scholarly journals A Novel In Vitro Method to Assess the Microbial Barrier Function of Tissue Adhesives Using Bioluminescence Imaging Technique

2022 ◽  
Vol 2022 ◽  
pp. 1-10
Author(s):  
Yalda Mirzaei ◽  
Kerstin Hagemeister ◽  
Martina Hüffel ◽  
Timo Schwandt ◽  
René H. Tolba ◽  
...  
Keyword(s):  
Barrier Function ◽  
Wound Closure ◽  
Bioluminescence Imaging ◽  
Imaging Technique ◽  
In Vitro Method ◽  
E Coli ◽  
Tissue Glue ◽  
Bioluminescence Signal

Background. Tissue glues can minimize treatment invasiveness, mitigate the risk of infection, and reduce surgery time; ergo, they have been developed and used in surgical procedures as wound closure devices beside sutures, staples, and metallic grafts. Regardless of their structure or function, tissue glues should show an acceptable microbial barrier function before being used in humans. This study proposes a novel in vitro method using Escherichia coli Lux and bioluminescence imaging technique to assess the microbial barrier function of tissue glues. Different volumes and concentrations of E. coli Lux were applied to precured or cured polyurethane-based tissue glue placed on agar plates. Plates were cultured for 1 h, 24 h, 48 h, and 72 h with bioluminescence signal measurement subsequently. Herein, protocol established a volume of 5 μL of a 1 : 100 dilution of E. coli Lux containing around 2 × 10 7  CFU/mL as optimal for testing polyurethane-based tissue glue. Measurement of OD600nm, determination of CFU/mL, and correlation with the bioluminescence measurement in p/s unit resulted in a good correlation between CFU/mL and p/s and demonstrated good reproducibility of our method. In addition, this in vitro method could show that the tested polyurethane-based tissue glue can provide a reasonable barrier against the microbial penetration and act as a bacterial barrier for up to 48 h with no penetration and up to 72 h with a low level of penetration through the material. Overall, we have established a novel, sensitive, and reproducible in vitro method using the bioluminescence imaging technique for testing the microbial barrier function of new tissue glues.

scholarly journals 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 ◽  
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.

scholarly journals An in vitro method for the determination of protein turnover in incubated proximal tubule segments

1993 ◽  
Vol 43 (5) ◽  
pp. 1156-1159 ◽  
Author(s):  
Robert May ◽  
Brian Logue ◽  
Byrad Edwards ◽  
Swati Patel
Keyword(s):  
Proximal Tubule ◽  
Protein Turnover ◽  
In Vitro Method ◽  
Proximal Tubule Segments

scholarly journals Synthesis of silver nanoparticles from extracts of Scytonema geitleri HKAR-12 and their in vitro antibacterial and antitumor potentials

2019 ◽  
Vol 8 (3) ◽  
pp. 576-585
Keyword(s):  
Silver Nanoparticles ◽  
Coli Strain ◽  
X Ray Diffraction ◽  
Xrd Pattern ◽  
E Coli ◽  
Vis Spectroscopy ◽  
Shape Structure ◽  
Actual Size

In the present study silver nanoparticles (AgNPs) have been synthesized through the cell-free extracts of the rooftop dwelling cyanobacterium Scytonema geitleri HKAR-12. UV-VIS spectroscopy, FTIR, X-ray diffraction, SEM and TEM were used for the determination of morphological, structural and optical properties of synthesized AgNPs. Extracts of Scytonema geitleri HKAR-12 have the ability to reduce AgNO3 to Ag0. Sharp peak at 422 nm indicated the rapid synthesis of AgNPs. FTIR results showed the presence of different groups responsible for the reduction of AgNO3 to AgNPs. XRD pattern confirmed the crystalline nature of AgNPs. SEM showed the bead shape structure of AgNPs. TEM confirmed the actual size of AgNPs to be ranging between 9-17 nm. AgNPs showed antibacterial activity against Pseudomonas aeruginosa, Escherichia coli strain1 and E. coli strain 2 and 11 μg/mL of AgNPs effectively inhibited the growth of MCF-7 cells. Hence, Scytonema geitleri HKAR-12, isolated from the rooftop could serve as a desirable biological candidate for convenient and cheap production of AgNPs having antimicrobial and anti-cancerous properties.

scholarly journals In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

2009 ◽  
Vol 76 (1) ◽  
pp. 264-274 ◽  
Author(s):  
M.-L. Foucault ◽  
L. Thomas ◽  
S. Goussard ◽  
B. R. Branchini ◽  
C. Grillot-Courvalin
Keyword(s):  
Escherichia Coli ◽  
Bioluminescence Imaging ◽  
Light Emission ◽  
Direct Method ◽  
Firefly Luciferase ◽  
Intestinal Colonization ◽  
Bacterial Populations ◽  
Bioluminescence Signal

ABSTRACT Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 = 0.98) or transcutaneously in the abdominal region of whole animals (R 2 = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

Screening of Antimicrobial Activity and Cytotoxic Effects of Two Cladonia Species

