LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

Autophagy in protists and their hosts: When, how and why?

Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.

Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P-mediated autophagy-independent pathway.

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterised by a long-lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3-phosphate (PI3P)-labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P-labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3-labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.

Recruitment of Host Nuclear Pore Components to the Vicinity of Theileria Schizonts.

Parasitic protozoans of the genus Theileria are intracellular pathogens that induce the cellular transformation of leukocytes, causing uncontrolled proliferation of the infected host cell. The transforming stage of the parasite has a strictly intracellular lifestyle and ensures its distribution to both daughter cells during host cell cytokinesis by aligning itself across the metaphase plate and by binding tightly to central spindle and astral microtubules. Given the importance of the parasite surface in maintaining interactions with host microtubules, we analyzed the ultrastructure of the host-parasite interface using transmission electron microscopy combined with high-resolution fluorescence microscopy and live-cell imaging. We show that porous membranes, termed annulate lamellae (AL), closely associate with the Theileria surface in infected T cells, B cells, and macrophages and are not detectable in noninfected bovine cell lines such as BL20 or BoMACs. AL are membranous structures found in the cytoplasm of fast-proliferating cells such as cancer cells, oocytes, and embryonic cells. Although AL were first observed more than 60 years ago, the function of these organelles is still not known. Indirect immunofluorescence analysis with a pan-nuclear pore complex antibody, combined with overexpression of a panel of nuclear pore proteins, revealed that the parasite recruits nuclear pore complex components close to its surface. Importantly, we show that, in addition to structural components of the nuclear pore complex, nuclear trafficking machinery, including importin beta 1, RanGAP1, and the small GTPase Ran, also accumulated close to the parasite surface.IMPORTANCETheileria schizonts are the only known eukaryotic organisms capable of transforming another eukaryotic cell; as such, probing of the interactions that occur at the host-parasite interface is likely to lead to novel insights into the cell biology underlying leukocyte proliferation and transformation. Little is known about how the parasite communicates with its host or by what route secreted parasite proteins are translocated into the host, and we propose that nuclear trafficking machinery at the parasite surface might play a role in this. The function of AL remains completely unknown, and our work provides a basis for further investigation into the contribution that these porous, cytomembranous structures might make to the survival of fast-growing transformed cells.

Evaluation of offline adaptive planning techniques in image-guided brachytherapy of cervical cancer.

Modern three-dimensional image-guided intracavitary high dose rate (HDR) brachytherapy is often used in combination with external beam radiotherapy (EBRT) to manage cervical cancer. Intrafraction motion of critical organs relative to the HDR applicator in the time between the planning CT and treatment delivery can cause marked deviations between the planned and delivered doses. This study examines offline adaptive planning techniques that may reduce intrafraction uncertainties by shortening the time between the planning CT and treatment delivery. Eight patients who received EBRT followed by HDR boosts were retrospectively reviewed. A CT scan was obtained for each insertion. Four strategies were simulated: (A) plans based on the current treatment day CT; (B) plans based on the first fraction CT; (C) plans based on the CT from the immediately preceding fraction; (D) plans based on the closest anatomically matched previous CT, using all prior plans as a library. Strategies B, C, and D allow plans to be created prior to the treatment day insertion, and then rapidly compared with the new CT. Equivalent doses in 2 Gy for combined EBRT and HDR were compared with online adaptive plans (strategy A) at D90 and D98 for the high-risk CTV (HR-CTV), and D2 cc for the bladder, rectum, sigmoid, and bowel. Compared to strategy A, D90 deviations for the HR-CTV were -0.5 ± 2.8 Gy, -0.9 ± 1.0 Gy, and -0.7 ± 1.0 Gy for Strategies B, C, and D, respectively. D2 cc changes for rectum were 2.7 ± 5.6 Gy, 0.6 ± 1.7 Gy, and 1.1 ± 2.4 Gy for Strategies B, C, and D. With the exception of one patient using strategy B, no notable variations for bladder, sigmoid, and bowel were found. Offline adaptive planning techniques can shorten time between CT and treatment delivery from hours to minutes, with minimal loss of dosimetric accuracy, greatly reducing the chance of intrafraction motion.

