Streptomycin sulphate

The in vitro activity against N. fowleri trophozoites was evaluated in two different strains from the American Type Culture Collection (LG Promochem, Barcelona, Spain), ATCC^® 30808™ and ATCC^® 30215™. Cells were axenically grown in 2% Bactocasitone (w/v) medium (Thermo Fisher Scientific, Madrid, Spain) at 37 °C. Moreover, 10% (v/v) of fetal bovine serum (FBS) 0.3 μg/mL of Penicillin G Sodium Salt and 0.5 mg/mL of Streptomycin sulphate (Sigma-Aldrich, Madrid, Spain) were also added to the Bactocasitone medium.
Murine macrophages from the J774A.1 cell line (ATCC^® TIB-67) were maintained in in Dulbecco’s Modified Eagle’s medium (DMEM) in order to evaluate the toxicity of the compounds. In addition, 10% (v/v) FBS and 10 μg/mL gentamicin (Sigma-Aldrich, Madrid, Spain) were added to the growth medium. Cells were grown in a 5% CO₂ atmosphere at 37 °C.

HCT116 cells (CCL-247™, originally obtained from ECACC) were cultured in McCoy’s medium (Sigma-Aldrich, Taufkirchen, Germany). The medium solution was supplemented with 10% foetal bovine serum (Biowest, Riverside, MO, USA), non-essential amino acids (Sigma), 100 U/mL of penicillin G (Sigma-Aldrich, Taufkirchen, Germany) and 0.1 mg/mL of streptomycin sulphate (Sigma-Aldrich, Taufkirchen, Germany). The cells were maintained at 37 °C in the atmosphere of 5% carbon dioxide. Genotoxic stress was generated by the addition of doxorubicin to a final concentration of 0.1 or 0.5 μg/mL (Sigma-Aldrich, Taufkirchen, Germany). Rapamycin was added to a final concentration of 50 nM (Sigma-Aldrich, Taufkirchen, Germany). The cells were exposed to stress conditions for 2 or 24 h, then harvested.

Primary cells were isolated from the ascites of advanced stage HGSOC patients (n = 2), with patient consent and approval, by the Royal Adelaide Hospital RAH and Central Adelaide Local Health Network Human Ethics Committees (RAH # 140201) (CALHN # R20181215). All primary cells were grown in Advanced RPMI 1640 medium (Life Technologies, Mulgrave, VIC, Australia) supplemented with 4 mM L-glutamine, 10% FBS (Sigma Aldrich, St. Louis, MO, USA), and antibiotics (100 U penicillin G, 100 µg/mL streptomycin sulphate, and 100 µg/mL amphotericin B, Sigma Aldrich). Patients that experienced a recurrence within six months after finishing first-line CBP+paclitaxel chemotherapy were classified as being chemoresistant. Patients that remained in full remission for longer than 6 months were classified as being chemosensitive. Here, we investigated the proteome of cells derived from one patient who was deemed chemoresistant and one who was deemed chemosensitive using the above-mentioned classifications. Patient diagnoses and chemotherapy responses are outlined in Supplementary Figure S1.

Eugenol, RPMI 1640 medium, M-199 medium, penicillin G sodium salt, streptomycin sulphate, 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT), carboxyfluoresceinsuccinimidyl ester (CFSE), AmB, anti-mouse IgG and isotype antibodies, o-phenylenediaminedihydrochloride (OPD) were procured from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Gibco-BRL, DMSO from SRL, methanol from Merck, limulus amebocyte lysate (LAL) kit from Pierce, Thermo Scientific. Fluorochrome conjugated anti-mouse antibodies such as CD4-phycoerythin (PE), CD8-fluorescein isothiocyanate (FITC), CD80-allophycocyanin (APC), CD86-phycoerythin cyanine dye 7, APC-CD4 and PE-CD8, FITC-IFN-γ, CD8-APC, CD62L-PE and CD44-FITC, isotype controls and Brefeldin A and cytokine bead array kit (CBA) were procured from BD Pharmingen, USA. Aspartate aminotransaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), creatinine and urea kits were purchased from Span Diagnostics Ltd (Surat, Gujarat, India). Besides these, the analytical grade reagents were used.

The disinfection methods were evaluated against the type strain of N. fowleri (ATCC 30808™) of the American Type Culture Collection (LG Promochem, Barcelona, Spain). The amoebae were axenically cultured at 37 °C in 2% Bactocasitone medium (Thermo Fisher Scientific, Madrid, Spain) supplemented with 10% (v/v) of foetal bovine serum (FBS), 0.5 mg/ml of streptomycin sulphate and 0.3 μg/ml of penicillin G (Sigma-Aldrich, Madrid, Spain). This N. fowleri strain was cultured in a biological security facility level 3 at the Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna as required by the Spanish Government biosafety guidelines for this pathogen.

A total of 46 Fusarium oxysporum strains (Table 1) isolated from vascular tissues of decayed asparagus plants in different fields in three EU countries (20 from Spain, 21 from Poland, and 5 from The Netherlands) were used in this study. The analysis of the plant samples consisted of the superficial disinfection of secondary and storing roots with 1.5% sodium hypochlorite solution for 1 min, followed by two successive washings with sterile distilled water. After drying, 1 cm pieces were sown in plates with a potato dextrose agar (PDA) culture medium supplemented with 0.5 g/L of streptomycin sulphate (Sigma-Aldrich, St. Louis, MO, USA) (PDAS) and incubated for 5–7 days at laboratory temperature (25 °C) under continuous fluorescent light. Fungal single-spore cultures were obtained from the different Fusarium colonies recovered. Isolations were carried out in the country of origin where the strains were properly stored in fungal collections on potato dextrose agar (PDA; Merck, Darmstadt, Germany) medium at 4 °C in the dark. Some of the Spanish strains had already been investigated in a previous study [3 (link)] and were included for further characterisation in a wider geographical context.