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The World Health Organization has designated Pseudomonas aeruginosa as a critical pathogen for the development of new antimicrobials. Bacterial viruses, or bacteriophages, have been used in various clinical settings, commonly called phage therapy, to address this growing public health crisis. Here, we describe a high-resolution structural atlas of a therapeutic, contractile-tailed Pseudomonas phage, Pa193. We used bioinformatics, proteomics, and cryogenic electron microscopy single particle analysis to identify, annotate, and build atomic models for 21 distinct structural polypeptide chains forming the icosahedral capsid, neck, contractile tail, and baseplate. We identified a putative scaffolding protein stabilizing the interior of the capsid 5-fold vertex. We also visualized a large portion of Pa193 ~ 500 Å long tail fibers and resolved the interface between the baseplate and tail fibers. The work presented here provides a framework to support a better understanding of phages as biomedicines for phage therapy and inform engineering opportunities. Leveraging bioinformatics, proteomics, and cryogenic electron microscopy, this study deciphers the architecture and design principles of the therapeutic Pseudomonas phage Pa193.

Introduction For gynaecological cancers, volumetric modulated arc therapy (VMAT) offers comparable plan quality with shorter treatment delivery times when compared to intensity modulated radiation therapy (IMRT). Methods The clinical IMRT plans of twenty gynaecological cancer patients were compared with a retrospectively generated VMAT plan. Planning target volume (PTV) metrics compared were D95 > 99%, homogeneity index, and conformity index. Organs at risk (OAR) doses compared were bladder V45 < 35%, bowel V40 < 30%, femoral head and neck (FHN) V30 < 50%, V44 < 35% and V44 < 5%. Plan quality was also assessed by comparing the monitor units (MU), treatment time and the patient‐specific quality assurance results. Results VMAT and IMRT resulted in comparable PTV coverage with D95 values of 98.92% ± 0.69% and 98.91% ± 1.43% respectively, and homogeneity index values of 0.08 ± 0.02 (VMAT) and 0.08 ± 0.03 (IMRT). The conformity index for VMAT was 0.93 ± 0.04 and IMRT 0.85 ± 0.06 (P < 0.001). For the bowel tolerance (40 Gy < 30%) VMAT resulted in 22.39% ± 12.5% compared to 28.8% ± 16.78% for IMRT, with bladder and FHN VMAT doses also lower. VMAT MU were 694.35 ± 126.56 compared to 606.8 ± 96.16 for IMRT (P < 0.01). Treatment times of 6.6 ± 0.82 min and 2.47 ± 0.35 min were achieved for IMRT and VMAT respectively. Conclusion VMAT showed improvements in sparing OAR compared to IMRT. Target volume coverage with VMAT was equivalent or better than that of IMRT. These results in conjunction with the confirmed shorter treatment delivery time, have led to the development and implementation of a clinical protocol. This study developed a twin arc volumetric modulated arc therapy (VMAT) technique for gynaecological cancer patients. Comparisons between the VMAT and previously implemented 7–9 intensity modulated radiation therapy technique found comparable planning target volume coverage and improved organs at risk sparing.

