Thiazolyl blue tetrazolium bromide

Human RSV strain A2 (ATCC, Manassas, VA; #VR-1540) was infected at a multiplicity of 0.1 into Hep2 cells. The virus was allowed to grow for 5 days at 37°C in a 5% CO₂ atmosphere. The infected Hep2 monolayers were collected and the virus was released by sonication. Cell debris was removed by centrifugation at 2500 g for 5 minutes at 4°C. Virus was collected by centrifuging the supernatant for 2 hours at 22000×g at 4°C. Virus were suspended in culture media and snap frozen and maintained at −80°C. Infectious virus titers were determined on Hep2 cells by performing serial dilutions of the RSV stocks and counting infected cells stained for indirect immunofluorescence with an RSV F-specific monoclonal antibody (Abcam, Cambridge, MA). Additionally, plaque assays were performed as previously described [25] on Hep2 cells using methyl cellulose overlay media (R&D Systems) and staining with 0.5 mg/ml thiazolyl blue tetrazolium bromide (MTT; Sigma Aldrich) solution in PBS for 3 hours at 37°C. Non-infected Hep2 cell cultures were processed in the same manner as RSV infected cells and the resulting sample collection was used as a mock control.

Bacterial strains and growth conditions.Micrococcus luteus C01 was isolated from the skin of a healthy volunteer and identified by 16S rRNA sequencing, as described previously [30 (link)]. The bacterium was conserved at room temperature (RT) in glass tubes filled with 5 mL lysogeny broth (LB, Lennox, Dia-M, Moscow, Russia) with the addition of 0.3% agar (BD, USA), and cultures were grown on the surface of semisolid agar and covered with sterile mineral oil. For experiments examining biofilm growth, cultures were plated onto reinforced clostridial medium (RCM) with 1.5% agar, and single colonies were obtained. The RCM composition (g/L) was yeast extract 13, peptone– 10, sodium chloride – 5, sodium acetate– 3, glucose – 5, starch – 1, and L-cysteine-HCl – 0.5, pH 7.0. All reagents were obtained in Dia-M, Russia except L-cys-HCl (Biomerieux, France). The RCM medium was tested previously (data not shown) and demonstrated the optimal conditions for M. luteus C01 biofilm experiments with medium-level biofilm growth in comparison to lysogeny broth (LB) and tryptic soy broth (TSB) (not too abundant but significant biofilms growth to track the epinephrine effect). The RCM medium was sterilized in mild conditions at 112°C for 30 min to avoid sugar caramelization and destruction of other medium components? such asyeast extract. For each experiment, a single colony was inoculated in a 50 mL conical flask with 15 mL of LB and cultivated overnight at 33°C (close to skin physiological temperature [7 (link)]) and shaker speed 180 rpm. The resulting M. luteus cell suspension was adjusted with sterile physiological saline (PS, 0.9% NaCl in distilled water, pH 7.0) to a final OD_{540 of} 0.5 (3 × 10⁷ CFU/mL) for polytetrafluoroethylene (PTFE) cube experiments and confocal microscopy studies or 0.1 (6 × 10⁶ CFU/mL) for assessments of biofilms on glass fiber filters.
Stock solutions of epinephrine. Epinephrine (Sigma–Aldrich, USA) was dissolved in sterile Milli-Q water, and a series of stock solutions in sterile Milli-Q water were prepared for use in experiments. Under these conditions epinephrine is stable for a sufficiently long time [31 (link)]. Stock solutions were stored at −18 °C in 15 mL conical centrifuge tubes (ThermoFisher, USA), in the dark to avoid potential light-caused destruction of the hormone. Because of relatively unstable liquid homeostasis in skin (due to the alternation of sweating and drying periods), it is difficult to define the concentrations of epinephrine in M. luteus inhabiting microniches in skin, such as hair follicles and