scholarly journals Synthesis of Ferulenol by Engineered Escherichia coli: Structural Elucidation by Using the In Silico Tools

Molecules ◽  
2021 ◽  
Vol 26 (20) ◽  
pp. 6264
Author(s):  
Anuwatchakij Klamrak ◽  
Jaran Nabnueangsap ◽  
Ploenthip Puthongking ◽  
Natsajee Nualkaew
Keyword(s):  
In Silico ◽  
Molecular Formula ◽  
Bioactive Substances ◽  
E Coli ◽  
Isomeric Structure ◽  
Anti Cancer ◽  
Genes Encoding ◽  
Derivatives Of ◽  
In Silico Tools

4-Hydroxycoumarin (4HC) has been used as a lead compound for the chemical synthesis of various bioactive substances and drugs. Its prenylated derivatives exhibit potent antibacterial, antitubercular, anticoagulant, and anti-cancer activities. In doing this, E. coli BL21(DE3)pLysS strain was engineered as the in vivo prenylation system to produce the farnesyl derivatives of 4HC by coexpressing the genes encoding Aspergillus terreus aromatic prenyltransferase (AtaPT) and truncated 1-deoxy-D-xylose 5-phosphate synthase of Croton stellatopilosus (CstDXS), where 4HC was the fed precursor. Based on the high-resolution LC-ESI(±)-QTOF-MS/MS with the use of in silico tools (e.g., MetFrag, SIRIUS (version 4.8.2), CSI:FingerID, and CANOPUS), the first major prenylated product (named compound-1) was detected and ultimately elucidated as ferulenol, in which information concerning the correct molecular formula, chemical structure, substructures, and classifications were obtained. The prenylated product (named compound-2) was also detected as the minor product, where this structure proposed to be the isomeric structure of ferulenol formed via the tautomerization. Note that both products were secreted into the culture medium of the recombinant E. coli and could be produced without the external supply of prenyl precursors. The results suggested the potential use of this engineered pathway for synthesizing the farnesylated-4HC derivatives, especially ferulenol.

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

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.

scholarly journals Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis in Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 4975-4983 ◽  
Author(s):  
Blaine A. Legaree ◽  
Calvin B. Adams ◽  
Anthony J. Clarke
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Morphological Changes ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Prolonged Incubation ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Derivatives Of

ABSTRACT Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction

1999 ◽  
Vol 181 (22) ◽  
pp. 7143-7148 ◽  
Author(s):  
F. Martinez-Morales ◽  
A. C. Borges ◽  
A. Martinez ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Helper Plasmid ◽  
Flp Recombinase ◽  
E Coli ◽  
Homologous Fragment ◽  
Genes Encoding ◽  
Dna Insertion ◽  
K 12 ◽  
Multiple Cloning Sites

ABSTRACT A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain twoFRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomaladhE gene in strains of E. coli K-12 andE. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

scholarly journals Evaluation of 2-Thioxoimadazolidin-4-One Derivatives as Potent Anti-Cancer Agents through Apoptosis Induction and Antioxidant Activation: In Vitro and In Vivo Approaches

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 83
Author(s):  
Mohamed S. Nafie ◽  
Ahmed I. Khodair ◽  
Hebat Allah Y. Hassan ◽  
Noha M. Abd El-Fadeal ◽  
Hanin A. Bogari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
In Silico ◽  
Hepg2 Cells ◽  
Flow Cytometric Analysis ◽  
Apoptosis Induction ◽  
Rt Pcr ◽  
Anti Cancer ◽  
Ic50 Values

Background: Hepatocellular carcinoma (HCC) is one of the most widespread malignancies and is reported as the fourth most prevalent cause of cancer deaths worldwide. Therefore, we aimed to investigate the probable mechanistic cytotoxic effect of the promising 2-thioxoimidazolidin-4-one derivative on liver cancer cells using in vitro and in vivo approaches. The compounds were tested for the in vitro cytotoxic activity using MTT assay, and the promising compound was tested in colony forming unit assay, flow cytometric analysis, RT-PCR, Western blotting, in vivo using SEC-carcinoma and in silico to highlight the virtual mechanism of action. Both compounds 4 and 2 performed cytotoxic effects against HepG2 cells with IC50 values of 0.017 and 0.18 μM, respectively, compared to Staurosporine and 5-Fu as reference drugs with IC50 values of 5.07 and 5.18 µM, respectively. Compound 4 treatment revealed apoptosis induction by 19.35-fold (11.42% compared to 0.59% in control), arresting the cell cycle at G2/M phase. Moreover, studying gene expression that plays critical roles in cell cycle and apoptosis by RT-PCR demonstrated that compound 4 enhances the expression of the pro-apoptotic genes p53, PUMA, and Caspase 3, 8, and 9, and impedes the anti-apoptotic Bcl-2 gene in the HepG2 cells. It can also inhibit the PI3K/AKT pathway at both gene and protein levels, which was reinforced by the in silico predictions of the molecular docking simulations towards the PI3K/AKT proteins. Finally, in vivo study verified that compound 4 has a promising anti-cancer activity through activating antioxidant levels (CAT, SOD and GSH) and ameliorating hematological, biochemical, and histopathological findings.