Introduction Paediatric patients (individuals below 18 years of age) requiring cranial‐spinal irradiation (CSI) at our institution are commonly planned and treated using a three isocentre (3‐ISO) volumetric modulated arc therapy (VMAT) technique. A modified two isocentre (2‐ISO) VMAT technique was investigated with the aim to improve workflow and reduce planning and treatment time. Methods Five CSI paediatric patients previously treated with a 3‐ISO VMAT technique were retrospectively replanned using a 2‐ISO VMAT technique. The 2‐ISO VMAT plans were reviewed and approved by a radiation oncologist (RO) before undergoing patient‐specific quality assurance (QA) procedures, performed by a radiation oncology medical physicist (ROMP). Planning target volume (PTV) coverage, organ‐at‐risk (OAR) dose as well as planning and treatment durations of the first five patients utilising 2‐ISO technique were compared with 3‐ISO technique. Results The average percentage difference in PTV coverage by 95% reference dose between the 2‐ISO and 3‐ISO is 0.14%, and the average difference in OAR median dose is 0.68 Gy. Conformity and homogeneity indices have the same averages at 1.18 and 0.4 respectively. Patient‐specific physics QA results were all comparable with the 3‐ISO averages at 98.84% and the 2‐ISO at 98.71%. Planning duration for the 2‐ISO was reduced by up to 75%, and daily treatment duration was reduced by up to 50%. Of all the previously treated CSI patients using a 3‐ISO technique, 45% were suitable for the 2‐ISO technique. Conclusion The 2‐ISO VMAT technique provided comparable dose distribution based on PTV coverage, OAR dose and plan metric indices. Reduced planning and treatment duration with the 2‐ISO technique facilitated improved workflow with decreased sedation time for paediatric patients requiring a general anaesthesia. A modified two isocentre (2‐ISO) volumetric modulated arc therapy (VMAT) technique was investigated to determine whether it was dosimetrically equivalent to a 3‐ISO VMAT technique and whether it can provide for an improved workflow process for planning and treatment of CSI patients. The new 2‐ISO VMAT technique provided comparable dose distribution and patient‐specific QA results to the 3‐ISO technique. Its reduced planning and treatment times allows patients to commence treatment earlier, reduces sedation time during treatment if it is required and assists with limiting intra‐fraction motion while potentially increasing treatment accuracy.

Total body irradiation (TBI) is a complex treatment technique, which has been slow to transition to a three‐dimensional (3D) planning approach. There is limited literature available providing a detailed description on methods to plan TBI on a 3D planning system. 3D planning using the modulated arc TBI (MATBI) technique is a complex process involving a significant number of quality assurance processes and scripts, due to more than 40 treatment beams and two patient positions. This article will focus on the workflow and technical planning aspects of our institution’s MATBI technique and identify reasons for modifications made to the developing institution’s original MATBI approach. Included is a description of specific simulation equipment, detailed explanation of the four‐stage computing process including the role of scripting to standardise and streamline what is otherwise a complex number of steps. The information provided is specific to one centre’s approach but shows the fundamental planning process and demonstrates a streamlined method, which can be adapted to other planning systems. Overall, the ability to accurately represent the TBI technique in 3D on a planning system will be shown. This article will focus on the workflow and technical planning aspects of our institution’s MATBI technique and identify reasons for modifications made to the developing institution’s original MATBI approach. Included is a description of specific simulation equipment, detailed explanation of the four‐stage computing process including the role of scripting to standardise and streamline what is otherwise a complex number of steps. Alterations to the original technique will be discussed.

Introduction To implement the modulated arc total body irradiation (MATBI) technique within the existing infrastructure of a radiation oncology department. The technique needed to treat paediatric patients of all ages, some of whom would require general anaesthesia (GA). Methods The MATBI technique required minor modifications to be incorporated within existing departmental infrastructure. Ancillary equipment essential to the technique were identified and in some cases custom designed to meet health and safety criteria. GA equipment was also considered. To evaluate the effectiveness of the implemented technique, an audit of the cases clinically treated was conducted. Results A motorised treatment couch was designed to allow the patient to be positioned in stabilisation equipment at a height, then lowered to the floor to accommodate source‐to‐skin‐distances from 180 cm to 198 cm to treat the fixed 40 cm × 40 cm field size. Treatment couch design also facilitated positioning of the bespoke two‐part spoiler. While organ at risk dose is limited using a beam weight optimisation technique, the dose is further reduced using compensators placed close to the patient's skin on a 3D printed custom‐made support bridge. A digital radiography system is used to verify compensator position. Fifteen patients have been treated to date for various diseases using a variety of dose fractionations ranging from 2 Gy in a single fraction to 12 Gy in 6 fractions. Five patients have required GA due to age or behavioural issues. Conclusion The modified MATBI technique and the equipment required for treatment delivery has been found to be well tolerated by all patients. Implementation and modification of the modulated arc total body irradiation technique within an established department. Paediatric patients of all ages need to be accommodated within the technique on custom made equipment.

