Using SuperClomeleon to measure changes in intracellular chloride during development and after early life stress

Intraneuronal chloride concentrations ([Cl- ]i) decrease during development resulting in a shift from depolarizing to hyperpolarizing γ-aminobutyric acid (GABA) responses via chloride-permeable GABAA receptors. This GABA shift plays a pivotal role in postnatal brain development, and can be strongly influenced by early life experience. Here, we assessed the applicability of the recently developed fluorescent SuperClomeleon (SClm) sensor to examine changes in [Cl- ]i using two-photon microscopy in brain slices. We used SClm mice of both sexes to monitor the developmental decrease in neuronal chloride levels in organotypic hippocampal cultures. We could discern a clear reduction in [Cl-]i between DIV3 and DIV9 (equivalent to the second postnatal week in vivo) and a further decrease in some cells until DIV22. In addition, we assessed alterations in [Cl- ]i in the medial prefrontal cortex (mPFC) of P9 male SClm mouse pups after early life stress (ELS). ELS was induced by limiting nesting material between P2 and P9. ELS induced a shift towards higher (i.e. immature) chloride levels in layer 2/3 cells in the mPFC. Although conversion from SClm fluorescence to absolute chloride concentrations proved difficult, our study underscores that the SClm sensor is a powerful tool to measure physiological changes in [Cl- ]i in brain slices

A hybrid 2D/4D-MRI methodology using simultaneous multi-slice imaging for radiotherapy guidance

Purpose: Respiratory motion management is important in abdominothoracic radiotherapy. Fast imaging of the tumor can facilitate multileaf collimator (MLC) tracking that allows for smaller treatment margins, while repeatedly imaging the full field-of-view is necessary for 4D dose accumulation. This study introduces a hybrid 2D/4D-MRI methodology that can be used for simultaneous MLC tracking and dose accumulation on a 1.5 T Unity MR-linac (Elekta AB, Stockholm, Sweden). Methods: We developed a hybrid 2D/4D-MRI methodology that uses a simultaneous multislice (SMS) accelerated MRI sequence, which acquires two coronal slices simultaneously and repeatedly cycles through slice positions over the image volume. As a result, the fast 2D imaging can be used prospectively for MLC tracking and the SMS slices can be sorted retrospectively into respiratory-correlated 4D-MRIs for dose accumulation. Data were acquired in five healthy volunteers with an SMS-bTFE and SMS-TSE MRI sequence. For each sequence, a prebeam dataset and a beam-on dataset were acquired simulating the two phases of MR-linac treatments. Prebeam data were used to generate a 4D-based motion model and a reference mid-position volume, while beam-on data were used for real-time motion extraction and reconstruction of beam-on 4D-MRIs. In addition, an in-silico computational phantom was used for validation of the hybrid 2D/4D-MRI methodology. MLC tracking experiments were performed with the developed methodology, for which real-time SMS data reconstruction was enabled on the scanner. A 15-beam 8× 7.5 Gy intensity-modulated radiotherapy plan for lung stereotactic body radiotherapy with isotropic 3 mm GTV-to-PTV margins was created. Dosimetry experiments were performed using a 4D motion phantom. The latency between target motion and updating the radiation beam was determined and compensated. Local gamma analyses were performed to quantify dose differences compared to a static reference delivery, and dose area histograms (DAHs) were used to quantify the GTV and PTV coverage. Results: In-vivo data acquisition and MLC tracking experiments were successfully performed with the developed hybrid 2D/4D-MRI methodology. Real-time liver–lung interface motion estimation had a Pearson's correlation of 0.996 (in-vivo) and 0.998 (in-silico). A median (5th–95th percentile) error of 0.0 (−0.9 to 0.7) mm and 0.0 (−0.2 to 0.2) mm was found for real-time motion estimation for in-vivo and in-silico, respectively. Target motion prediction beyond the liver–lung interface had a median root mean square error of 1.6 mm (in-vivo) and 0.5 mm (in-silico). Beam-on 4D MRI reconstruction required a median amount of data equal to an acquisition time of 2:21–3:17 min, which was 20% less data compared to the prebeam-derived 4D-MRI. System latency was reduced from 501 ± 12 ms to −1 ± 3 ms (SMS-TSE) and from 398 ± 10 ms to −10 ± 4 ms (SMS-bTFE) by a linear regression prediction filter. The local gamma analysis agreed within (Formula presented.) to 3.3% (SMS-bTFE) and (Formula presented.) to 10% (SMS-TSE) with a reference MRI sequence. The DAHs revealed a relative D 98% GTV coverage between 97% and 100% (SMS-bTFE) and 100% and 101% (SMS-TSE) compared to the static reference. Conclusions: The presented 2D/4D-MRI methodology demonstrated the potential for accurately extracting real-time motion for MLC tracking in abdominothoracic radiotherapy, while simultaneously reconstructing contiguous respiratory-correlated 4D-MRIs for dose accumulation