A hybrid planning strategy for stereotactic body radiation therapy of early stage non-small-cell lung cancer.

Currently dynamic conformal arcs (DCA) and volumetric modulated arc therapy (VMAT) are two popular planning techniques to treat lung stereotactic body radiation therapy (SBRT) patients. Of the two, DCA has advantages in terms of multi-leaf collimator (MLC) motion, positioning error, and delivery efficiency. However, VMAT is often the choice when critical organ sparing becomes important. We developed a hybrid strategy to incorporate DCA component into VMAT planning, results were compared with DCA and VMAT plans. Four planning techniques were retrospectively simulated for 10 lung SBRT patients: DCA, Hybrid-DCA (2/3 of the doses from DCA beams), Hybrid-VMAT (2/3 of the doses from VMAT beams) and VMAT. Plan complexity was accessed by modulation complexity score (MCS). Conformity index (CI) for the planning target volume (PTV), V20 and V5 for the lung, V30 for the chestwall, and maximum dose to all other critical organs were calculated. Plans were compared with regard to these metrics and measured agreement between the planned and delivered doses. DCA technique did not result in acceptable plan quality due to target location for five patients. Hybrid-DCA produced one unacceptable plan, and Hybrid-VMAT and VMAT produced no unacceptable plans. The CI improved with increasing VMAT usage, as did the dose sparing to critical structures. Compared to the VMAT technique, a total MU reduction of 14%, 25% and 37% were found for Hybrid-VMAT, Hybrid-DCA and DCA techniques for 54 Gy patient group, and 9%, 23% and 34% for 50 Gy patient group, suggesting improvement in delivery efficiency with increasing DCA usage. No significant variations of plan complexity were observed between Hybrid-DCA and Hybrid-VMAT (P = 0.46 from Mann-Whitney U-test), but significant differences were found among DCA, Hybrid and VMAT (P < 0.05). Better agreements between the planned and delivered doses were found with more DCA contributions. By adding DCA components to VMAT planning, hybrid technique offers comparable dosimetry to full VMAT, while increasing delivery efficiency and minimizing MLC complexity.

Comparison of the progressive resolution optimizer and photon optimizer in VMAT optimization for stereotactic treatments.

The photon optimization (PO) algorithm was recently released by Varian Medical Systems to improve volumetric modulated arc therapy (VMAT) optimization within Eclipse (Version 13.5). The purpose of this study is to compare the PO algorithm with its predecessor, progressive resolution optimizer (PRO) for lung SBRT and brain SRS treatments. A total of 30 patients were selected retrospectively. Previously, all the plans were generated with the PRO algorithm within Eclipse Version 13.6. In the new version of PO algorithm (Version 15), dynamic conformal arcs (DCA) were first conformed to the target, then VMAT inverse planning was performed to achieve the desired dose distributions. PTV coverages were forced to be identical for the same patient for a fair comparison. SBRT plan quality was assessed based on selected dose-volume parameters, including the conformity index, V20 for lung, V30 Gy for chest wall, and D0.035 cc for other critical organs. SRS plan quality was evaluated based on the conformity index and normal tissue volumes encompassed by the 12 and 6 Gy isodose lines (V12 and V6 ). The modulation complexity score (MCS) was used to compare plan complexity of two algorithms. No statistically significant differences between the PRO and PO algorithms were found for any of the dosimetric parameters studied, which indicates both algorithms produce comparable plan quality. Significant improvements in the gamma passing rate (increased from 97.0% to 99.2% for SBRT and 96.1% to 98.4% for SRS), MCS (average increase of 0.15 for SBRT and 0.10 for SRS), and delivery efficiency (MU reduction of 29.8% for SBRT and 28.3% for SRS) were found for the PO algorithm. MCS showed a strong correlation with the gamma passing rate, and an inverse correlation with total MUs used. The PO algorithm offers comparable plan quality to the PRO, while minimizing MLC complexity, thereby improving the delivery efficiency and accuracy.

