Four pure molecules were tested to evaluate their antibacterial activity: pentadecanal, pentadecanoic acid (carboxylic acid), pentadecanoic acid methyl ester (ester), and 1,1-dimethoxypentadecane (acetal). The molecules were synthesized from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 at the Department of Chemical Sciences (University of Naples Federico II, Naples, Italy) as described in Ricciardelli et al. (2019). The compounds, supplied in lyophilized form, were resuspended at the Department of Veterinary Medicine and Animal Production using 99.9% pure Dimethyl Sulfoxide (DMSO, Biofroxx, Germany) with a final concentration of 40 mg/mL.
Dimethyl sulfoxide (dmso)
Manufactured by neoFroxx
Sourced in Germany, United States, China
DMSO (Dimethyl Sulfoxide) is a versatile organic solvent used in various laboratory applications. It has a high dielectric constant, making it an effective solvent for a wide range of polar and non-polar compounds. DMSO is known for its ability to penetrate biological membranes, making it a useful component in cell culture and biological research.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by neoFroxx (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Phosphate buffer, by Sinopharm (1 mentions)
Nah2po4 2h2o, by Sinopharm (1 mentions)
Hydrogen peroxide, by Sinopharm (1 mentions)
Na2hpo4 12h2o, by Sinopharm (1 mentions)
Na2hpo4 12h2o, by Tianjin Damao (1 mentions)
P srctyr416, by Abcam (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Matrigel coated transwell chamber, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by Biosharp (1 mentions)
Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Tbk1 antibody, by ABclonal (1 mentions)
C myc antibody, by Beyotime (1 mentions)
Coumarin 6, by Merck Group (1 mentions)
β actin, by ABclonal (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
16 protocols using dimethyl sulfoxide (dmso)
1
Antibacterial Activity of Antarctic Molecules
2
Intracellular Oxidative Stress Analysis
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ARS and CAT from bovine liver were purchased from Beijing Solarbio Ltd. Na2HPO4·12H2O and NaH2PO4·2H2O were purchased from Tianjin Damao Chemical Reagent Factory. Phosphate buffered solution (PBS, pH 7.2–7.4) for intracellular oxidative stress analysis was from Beijing Macgene Ltd., Beijing, China. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and penicillin/streptomycin were all purchased from Thermo Fisher Scientific, Waltham, MA, USA. Dimethyl sulfoxide (DMSO) was provided by from Biofroxx (Germany). Hydrogen peroxide was bought from Sinopharm Chemical Reagent Co., Ltd. Phosphate buffer (a mixture of Na2HPO4·12H2O and NaH2PO4·2H2O solution, pH 7.4) was chosen to control the solution pH. All the water solutions involved in this work were prepared by using ultrapure water, and stock solutions were preserved at 0–4 °C.
3
Evaluating β-elemene's Anti-Metastatic Potential
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β-elemene was obtained from CSCP Pharmaceutical Group Ltd. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM/H) were purchased from Cytvia. Dimethyl sulfoxide (DMSO) was purchased from BioFROXX and 3-(4,5-dimethylthiazol-2-yl)-2, 5-dephenyltetrazolium bromide (MTT) was obtained from (Shanghai Ica Biotechnology Co., Ltd.; cat. no. MO105-1G). A Matrigel-coated Transwell chamber was purchased from BD Biosciences. Antibodies against phosphorylated (p)-FAKTyr397, total (t)-FAK, p-SrcTyr416, p-SrcTyr527, and t-Src were obtained from Abcam. Crystal violet was obtained from Beyotime Institute of Biotechnology.
