Cadaverine

SCFAs including acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate were analysed as described previously67 (link). To ensure the homogenicity of the intestine content sample, the freeze-dried samples were prepared using a Vacuum freeze-dryer (Hrist ALPHA 2-4/LSC, Germany) at −80 °C. Briefly, freeze-dried samples (0.5–0.6 g) were weighed into 10 ml centrifuge tubes and mixed with 8 ml ddH₂O, homogenised, and centrifuged in sealed tube at 7,000 g and 4 °C for 10 min. A mixture of the supernatant fluid and 25% metaphosphoric acid solution (0.9 and 0.1 ml, respectively) was centrifuged at 20,000 g and 4 °C for 10 min after standing in a 2 ml sealed tube at 4 °C for over 2 h. The supernatant portion was then filtered through a 0.45-μm polysulfone filter and analysed using Agilent 6890 gas chromatography (Agilent Technologies, Inc, Palo Alto, CA, USA) with a flame ionisation detector and a 1.82 m × 0.2 mm I.D. glass column that was packed with 10% SP-1200/1% H₃PO₄ on the 80/100 Chromosorb W AW (HP, Inc., Boise, ID, USA). The concentration of NH₃-N in the supernatant fluid was measured at 550 nm using a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan)68 . The bioamines including 1,7-heptyl diamine, cadaverine, phenylethylamine, putrescine, trytamine, tyramine, spermidine, and spermine, as well as the indoles and skatoles, were analysed as described previously69 .

The plasmid pNL4-3_V1Q3_V4A1 ΔRT encoding for HIV-1_NL4-3 Env and plasmid Q23_BG505_V1Q3-V4A1 ΔRT encoding for HIV-1_BG505 Env carrying the Q3 (GQQQLG) and A1 (GDSLDMLEWSLM) labeling peptides (Wu et al., 2006 (link); Yin et al., 2006 (link); Zhou et al., 2007 (link)) in V1 and V4 loops of full-length pNL4-3 ΔRT construct and full-length Q23_BG505 ΔRT construct, respectively; and the Env-expressing plasmid pCAGGS_JR-FL_V1Q3_V4A1 containing the peptides in analogous positions were previously described (Munro et al., 2014 (link)). The D368R point mutation (Olshevsky et al., 1990 (link)) was introduced into untagged constructs and V1Q3_V4A1 tagged constructs of full-length NL4-3, BG505 and Env-expressing JR-FL by overlap-extension PCR. All viruses were produced by co-transfecting HEK293 cells using the following ratios of plasmids:
HIV-1_NL4-3: 40:1 of pNL4-3 ΔRT to pNL4-3_V1Q3_V4A1 ΔRT;
HIV-1_BG505: 40:1 of Q23_BG505 ΔRT to Q23_BG505_V1Q3_V4A1 ΔRT;
HIV-1_JR-FL: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 40:1 for pCAGGS_JR-FL to pCAGGS_JR-FL_V1Q3_V4A1.
HIV-1_NL4-3_D368R: 40:1 of pNL4-3_D368R ΔRT to pNL4-3_V1Q3_V4A1_D368R ΔRT.
HIV-1_BG505_D368R: 40:1 of Q23_BG505_D368R ΔRT to Q23_BG505_V1Q3_V4A1_D368R ΔRT.
HIV-1_JR-FL_D368R: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 40:1 for pCAGGS_JR-FL_D368R to pCAGGS_JR-FL_V1Q3_V4A1_D368R.
HIV-1_NL4-3 mixed trimer 1: 40:1 of pNL4-3_D368R ΔRT to pNL4-3_V1Q3_V4A1 ΔRT.
HIV-1_BG505 mixed trimer 1: 40:1 of Q23_BG505_D368R ΔRT to Q23_BG505_V1Q3_V4A1 ΔRT.
HIV-1_JR-FL mixed trimer 1: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 40:1 for pCAGGS_JR-FL_D368R to pCAGGS_JR-FL_V1Q3_V4A1.
HIV-1_NL4-3 mixed trimer 2: 20:20:1 of pNL4-3 ΔRT to pNL4-3_D368R ΔRT to pNL4-3_V1Q3_V4A1_D368R ΔRT.
HIV-1_BG505 mixed trimer 2: 20:20:1 of Q23_BG505 ΔRT to Q23_BG505_D368R ΔRT to Q23_BG505_V1Q3_V4A1_D368R ΔRT.
HIV-1_JR-FL mixed trimer 2: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 20:20:1 for pCAGGS_JR-FL to pCAGGS_D368R to pCAGGS_JR-FL_V1Q3_V4A1_D368R.
Statistically, the 20:20:1 ratio for mixed trimer 2 will yield 50% trimers with the desired mixed trimer two shown in Figure 2B.
HIV-1_NL4-3 mixed trimer 3: 40:1 of pNL4-3 ΔRT to pNL4-3_V1Q3_V4A1_D368R ΔRT.
HIV-1_BG505 mixed trimer 3: 40:1 of Q23_BG505 ΔRT to Q23_BG505_V1Q3_V4A1_D368R ΔRT.
HIV-1_JR-FL mixed trimer 3: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 40:1 for pCAGGS_JR-FL to pCAGGS_JR-FL_V1Q3_V4A1_D368R.
Virus was harvested 40 hr post transfection and concentrated by centrifugation over a 15% sucrose cushion at 20,000 x g for 2 hr. Virus pellets were resuspended in the labeling buffer containing 50 mM HEPES, 10 mM MgCl2, 10 mM CaCl2. Resuspended virus was enzymatically labeled with fluorophores in a reaction mixture containing Cy3B(3S)-cadaverine (0.5 μM), transglutaminase (0.65 μM; Sigma Aldrich), LD650-CoA (0.5 μM) (Lumidyne Technologies), and AcpS (5 μM) (Zhou et al., 2007 (link)). The labeling reaction was incubated at room temperature overnight. 0.1 mg/ml PEG2000-biotin (Avanti Polar Lipids) was added to the labeling reaction and incubated for 30 min at room temperature. Under these conditions, the labeling efficiencies of the Q3 and A1 tags are 40% and 55%, respectively (Munro et al., 2014 (link)). The virus was then purified away from excess fluorophore and lipid by ultracentrifugation (1 hr at 150,000 x g) over a 6–18% Optiprep (Sigma Aldrich) gradient containing 50 mM Tris pH 7.4 and 50 mM NaCl. The fractions containing viral particles were identified by p24 western blot and stored at −80°C.

