Staphylococcus aureus

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Staphylococcus aureus is a species of Gram-positive bacteria that is commonly found in the human respiratory tract and on the skin. It is a spherical-shaped bacterium that forms clusters resembling grapes. Staphylococcus aureus is widely used in laboratory settings for various research and diagnostic purposes.

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28 protocols using staphylococcus aureus

Antioxidant and Antibacterial Evaluation

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M ethanol, hexane, chloroform, ethyl acetate and ethanol were purchased from Junsei Chemical Co., Ltd., Tokyo, Japan. Potassium phosphate monobasic and dibasic, xanthine, xanthine oxidase, allopurinol, and hydrochloric acid were obtained from Sigma-Aldrich Corp., St. Louis, MO, USA. Reagents including 1,1-diphenyl-2-picrylhydrazyl (DPPH), sodium acetate, acetic acid, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium peroxodisulfate, and dibutyl hydroxytoluene (BHT) were supplied by Kanto Chemical Co. Inc., Tokyo, Japan. Four bacteria including Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Proteus mirabilis were provided by Sigma-Aldrich Corp., St. Louis, MO USA. All chemicals used were of analytical grade.

Quy T.N, & Xuan T.D. (2019). Xanthine Oxidase Inhibitory Potential, Antioxidant and Antibacterial Activities of Cordyceps militaris (L.) Link Fruiting Body. Medicines, 6(1), 20.

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Muscle Fibre Superfusion with SMase

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Muscle fibres were superfused with Tyrode solution (mM): NaCl, 121; KCl, 5.0; CaCl₂, 1.8; MgCl₂, 0.5; NaH₂PO₄, 0.4; NaHCO₃, 24.0; EDTA, 0.1; glucose, 5.5. FBS (0.2%) was added to the solution. The solution was bubbled with a mixture of 5% CO₂ and 95% O₂, which gives an extracellular pH of 7.4. For single fibre SMase experiments, fibres were exposed to 0.05 U/mL bacterial SMase (Staphylococcus aureus; Sigma‐Aldrich, St. Louis, MO). The temperature of the solution flowing through the muscle bath was kept constant at 37°C by passing it through the inner glass coil of a heated Graham condenser. The temperature of the bath solution was routinely measured in front of the intact fibre at a point furthest from the solution inflow. Caffeine (5 mM) was prepared fresh in Tyrode each day. For the RNA‐sequencing experiments, dissected fibre bundles were exposed to 0.25 U/mL bacterial SMase in Tyrode, or control solution without SMase, for 1 h at 37°C.

Olsson K., Cheng A.J., Al‐Ameri M., Tardif N., Melin M., Rooyackers O., Lanner J.T., Westerblad H., Gustafsson T., Bruton J.D, & Rullman E. (2022). Sphingomyelinase activity promotes atrophy and attenuates force in human muscle fibres and is elevated in heart failure patients. Journal of Cachexia, Sarcopenia and Muscle, 13(5), 2551-2561.

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Cytokine production in dendritic cells

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DCs were treated with AC PSs (crude extract or various fractions), LPS (100 ng/mL, from Escherichia coli serotype O26:B6, Sigma, St. Louis, MO, USA), or lipoteichoic acid (LTA, 1 μg/mL, from Staphylococcus aureus, Sigma) for 24 h. The production of cytokines (TNF-α, IL-6, and IL-12) was quantified by ELISA (eBioscience and R&D systems) as described previously [18 (link)].

Lin C.C., Pan I.H., Li Y.R., Pan Y.G., Lin M.K., Lu Y.H., Wu H.C, & Chu C.L. (2015). The Adjuvant Effects of High-Molecule-Weight Polysaccharides Purified from Antrodia cinnamomea on Dendritic Cell Function and DNA Vaccines. PLoS ONE, 10(2), e0116191.

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Evaluating Bacterial Interactions with Mesothelial Cells

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Confluent cells were serum starved in the absence of antibiotics for 18 hours prior to incubation with bacteria. Standardised bacterial suspensions (~1 x 10⁸ cfu/mL) were diluted ¹/₁₀ in the appropriate antibiotic-free cell culture media to give ~1 x 10⁷ cfu/mL, of which 2 mL was co-incubated with cells for 1 hour at 37°C/5% CO₂. For dose-response experiments, bacterial suspensions were standardized to ~1 x 10⁹ cfu/mL, serially diluted in LB broth then diluted ¹/₁₀ in cell-culture media, as described above. Met-5A cells were also exposed to lipoteichoic acid (LTA) from Staphylococcus aureus (Sigma; Cat. No. L2515), the primary component of the Gram positive cell wall, at 10 μg/mL in antibiotic-free DMEM. All test conditions were set up in triplicate in 6 well plates (Falcon^® by Corning, Corning NY USA). Control wells contained mesothelial cells with media alone, or media containing 10% LB.