2013 ◽  
Vol 68 (5-6) ◽  
pp. 191-197 ◽  
Author(s):  
Birkan Açıkgöz ◽  
İskender Karaltı ◽  
Melike Ersöz ◽  
Zeynep M. Coşkun ◽  
Gülşah Çobanoğlu ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Activities ◽  
Chloroform Extract ◽  
Cytotoxic Effects ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Methanol Extracts ◽  
Gram Positive Bacteria

The present study explores the antimicrobial activity and cytotoxic effects in culture assays of two fruticose soil lichens, Cladonia rangiformis Hoffm. and Cladonia convoluta (Lamkey) Cout., to contribute to possible pharmacological uses of lichens. In vitro antimicrobial activities of methanol and chloroform extracts against two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), two Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), and the yeast Candida albicans were examined using the paper disc method and through determination of minimal inhibitory concentrations (MICs). The data showed the presence of antibiotic substances in the chloroform and the methanol extracts of the lichen species. The chloroform extracts exhibited more signifi cant antimicrobial activity than the methanol extracts. However, a higher antifungal activity was noted in the methanol extract of C. rangiformis. The maximum antimicrobial activity was recorded for the chloroform extract of C. convoluta against E. coli. The cytotoxic effects of the lichen extracts on human breast cancer MCF-7 cells were evaluated by the trypan blue assay yielding IC50 values of ca. 173 and 167 μg/ml for the extracts from C. rangiformis and C. convoluta, respectively.

An in vitro method for the quantitative determination of the antimicrobial efficacy of silver-containing wound dressings

2009 ◽  
Vol 366 (1-2) ◽  
pp. 111-116 ◽  
Author(s):  
Simon Gaisford ◽  
Anthony E. Beezer ◽  
Alistair H. Bishop ◽  
Michael Walker ◽  
David Parsons
Keyword(s):  
Quantitative Determination ◽  
Wound Dressings ◽  
Antimicrobial Efficacy ◽  
In Vitro Method

scholarly journals Development and validation of a method for the determination of the specific activity of recombinant monoclonal antibody eculizumab

2020 ◽  
Vol 15 (2) ◽  
pp. 77-85
Author(s):  
D. I. Zybin ◽  
A. S. Seregin ◽  
A. D. Askretkov ◽  
N. V. Orlova ◽  
Yu. A. Seregin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Pharmaceutical Analysis ◽  
Comparative Evaluation ◽  
Specific Activity ◽  
In Vitro Method ◽  
Recombinant Monoclonal Antibody ◽  
Development And Validation ◽  
First Time

Objectives. Developing reliable and accurate analytical methods is necessary for comparative pharmaceutical analysis using physicochemical, biological (in vitro), preclinical, and clinical trials. The main objective of this study was to develop and validate an in vitro method for determining the specific activity of the recombinant monoclonal antibody eculizumab.Methods. The method of indirect enzyme immunoassay was used in the study.Results. A method for determining the specific activity of the humanized recombinant monoclonal antibody eculizumab was described and validated for the first time. A comparative evaluation of the specific activity of Soliris® (Alexion Pharmaceuticals Inc., USA), and its biosimilar PRK-001 (Pharmapark, Russia) was performed using the developed method.Conclusions. The similarity of PRK-001 and the original Soliris® in relation to their specific activity, that is, binding to the human complement system C5 protein, was proved. 

scholarly journals Pyroptosis engagement and bladder urothelial cell-derived exosomes recruit mast cells and induce barrier dysfunction of bladder urothelium after uropathogenic E. coli infection

2019 ◽  
Vol 317 (3) ◽  
pp. C544-C555 ◽  
Author(s):  
Zonglong Wu ◽  
Yan Li ◽  
Qinggang Liu ◽  
Yaxiao Liu ◽  
Lipeng Chen ◽  
...  
Keyword(s):  
Mast Cells ◽  
Barrier Function ◽  
Urothelial Cells ◽  
Barrier Dysfunction ◽  
Epithelial Barrier Function ◽  
E Coli ◽  
Bladder Urothelium ◽  
Protease Activated Receptor 2

The specific regulatory mechanism of bladder urothelial barrier dysfunction after infection with uropathogenic Escherichia coli (UPEC) is still unclear. The cross talk between bladder urothelial cells and mast cells may play an important role during UPEC infection. In this study, the pyroptosis of urothelial cells was investigated after UPEC infection both in vivo and in vitro. The levels of IL-1β and IL-18 in exosomes derived from bladder urothelial cells after UPEC infection were detected. The role of these processes in the recruitment and activation of mast cells was measured. The mechanism of mast cell-induced disruption of bladder epithelial barrier function was also assessed. We found that UPEC infection induced pyroptosis of bladder urothelial cells and led to the release of IL-1β and IL-18 in the form of exosomes, which promoted the migration of mast cells. Tryptase secreted by mast cells aggravated the damage to the barrier function of the bladder urothelium by acting on protease-activated receptor 2 (PAR2). Inhibition of pyroptosis or the tryptase-PAR2 axis reduced the disruption of bladder urothelial barrier function and decreased the bacterial burden. The present study supports a novel mechanism by which pyroptosis-dependent release of exosomes from bladder urothelial cells activates mast cells and regulates bladder urothelial barrier function during UPEC infection.