4
Evaluating CPI-613 and 3-BPA Cytotoxicity
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3-BPA (bromopyruvic acid) and CPI-613(6,8-bis-benzylsulfanyl-octanoic acid) were purchased from Acros (UK). PLGA (Mw = 38–54 kg/mol) was acquired from Jinan Dai Gang Biomaterial Co., Ltd (China). Poly(vinyl alcohol) 1788 (PVA, low molecular weight), d -(+)-trehalose anhydrous and dichloromethane were obtained from Aladdin Reagent (China). DMEM was purchased from biosharp (China). Fetal bovine serum (FBS) was acquired from Cellma (China). Dimethyl sulfoxide (DMSO) were obtained from BioFroxx (China). 3-(4,5-Dimethylthiazol-2-yl)-thiazolyl blue tetrazolium bromide (MTT) was provided by GoldBio. Coumarin 6 was acquired from Sigma-Aldrich (USA). Alexa Fluor® 488 annexin V/Dead Cell Apoptosis Kit was purchased from Thermo Fisher Scientific (USA). Mitochondrial Membrane Potential Assay Kit with JC-1 was obtained from Solarbio life sciences Co., Ltd (China). Pyruvate assay kit and glucose kit were obtained from Nanjing jiancheng Co., Ltd (China). ATP Assay Kit was obtained from Beyotime. Lactic acid kit, β-actin, GAPDH and TBK1 antibody were obtainted from Abclonal (China). HK-II antibody was purchased from proteintech. c-Myc antibody was purchased from Beyotime (China). P-TBKI antibody were provided by Cell Signaling Technology. STING antibody was donated by HongbinShu’s laboratory.
5
Antimicrobial and Cytotoxic Nanoparticle Evaluation
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DSPE-PEG-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly (ethylenegly-col)]-hydroxy succinimide], L-α-phosphatidylcholine, curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], and cholesterol were obtained from Aladdin Bio-Chem Technology (Shanghai, China). S. mutans Ingbritt and MC3T3-E1 osteoblast precursor cells were provided by the School of Stomatology, Wuhan University (China). Brain heart infusion (BHI) broth was purchased from Beijing Land Bridge Technology Co., Ltd. (China). Dimethyl sulfoxide (DMSO) was obtained from BioFroxx (Germany). Agar was obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China). The crystal violet staining solution and lactate dehydrogenase (LDH) cytotoxicity assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Methanol, ethanol, dichloromethane, glutaraldehyde, and sucrose were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Cell counting kit-8 (CCK-8) was purchased from Dojindo Kagaku (Kumamoto, Japan). The α-modified essential medium (α-MEM) was obtained from HyClone (Logan, UT, United States). All reagents and chemicals were utilized as received.
6
Rabbit Model for I3C and LPS Effect
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I3C (CAS: 700-06-1) and LPS (Escherichia coli O55:B5) were obtained from Sigma Chemical (St Louis, MO, USA), and Dimethyl sulfoxide (DMSO) from BioFroxx (Germany).
Animal experiments 6-week-old male Hyla rabbits were raised in stain-steel cages with a temperature regulation system, and all the groups were fed a basal diet with no antibiotic additives (Table 1). Water and diets were provided ad libitum. After a week pre-experiment, 40 rabbits were randomly and averagely assigned to four groups: control, I3C (40 mg•kg -1 •BW I3C, OP), LPS (0.5 mg•kg -1 •BW LPS, IP injection), I3C+LPS (40 mg•kg -1 •BW I3C, OP; 0.5 mg•kg -1 •BW LPS, IP injection). In the analysis of RNA-seq and serum metabolome, control, I3C, LPS, I3C+LPS group were named as C, I, L, IL, respectively. Rabbits were administrated 40 mg•kg -1 •BW I3C (dissolved in a solution of 92% corn oil and 8% DMSO) or vehicle (92% corn oil and 8% DMSO) orally for two weeks. At the end of the experiment, rabbits were intraperitoneally injected 0.5 mg•kg -1 •BW LPS (dissolved in saline) or saline.
Rabbits were sacri ced four hours later after injection, and the blood and organs were collected for the next assay. All the procedures of animal experiments were approved by the Ethics Committee of Animal Welfare at Southwest University (IACUC Issue No. 20210122-1).
Animal experiments 6-week-old male Hyla rabbits were raised in stain-steel cages with a temperature regulation system, and all the groups were fed a basal diet with no antibiotic additives (Table 1). Water and diets were provided ad libitum. After a week pre-experiment, 40 rabbits were randomly and averagely assigned to four groups: control, I3C (40 mg•kg -1 •BW I3C, OP), LPS (0.5 mg•kg -1 •BW LPS, IP injection), I3C+LPS (40 mg•kg -1 •BW I3C, OP; 0.5 mg•kg -1 •BW LPS, IP injection). In the analysis of RNA-seq and serum metabolome, control, I3C, LPS, I3C+LPS group were named as C, I, L, IL, respectively. Rabbits were administrated 40 mg•kg -1 •BW I3C (dissolved in a solution of 92% corn oil and 8% DMSO) or vehicle (92% corn oil and 8% DMSO) orally for two weeks. At the end of the experiment, rabbits were intraperitoneally injected 0.5 mg•kg -1 •BW LPS (dissolved in saline) or saline.