the stratum corneum [2 (link)]. Thus, we have taken 4.9 × 10⁻⁹ M (physiological) of epinephrine as the baseline concentration because, according to a recent review, it is close to the normal physiological concentration in blood plasma [28 (link)]. In experiments with PTFE cubes, the range of epinephrine concentrations (4.9 × 10⁻¹², 4.9 × 10⁻¹¹, 4.9 × 10⁻¹⁰, 2.4 × 10⁻⁹, 4.9 × 10⁻⁹, 9.8 × 10⁻⁹, 4.9 × 10⁻⁷, 4.9 × 10⁻⁶ M) was also tested. Higher concentrations of epinephrine were chosen to simulate different stress conditions when level of epinephrine in bloodstream increases or when there is a disorder in the human organism leading to a permanent increase in the epinephrine concentration [28 (link)], or potentially lower concentrations of the hormone which may be present in skin. Next, a concentration of 4.9 × 10⁻⁹ M was employed in the transcriptomic analysis, confocal microscopy studies and glass fiber filters assay × . All tubes, Petri plates and glass-bottom plates with biofilms of M. luteus C01 were also incubated in the dark when epinephrine was added. Due to the lack of information on the probable consumption of epinephrine by M. luteus C01 during its growth, and because of technical difficulties in measuring of epinephrine concentration in the small volume of the rich medium at each time point of an experiment, we did not inoculate any additional portions of epinephrine during the incubation. The hormone was inoculated once at the beginning of the experiment to make conditions standard.
Biofilm growth on PTFE cubes. Biofilms on cubes were grown as described in previous studies [30 (link),32 (link),33 (link)] with modifications. Briefly, in glass tubes of a standard volume 22 mL with screw plugs, 21 chemically pure cubes with a size of 4 × 4x4 mm were placed. After addition of 3 mL of the RCM to each tube, the tubes were capped loosely with screw plugs and autoclaved at 112 °C. After sterilization and cooling, appropriate epinephrine stock solutions were added to each tube to obtain different final epinephrine concentrations in the medium. Tubes without epinephrine addition were used as a positive control. Then, 50 μL of prepared cell suspension was added to each tube, and at least two tubes were used as negative controls without bacterial inoculation. The tubes were incubated at 33 °C at 180 rpm for 24 h or 72 h to obtain immature and mature biofilms, respectively [29 (link)]. After incubation, the OD₅₄₀ was measured using of empty controls without bacterial inoculation, and biofilm CV or MTT staining was subsequently performed.
Biofilm staining on PTFE cubes. To analyze the total amount of biofilms on the PTFE surface, CV staining was used. Biofilms were stained as described previously [32 (link)]. Briefly, cubes were washed twice gently with room temperature tap water to remove the planktonic culture and fixed with 3 mL of 96% ethanol for 20 min. After fixation, ethanol was removed, cubes were dried, and 2 mL of the 0.5% CV solution in distilled water was added to each tube and incubated for 20 min at room temperature. Next, the CV was removed, and cubes were washed 6 times gently with RT tap water and placed into new clear glass tubes to be covered with 3 mL of 96% ethanol for dye extraction. The OD₅₉₀ was measured after 30 min of extraction using of negative controls. OD measurements were performed using a spectrophotometer PE-5400VI (Ecroskhim, Russia).