scholarly journals Recent study on the anticancer activity of nobiletin and its metabolites

2021 ◽  
Vol 14 ◽  
Author(s):  
Ke Lv ◽  
Lingling Zhang ◽  
Hui Zhao ◽  
Chi-Tang Ho ◽  
Shiming Li
Keyword(s):  
Cancer Prevention ◽  
Anticancer Activity ◽  
Promising Candidate ◽  
Naturally Occurring ◽  
Antitumor Potential ◽  
Anti Cancer ◽  
Citrus Peels ◽  
Derivatives Of ◽  
Cancer Activity

The exploration of naturally occurring phytochemicals with antitumor potential has been the focus of many studies. Nobiletin, a polymethoxyflavone (PMF) exclusively derived from citrus peels, has been reported as a promising candidate for the prevention and/or treatment of cancers. Additionally, multiple demethylated derivatives of nobiletin from in vivo biotransformation, including 3′-demethylnobiletin (3′-DMN), 4′-demethylnobiletin (4′-DMN), 3′,4′-didemethylnobiletin (3′,4′-DDMN) and 5-demethylnobiletin (5-DMN), among others, have been found to show anti-cancer activity. In this review, the anticancer activity of nobiletin and its derivatives in cancer prevention are comprehensively described.

scholarly journals Functional Differences between Heme Permeases: Serratia marcescens HemTUV Permease Exhibits a Narrower Substrate Specificity (Restricted to Heme) Than the Escherichia coli DppABCDF Peptide-Heme Permease

2008 ◽  
Vol 190 (6) ◽  
pp. 1866-1870 ◽  
Author(s):  
Sylvie Létoffé ◽  
Philippe Delepelaire ◽  
Cécile Wandersman
Keyword(s):  
Escherichia Coli ◽  
Serratia Marcescens ◽  
Outer Membrane Receptor ◽  
Heme Uptake ◽  
Periplasmic Binding Protein ◽  
E Coli ◽  
Iron Source ◽  
Functional Differences ◽  
Genes Encoding

ABSTRACT Serratia marcescens hemTUV genes encoding a potential heme permease were cloned in Escherichia coli recombinant mutant FB827 dppF::Km(pAM 238-hasR). This strain, which expresses HasR, a foreign heme outer membrane receptor, is potentially capable of using heme as an iron source. However, this process is invalidated due to a dppF::Km mutation which inactivates the Dpp heme/peptide permease responsible for heme, dipeptide, and δ-aminolevulinic (ALA) transport through the E. coli inner membrane. We show here that hemTUV genes complement the Dpp permease for heme utilization as an iron source and thus are functional in E. coli. However, hemTUV genes do not complement the Dpp permease for ALA uptake, indicating that the HemTUV permease does not transport ALA. Peptides do not inhibit heme uptake in vivo, indicating that, unlike Dpp permease, HemTUV permease does not transport peptides. HemT, the periplasmic binding protein, binds heme. Heme binding is saturable and not inhibited by peptides that inhibit heme uptake by the Dpp system. Thus, the S. marcescens HemTUV permease and, most likely, HemTUV orthologs present in many gram-negative pathogens form a class of heme-specific permeases different from the Dpp peptide/heme permease characterized in E. coli.

scholarly journals Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

2015 ◽  
Vol 83 (4) ◽  
pp. 1384-1395 ◽  
Author(s):  
Aimee Tan ◽  
Nicola K. Petty ◽  
Dianna Hocking ◽  
Vicki Bennett-Wood ◽  
Matthew Wakefield ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Gene Repertoire ◽  
Citrobacter Rodentium ◽  
Environmental Strain ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogenCitrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and thegrlRAoperon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genusEscherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated thatregAhas been horizontally transferred toEscherichiaspp. andC. rodentium. Comparative studies of two RegA homologues, namely, those fromC. rodentiumandE. coliSMS-3-5, a multiresistant environmental strain ofE. coli, showed that the two regulators acted similarlyin vitrobut differed in terms of their abilities to activate the virulence ofC. rodentiumin vivo, which evidently was due to their differential activation ofgrlRA. Our data indicate that RegA fromC. rodentiumhas strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence byC. rodentiumand on the evolution of virulence-regulatory genes of bacterial pathogens in general.