Do we need a new CT scan for retreatment of intracranial SRS patients?

PURPOSE: To determine if the treatment planning computed tomography scan (CT) from an initial intracranial stereotactic radiosurgery (SRS) treatment can be used for repeat courses of SRS. METHODS AND MATERIALS: Twenty-five patients with 40 brain metastases that received multiple courses of SRS were retrospectively studied. Magnetic resonance scans from repeat SRS (rMR) courses were registered to CT scans from the initial SRS (iCT) and repeat SRS (rCT). The CT scans were then registered to find the displacement of the rMR between iCT and rCT registrations. The distance from each target to proximal skull surface was measured in 16 directions on each CT scan after registration. The mutual information (MI) coefficients from the registration process were used to evaluate image set similarity. Targets and plans from the rCTs were transferred to the iCTs, and doses were recalculated on the iCT for repeat plans. The two dose distributions were compared through 3D gamma analysis. RESULTS: The magnitude of the mean linear translations from the MR registrations was 0.6 ± 0.3 mm. The mean differences in distance from target to skull on a per target basis were 0.3 ± 0.2 mm. The MI was 0.582 ± 0.042. Registration between a comparison group of 30 CT scans that had the same data resampled and 30 scans that were intercompared with different patients gave MI = 0.721 ± 0.055 and MI = 0.359 ± 0.031, respectively. The mean gamma passing rates were 0.997 ± 0.007 for 1 mm/1% criteria. CONCLUSIONS: The rMR can be aligned to the iCT to accurately define targets. The skull shows minimal change between scans so the iCT can be used for set-up at repeat treatments. The dosimetry provided by the iCT dose calculation is adequate for repeat SRS. Treatment based on iCT is feasible.

Inverted recruitment of autophagy proteins to the Plasmodium berghei parasitophorous vacuole membrane.

Selective autophagy and related mechanisms can act as variable defense mechanisms against pathogens and can therefore be considered as intracellular immune responses. When in hepatocytes, Plasmodium parasites reside in a parasitophorous vacuole (PV) and the PV membrane (PVM) is the main contact site between host cell and parasite. Early in infection, the PVM is directly labeled with host cell autophagy proteins LC3B and p62 (nucleoporin 62). We investigated the recruitment of different selective autophagy receptors and could show that mainly p62 and NBR1 (neighbour of BRCA1 gene 1) and to a lesser extent NDP52 (nuclear dot protein 52) associate with the PVM. To investigate the recruitment of these receptors to the PVM in Plasmodium-infected cells, we generated LC3B knock out HeLa cells. In these cell lines, autophagosome formation and autophagic flux are not different to those in WT cells. Unexpectedly, p62 and NBR1 recruitment to the PVM was strongly impaired in LC3B-negative host cells, suggesting that LC3B recruits both receptors to the PVM of Plasmodium parasites. We also noticed that LC3B recruited ubiquitin to the PVM. This indicates that, in comparison to classical selective autophagy, in P. berghei-infected cells the order of membrane labeling with autophagy proteins appears to be inverted from canonical ubiquitin-receptor-LC3B recruitment to LC3B-receptor and possibly ubiquitin.

LC3-association with the parasitophorous vacuole membrane of Plasmodium berghei liver stages follows a noncanonical autophagy pathway.

Eukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with P. berghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in P. berghei-infected ATG5-/- host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3-independent pathway.

A novel method for radiotherapy patient identification using surface imaging.

