All work involving animals was performed in accordance with the Animal (Scientific procedures) Act 1986. Protocols were produced in line with PREPARE guidelines and FRAME recommendations and were reviewed by the University of Sheffield’s Animal Welfare Committee. C57BL/6 mice (female, 9–10 weeks old) were nebulised with LPS (3 mg per group of 8 mice) (Pseudomonas aeruginosa, Sigma-Aldrich) and immediately injected intraperitoneally (i.p.) with either Tyrphostin AG825 (Tocris Bioscience) at 20 mg/Kg in 10% DMSO v/v in vegetable oil (eight mice, treatment group) or an equivalent volume of 10% DMSO v/v in vegetable oil (eight mice, control group) (Kedrin et al., 2009 (link); Roos et al., 2014 (link)). After 48 hr the mice were sacrificed by terminal anaesthesia by i.p. pentobarbitone and subjected to bronchoalveolar lavage (BAL, 4 × 1 mL of saline). BAL samples were microcentrifuged and the cellular fraction counted by a hemocytometer and cytocentrifuged. Neutrophil apoptosis and macrophage efferocytosis of apoptotic neutrophils was quantified by oil immersion light microscopy (Nikon Eclipse TE300, Japan).
Pseudomonas aeruginosa
Manufactured by Merck Group
Sourced in United States, United Kingdom
Pseudomonas aeruginosa is a bacterial strain commonly used in laboratory settings. It is a Gram-negative, rod-shaped bacterium that can be cultivated for various research and testing purposes. This product provides a stable and reliable source of the Pseudomonas aeruginosa strain for use in controlled experiments and analyses.
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Lab products found in correlation
Staphylococcus aureus, by Merck Group (4 mentions)
Klebsiella pneumoniae, by Merck Group (3 mentions)
Escherichia coli, by Merck Group (2 mentions)
Streptococcus pyogenes, by Merck Group (2 mentions)
Eclipse te300, by Nikon (1 mentions)
S 6 methoxy 2 5 7 8 tetramethylchromane 2 carboxylic acid trolox, by Merck Group (1 mentions)
Luria agar, by Merck Group (1 mentions)
S aureus, by Merck Group (1 mentions)
S typhosa, by Merck Group (1 mentions)
S marcescens, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (1 mentions)
Staphylococcus aureus, by American Type Culture Collection (1 mentions)
Bacillus subtilus, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Thp 1 cell line, by American Type Culture Collection (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Tryptic soy agar, by Merck Group (1 mentions)
Rhamnolipid biosurfactant, by Merck Group (1 mentions)
14 protocols using pseudomonas aeruginosa
1
Modulating Neutrophil Apoptosis in LPS-Induced Lung Injury
2
Grasshopper Bioactive Compounds Extraction and Characterization
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Grasshopper samples were taken from maize fields in Coronango, Puebla, Mexico. The geographical coordinates are the parallels 19° 06′36″ and 19° 10′42″ of north latitude and the meridians 98° 14′54″ and 98° 19′40″ of western longitude to 2,180 m above sea level. The recollection took place in September 2020 for early grasshopper (EG) and November 2020 for adult grasshopper (AG). Grasshoppers were collected and transported alive, and later, they were cleaned, not purged, and washed with distilled water, and frozen at −80°C. The samples were freeze-dried, blended (NutriBullet NBR-0601), and stored at room temperature until use.
Folin–Ciocalteu reagent, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+), (S)-6-methoxy-2,5,7,8-tetramethylchromane-2-carboxylic acid trolox (Sigma Aldrich), serine endoprotease from Bacillus licheniformis 2.4L E.C.3.4.21.14 (Sigma Aldrich), Luria Agar, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Enterobacter aerogenes ATCC 13048 Salmonella sp., and Pseudomonas aeruginosa ATCC 77853 were used in this study. Samples of EG, AG, early grasshopper extract (EGE), adult grasshopper extract (AGE), early grasshopper hydrolysate (EGH), adult grasshopper hydrolysate (AGH), and hydrolyzed fractions were tested in this research.