scholarly journals Redundancy and specificity of multiple trigger factor chaperones in Desulfitobacteria

Microbiology ◽  
2011 ◽  
Vol 157 (8) ◽  
pp. 2410-2421 ◽  
Author(s):  
Julien Maillard ◽  
Pierre Genevaux ◽  
Christof Holliger
Keyword(s):  
Trigger Factor ◽  
Organohalide Respiration ◽  
Macromolecular Complexes ◽  
Reductive Dehalogenase ◽  
E Coli ◽  
Twin Arginine Translocation ◽  
Growth Defects ◽  
Heterologous System ◽  
Genes Encoding

The ribosome-bound trigger factor (TF) chaperone assists folding of newly synthesized polypeptides and participates in the assembly of macromolecular complexes. In the present study we showed that multiple distinct TF paralogues are present in genomes of Desulfitobacteria, a bacterial genus known for its ability to grow using organohalide respiration. Two full-length TF chaperones and at least one truncated TF (lacking the N-terminal ribosome-binding domain) were identified, the latter being systematically linked to clusters of reductive dehalogenase genes encoding the key enzymes in organohalide respiration. Using a well-characterized heterologous chaperone-deficient Escherichia coli strain lacking both TF and DnaK chaperones, we demonstrated that all three TF chaperones were functional in vivo, as judged by their ability to partially suppress bacterial growth defects and protein aggregation in the absence of both major E. coli chaperones. Next, we found that the N-terminal truncated TF-like protein PceT functions as a dedicated chaperone for the cognate reductive dehalogenase PceA by solubilizing and stabilizing it in the heterologous system. Finally, we showed that PceT specifically interacts with the twin-arginine signal peptide of PceA. Taken together, our data define PceT (and more generally the new RdhT family) as a class of TF-like chaperones involved in the maturation of proteins secreted by the twin-arginine translocation pathway.

scholarly journals Screening of phytochemicals from Golden apple of discord, Cydonia oblonga against the pTen and HBx using in silico-based tools.

2021 ◽  
Author(s):  
Elhan Khan ◽  
Iffat Zareen Ahmad
Keyword(s):  
Hepatocellular Carcinoma ◽  
In Silico ◽  
Experimental Studies ◽  
Docking Study ◽  
Cydonia Oblonga ◽  
Standard Drug ◽  
Anti Cancer ◽  
Anti Oxidant ◽  
Inhibitory Potential ◽  
Derivatives Of

Abstract Background Phytochemicals derived from Cydonia oblonga have been investigated for their anti-oxidant and anti-cancer activities in many cancer cells lines. The reported bioactive compounds are evaluated in-silico to develop a novel antagonist against pTEN and HBx to target hepatocellular carcinoma. Lower expression of pTEN or higher expression of HBx represents the progression of hepatocellular carcinoma (HCC). Objective This research is intended to identify the best candidate which interacts with our target proteins (pTEN and HBx) from the quince seeds by using computational methodologies. Materials and Methods The ternary structures of protein and phytochemicals reported in quince like derivatives of caffeoylquinic acid, kaempferol and quercetin, Chrysoeriol, Catechin and other compounds are retrieved from the online databanks. Druglikness analysis and ADMET profiling was done, followed by docking analysis. Lastly, molecular dynamics study was done to determine the complexes stability. Results Docking study revealed that CQA derivatives have the significant inhibitory potential of 3CQA and 5CQA with binding affinity of -7.53 and -7.49 against pTEN and -5.94 and -6.01 against HBx in comparison to the standard drug doxorubicin. The average RMSD and RMSF value for protein-ligand complexes were found quite stable comparative to the standard, while parameters like gyration and SASA supports the complexes having the significant binding and stability especially against pTEN. Conclusion The results obtained from the evaluation depict 3CQA and 5CQA shows best stability especially with the p10 protein target. Hence, these compounds have to be considered for detailed experimental studies to understand its biological function against HCC.

scholarly journals Isolation of RNase H Genes That Are Essential for Growth of Bacillus subtilis 168

1999 ◽  
Vol 181 (7) ◽  
pp. 2118-2123 ◽  
Author(s):  
Mitsuhiro Itaya ◽  
Akira Omori ◽  
Shigenori Kanaya ◽  
Robert J. Crouch ◽  
Teruo Tanaka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rnase H ◽  
Positive Bacterium ◽  
E Coli ◽  
Cellular Processes ◽  
Dna Library ◽  
Bacillus Subtilis 168 ◽  
Genes Encoding

ABSTRACT Two genes encoding functional RNase H (EC 3.1.26.4 ) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilisRNase HIII. The B. subtilis genome has anrnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB andrnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both thernhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the singlernhC homologue in the small genome ofMycoplasma species.

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