McGuire A.L., Mulroney K.T., Carson C.F., Ram R., Morahan G, & Chakera A. (2017). Analysis of early mesothelial cell responses to Staphylococcus epidermidis isolated from patients with peritoneal dialysis-associated peritonitis. PLoS ONE, 12(5), e0178151.

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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.

Marín-Morales M.S., Ibarra-Herrera C.C., Luna-Vital D.A., Monribot-Villanueva J.L, & Guerrero-Analco J.A. (2022). Biological activity of extracts and hydrolysates from early- and adult-stage edible grasshopper Sphenarium purpurascens. Frontiers in Nutrition, 9, 1028543.

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Synthesis of Synthetic Amorphous Magnesium-Substituted Calcium Phosphate Particles

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Synthetic amorphous magnesium-substituted calcium phosphate (sAMCP) nanomineral particles were prepared as previously described (20 (link)). Briefly, for the preparation of particles incorporating PGN (Staphylococcus aureus, Fluka), PGN was added to phosphate (PO₄) solution [containing 39 mM (NH₄)HPO₄ (Sigma-Aldrich) in 0.15 M Tris buffer, adjusted to pH 8 with hydrochloric acid], prior to mixing in equal parts with calcium–magnesium–BSA solution (BSA; Sigma-Aldrich Company Ltd., Dorset, UK) dissolved in a Ca/Mg solution [containing 35 mM CaCl₂ (Sigma-Aldrich), 7.2 mM MgCl₂ (Sigma-Aldrich) in 0.15 M Tris buffer (Sigma-Aldrich)] at 1 mg/mL for a final PGN concentration of 50 μg/mL mixed solution. For particles incorporating protein purified derivative (PPD) of tuberculin (Statens Serum Institute, Denmark), PPD was added to the phosphate solution prior to mixing with the calcium–magnesium–BSA solution for a final PPD concentration of 100 μg/mL, which was then incubated with gentle rotation at room temperature for 1 h to allow particle formation. Particles were precipitated by centrifugation (1,500 rpm for 5 min), washed in pH 10 water, followed by washing in tissue culture grade water before re-suspending in tissue culture media at half of the original particle solution volume.

Hewitt R.E., Robertson J., Haas C.T., Pele L.C, & Powell J.J. (2017). Reduction of T-Helper Cell Responses to Recall Antigen Mediated by Codelivery with Peptidoglycan via the Intestinal Nanomineral–Antigen Pathway. Frontiers in Immunology, 8, 284.

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Modeling Vascular Permeability in HRECs

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HRECs were obtained from Cell Systems (Kirkland, WA). HRECs were cultured and maintained in classic cell culture medium (Cell Systems) supplemented with 10% FBS and 2% animal-derived growth factors at 37 °C in a 95% humidified incubator with 5% CO₂. Only low-passage HRECs (<6–9 passages) were used in the described experiments. Approximately 50,000 cells/well were seeded onto the chambered slides and allowed to grow in culture until reaching 80–90% confluence. TLR-2, MyD88, ARNO, ARF6 and p120 catenin staining was preformed when the cells reached 100% confluence. Purified peptidoglycan from Staphylococcus aureus (#77140, Sigma-Aldrich, St. Louis, MO) and NAV2729, an inhibitor of ARF6 (#5986, Tocris Bio-Techne Corporation, Minneapolis, MN), were used to determine the role of PGN on vascular permeability. HRECs were treated with PGN in a dose-dependent manner (40 and 100μg/mL) for 24 hours. In select experiments, HRECs were pre-treated with NAV2729 (10μM) for one hour followed by PGN treatment for 24 hours. The PGN and NAV2729 were dissolved in dimethyl sulfoxide (DMSO) before adding to the culture medium. The final concentration of DMSO in the culture medium did not exceed 0.1% (v/v), and an equivalent amount of DMSO was added to the culture media of control (untreated) groups.