The bacterial strains and plasmids used in this study are listed in Table 1. Wild-type S. meliloti strain Rm8530 is identical to strain 1021 except that it has a functional copy of the transcriptional regulator gene expR, which is required for QS [20 (link)]. PY (peptone-yeast extract) and LB (Luria broth) complex media and MMSN (minimal medium succinate ammonium) were described previously [7 (link)] and solidified with 1.5 % agar when necessary. Bromfield medium containing 0.5 % or 0.3 % Difco Noble Agar (Beckman, Dickinson and Co., Sparks, MD, USA) were prepared as described by Bahlawane [21 (link)]. Putrescine ·2HCl [Put; H₂N(CH₂)₄NH₂], cadaverine [Cad; H₂N(CH₂)₅NH₂], spermine [Spm; H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂], spermidine [Spd; H₂N(CH₂)₃NH(CH₂)₄NH₂], 1,3-diaminopropane [DAP; H₂N(CH₂)₃NH₂] and norspermidine [NSpd; H₂N(CH₂)₃NH(CH₂)₃NH₂] were purchased from Sigma (St. Louis, MO, USA) and homospermidine·3HCl [HSpd; H₂N(CH₂)₄NH(CH₂)₄NH₂] was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Aqueous 200 mM PA stock solutions were adjusted to pH 6.8, filter sterilized and added to cultures to a final concentration of 0.1 mM. When required, antibiotics were used at the following concentrations (µg ml⁻¹): gentamicin (Gm), 15; kanamycin (Km), 50; spectinomycin (Sp), 100; and streptomycin (Sm), 200.

A tuna extract (from a local supermarket) was prepared and analyzed by HPLC–MS by the Laboratorio de Salud Pública of Aragón (LSPA) using a previously validated method [36 ]. 2.5 g tuna were treated with 20 mL 5% trichloroacetic acid; the samples were shaken in a vortex for 30 s. Then, the mixture was submitted to ultracentrifugation for 10 min at 4000 rpm (4 °C); this operation was repeated twice. The filtrated was taken to 50 mL.
The following concentrations were obtained (found ± sd, in mg kg⁻¹): 100 ± 11 putrescine, 380 ± 19 cadaverine, 900 ± 40 histamine, 300 ± 22 tyramine.
A fraction of this extract was analyzed by the procedure previously described.

Phosphate buffer solutions (0.1 M, pH 6.0, 7.0, and 8.0) were prepared from Na₂HPO₄ and NaH₂PO₄ solids (Sigma S9638 and S9763). Carbonate buffer solution (0.1 M, pH 9.0) was prepared from Na₂CO₃ (Sigma 222,321) and NaHCO₃ (Sigma S5761).
Hydrogen peroxide stock solution (33% w/v) was supplied by Panreac (131,077.1211); the exact concentration was established and periodically checked by titration using potassium permanganate (oxalic acid as primary standard). Peroxidase from Horseradish (HRP EC 1.11.1.7) was obtained from Sigma (P8125 88.6 U mg⁻¹). Diamine oxidase from Lathirus cicera 280 U mL⁻¹ (DAO EC 1.4.3.22) was purchased from Molirom P021. Tyramine oxidase (TAO EC 1.4.3.9) from Arthrobacter sp. (T-25) 4600 U mg⁻¹ was purchased from Asahi Kasei Pharma Corporation.
Cadaverine (C8561), putrescine (P7505), histamine (53,300), tyramine (T287998), 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (A1888), 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red™, AR) (90,101), and 3,3′,5,5′-Tetramethylbenzidine (TMB) (860,336) were supplied by Sigma. All solutions were daily prepared by weighing and dissolving in the buffer solution (minus TMB and AR, which was dissolved in dimethyl sulfoxide (Panreac131954.1611)). TMB, ABTS, and AR solutions were stored in darkness.
Cellulose microcrystalline of 20 μm of particle size and average degree of polymerization minor than 350 (Aldrich 310,697) was used to develop the biosensors.