Performing a procedure on the wrong patient or site is one of the greatest errors that can occur in medicine. The addition of automation has been shown to reduce errors in many processes. In this work we explore the use of an automated patient identification process using optical surface imaging for radiotherapy treatments. Surface imaging uses visible light to align the patient to a reference surface in the treatment room. It is possible to evaluate the similarity between a daily set-up surface image and the reference image using distance to agreement between the points on the two surfaces. The higher the percentage overlapping points within a defined distance, the more similar the surfaces. This similarity metric was used to intercompare 16 left-sided breast patients. The reference surface for each patient was compared to 10 daily treatment surfaces for the same patient, and 10 surfaces from each of the other 15 patients (for a total of 160 comparisons per patient), looking at the percent of points overlapping. For each patient, the minimum same-patient similarity score was higher than the maximum different-patient score. For the group as a whole a threshold was able to classify correct and incorrect patients with high levels of accuracy. A 10-fold cross-validation using linear discriminant analysis gave cross-validation loss of 0.0074. An automated process using surface imaging is a feasible option to provide nonharmful daily patient identification verification using currently available technology.

A prospective evaluation of open face masks for head and neck radiation therapy.

PURPOSE: Head and neck (HN) radiation therapy patients are typically immobilized with closed thermoplastic masks that cover the face and may cause discomfort. In this work, we examine the use of open masks for HN radiation therapy. METHODS AND MATERIALS: Fifty HN patients were prospectively randomized into 2 groups (25 closed masks, 25 open masks). The open-mask group was monitored with surface imaging to evaluate intrafraction motion. Both groups underwent daily volumetric imaging. All daily images were rigidly registered to their respective planning images to evaluate spinal canal and mandible position as a check for interfraction posture change. Posture changes were determined by the amount the spinal canal and mandible contours from the planning images had to be expanded to cover the structures on each daily image set. The vector length (VL) of the intrafraction linear translations, spine, and mandible positions for each open-mask patient were checked for correlation with fraction number using the Pearson r value. All patients were given a weekly survey ranking anxiety and claustrophobia from 0 to 10 (0 = no issue, 10 = extreme issue). RESULTS: The mean VL for all open-mask patients was 0.9 ± 0.5 mm (1 standard deviation). Only 1 patient showed significant correlation between VL and fraction number. The mean contour expansions to cover the spine and mandible were 1.5 ± 0.9 mm and 1.8 ± 1.3 mm for the closed-mask group, and 1.6 ± 0.8 mm and 1.8 ± 1.1 mm for the open-mask group. Both groups showed similar behavior relative to fraction number. The mean anxiety and claustrophobia scores were 1.63 and 1.44 for the closed-mask group, and 0.81 and 0.63 for the open-mask group. The groups were not significantly different. CONCLUSIONS: Open masks provide comparable immobilization and posture preservation to closed masks for HN radiation therapy.

Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms.

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.

Surface imaging-based analysis of intrafraction motion for breast radiotherapy patients.

Breast treatments are becoming increasingly complex as the use of modulated and partial breast therapies becomes more prevalent. These methods are predicated on accurate and precise positioning for treatment. However, the ability to quantify intrafraction motion has been limited by the excessive dose that would result from continuous X-ray imaging throughout treatment. Recently, surface imaging has offered the opportunity to obtain 3D measurements of patient position throughout breast treatments without radiation exposure. Thirty free-breathing breast patients were monitored with surface imaging for 831 monitoring sessions. Mean translations and rotations were calculated over each minute, each session, and over all sessions combined. The percentage of each session that the root mean squares (RMS) of the linear translations were outside of defined tolerances was determined for each patient. Correlations between mean translations per minute and time, and between standard deviation per minute and time, were evaluated using Pearson's r value. The mean RMS translation averaged over all patients was 2.39 mm ± 1.88 mm. The patients spent an average of 34%, 17%, 9%, and 5% of the monitoring time outside of 2 mm, 3 mm, 4 mm, and 5 mm RMS tolerances, respectively. The RMS values averaged over all patients were 2.71 mm ± 1.83 mm, 2.76 ± 2.27, and 2.98 mm ± 2.30 mm over the 5th, 10th, and 15th minutes of monitoring, respectively. The RMS values (r = 0.73, p = 0) and standard deviations (r = 0.88, p = 0) over all patients showed strong significant correlations with time. We see that the majority of patients' treatment time is spent within 5 mm of the isocenter and that patient position drifts with increasing treatment time. Treatment length should be consid- ered in the planning process. An 8 mm margin on a target volume would account for 2 SDs of motion for a treatment up to 15 minutes in length. 

