Fiducial Markers

We used previously analysed MEG data from 13 healthy subjects, where they formed part of studies on Parkinson's disease for which approval was obtained from the medical ethics committee of the VU University Medical Center. In these studies oscillatory power, as well as functional connectivity and network characteristics at the sensor level, were estimated and compared between healthy controls and demented and non-demented patients with Parkinson's disease (Bosboom et al., 2006, 2009 ).
All subjects gave written informed consent prior to participating. MEG data were acquired in the morning, using a 151-channel whole head MEG system (CTF Systems Inc., Port Coquitlam, Canada), situated in a magnetically shielded room (Vacuum-schmelze GmbH, Hanau, Germany). The data were sampled at 312.5 Hz, with a recording pass-band of 0–125 Hz, and a third-order software gradient was applied (Vrba and Robinson, 2002 ). Each session started with an approximately 5 minutes eyes-closed (EC) resting-state recording, followed by an approximately 5 minutes eyes-open (EO) recording. We only analysed the data recorded during the eyes-closed resting-state. Due to technical problems, 1–3 channels were discarded from the analysis (3, 3, and 7 datasets contained 148, 149, and 150 channels, respectively). For the construction of the beamformer weights, the eyes-closed data were band-pass filtered from 0.5 to 48 Hz, and after visual inspection, trials containing artefacts were removed. A time-window of, on average, 264.2 seconds (range: 175–360 s.) was used for the computation of the data covariance matrix. Broadband data were used for the estimation of the beamformer weights as this avoids overestimation of covariance between channels (Barnes and Hillebrand, 2003 (link)).
For each subject, an anatomical MRI of the head was obtained at 1 T (Impact, Siemens, Erlangen, Germany), with an in-plane resolution of 1 mm and slice thickness of 1.5 mm. Vitamin E capsules were placed at anatomical landmarks, the pre-auricular points and the nasion, to guide co-registration with the MEG data. In the MEG setting, three head position indicator coils were placed at the same fiducial locations, and these coils were activated at the start of each MEG acquisition. Head position and orientation were computed on the basis of the magnetic fields produced by these coils. Using these two corresponding sets of fiducial markers, the MEG and MRI coordinate systems were matched. The co-registered MRI was subsequently segmented, and the outline of the scalp was used to compute a multi-sphere head model (Huang et al., 1999 (link)) for the calculation of the lead-fields.

Plunge freezing was performed using an FEI Vitrobot (Thermo Fisher)^{53 (link)}. For cryoET sample preparation of bacterial cells, 10 nm colloidal gold fiducial markers (Sigma-Aldrich) were added to each sample at a ratio of 1:5 (v/v) to allow tilt image alignments. For Vitrobot setup, a filter paper (Whatman, 47 mm diameter) and a Teflon sheet were installed for single-sided blotting in a pre-cooled chamber (4 °C) with 100% humidity. EM grids (R2/2, Cu 200 mesh; Quantifoil Micro Tools) were glow-discharged for 45 s at 25 mA by PELCO easiGlow discharger. Sample aliquots (4 μl) were applied to each grid, incubated for 15 s and blotted for 6.5 s, followed by immediate plunge freezing in an ethane:propane mixture (37% v/v ethane:63% v/v propane)^{54 (link)}. Grids were stored in liquid nitrogen. Before loading of the samples into the cryo-electron microscope, the grids were clipped. For sample preparation, all bacterial samples were pelleted, and OD₆₀₀ was adjusted to 2–2.5 before blotting. If required, L. monocytogenes or E. faecalis cells were exposed to 1,024 nM purified Ply006 or Ply007, respectively, followed by plunge freezing at the desired timepoints. For imaging of phage adsorption, bacterial cultures were adjusted to an OD₆₀₀ of 0.1. Samples (95 µl) were then mixed with 5 µl of purified phage lysate (10¹¹ p.f.u. ml⁻¹), followed by 5 min incubation at room temperature.

Men in the experimental arm of 25 Gy in 5 fractions received a 5 Gy daily fraction starting in mid-week and given on a 7-day period followed by a HDR BB of 15 Gy in a single fraction. The MHF groups were comprised of men who received either 36 Gy in 12 fractions with a 3 Gy daily fraction or 37.5 Gy in 15 fractions with a 2.5 Gy daily fraction, all followed by the same HDR BB of 15 Gy in a single fraction. Biological doses in the UHF regimen were calculated to be equivalent to the standard treatment schedule assuming an alpha/beta ratio of 1.5 (see Supplementary Table 5). Short term androgen deprivation therapy (STADT), from 4 to 6 months, was administered per physician’s preference if Gleason score was 7 (4 + 3) or if there was presence of more locally extensive disease (>50 % positive biopsies) corresponding to RTOG protocol[15] .
IGRT technique using fiducial gold markers was required for all groups for daily match on prostate and first proximal cm of seminal vesicles. Intensity-modulated radiation techniques (IMRT) with volumetric-modulated arc therapy (VMAT) and inverse planning were used for all treatment groups. Dose constraints in the experimental arm were followed for organs at risk such as the bladder and rectum (see Supplementary Table 4). Minor deviations in the prescribed doses were permitted to meet those constraints. Energy used for EBRT was 6 MV. The clinical target volume (CTV) consisted of the prostate plus the first proximal cm of the seminal vesicles as identified on the planning CT scan at the time of treatment planning. The planning target volume was obtained by a 3D expansion of 5 mm of the previously described CTV. Pelvic lymph nodes were not included.
The HDR brachytherapy procedure has already been described before[16] (link). Under general anesthesia, 14 to 21 interstitial catheters were placed into the prostate gland through the perineum via ultrasound guidance. Dosimetric optimization was done using ultrasonographic-based planning (Oncentra Prostate v.4.2.2 brachytherapy software), allowing contouring of the prostate and organs at risk. The prescribed dose was 15 Gy. Details on dosimetric goals and constraints are provided in the supplementary appendix. Cystoscopy was performed to ensure the bladder and urethra integrity.
All EBRT plans were reviewed in a weekly quality assurance meeting with other radiation oncologists at our center. Target volumes, isodoses, organ doses constraints and DVH were validated by colleagues for compliance with protocol guidelines. A kilovoltage (KV) imaging marker match was performed daily and cone beam CT (CBCT) scans were acquired at each fraction in the experimental arm (weekly for the reference arms).