Lead shielding is commonly used in the delivery of superficial radiotherapy albeit that the toxicity of this substance is of concern. The feasibility of using a non-toxic alternative, AttenuFlex™, is assessed using Xstrahl and Sensus treatment units. A series of lead and AttenuFlex™ circular cut outs and applicators were used with superficial beams (1.0–8.5 mm Al HVL) to measure percentage depth dose (PDD), output factors (OF) and surface dose correction factors (DCF). X-ray transmission for each material was determined for each beam quality. For these measurements an Advanced Markus chamber either embedded within a virtual water phantom (PDD, OF, transmission) or placed on the surface of the phantom with entrance window downstream (DCF), was used. The depth of the phantom is 10 cm for PDD and surface OF measurements. DCF(t) measurements were obtained with underlying lead or AttenuFlex™ at depth t = 0.1–10 cm. Additionally, using EBT3 film fluorescent surface doses, to non-target tissue, due to underlying lead or AttenuFlex™ were compared. PDDs and OFs for both materials were within ± 1%. Lead and AttenuFlex™ transmission differences were clinically acceptable, all transmission values were < 5% and non-target doses were comparable. The variation of DCF(t) for lead and AttenuFlex™ exhibit a minima for all beams. In the minima region energy and applicator dependent differences between DCF(lead) and DCF(AttenuFlex™) are observed. These differences do not preclude the use of AttenuFlex™ as an alternative to lead in superficial therapy.

Total body irradiation (TBI) is an important treatment modality for the preparation of patients for bone marrow transplants. It is technically challenging and the actual delivery may vary from clinic to clinic. Knowledge of the pattern of practice may be helpful for clinics to determine future practice. We carried out an email survey from April to September 2019 sending 48 TBI related questions to all radiotherapy clinics in Australia and New Zealand via the Australasian College of Physical Scientists in Medicine email distribution list. Centres not performing TBI were not expected to complete the survey and centres that had participated in a previous survey, or that were known to perform the treatment, were followed up if no response was received. Of a total of approximately 70 centres, 14 clinics responded to the survey. The vast majority of clinics use conventional lateral and/or anterior–posterior beams at extended SSD for TBI treatment delivery. However, treatment planning, ancillary equipment (used for immobilisation/modulation), beam energy and prescribed lung doses vary considerably—with some clinics delivering the prescription dose to the lungs and some aiming to deliver a lung dose which is lower than the prescription dose. Only one clinic reported using an advanced delivery technique with modulated arcs at extended SSD. Centres either said they had no access to outcome data or did not answer this question. Compared with an earlier survey from 2005, 3 clinics have lowered their linac dose rate and 7 are the same or similar. The TBI practice in Australia and New Zealand remains varied, with considerable differences in treatment planning, beam energy, accepted lung doses and delivered dose rates.

Cryogenic electron microscopy (cryo-EM) analysis of bacteriophages is a valuable method for deciphering virus composition and conformational plasticity. In this study, we present a high-resolution structural atlas of the virus Pa223, a phage from the genus that has recently been used in clinical cocktails for treating cystic fibrosis and non-cystic fibrosis bronchiectasis, as well as for compassionate care. By combining bioinformatics, proteomics, cryo-EM single particle analysis, and localized reconstruction, we annotated and built atomic models for eight structural polypeptide chains that form the icosahedral capsid and noncontractile tail. We discovered that the Pa223 capsid is decorated by a spike protein that features a unique triple-β helix fold with no structural homologs in the database. The Pa223 tail features six trimeric tail fibers extending upwards, similar to, but shorter than, those found in phage T7. Unlike T7, the Pa223 tail is extended by two head-to-tail adaptors and sealed by a trimeric tail needle, similar to P22-like phages. We identified a protein bound around the outer perimeter of the portal protein, positioned similarly to the ejection protein gp72, which was identified in the phage DEV, a phage and member of the reclassified family. This structural hint led us to identify the Pa223 ejection proteins gp53, gp54, and gp56, which bioinformatically resemble those of T7-like phages more closely than . Thus, phage Pa223 contains diverse structural elements found in P22-like, T7-like, and phages, providing a framework for understanding the diversification and evolution of ejection proteins in . The high-resolution structure of Pa223 reveals hybrid structural features that are shared among P22-like, T7-like, and phages. The Pa223 capsid is decorated with a trimeric spike asymmetrically bound at the icosahedral 3-fold axes.The Pa223 tail features two quasi-equivalent conformations of the head-to-tail adaptor protein arranged into two coaxial rings. Identification of the ejection protein gp54 through structural similarity to gp72 from the DEV. Bioinformatic mapping of the Pa223 ejection proteins gp53 and gp56 validated through mass spectrometry analysis of infectious virions.