Duan Y., Prasad R., Feng D., Beli E., Calzi S.L., Longhini A.L., Lamendella R., Floyd J.L., Dupont M., Noothi S.K., Sreejit G.K., Athmanathan B., Wright J., Jensen A.R., Oudit G.Y., Markel T.A., Nagareddy P.R., Obukhov A.G, & Grant M.B. (2019). Bone Marrow-Derived Cells Restore Functional Integrity of the Gut Epithelial and Vascular Barriers in a Model of Diabetes and ACE2 Deficiency. Circulation research, 125(11), 969-988.

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Droplet-Interface Bilayer Microfluidics

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A zwitterionic lipid, PC (Avanti Polar Lipids, Alabaster, AL, USA), and a nonionic surfactant, sorbitan monooleate (Span80, Tokyo Chemical Industry Co., Tokyo, Japan), were dissolved in hexadecane (purity > 98%; Tokyo Chemical Industry Co., Tokyo, Japan) and used as the continuous oil phase. Milli-Q water (H20MB0124, Merck KGaA) was used as the aqueous phase. To visualize a lipid membrane covering a droplet, a fluorescent-labeled lipid, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt; Rho-PE, Avanti Polar Lipids) was used. Calcein (Sigma-AldrichSt. Louis, MO, USA), a water-soluble fluorescein, was used to analyze the function of α-hemolysin isolated from Staphylococcus aureus (Sigma-Aldrich) reconstituted in a DIB. The microfluidic device was developed using a photoresist (SU-8 3025, MicroChem Corp., Westborough, MA, USA) and a polydimethylsiloxane (PDMS) package (Sylgard 184, Dow Corning Toray Corp., Tokyo, Japan).

Fujiwara S., Shoji K., Watanabe C., Kawano R, & Yanagisawa M. (2020). Microfluidic Formation of Honeycomb-Patterned Droplets Bounded by Interface Bilayers via Bimodal Molecular Adsorption. Micromachines, 11(7), 701.

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Immunomodulatory Responses of BEAS-2B and THP-1 Cells

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SBDE, LPS (Escherichia coli O127:B8; Sigma), and PGN
(Staphylococcus aureus; Sigma) treatments were performed on
BEAS-2B and THP-1 cell lines. Ligand stimulants (SBDE, LPS, PGN) were prepared
by dissolving stock concentrations in a serum-free culture medium to achieve a
final concentration of 10 µg/ml (LPS and PGN) and 5% (SBDE), respectively. Five
percent SBDE is known to induce maximal pro-inflammatory response with limited cytotoxicity.^{13 (link)} Stimulant treatments were performed at the respective final
concentrations and media alone treated cells served as no treatment controls.
Stimulant and no treatment control cell pellets were prepared at 0 − and 24-h
post treatment for kinome and transcriptional analyses.

Nath Neerukonda S., Mahadev-Bhat S., Aylward B., Johnson C., Charavaryamath C, & Arsenault R.J. (2018). Kinome analyses of inflammatory responses to swine barn dust extract in human bronchial epithelial and monocyte cell lines. Innate Immunity, 24(6), 366-381.

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Antibody Immobilization Techniques on OWLS Chip

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The Abs were immobilized on three types of coatings prepared on the OWLS sensor chip surface prior to Ab deposition. The reagents used for coating preparation were the following. Mix&Go Biosensor coating agent (shortened as MG from now on) (Anteo Technologies Pty Ltd., Brisbane, Australia, A-PLSCO10) (noteL its new name is AnteoBind), was used for preparing the MG Ab immobilization layer.
GOPS ((3-glycidyloxypropyl)-triethoxysilane, Sigma-Aldrich Chemie GmbH, Darmstadt, Germany, catalog number: 50059) was used to silanize the chip surface for capturing a layer of protein A (from Staphylococcus aureus, Sigma-Aldrich Chemie GmbH, Darmstadt, Germany, catalog number: 539202).
In the case of the avidin–biotin method, the OWLS chip surface was coated with poly-(L-lysine)-graft-poly(ethylene-glycol)-20% biotin (PLL-g-PEG-20% biotin, SuSoS AG, Dübendorf, Switzerland, shortened as PP-b from now on) to which avidin (VWR, A2568.0010) was bound. This was followed by the addition of the biotinylated Abs (see Ab specification earlier).

Farkas E., Tarr R., Gerecsei T., Saftics A., Kovács K.D., Stercz B., Domokos J., Peter B., Kurunczi S., Szekacs I., Bonyár A., Bányai A., Fürjes P., Ruszkai-Szaniszló S., Varga M., Szabó B., Ostorházi E., Szabó D, & Horvath R. (2022). Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing. Biosensors, 12(2), 56.

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