Folin–Ciocalteu reagent, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+), (S)-6-methoxy-2,5,7,8-tetramethylchromane-2-carboxylic acid trolox (Sigma Aldrich), serine endoprotease from Bacillus licheniformis 2.4L E.C.3.4.21.14 (Sigma Aldrich), Luria Agar, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Enterobacter aerogenes ATCC 13048 Salmonella sp., and Pseudomonas aeruginosa ATCC 77853 were used in this study. Samples of EG, AG, early grasshopper extract (EGE), adult grasshopper extract (AGE), early grasshopper hydrolysate (EGH), adult grasshopper hydrolysate (AGH), and hydrolyzed fractions were tested in this research.
3
Nanofiber-Mediated Immunomodulation in Endodontic Infection
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Nanofibers were fixed to the bottom of the 96-well plates with porcine gelatin, as previously described, to guarantee their direct contact with human immune cells (PBMCs). After 24 h of incubation, the supernatant of the stimulated cultures was collected and stored at - 80 °C until the day of the experiment. As endodontic infection processes are polymicrobial, PBMCs were stimulated with LPS (Pseudomonas aeruginosa, Sigma Aldrich) and LTA (S. aureus, Sigma Aldrich, St. Louis, USA), mimicking an in vitro infection. Cytokines involved with the pro-inflammatory activity (IL-1β, IL-6 and TNF-α) or anti-inflammatory activity and tissue repair (IL-10 and TGF-β) were measured by Enzyme-linked immunosorbent assay (ELISA, eBiosciences and Invitrogen, Thermo Fisher). We compared the data with free ciprofloxacin, IDR-1002, or both molecules to confirm the immunomodulatory profile of nanoformulations.
4
Bacterial LPS and MIPS-9451 Synthesis
5
Antimicrobial Evaluation of M. oleifera Extracts
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The pathogens used were Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 9721), Neisseria gonorrhoeae (ATCC 49981), and Streptococcus pyogenes (ATCC 19615), purchased from Sigma-Aldrich (Pretoria, South Africa) at University of Pretoria. They were selected based on their pathogenicity, clinical relevance and the literature and were used to evaluate antimicrobial activity of crude extracts and proposed pure compounds isolated from M. oleifera Lam leaves’ hexane extract as well as ethanol extract. Stock bacterial cultures were sub-cultured into freshly prepared Mueller–Hinton agar (MHA) and incubated at 37 °C for 18–24 h to produce fresh bacterial culture. However, Neisseria gonorrhoeae (ATCC 49981) was incubated in a jar with carbon dioxide at the same temperature. To keep the bacterial strains alive, glycerol stock cultures of each organism were prepared and stored at 80 °C until needed.
6
Oropharyngeal Exposure of C57BL/6J Mice
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Six week old C57BL/6J female mice were
purchased from Envigo, UK. The mice treatment was randomized, and
animals were kept in groups of four in ventilated cages with ad libitum access to food and water in a controlled environment
(humidity, temperature, and light). All procedures were conducted
after ethical approval from the UK Home Office, under Project License
no. P089E2E0A. Following a week of acclimatization, the animals were
exposed to a solution of 1 μg/μL (30 μg of materials
in 30 μL of 0.5 % bovine serum albumin (BSA; Gibco, ThermoFisherScientific)
in water for injection (v/v)) or controls (negative: vehicle, Pseudomonas aeruginosa; Merck-Sigma) 0.5 mg/kg) by single
oropharyngeal aspiration. For the procedure, the mice were anesthetized
by inhalation of 3% isoflurane in 100% oxygen and then held on a slanted
board to deliver the materials or controls. The animals (n = 5) were kept for 1, 7, or 28 days after exposure. LPS 0.5 mg/kg
delivered via oropharyngeal aspiration was used as a positive control
to induce acute inflammation. Methylmethanesulfonate (MMS; Merck-Sigma;
3 × 150 mg/kg; n = 3; provided by gavage, 48,
24, and 4 h before sacrifice) was used as a positive control for DNA
damage.