To analyze the metabolic activity of biofilms on PTFE cubes, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Dia-M, Russia) was used [33 (link)]. Cubes were washed twice to remove planktonic suspension residues. After that step, 0.1% MTT solution in sterile LB medium was prepared, 3 mL of this solution was added to each tube, and biofilms were stained for 1 h at room temperature. Then, biofilms were washed three times gently with room temperature tap water, dried, and moved into new clear glass tubes, and 3 mL of dimethyl sulfoxide (DMSO, 99.9%, EKOS, Russia) was added to each tube for formazan extraction. Extraction was performed overnight in tubes sealed with Parafilm® (Amcor, Switzerland), and the OD₅₄₀ was measured after extraction.
DNAse I and Proteinase K succeptibility test. Biofilms of M. luteus were grown on PTFE cubes for 24 h and 72 h in the presence of physiological concentrations of epinephrine and treated with proteinase K (Dia-M, Russia) and DNAse I (NEB, USA). Both enzymes were used in concentration 5 μg/mL. Proteinase K treatment was performed according to Ref. [34 (link)]. DNAse I treatment was conducted according to Ref. [35 (link)] and the manufacturer's protocol. Briefly, biofilms on cubes were washed twice with water and treated with an appropriate enzyme. Subsequently, biofilms were washed twice again, fixed in 96% ethanol and stained with CV as described before.
Initial adhesion test and cell surface hydrophobicity in the presence of epinephrine. Epinephrine can potentially change the initial adhesion of M. luteus C01 cells to PTFE. To examine the potential changes in initial cell adhesion in the presence of 4.9 × 10⁻⁹ M epinephrine, the potential decrease of in the OD₅₄₀ of the M. luteus C01 cell suspension exposed to PTFE cubes in the system depicted above was measured. Briefly, a portion of the 24 h culture was diluted in the RCM with or without epinephrine up to an OD₅₄₀ = 0.1. Three milliliters of the suspension was inoculated into chemically clean tubes with 21 PTFE cubes and incubated at 33°C at 150 rpm. The OD₅₄₀ was measured at different time points (10, 20, 30 min) after the start of the experiment. Also, after 30 min of incubation cells were washed out from cubes and plated for CFU counts. Cells adherence to the cubes resulted in an OD decrease, and epinephrine could potentially alter this process. Additionally, cell surface hydrophobicity in the presence of epinephrine was measured as described by Gannesen et al., 2018 and [36 (link)] by the adhesion to hexadecane method.
Biofilm growth on glass filters. Glass microfiber filters (GMFF, Whatman® GF/F, USA) were used as carriers for biofilms of M. luteus to analyze the metabolic activity and colony forming unit (CFU) amounts. Biofilms were studied as described previously [37 (link)]. RCM with 1.5% agar was melted and stored in a 55°C water bath for the experiment. An appropriate volume of the epinephrine stock solution was dropped into a sterile 100 mL glass vial. Next, 20 mL of RCM-agar was addedto the vial, mixed properly for 5 s and plated onto a 90 mm Petri dish. RCM-agar without epinephrine was used as a control. After medium solidification, six sterile Ø 21 mm GMFFs were placed on the agar surface, and 20 μL of M. luteus suspension with OD₅₄₀ 0.1 was dropped on the center of each filter. A filter without inoculation was used as a negative control for MTT staining. Biofilms were grown for 24 h and 72 h, after which 3 filters were stained with MTT. Briefly, the filters wereplaced in a clean 6-well plate, with one filter per well, after addition 3 mL of LB containing 0.1% MTT (mass/volume percent) to each well and resting for 30 min at room temperature. Then, the filters were gently washed with distilled water to remove the resting MTT solution and stopthe reaction, placed into another 6-well plate and covered with 3 mL of DMSO per filter to extract the formazan. The remaining 3 filters from a plate were dispersed for CFU counts. Briefly, each filter was placed into a glass tube filled with 10 mL of PS, dispersed with a glass stick and