scholarly journals Affinity labelling in situ of the bL12 protein on E. coli 70S ribosomes by means of a tRNA dialdehyde derivative

2017 ◽  
Vol 162 (6) ◽  
pp. 437-448 ◽  
Author(s):  
Codjo Hountondji ◽  
Jean-Bernard Créchet ◽  
Jean-Pierre Le Caër ◽  
Véronique Lancelot ◽  
Jean A H Cognet ◽  
...  
Keyword(s):  
E Coli ◽  
Affinity Labelling

Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

2020 ◽  
Author(s):  
Feifei Jia ◽  
Jie Wang ◽  
Yanyan Zhang ◽  
Qun Luo ◽  
Luyu Qi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Single Cells ◽  
Time Of Flight ◽  
E Coli ◽  
Tof Sims ◽  
Ion Mass Spectrometry ◽  
Chemical Tags ◽  
Secondary Ion

<p></p><p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ </i>visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.</p><p></p>

scholarly journals DnaA, the Initiator of Escherichia coliChromosomal Replication, Is Located at the Cell Membrane

2000 ◽  
Vol 182 (9) ◽  
pp. 2604-2610 ◽  
Author(s):  
Gillian Newman ◽  
Elliott Crooke
Keyword(s):  
Cell Membrane ◽  
Spatial Organization ◽  
Active Form ◽  
Chromosomal Replication ◽  
E Coli ◽  
Dnaa Protein ◽  
Section Electron ◽  
Acidic Phospholipids

ABSTRACT Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.

scholarly journals Microchannelled alkylated chitosan sponge to treat noncompressible hemorrhages and facilitate wound healing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xinchen Du ◽  
Le Wu ◽  
Hongyu Yan ◽  
Zhuyan Jiang ◽  
Shilin Li ◽  
...  
Keyword(s):  
Wound Healing ◽  
Shape Recovery ◽  
Parenchymal Cell ◽  
Gelatin Sponge ◽  
Defect Model ◽  
Liver Parenchymal Cell ◽  
E Coli ◽  
Tissue Integration ◽  
3D Printed

AbstractDeveloping an anti-infective shape-memory hemostatic sponge able to guide in situ tissue regeneration for noncompressible hemorrhages in civilian and battlefield settings remains a challenge. Here we engineer hemostatic chitosan sponges with highly interconnective microchannels by combining 3D printed microfiber leaching, freeze-drying, and superficial active modification. We demonstrate that the microchannelled alkylated chitosan sponge (MACS) exhibits the capacity for water and blood absorption, as well as rapid shape recovery. We show that compared to clinically used gauze, gelatin sponge, CELOX™, and CELOX™-gauze, the MACS provides higher pro-coagulant and hemostatic capacities in lethally normal and heparinized rat and pig liver perforation wound models. We demonstrate its anti-infective activity against S. aureus and E. coli and its promotion of liver parenchymal cell infiltration, vascularization, and tissue integration in a rat liver defect model. Overall, the MACS demonstrates promising clinical translational potential in treating lethal noncompressible hemorrhage and facilitating wound healing.

scholarly journals Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

2016 ◽  
Vol 12 ◽  
pp. 2588-2601 ◽  
Author(s):  
Vladimir A Stepchenko ◽  
Anatoly I Miroshnikov ◽  
Frank Seela ◽  
Igor A Mikhailopulo
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Reaction Conditions ◽  
E Coli ◽  
No Formation ◽  
Structural Peculiarities ◽  
Substrate Properties ◽  
Nucleoside Phosphorylases ◽  
Derivatives Of

The trans-2-deoxyribosylation of 4-thiouracil (4SUra) and 2-thiouracil (2SUra), as well as 6-azauracil, 6-azathymine and 6-aza-2-thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP) for the in situ generation of 2-deoxy-α-D-ribofuranose-1-phosphate (dRib-1P) followed by its coupling with the bases catalyzed by either E. coli thymidine (TP) or uridine (UP) phosphorylases. 4SUra revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2’-deoxy-2-thiouridine (2SUd) was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, 2SU, 2SUd, 4STd and 2STd are good substrates for both UP and TP; moreover, 2SU, 4STd and 2’-deoxy-5-azacytidine (Decitabine) are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of 2SUra and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave 2SUd and 2’-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl- and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.

scholarly journals Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Nanomaterials ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1661
Author(s):  
Mei-Hsiu Chen ◽  
Tse-Ying Liu ◽  
Yu-Chiao Chen ◽  
Ming-Hong Chen
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Glioma Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
E Coli ◽  
Tumor Bearing ◽  
Nano Gold ◽  
Gl261 Cell

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.

scholarly journals Rapid Detection, Identification, and Enumeration ofEscherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

2001 ◽  
Vol 67 (1) ◽  
pp. 142-147 ◽  
Author(s):  
Henrik Stender ◽  
Adam J. Broomer ◽  
Kenneth Oliveira ◽  
Heather Perry-O'Keefe ◽  
Jens J. Hyldig-Nielsen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Sensitivity And Specificity ◽  
Bacterial Species ◽  
Rdna Sequence ◽  
Sequence Information ◽  
Sequence Alignments ◽  
E Coli ◽  
Municipal Water

ABSTRACT A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturableEscherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coliwere detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targetingP. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other thanE. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

Antibacterial Activity of Surface-Modified Fabric with Ag/AgCl-Doped Quaternary Ammonium-Modified Silicate Hybrid

2019 ◽  
Vol 19 (11) ◽  
pp. 7285-7293 ◽  
Author(s):  
Kuo-Hui Wu ◽  
Yin-Chiung Chang ◽  
Ken-Fa Cheng ◽  
Je-Chuang Wang
Keyword(s):  
Colloidal Suspension ◽  
Sol Gel ◽  
Antimicrobial Properties ◽  
Woven Fabrics ◽  
Dipolar Interactions ◽  
Zone Of Inhibition ◽  
E Coli ◽  
Antibacterial Material ◽  
Surface Modified

An organic–inorganic hybrid antibacterial material based on Ag/AgCl and quaternary ammoniummodified silicate (Ormosil (NR+4 Cl-)) was prepared by sol–gel processes and an in situ reduction method, in which silver nitrate, tetraethoxysilane (TEOS), N-trimethoxysilylpropyl-N,N,Ntrimethylammonium chloride (TMAPS) and N-[3-(trimethoxysilyl)propyl]diethylenetriamine (ATS) acted as precursor, linker and colloidal suspension stabilizer, respectively. The physical properties of the Ormosil (NR+4 Cl-) and Ormosil (NR+4 Cl-)/Ag hybrids were examined using XPS and TGA spectroscopy, the results of which indicated that Ag/AgCl was immobilized in channels by dipolar interactions with the Ormosil (NR+4 Cl-) matrix. Ormosil (NR+4 Cl-) and Ormosil (NR+4 Cl-)/Ag sol solutions were used to functionalize non-woven fabrics by simply immersing the textiles into the dispersions in order to obtain antimicrobial fabrics. The antibacterial effects of the Ormosil/Ag- and Ormosil (NR+4 Cl-)/Ag-modified fabrics against Gram-negative P. aeruginosa and E. coli, and Grampositive S. aureus and B. subtilis, were assessed using the zone of inhibition and the plate-counting method. The antibacterial efficacy was maintained even after 20 laundry cycles. The results indicated that the Ormosil/Ag and Ormosil (NR+4 Cl-)/Ag hybrids are useful as non-leaching agents imparting durable antimicrobial properties to fabrics.

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