On the validity of density overrides for VMAT lung SBRT planning.

PURPOSE: Modeling dose to a moving target in lung is a very difficult task. Current approaches to planning lung stereotactic body radiotherapy (SBRT) generally calculate dose on either free breathing or average computed tomography (CT) scans, which do not always accurately predict dose to parts of the target volume not occupied by tumor on the planning scan. In this work, the authors look at using density overrides of the target volumes to more accurately predict dose for lung SBRT using the analytic anisotropic algorithm (AAA). METHODS: Volumetric modulated arc therapy plans were created on free breathing scans (FBP), time average scans (AVGP), free breathing scans with the internal target volume overridden to tumor density (ITVP), free breathing scans with the planning target volume overridden to tumor density (PTVP), and free breathing scan using a hybrid scheme with the internal target volume set to tumor density and the planning target volume minus the internal target volume set to a density intermediate between lung and tumor (HP) for the case of a 4D motion phantom and five patient cases. Radiochromic film measurements were made for the phantom plans, with gamma analysis used to compare the planned to delivered dose. The patient plans were recalculated on each of the phases of a 4DCT to evaluate tumor coverage and conformity index (CI). A modified modulation complexity score (MCSv) and average open area per control point (AA) metrics were used to evaluate multileaf collimator (MLC) modulation for each of the plans. RESULTS: The HP plans showed significantly higher gamma passing rates (p < 0.05) than the FBP, AVGP, and ITVP for criteria of 2 mm/2% and 1 mm/1%. No significant correlation was observed between gamma values and AA or MCSv. The tumor volume was covered by the prescription dose on all phases of the 4DCT for all patient plans. The PTVP and HP yielded lower mean CI than the other plans for all five patients, with three of the cases showing statistically significant differences (p < 0.05). No meaningful correlation was observed between the mean CI and AA or MCSv. CONCLUSIONS: These measurements suggest that the HP planning method may provide more accurate dose modeling and decreased normal lung irradiation for lung SBRT compared to the commonly used FBP and AVG planning methods when used with the AAA. The HP method does not appear to have a strong relationship with MLC modulation.

Commissioning and validation of BrainLAB cones for 6X FFF and 10X FFF beams on a Varian TrueBeam STx.

Small field dosimetry is a challenging task. The difficulties of small field measurements, particularly stereotactic field size measurements, are highlighted by the large interinstitution variability that can be observed for circular cone collimator commissioning measurements. We believe the best way to improve the consistency of small field measurements is to clearly document and share the results of small field measurements. In this work we report on the commissioning and validation of a BrainLAB cone system for 6 MV and 10 MV flattening filter-free (FFF) beams on a Varian TrueBeam STx. Commissioning measurements consisted of output factors, percent depth dose, and off-axis factor measurements with a diode. Validation measurements were made in a polystyrene slab phantom at depths of 5 cm, 10 cm, and 15 cm using radiochromic film. Output factors for the 6xFFF cones are 0.689, 0.790, 0.830, 0.871, 0.890, and 0.901 for 4 mm, 6 mm, 7.5 mm, 10 mm, 12.5 mm, and the 15 mm cones, respectively. Output factors for the 10xFFF cones are 0.566, 0.699, 0.756, 0.826, 0.864, and 0.888 for 4 mm, 6 mm, 7.5 mm, 10 mm, 12.5 mm, and the 15 mm cones, respectively. The full width half maximum values of the off-axis factors agreed with the nominal cone size to within 0.5 mm. Validation measurements showed an agreement of absolute dose between calculation and plan of < 3.6%, and an agreement of field sizes of ≤ 0.3 mm in all cases. Radiochromic film validation measurements show reasonable agreement with beam models for circular collimators based on diode commissioning measurements.