Rabbits were sacri ced four hours later after injection, and the blood and organs were collected for the next assay. All the procedures of animal experiments were approved by the Ethics Committee of Animal Welfare at Southwest University (IACUC Issue No. 20210122-1).
7
Antioxidant Activity of TTR Extract
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TTR extract (purity > 50%) was prepared by our laboratory. Sildenafil was purchased from Pfizer Pharmaceuticals Limited. SOD, glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) detection kits were obtained from Nanjing Jiancheng Bioengineering Institute. Enhanced RIPA lysate, broad-spectrum phosphatase inhibitor, and broad-spectrum protease inhibitor were obtained from Boster Bio Co., Ltd. BCA protein quantitation kits were purchased from Mei5 Biotech Co., Ltd. The following antibodies were used in the experiments: anti-Nrf2 (Wanleibio), anti-HO-1, anti-Bax, and anti-Bcl-2 (Abcam, USA), anti-β-actin (Beijing Affinity Biosciences), and HRP-conjugated AffiniPure goat anti-rabbit IgG (H + L) (Proteintech, Cat No. SA00001-2). Fetal calf serum was obtained from Gibco, USA. Primary endothelial cell basal culture medium, cell growth factor, and penicillin‒streptomycin double antibiotic were obtained from iCell Bioscience Inc. Pancreatin (0.25%) was purchased from HyClone, USA. Dimethyl sulfoxide was purchased from BIOFROXX, USA. Cell proliferation and toxicity detection kits were obtained from Bioss Biotech; apoptosis detection kits were obtained from USA BD, and reactive oxygen species (ROS) detection kits were obtained from Beijing Solarbio.
8
TNF-α and NF-κB Inhibitor Treatment
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The TNF-α (Sangon Biotech，China) was diluted to 100 ng/µL with nuclease-free water according to the instructions and was stored at −20°C. For TNF-α treatment, TNF-α was diluted to 25 ng/mL, and then cells were stimulated for 12 h for Western blots and dual-luciferase reporter gene assays. For the immunofluorescence assays, cells were stimulated for 1 h. The NF-κB inhibitor BAY 11-7082 (Beyotime, China) was diluted to 20 mM with dimethyl sulfoxide (Biofroxx, Germany) according to the instructions and was stored at −20°C. For BAY 11-7082 treatment, BAY 11-7082 was added to the cells to get the target final concentrations for 1 h. Then the cells were washed three times with PBS and replaced with fresh medium for further experimental treatments.
9
Zebrafish Embryo Membrane Dynamics
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For these tests, solutions of 100 nM bafilomycin A1 (Cayman Chemical, USA), 1.5 μM cathepsin L inhibitor (Cayman Chemical, USA), 5 μM cyclosporine A (Cayman Chemical, USA), and 200 μM DIDS (Sigma-Aldrich, Germany) were prepared in 0.05% dimethyl sulfoxide (DMSO) (BioFroxx, Germany), to inhibit the activities of V-ATPase, cathepsin L, cyclophilin D, and voltage-dependent anion-selective channel protein, respectively [63 (link)–66 (link)]. Mature female zebrafish were treated with 10 μL of 0.05% DMSO or the inhibitor solution by intraperitoneal injection (n = 3). After 17 h, zebrafish embryos (as a semibuoyant model, remarkable hydration, and identical to other semibuoyant eggs at the biochemical level, Figure S11 ) were obtained by artificial insemination and then immersed in 0.05% DMSO or inhibitor solutions. Immediately thereafter, photos of eggs were taken with a stereomicroscope (MODEL C-BD230, Nikon, Japan) at 1, 5, 10, 15, 20, 25, 30, and 60 min post fertilization and the membrane diameter was measured using ImageJ (k 1.45, National Institutes of Health, USA).
10
Photosensitizer-Induced Cell Death
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All of the stock solutions (Ce6 [J&K Scientific, Ltd.], apoptozole [MedChemExpress, USA], and nonactin [MKbio, China]) were prepared in dimethyl sulfoxide (DMSO; Biofroxx, Germany) and stored at −80°C in the dark. A laser (LWRPD-1.5F, Laserwave Co., Ltd., China) with red light (660 nm) was chosen as the light source.
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