK, PhoP and MDR Proteins in M. tuberculosis, V. cholerae and Pathogenic E. coli: An in silico and in vitro Study

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190121105851 ◽

2019 ◽

Vol 18 (31) ◽

pp. 2731-2740 ◽

Cited By ~ 3

Author(s):

Sandeep Tiwari ◽

Debmalya Barh ◽

M. Imchen ◽

Eswar Rao ◽

Ranjith K. Kumavath ◽

...

Keyword(s):

Broad Spectrum ◽

In Silico ◽

Pathogenic Bacteria ◽

In Vitro Study ◽

Docking Studies ◽

Acetate Kinase ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

Novel Target

Background: Mycobacterium tuberculosis, Vibrio cholerae, and pathogenic Escherichia coli are global concerns for public health. The emergence of multi-drug resistant (MDR) strains of these pathogens is creating additional challenges in controlling infections caused by these deadly bacteria. Recently, we reported that Acetate kinase (AcK) could be a broad-spectrum novel target in several bacteria including these pathogens. Methods: Here, using in silico and in vitro approaches we show that (i) AcK is an essential protein in pathogenic bacteria; (ii) natural compounds Chlorogenic acid and Pinoresinol from Piper betel and Piperidine derivative compound 6-oxopiperidine-3-carboxylic acid inhibit the growth of pathogenic E. coli and M. tuberculosis by targeting AcK with equal or higher efficacy than the currently used antibiotics; (iii) molecular modeling and docking studies show interactions between inhibitors and AcK that correlate with the experimental results; (iv) these compounds are highly effective even on MDR strains of these pathogens; (v) further, the compounds may also target bacterial two-component system proteins that help bacteria in expressing the genes related to drug resistance and virulence; and (vi) finally, all the tested compounds are predicted to have drug-like properties. Results and Conclusion: Suggesting that, these Piper betel derived compounds may be further tested for developing a novel class of broad-spectrum drugs against various common and MDR pathogens.

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Predicting the bactericidal efficacy of solar disinfection (SODIS): from kinetic modeling of in vitro tests towards the in silico forecast of E. coli inactivation

Chemical Engineering Journal ◽

10.1016/j.cej.2021.130866 ◽

2021 ◽

pp. 130866

Author(s):

Sofia Samoili ◽

Giulio Farinelli ◽

José Ángel Moreno-SanSegundo ◽

Kevin G. McGuigan ◽

Javier Marugán ◽

...

Keyword(s):

Kinetic Modeling ◽

In Silico ◽

In Vitro Tests ◽

Solar Disinfection ◽

E Coli ◽

Bactericidal Efficacy

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ANTIMICROBIAL AND ANTIOXIDANT EFFECT OF NATURAL EXTRACTS FROM LEAVES, ROOT, STEM AND FLOWERS OF BACCHARIS LATIFOLIA FROM ECUADOR

International Journal of Current Pharmaceutical Research ◽

10.22159/ijcpr.2020v12i2.37495 ◽

2020 ◽

pp. 78-84

Author(s):

FAVIAN BAYAS-MOREJON ◽

ANGELICA TIGRE ◽

RIVELINO RAMON ◽

DANILO YANEZ

Keyword(s):

Antimicrobial Activity ◽

Antioxidant Activity ◽

Potassium Phosphate ◽

Natural Antioxidants ◽

Butylated Hydroxytoluene ◽

Penicillin G ◽

Diffusion Method ◽

E Coli ◽

Antibacterial And Antioxidant Activity

Objective: The increase in chronic and degenerative diseases and the use of synthetic antioxidants such as (butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT)) are being restricted because they can be considered carcinogenic. Therefore, there is a growing interest in the search for natural antioxidants, especially from plants, due to their content in different bioactive compounds, such as antioxidants and antimicrobials. To evaluate the antibacterial and antioxidant activity of Baccharislatifolia extracts. Methods: For the determination of the antimicrobial activity of extracts of leaves, root, stem and flowers of Baccharislatifolia (Bl), the disk plate diffusion method was used, the strains of Listeria, Salmonella and E. coli were studied; antibiotics Penicillin G and Ciprofloxacin were the controls. For the antioxidant activity, a solution of H2O2 (Abs at 230 nm) was prepared in Potassium Phosphate Monobasic-Sodium Hydroxide buffer. Results: The antimicrobial activity against Listeria and Salmonella, showed that the extracts of leaves and flowers were more effective with inhibition zones>15 mm and>20 mm respectively. In front of E. coli, the extracts of flowers and stem were the best with zones>7.0 mm. Antibiotics studied inhibited the development of Listeria and Salmonella. However, E. coli isolates were resistant. In the antioxidant activity, the flower extract of Bl in 60 mg/ml presents a higher effect with 47.25%. Conclusion: Bl extracts from leaves and flowers were more efficient both in their antimicrobial and antioxidant capacity.

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Determination of genotoxic and antigenotoxic properties of essential oil from Ferula orientalis L. using Ames/Salmonella and E. coli WP2 bacterial test systems

Toxicology and Industrial Health ◽

10.1177/0748233712462479 ◽

2012 ◽

Vol 30 (8) ◽

pp. 714-723 ◽

Cited By ~ 3

Author(s):

Hakan Ozkan ◽

Derya Yanmis ◽

Mehmet Karadayi ◽

Tugba Bal ◽

Ozlem Baris ◽

...

Keyword(s):

Essential Oil ◽

E Coli ◽

Test Systems

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Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.7880-7887.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 7880-7887 ◽

Cited By ~ 228

Author(s):

Sang Jun Lee ◽

Dong-Yup Lee ◽

Tae Yong Kim ◽

Byung Hun Kim ◽

Jinwon Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Succinic Acid ◽

In Silico ◽

Acid Production ◽

Gene Knockout ◽

Fermentation Products ◽

E Coli ◽

Metabolic Analysis ◽

Enhanced Production ◽

Succinic Acid Production

ABSTRACT Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.

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ELISA- and Activity Assay-Based Quantification of BMP-2 Released In Vitro Can Be Biased by Solubility in “Physiological” Buffers and an Interfering Effect of Chitosan

Pharmaceutics ◽

10.3390/pharmaceutics13040582 ◽

2021 ◽

Vol 13 (4) ◽

pp. 582

Author(s):

Julius Sundermann ◽

Steffen Sydow ◽

Laura Burmeister ◽

Andrea Hoffmann ◽

Henning Menzel ◽

...

Keyword(s):

Matrix Effects ◽

In Vitro Release ◽

Tween 20 ◽

Activity Assay ◽

E Coli ◽

Reversible Aggregation ◽

Aggregation Tendency ◽

Bio Assay

Chitosan nanogel-coated polycaprolactone (PCL) fiber mat-based implant prototypes with tailored release of bone morphogenic protein 2 (BMP-2) are a promising approach to achieve implant-mediated bone regeneration. In order to ensure reliable in vitro release results, the robustness of a commercially available ELISA for E. coli-derived BMP-2 and the parallel determination of BMP-2 recovery using a quantitative biological activity assay were investigated within a common release setup, with special reference to solubility and matrix effects. Without bovine serum albumin and Tween 20 as solubilizing additives to release media buffed at physiological pH, BMP‑2 recoveries after release were notably reduced. In contrast, the addition of chitosan to release samples caused an excessive recovery. A possible explanation for these effects is the reversible aggregation tendency of BMP-2, which might be influenced by an interaction with chitosan. The interfering effects highlighted in this study are of great importance for bio-assay-based BMP-2 quantification, especially in the context of pharmaceutical release experiments.

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