scholarly journals In Vitro Activities and Inoculum Effects of Ceftazidime-Avibactam and Aztreonam-Avibactam against Carbapenem-Resistant Enterobacterales Isolates from South Korea

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 912
Author(s):  
Taeeun Kim ◽  
Seung Cheol Lee ◽  
Moonsuk Bae ◽  
Heungsup Sung ◽  
Mi-Na Kim ◽  
...  
Keyword(s):  
Resistance Mechanism ◽  
Fold Increase ◽  
Multidrug Resistant ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Carbapenem Resistant ◽  
High Inoculum ◽  
Inoculum Effect

Ceftazidime-avibactam (CAZ-AVI) and aztreonam-avibactam (AZT-AVI) are novel antibiotic combinations active against multidrug-resistant Gram-negative pathogens. This study aimed to evaluate their in vitro activities and inoculum effects in carbapenem-resistant Enterobacterales (CRE), including carbapenemase-producing (CP)-CRE and non-CP-CRE. A total of 81 independent clinical isolates of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae were collected. CAZ-AVI and AZT-AVI minimal inhibitory concentrations (MICs) were evaluated by broth microdilution using standard and high inocula. The inoculum effect was defined as an ≥8-fold increase in MIC with high inoculum. Phenotypic determination of β-lactam resistance mechanism and PCR for carbapenemase genes were performed. Of the 81 CRE isolates, 35 (43%) were CP-CRE. Overall, 73% of the isolates were susceptible to CAZ-AVI, and 95% had low AZT-AVI MICs (≤8 µg/mL). The MIC50/MIC90s of CAZ-AVI and AZT-AVI were 4/≥512 µg/mL and 0.5/4 µg/mL, respectively. CAZ-AVI was more active against non-CP-CRE than against CP-CRE (susceptibility 80% vs. 63%, p = 0.08; MIC50/MIC90, 2/16 μg/mL vs. 4/≥512 μg/mL), whereas AZT-AVI was more active against CP-CRE (MIC50/MIC90, 0.25/1 μg/mL vs. 0.5/8 μg/mL). All four isolates with high AZT-AVI MIC (≥16 μg/mL) were resistant to CAZ-AVI, but only 18% (4/22) of CAZ-AVI-resistant isolates had high AZT-AVI MIC. The rates of the inoculum effect for CAZ-AVI and AZT-AVI were 18% and 47%, respectively (p < 0.001). Interestingly, the frequency of the AZT-AVI inoculum effect was higher in K. pneumoniae than E. coli (64% vs. 8%, p < 0.001). AZT-AVI is more active against CRE than CAZ-AVI, even in CP-CRE and CAZ-AVI-resistant isolates. The presence of a substantial inoculum effect may contribute to clinical failure in high-inoculum infections treated with AZT-AVI.

scholarly journals EFFECTS OF ETHANOLIC EXTRACT OF RHINACANTHUS NASUTUS (L) KURZ AS AN ANTIMICROBIAL AGAINST ESCHERICHIA COLI BACTERIA USING IN VITRO METHOD

2019 ◽  
pp. 69-72
Author(s):  
SUNARTI M.BIOMED ◽  
DEBORA PANINSARI
Keyword(s):  
Escherichia Coli ◽  
Treatment Group ◽  
Ethanolic Extract ◽  
Inhibition Zone ◽  
Control Group ◽  
Control Groups ◽  
In Vitro Method ◽  
E Coli ◽  
Escherichia Coli Bacteria

Objective: The objective of this study was to discover of the ethanolic extract of Rhinacanthus nasutus (L) Kurz in inhibiting Escherichia coli bacteria using an in vitro method. Methods: This is an experimental study using a laboratory test with Kirby-Bauer or paper disc method by observing and measuring the inhibition zone of the ethanolic extract of R. nasutus against E. coli bacteria with extract concentrations of 15%, 30%, and 60% consisting of control groups and treatment group. The positive control group used chloramphenicol antibiotics and negative control groups used Aquadest. E. coli was incubated at 37°C for 24 h. Then, the plates were incubated for 24 h at 37°C and the diameter of the inhibition zone was observed until the 3rd day with three repetitions. Results: The results of the study showed that the mean inhibition zone of E. coli bacteria was 10.93 mm, 12.09 mm, and 18.90 mm. The results of the Shapiro–Wilk test were p=0.199. The results of the one-way analysis of variance test were p<0.05 and that of the post hoc test indicated a significant value of p<0.05. Based on the results of the research, there were significant differences in the inhibition zone between the control group and the treatment group at a concentration of 15%, 30%, and 60%. Conclusion: R. nasutus extract was effective to inhibit the growth of E. coli bacteria at concentrations of 15%, 30%, and 60%, so R. nasutus is effective as an antimicrobial.

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