Using a Pantak Therapax SXT 150 system HVL values for clinical beams generated with filters 4–8, were determined as a function of FCD (30–130 cm). Aluminium absorbers were placed midway between the focus and chamber with collimation to define both narrow and broad beam geometries. For filters 4–7 with broad beam geometry the HVL initially decreases as the FCD is increased from 30 cm and then increases as the FCD approaches 130 cm. In contrast filter 8 exhibits a reduction in HVL with increasing FCD attributed to the decreasing influence of absorber scatter. With narrow beam geometry the HVL of filter 4 increases as the FCD is increased. For other filters the HVL variation is similar to that for the broader beam albeit that for a given FCD the HVL is smaller, a consequence of reduced absorber scatter. Monte Carlo BEAMnrc simulations of filter 4–8 beams demonstrated a quality dependent air attenuation effect associated with an increase in HVL for lower quality beams with increasing FCD. Thus for the beams investigated in this work the variation of HVL with FCD can be interpreted in terms of the competing influences of absorber scatter, which tends to decrease the measured HVL, and a quality dependent in air attenuation that tends to increase the HVL with increasing FCD. In terms of an absorbed dose determination it is shown that changes of HVL with FCD resulted in variations of D w , z   =  0  < ±0.5 %.

Treponema denticola is a Gram-negative, oral spirochete identified as one of the predominant groups of microorganisms in periodontal diseases. The chemotaxis signaling pathway of T. denticola, composed of the cheA, chew, cheX and cheY genes and twenty putative chemoreceptors, the MCPs (methyl-accepting chemotaxis proteins), regulates motility through a complex series of conserved protein interactions that modulate flagellar motor function in response to environmental changes. Homologues to CheX, originally described as a novel spirochete chemotaxis protein of unknown function, have recently been demonstrated to exhibit phosphatase activity. The potential interactions of CheX with other components of the chemotaxis pathway as well as well-established interactions were investigated with the yeast-two hybrid system. CheX was found to interact with CheA and with itself. In agreement with homologous systems in other species, CheA forms a complex with the MCPs, and CheW. Interactions of CheA with itself and its cognate response regulator were also confirmed. Development of allelic replacement mutagenesis techniques for T. denticola has permitted targeted construction of gene inactivation mutants. Inactivation of cheA resulted in defective chemotaxis behavior. Mutants lacking cheA exhibited coordinated non-reversing cell movements confirming that CheA is involved in regulating cellular reversal frequency. In the last part of this dissertation cheX and cheY genes were inactivated individually and in combination by insertion of an ermF-ermAM resistance cassette. Reduced expression of the cheY gene in the cheX mutant resulted in the construction of an additional cheX mutant with a chemotaxis promoter in between the ermF-ermAM cassette and the cheY gene but could not remedy the problem. cheX cheY and cheAY deletion mutants exhibited chemotaxis defects and altered motility behavior. Interestingly, majority of the cheX and cheXY mutants failed to change direction of rotation whereas about half of the cheY mutants still reversed their direction of movement albeit at a reduced frequency compared to wild-type. The other half of the observed mutant cells moved in one direction but stopped frequently. Additionally, all chemotaxis mutant strains tested in this study showed reduced tissue penetration, a feature considered important for pathogenesis of T. denticola.