Vertex 70 infrared spectrometer

For Fourier-Transform Infrared Spectroscopy, 5 ml volumes of the S. kujiense spent medium experiment and of the abiotic control were pelleted and rinsed three times with DI water, and concentrated suspensions were deposited and air-dried on aluminum foils. FT-IR measurements were conducted on a vertex 70 infrared spectrometer (Bruker Optics) equipped with a deuterated triglycerine sulfate (DTGS) detector and high intensity water cooled globar source. Spectra were collected at 5 cm⁻¹ resolution (2.5 mm aperture) as an average of 100 scans using MVP PRO (Harrick Scientific) diamond attenuated total reflectance (ATR) accessory set at a fixed incident angle of 45°. The instrument was purged for 30 min before the first measurement to ensure baseline stability. Data were baseline corrected using the “Rubber Band” algorithm within the OPUS 2.2 software. The experimental spectra were plotted with a hydroxyapatite reference spectrum (RRUFF database; Lafuente et al., 2015 ). Except when otherwise noted, tentative band and peak assignments were performed according to Bellamy (1975) (link) and Socrates (2004) .

Optical rotations were carried out on an Autopol VI automatoc polarimeter. UV spectra were recorded on a Shimadzu UV-2401 PC spectrophotometer. IR spectra (KBr) were determined on a Bruker Vertex 70 infrared spectrometer. ESI and HRESIMS were performed on an UPLCIT-TOF spectrometer. ECD spectra were obtained on a Chirascan-plus CD spectrometer (Applied Photophysics Ltd., UK). NMR spectral data were measured on a Bruker DRX-600 spectrometer. Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., China) was used for column chromatography. Semi-preparative HPLC was performed on an Agilent 1260 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 150 mm) column.

Tissues samples were processed and analyzed as described previously [23 (link), 25 (link)] and in the Supplementary Information. Briefly, samples were de-identified and two adjacent sections, each ~ 4 μm thick, were cut from each tissue microarray block. One section was H&E stained and graded according to the Elston–Ellis method for NPI scoring. The second serial section was mounted on an infrared-transmitting calcium fluoride microscope slide and, after deparaffinization, was placed in a Bruker Vertex70 Infrared Spectrometer equipped with a Hyperion 2000 microscope. The microscope aperture was set to sample an area of 500 μm by 500 μm (smaller than the area of the core). The aperture was centered over each core and an average of 64 interferograms was then recorded for each unstained core section on the slide. The resulting averaged interferogram for each sample was Fourier Transformed and thus converted to an absorption spectrum using Bruker’s OPUS software. DI was quantified using proprietary software written in MATLAB version R2022b (Fig. 1).
Considering that DNA aneuploidy has been shown to correlate with a high malignancy grade, frequent mitoses and a high degree of nuclear pleomorphism, as well as the difficulties in assessing aneuploidy in tissue sections, we used pleomorphism as a surrogate for aneuploidy and examined its relationship to DI.