vortexed for 1 min at medium speed. A series of 10X dilutions was made from the resulting suspension and then 20 μL of a final suspension was plated onto a Petri dish. Additionally, 10 μL of each nondiluted filter suspension was fixed and stained with CV for cell aggregation analysis using light microscopy (Carl Zeiss Jena, Germany) analysis at 900x magnification with immersion oil (Merck, Germany). The samples were visually evaluated in 50–100 fields. For each sample, at least 5 of the most representative photos were taken (microscope-attached camera ToupView, China), and single cells, cell aggregates and aggregate ratios were calculated manually on photos. Additionally, the change in the amount of aggregates in the presence of the hormone was analyzed..
Confocal laser scanning microscopy (CLSM). CLSM was performed as described previously [38 (link)]. Briefly, biofilms were grown in 24-well black plates with flat glass bottoms (Eppendorf, Germany). One mLmilliliter per well of RCM with or without 4.9 × 10⁻⁹ M epinephrine was added to a plate, and 17 μL of prepared M. luteus cell suspension with OD₅₄₀ 0.5 was added to each well. The plates were incubated for 24 and 72 h at 33°C at a shaker speed of 180 rpm to obtain well-established biofilms on the flat glass surface. After incubation, the biofilms in wells were washed with sterile PS to remove unattached cells. Then, the samples were stained with SYTO 9 Green dye (Molecular Probes (Thermo Fisher Scientific)): the manufacturer-produced dye solution in DMSO was diluted 1000 times in PS, and 200 μL of the obtained solution was applied to the wells. Staining was performed for 20 min at room temperature in the dark, whereupon the liquid was discarded and the wells were washed two times with PS. ProLong Gold Antifade Mountant liquid (1–2 drops, Molecular Probes (Thermo Fisher Scientific)) was added, and plates were incubated overnight at 4°С. The samples were analyzed with an LSM 510 Meta inverted confocal microscope (Carl Zeiss, Germany) at an argon laser wavelength of 488 nm with a 63 × /1.2 water immersion lens. To limit the spectral range of fluorescence, a longpass filter with a transmission above 505 nm was used. The optical resolution of the system in routine measurements is 0.3 μm along the focal plane and 0.7 μm along the optical axis of the lens. The pixel size of the digitized image is 0.12 × 0.12 μm, the size of the area is 146.4 × 146.4 μ m, and the scanning step along the z axis is 1 μm. While obtaining more detailed 3-D images, the confocal aperture was close to a size corresponding to 0.7 Airy disk, and the z-scan was reduced to 0.5 μm. The microscopic studies were conducted, and the sample data files were obtained using Carl Zeiss LSM 510 Software, Version 3.2 (Carl Zeiss, Germany). The data obtained were processed using the ImageJ package in the Comstat2 plug-in software (based on predesigned computational algorithms). For each well, at least 5 3D-photos were taken for quantitative analysis. Four parameters were determined: biofilm average thickness (μm); average biomass distribution per area unit (ABD, μm³/μm²); and biofilm surface square (BSS, μm²) Images of 3-dimensional biofilm structure were obtained using the Zen 2.3 Blue Edition (Carl Zeiss microscopy GmbH). Experiments were conducted in triplicate.
Total RNA isolation from M. luteus biofilms. Before RNA isolation, crushed glass for cell disruption was prepared of typical filament lamp glass: four lamps were broken in the kettle and the glass was ground up, washed to chemical purity with bichrome solution, treated with 3% H₂O₂ to avoid any residual RNAse activity and sterilized by autoclaving. All vessels, pestles and nonmetal instruments were prepared chemically pure, pretreated with H₂O₂ and autoclaved similarly to crushed glass. The electrophoresis cell, gel combs and plates were also pretreated with H₂O₂ solution.
