Acetic acid

Peptides were separated by an Ultimate 3000 nanoLC‐MS/MS system (Dionex LC‐Packings, Amsterdam, the Netherlands) equipped with a 20 cm × 75 μm ID fused silica column custom packed with 1.9 μm 120 Å ReproSil Pur C18 aqua (Dr Maisch GMBH, Ammerbuch‐Entringen, Germany). After injection, peptides were trapped at 6 μl/min on a 10 mm × 100 μm ID trap column packed with 5 μm 120 Å ReproSil Pur C18 aqua in 0.05% formic acid. Peptides were separated at 300 nl/min in a 10–40% gradient (buffer A: 0.5% acetic acid (Fisher Scientific), buffer B: 80% ACN, 0.5% acetic acid) in 60 min (90‐min inject‐to‐inject). Eluting peptides were ionized at a potential of +2 kVa into a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Intact masses were measured at resolution 70,000 (at m/z 200) in the orbitrap using an AGC target value of 3E6 charges. The top 10 peptide signals (charge‐states 2+ and higher) were submitted to MS/MS in the HCD (higher‐energy collision) cell (1.6 amu isolation width, 25% normalized collision energy). MS/MS spectra were acquired at resolution 17,500 (at m/z 200) in the orbitrap using an AGC target value of 1E6 charges, a maxIT of 60 ms, and an underfill ratio of 0.1%. Dynamic exclusion was applied with a repeat count of 1 and an exclusion time of 30 s.

On days 7 and 21, the samples were fixed in PFA 4% overnight. The samples were then incubated in 3% acetic acid (Fisher, Loughborough, UK) for 3 min. Alcian blue solution (1%, in 3% acetic acid) (Fisher, Loughborough, UK) was added to the samples for 60 min, and excess solution was removed by rinsing with deionised water. Subsequently, guanidine hydrochloride solution (8 M) (Merck Life Science, Gillingham, UK) was added, and the samples were incubated overnight at 4 °C. An aliquot from each well was transferred to a 96-well plate. The absorbance at 600 nm was measured using a spectrophotometer (SpectraMax i3x, Molecular Devices) to quantify the sGAGs content.
For DNA content quantification, nuclei were stained with Harris Haematoxylin (Merck Life Science, Gillingham, UK) for 5 min, and excess staining was removed with 2 washes in deionised water. Subsequently, a mixture of 100% ethanol and 99% acetic acid (75:25) (Fisher, Loughborough, UK) was added, and the samples were incubated at 45 °C (4 h). Aliquots from each sample were transferred to a 96-well plate. The absorbance was read at 450 nm using a spectrophotometer to quantify DNA content.

The lipid metabolites from testicular contents were determined via UPLC–MS/MS. In short, the UPLC (Shim‐pack UPLC Shimadzu CBM30A) and MS/MS (QTRAP 6500+) system was configured with a resolution of 30,000 in order to acquire UPLC–MS/MS data. The analysis was conducted in positive ion mode using a spray voltage of 5.5 kV and in negative ion mode using a spray voltage of −4.5 kV, while maintaining a capillary temperature of 500°C. The mass spectrometer was set to scan 50–1500 m/z. The nitrogen sheath and nitrogen auxiliary gas were adjusted to flow at rates of 30 and 10 L/min, respectively. Solvent A consisted of 0.04% acetic acid (Fisher Scientific) diluted with water (Millipore) in a volume‐to‐volume ratio, while solvent B was prepared by combining 0.04% acetic acid (Fisher Scientific) with acetonitrile (Fisher Scientific) in a volume‐to‐volume ratio. The rate of gradient flow was adjusted to 0.4 mL/min, while maintaining a column temperature of 40°C. The linear gradient was as follows: 5% B at 0 min, 95% B at 11.0 min, 95% B at 12.0 min, 5% B at 12.1 min, and 5% B at 14 min. To guarantee system stability, the QC samples were injected four times initially. For all analyses, a Waters ACQUITY UPLC HSS T3 C18 column measuring 100 mm × 2.1 mm with a particle size of 1.8 μm was utilized. The contents of lipid metabolites were detected using MetWare (http://www.metware.cn/).

12 μg of total protein (same preparation as Western blot) was loaded on Novex® 10% Zymogram (Gelatin) Protein gels (Invitrogen) for electrophoresis. Gels were then renatured using Renaturing Buffer (Invitrogen) for 30 min at room temperature and equilibrated in Developing Buffer (Invitrogen) for 30 min at room temperature. After that, gels were incubated in Developing Buffer at 37 °C overnight. After a few brief rinses with distilled water, gels were stained with 0.1% Coomassie Blue (Sigma) in 40% methanol (Fisher Scientific) and 10% acetic acid (Fisher Scientific) for 30 min at room temperature and destained in 10% methanol and 7.5% acetic acid for 1–2 h at room temperature. Tap water was used to stop destaining. Gels were pictured on Image Quant™ LAS 4000 and results were analyzed with Image Quant TL 7.0 software. Recombinant human MMP-9, MMP-2 and purified neutrophil MMP-9/NGAL complex were used as positive controls.

To quantify biofilms, plates or pegs were gently immersed in water to remove non-adhered cells, placed in 0.1 % crystal violet (w/v) (Sigma, Dorset) for 10 min then gently washed in water, twice, to remove excess stain. The plates for temperature-dependent biofilm analysis had 200 µl of 30 % acetic acid (v/v) added to each well to solubilize stain, whereas the lid with stained pegs was placed into a 96-well plate containing 200 µl per well of 30 % acetic acid (Fisher Scientific, Loughborough) for 10 min with agitation (110 rpm). Optical density of each well was measured at 540nm using a FLUOstar Omega plate reader (BMG Labtech, Aylesbury).

All the chemicals, solvents, and other consumable used were of analytical grade. All inorganic chemicals and salts were purchased from Sigma‐Aldrich. Methanol and acetic acid were purchased from Acros Organics. LB broth and agar for culturing bacteria were purchased from Fisher Scientific: Janssen Pharmaceuticalaan. The distilled water used in this work was provided by the Thermo Scientific.

To synthesize star-shaped poly(_L-lysine) homopolypeptides (s-PLL), the Z groups were removed by using hydrogen bromide (HBr, 33.0 wt% in acetic acid, Acros, Yehud, Israel) (Scheme 1a) [21 (link),22 (link),27 (link)]. In detail, each 1.0 g of s-PZLL was dissolved in 50 mL of trifluoroacetic acid (TFA, Alfa Aesar, Lancashire, England) before HBr was dropped into the mixture slowly (molar ratio of HBr:Z group = 5:1). The reaction occurred at RT during stirring for 1 h. After completion of reaction, the solution was poured into diethyl ether for precipitation and the solvent was removed by centrifugation. The product was washed with diethyl ether twice and dried completely under vacuum overnight. The dried solid was dissolved in DIW and dialyzed against DIW for 72 h using a cellulose membrane with MWCO of 6000–8000 g mol⁻¹. Lyophilization was employed for the removal of water to obtain the final product with a 90.0–97.0% of yield. The polypeptide sample after deprotection was dissolved in D₂O for ¹H NMR-600NMR analysis.