purchased from Envigo, UK. The mice treatment was randomized, and
animals were kept in groups of four in ventilated cages with ad libitum access to food and water in a controlled environment
(humidity, temperature, and light). All procedures were conducted
after ethical approval from the UK Home Office, under Project License
no. P089E2E0A. Following a week of acclimatization, the animals were
exposed to a solution of 1 μg/μL (30 μg of materials
in 30 μL of 0.5 % bovine serum albumin (BSA; Gibco, ThermoFisherScientific)
in water for injection (v/v)) or controls (negative: vehicle, Pseudomonas aeruginosa; Merck-Sigma) 0.5 mg/kg) by single
oropharyngeal aspiration. For the procedure, the mice were anesthetized
by inhalation of 3% isoflurane in 100% oxygen and then held on a slanted
board to deliver the materials or controls. The animals (n = 5) were kept for 1, 7, or 28 days after exposure. LPS 0.5 mg/kg
delivered via oropharyngeal aspiration was used as a positive control
to induce acute inflammation. Methylmethanesulfonate (MMS; Merck-Sigma;
3 × 150 mg/kg; n = 3; provided by gavage, 48,
24, and 4 h before sacrifice) was used as a positive control for DNA
damage.
7
Antimicrobial Chitosan Nanocomposite Synthesis
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CS (degree of deacetylation, 85%; Mw, around 600 kDa), silver nitrate, sodium periodate, glycerol, acetic acid, sodium tri-polyphosphate and Sulforhodamine B were purchased from Shanghai branch of Sigma Aldrich Chemical Co., Ltd (Shanghai, China). CNC aqueous suspension (1 mg/mL) was prepared by our group according to a procedure modified from the literature [17 (link)]. All the other chemicals and reagents were of analytical grade and double distilled water was used throughout. Dulbecco's modified eagle medium (DMEM) and beef extract peptone medium used for the cultivation of bacteria were obtained from Sigma Co. Ltd. The clinical strains (C) of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, Streptococcus pneumoniae and Pseudomonas aeruginosa, Candida albicans, Candida glabrata and Candida krusei were donated by the Second Affiliated Hospital of Qiqihar Medical College (Qiqihar, China). Staphylococcus aureus standard strain (S) (ATCC25923), Escherichia coli (S) (ATCC25922), Pseudomonas aeruginosa (S) (ATCC27853)and NIH3T3 cell lines were obtained from Laboratory of Biochemistry of Qiqihar University (Qiqihar, China).
8
Bacterial Cultures for Immune Induction
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Standard reference bacteria Escherichia coli (ATCC 10536), Micrococcus luteus (National Collection of Type Cultures NCTC 2665; Sigma, UK), Staphylococcus aureus (ATCC 6538), Bacillus subtilus (ATCC 6051), and Pseudomonas aeruginosa (ATCC 9027) were purchased from Sigma, UK. Locally isolated AFB bacteria, P. larvae (ksuPL5) (Ansari et al., 2017a , 2017b), were used for immune induction in the third instar and for in vitro and in vivo immune-verification experiments in the first instar. All bacteria were kept at −20 °C in 20% glycerol (v/v) added brain heart infusion broth. Bacteria were propagated and prepared for experiments according Al-Ghamdi et al. (2020) .
9
Differentiation and Treatment of THP-1 Macrophages
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The human acute monocytic leukaemia THP-1 cell line (ATCC, Manassas, USA) was routinely cultured (P2-30) in RPMI 1640 medium with ʟ-glutamine (2 mM) (Gibco, ThermoFisher Scientific, UK) supplemented with 10% (v/v) heat-inactivated foetal calf serum (FCS; Gibco, ThermoFisher Scientific, UK) and 1% (v/v) penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Austria). Cells were seeded at a density of 2.5 x 105 cells/well in 24 well plates and were differentiated to macrophages by incubating with 2 ml of medium with 162 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich, UK) for 72 hrs, then rested in fresh media (PMA-free) for 24 hrs before use. Cells were incubated with peptides (20 μg/ml) and/or LPS from Pseudomonas aeruginosa (100 ng/ml, Serotype 10, Source strain ATCC 27316; Sigma Aldrich, UK) in media for 16 hrs.
10
Burn Wound Infection by Pseudomonas
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