Petri dishes with RCM-agar with and without the addition of 4.9 × 10⁻⁹ M epinephrine were prepared as described above. Two sterile Ø 21 mm GMFFs (one filter was in reserve) were placed onto the agar surface per dish. Beforehand, the prepared M. luteus cell suspension with an OD₅₄₀ 0.5 was diluted 100 times with sterile PS to a final CFU count of 3 × 10⁵ CFU/mL, and 25 μL of this suspension was inoculated on the center of each filter. Biofilms were grown for 24 h. A Qiagen RNeasy® Mini Kit (Qiagen, Germany) was used for total RNA extraction. The manufacturer's protocol was performed with changes. After incubation, the filter with the biomass was placed into the porcelain mortar. Twenty-five microlitersof RLT buffer (with addition of mercaptoethanol according to the manufacturer's protocol) was applied onto the biofilm, and 0.5 cm³ of crushed glass was placed onto the filter. The mortar was filled top to bottom with liquid N₂[39] , and the pestle was also cooled in liquid N₂. When 3/4 of the N₂ volume was evaporated, the mass in the mortar was vigorously smashed using the pestle until N₂ was completely gone and before the moment of ice melting. The cycle with N₂ addition and smashing was then repeated 4 times. Ultimately, 1 mL of RLT buffer was added to the ice-cold mortar with the resulting frozen homogenous powder of glass with disrupted cells, mL, and the mass was vigorously mixed until the ice was melted. mLThe suspension was then transferred into a sterile 2 mL Eppendorf tube, and the glass powder was pelleted in an Eppendorf Minispin centrifuge (Germany) at 13000 rpm (11700 g) and room temperature for 15 s. The supernatant was transferred into RNeasy columns, and all subsequent manipulations were conducted according to the manufacturer's protocol.
Agarose gel electrophoresis was performed to check the quality of the total RNA extracted from biofilms. A 1% agarose (Sigma, USA) gel was used, and 0.01% v/v ethidium bromide (Sigma, USA) was inoculated into the gel. For each experiment, fresh 1X TAE buffer was prepared to fill the electrophoresis cell. The RNA samples were separated at 65 V for 70 min. Ribosomal RNA bands were the main marker of successful extraction and other RNAs were visualized using of Bio–Rad Gel Doc XR System w/Universal Hood II (Bio–Rad, USA) and Gel Doc XR software. Samples of RNA were stored at −80 °C. Experiments were conducted in duplicate.
Total RNA sequencing. The concentration of RNA in the samples was measured with Qubit 2.0 (Invitrogen, USA). Ribosomal RNA depletion was conducted using the Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina, USA). Depletion was performed according to the manufacturer's protocol with 120 ng of total RNA for each sample. Next, RNA libraries were prepared using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs® Inc., USA) according to the manufacturer's protocol. RNA was fragmented for 5 min. Libraries were indexed using of the index primers set NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 2) from New England Biolabs® Inc., USA. Library amplification was carried out in 15 PCR cycles. Sequencing was conducted in single-end mode in three runs: the first on a HiSeq 4000 with read length of 151 base pairs (bp), the second on MiSeq with a read length of 301 bp and the third on HiSeq 4000 with read length of 51 bp. Reads were generated by bcl2fastq 2.20 [40] without allowing mismatches in sequencing indexes (“--barcode-mismatches = 0″).
Read preprocessing and quality control. Reads were preprocessed by Trimmomatic 0.39 [41 (link)] performing the following procedures consecutively:1.

