Tuberculin

The ocular availability of LZ was determined, after the topical application of LZ–containing formulations in the eyes of healthy rabbits. Six animals were divided into two groups (the first group for LZ–CSNPs and the second group for LZ–AqS). Around 40 μL of sterilized LZ–containing formulations (equivalent to 40 µg of LZ) were instilled in the right eyes of the respective group rabbits. After 1 h of dosing, the animals were sedated with intravenous administration of the mixture of Ketamine. HCl (15 mg/kg of b. wt.) and Xylazine (3 mg/kg of b. wt.) [3 (link),16 (link),35 (link),47 (link),50 (link)]. Successively,50 µL of AqH was taken out by a 29-gauge needle attached to a tuberculin syringe at predetermined time intervals. The obtained AqS samples were kept at −70 °C till the analysis was completed. The samples for the analysis were prepared and the drug concentrations in the samples were analyzed by the HPLC–UV method [31 (link),33 ].

Samples of the bottom sediments were collected in October 2018 in the Laptev Sea during the 73rd cruise of RV Akademik Mstislav Keldysh. Upper sediments were collected at three most representative stations at the shelf edge (Figure 1). Two of them (AMK73-6027 and AMK73-6045) were located at the sites of methane seeps, while station AMK73-6053, not affected directly by the river flow or methane seeps, was used as the reference one.
At each station, 6–7 sediment horizons (from the surface to 20–30 cm deep) were investigated. The samples were obtained with a plastic tube (15 cm in diameter) inserted into the sample collected with a box corer grab immediately after its retrieval on board the ship. Samples of the sediment layers were prepared in the onboard laboratory for radiotracer, chromatographic, biogeochemical, microbiological, and molecular genetic research. All experiments with the sediments were carried out within several hours after sampling at the temperatures close to in situ values. The samples for DNA isolation were collected from the same horizons, frozen, and stored at –20 °C.
To determine methane content, the head-space method of sampling was used. Methane concentration was measured by the phase-equilibrium degassing method on a Kristall-2000-M gas chromatograph (Russia) equipped with a flame ionization detector. The measurement error did not exceed ±5%.
Pore water was obtained from the sediments by centrifugation at 5000× g on a TsUM-1 centrifuge (Russia). The alkaline reserve was determined with the relevant reagent kit (Merck, Germany). The concentrations of sulfate and chloride ions in pore water were determined on a Staier ion chromatograph (Russia).
The rates of microbial processes: dark CO₂ assimilation (DCA), hydrogenotrophic methanogenesis (MG), methane oxidation (MO), and sulfate reduction (SR) was determined by radiotracer analysis with ¹⁴C- and ³⁵S-labeled substrates. For this purpose, bottom sediment samples (2.5 cm³) were collected with a cut-off syringe with a rubber plunger and sealed with a gas-tight butyl rubber stopper. The labeled substrate (0.2 mL) was injected with a tuberculin syringe by piercing the stopper at the center and distributing the substrate uniformly along the syringe length. Methane oxidation rate was determined with ¹⁴C-labeled methane dissolved in sterile distilled water (1 μCi per sample). Sulfate reduction rate was determined with ³⁵S-labeled sulfate (2.5 μCi per sample). The rates of methanogenesis and microbial CO₂ assimilation were determined using ¹⁴C-labeled bicarbonate (4.0 μCi per sample). The samples were incubated for 24 h at the temperature close to in situ values and then fixed with 1.0 mL of 1 M KOH. Sediment samples fixed with KOH prior to addition of the labeled substrates were used as the controls. Subsequent sample processing and calculation of the rates of microbial processes were carried out as described previously [19 ]. Radioactivity (¹⁴C and ³⁵S) of the products of microbial processes was measured on a PackardTRI-CarbTR 2400 liquid scintillation counter (USA). Numerical values of the rates of microbial processes (DCA, MG, MO, and SR) were calculated using the averages for two replicate measurements per each sample.
C_org content in the sediments was determined after removal of carbonates on an AN-7560 express analyzer (Russia) by registering the amount of CO₂ released after incineration of the sample at ~900 °C in the flow of CO₂-free air.