Analysis of liver stage development in and merozoite release from hepatocytes.

Exoerythrocytic Plasmodium parasites infect hepatocytes and develop to huge multinucleated schizonts inside a parasitophorous vacuole. Finally, thousands of merozoites are formed and released into the host cell cytoplasm by complete disintegration of the parasitophorous vacuole membrane. This, in turn, results in death and detachment of the infected hepatocyte, followed by the formation of merosomes. The fast growth of the parasite and host cell detachment are hallmarks of liver stage development and can easily be monitored. Here, we describe how to translate these observations into assays for characterizing parasite development. Additionally, other recently introduced techniques and tools to analyze and manipulate liver stage parasites are also discussed.

Potential underestimation of the internal target volume (ITV) from free-breathing CBCT.

PURPOSE: Localization prior to delivery of SBRT to free-breathing patients is performed by aligning the planning internal target volume (ITV) from 4DCT with an on-board free-breathing cone-beam CT (FB-CBCT) image. The FB-CBCT image is assumed to also generate an ITV that captures the full range of motion, due to the acquisition spanning multiple respiratory cycles. However, the ITV could potentially be underestimated when the ratio of time spent in inspiration versus time spent in expiration (I/E ratio) deviates from unity. Therefore, the aim of this study was to investigate the effect of variable I/E ratios on the FB ITV generated from a FB-CBCT scan. METHODS: This study employed both phantom and patient imaging data. For the phantom study, five periodic respiratory cycles were simulated with different I/E ratios. Six patient respiratory cycles with variable I/E ratios were also selected. All profiles were then programmed into a motion phantom for imaging and modified to exhibit three peak-to-peak motion amplitudes (0.5, 1.0, and 2.0 cm). Each profile was imaged using two spherical targets with 1.0 and 3.0 cm diameters. 2D projections were acquired with full gantry rotation of a kiloVoltage (kV) imager mounted onto the gantry of a modem linear accelerator. CBCT images were reconstructed from 2D projections using a standard filtered back-projection reconstruction algorithm. Quantitative analyses for the phantom study included computing the change in contrast along the direction of target motion as well as determining the area (which is proportional to the target volume) inside of the contour extracted using a Canny edge detector. For the patient study, projection data that were previously acquired under an investigational 4D CBCT slow-gantry imaging protocol were used to generate both FB-CBCT and 4D CBCT images. Volumes were then manually contoured from both datasets (using the same window and level) for quantitative comparison. RESULTS: The phantom study indicated a reduction in contrast at the inferior edge of the ITV (corresponding to inspiration) as the ratio decreased, for both simulated and patient respiratory cycles. For the simulated phantom respiratory cycles, the contrast reduction of the smallest I/E ratio was 27.6% for the largest target with the smallest amplitude and 89.7% for the smallest target with the largest amplitude. For patient respiratory cycles, these numbers were 22.3% and 94.0%, respectively. The extracted area from inside of the target contours showed a decreasing trend as the I/E ratio decreased. In the patient study, the FB-CBCT ITVs of both lung tumors studied were underestimated when compared with their corresponding 4D CBCT ITV. The underestimations found were 40.1% for the smaller tumor and 24.2% for the larger tumor. CONCLUSIONS: The ITV may be underestimated in a FB-CBCT image when a patient's respiratory pattern is characterized by a disparate length of time spent in inspiration versus expiration. Missing the full target motion information during on-board verification imaging may result in localization errors.

The transforming parasite Theileria co-opts host cell mitotic and central spindles to persist in continuously dividing cells.

The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival.

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata.

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite-host interactions during host cell division.