Adapter trimming.

2.

Removal of bases with a quality below 3 from the 3′ ends of reads.

3.

Removal of 3′ ends of reads starting with 4 bp-long regions with an average quality below 15 (option “SLIDINGWINDOW:4:15″).

4.

Removal of reads with average quality below 20.

5.

Removal of reads shorter than 30 bp.

To analyze the level of contamination in reads, 1000 reads from each library were aligned by BLASTN 2.9.0 [42 (link)] to the NCBI nt database with a maximum e-value of 10⁻⁵. The taxonomy of the best BLAST hit according to the NCBI Taxonomy database was used to infer the taxonomy of the read source. The NCBI nt and NCBI Taxonomy databases were current as of 24 April 2020. The analysis showed that the level of contamination is negligible (Table S1.)
Differential expression analysis. To analyze differential expression, reads were aligned to the genome of M. luteus NCTC 2665 (NCBI accession NC_012803.1) by BWA 0.7.17 [43 (link)] using the BWA-MEM algorithm. The numbers of reads belonging to different genes were calculated by Salmon 1.3.0 [44 (link)] with 20 Gibbs samples and a correction for GC bias. Then, the differential expression was analyzed by DeSeq2 1.22.2 with the default parameters. Hierarchical clustering was performed using the hclust function of the R programming language with the complete linkage clustering algorithm. Principal component analysis was performed using the plotPCA function of DeSeq2.
Quantitative RT–PCR. To confirm, the results of differential expression analysis. RT–PCR was conducted. Newly extracted total RNA samples were obtained from three independent experiments as described above. First strand complementary DNA (cDNA) synthesis for real-time qPCR was performed using Moloney Mouse Leukemia Virus reverse transcriptase according to the manufacturer's protocol (Evrogen, Russia). Specific primers (Supplementary data Table S3) were applied for the synthesis of unique cDNA fragments. At least three pairs of primers for each gene with differential expression were selected using Unipro UGENE v.38.1 (Okonechnikov et al., 2012) with the built-in Primer3 module and the primer selection function for RT-PCR. The primers were checked in silico using the web resource insilico [45 (link)]. The annotated M. luteus genome NTCT 2665 was used as a reference. To find the optimal primer pairs, hybridization with total DNA of M. luteus C01 was performed once before the RT–PCR experiments. Total DNA was extracted from 24 h suspension cultures of M. luteus C01 using of Wizard® Genomic DNA Purification Kit (Promega, USA). For improved cell wall disruption, the pellet was previously frozen with liquid nitrogen and milled with glass as described for the total RNA extraction protocol.
RT-PCR was performed in PB PCR buffer (Syntol, Russia) in the presence of SYBR Green I and the passive reference dye ROX for fluorescent signal normalization. For each sample, detection was conducted twice. ddH2O (Syntol, Russia) was used as a negative control. Amplification was carried out with the CFX96 Touch™ RT–PCR detection system (Bio-Rad, USA) in the following reaction regime: polymerase activation for 5 min at 95°C followed by 40 cycles of 15 s at 95 °C–20 s at 55 °C–40 s at 62 °C. The differential expression of selected genes was measured in comparison to a control nonprocessed sample. Average means of target genes were normalized in comparison with the reference gene MLUT_08120 (F0 subunit of the conserved ATP synthase). The amount of a target normalized to an endogenic control and a calibrator was determined using the Ct (ΔΔСt) comparison method with formula 2^−ΔΔCt. Data analysis was performed using CFX ManangerTM Software v. 1.6.
In silico protein sequence analysis. Protein homologs searches, alignment analyses, and building similarity trees were performed using NCBI BLAST tools [46 ] (https://blast.ncbi.nlm.nih.gov/Blast.cgi), the NCBI protein database [47 ] (https://www.ncbi.nlm.nih.gov/protein) and the UniProt database [48 (link)].
Microbiological statistics. All experiments were conducted at least in triplicate. RNA extraction was performed in duplicate. Statistical analysis of the data (except the RNA sequencing described above) was performed using the nonparametric Mann–Whitney U test. Analysis of the CFU counts was performed using the nonparametric Wilcoxon Z-test. q-values were calculated from p-value in each experiment using false discovery rate correction, as proposed previously [49 ]. q-values are indicated on the data plots.
All microbiological data plots were designed using Microsoft EXCEL 2007 Software. Average relative values (the control without addition of epinephrine was designated as 100%) were plotted on the graphs, and the standard error of the mean was depicted as error bars.

The cytotoxicity of different ADPs was evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, thiazolyl blue tetrazolium bromide) assay and flow cytometry. The cells were treated as above mentioned. Subsequently, Annexin V fluorescein isothiocyanate/propidium iodide double-staining kit was used to assess the cell apoptosis and the MTT was performed to assess the cell viability.

Cell viability was determined using the MTT assay. PC12 cells were seeded in 96-well plates (WHB Scientific, China) at a density of 5×10³ cells/well, treated by compounds for 18 h, and subjected to oxidative damage for 24 h with H₂O₂. MTT (20 μL) (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide, Solarbio, China) at a concentration of 5 mg/mL was added in each well and incubated under dark conditions for 4 h. Subsequently, 120 μL of dimethyl sulfoxide (DMSO) was added and the absorbance of each well was measured at 490 nm using a microorifice plate detector (Thermo Fisher Scientific, USA).