Dosimetric evaluation of MRI-guided multi-leaf collimator tracking and trailing for lung stereotactic body radiation therapy

Purpose: The treatment margins for lung stereotactic body radiotherapy (SBRT) are often large to cover the tumor excursions resulting from respiration, such that underdosage of the tumor can be avoided. Magnetic resonance imaging (MRI)-guided multi-leaf collimator (MLC) tracking can potentially reduce the influence of respiration to allow for smaller treatment margins. However, tracking is accompanied by system latency that may induce residual tracking errors. Alternatively, a simpler mid-position delivery combined with trailing can be used. Trailing reduces influences of respiration by compensating for baseline motion, to potentially improve target coverage. In this study, we aim to show the feasibility of MRI-guided tracking and trailing to reduce influences of respiration during lung SBRT. Methods: We implemented MRI-guided tracking on the MR-linac using an Elekta research tracking interface to track tumor motion during intensity modulated radiotherapy (IMRT). A Quasar (Formula presented.) phantom was used to generate Lujan motion ((Formula presented.), 4 s period, 20 mm peak-to-peak amplitude) with and without 1.0 mm/min cranial drift. Phantom tumor positions were estimated from sagittal 2D cine-MRI (4 or 8 Hz) using cross-correlation-based template matching. To compensate the anticipated system latency, a linear ridge regression predictor was optimized for online MRI by comparing two predictor training approaches: training on multiple traces and training on a single trace. We created 15-beam clinical-grade lung SBRT plans for central targets (8 × 7.5 Gy) and peripheral targets (3 × 18 Gy) with different PTV margins for mid-position based motion management (3–5 mm) and for MLC tracking (3 mm). We used a film insert with a 3 cm spherical target to measure the spatial distribution and quantity of the delivered dose. A 1%/1 mm local gamma-analysis quantified dose differences between motion management strategies and reference cases. Additionally, a dose area histogram (DAH) revealed the target coverage relative to the reference scenario. Results: The prediction filter gain was on average 25% when trained on multiple traces and 44% when trained on a single trace. The filter reduced system latency from 313 ± 2 ms to 0 ± 5 ms for 4 Hz imaging and from 215 ± 3 ms to 3 ± 3 ms for 8 Hz. The local gamma analysis for the central delivery showed that tracking improved the gamma pass-rate from 23% to 96% for periodic motion and from 14% to 93% when baseline drift was applied. For the peripheral delivery during periodic motion, delivery pass-rates improved from 22% to 93%. Comparing mid-position delivery to trailing for periodic+drift motion increased the local gamma pass rate from 15% to 98% for a central delivery and from 8% to 98% for a peripheral delivery. Furthermore, the DAHs revealed a relative (Formula presented.) GTV coverage of 101% and 97% compared to the reference scenario for, respectively, central and peripheral tracking of periodic+drift motion. For trailing, a relative (Formula presented.) of 99% for central and 98% for peripheral trailing was found. Conclusions: We provided a first experimental demonstration of the technical feasibility of MRI-guided MLC tracking and trailing for central and peripheral lung SBRT. Tracking maximizes the sparing of healthy tissue, while trailing is highly effective in mitigating baseline motion

Experimental demonstration of real-time cardiac physiology-based radiotherapy gating for improved cardiac radioablation on an MR-linac

Background: Cardiac radioablation is a noninvasive stereotactic body radiation therapy (SBRT) technique to treat patients with refractory ventricular tachycardia (VT) by delivering a single high-dose fraction to the VT isthmus. Cardiorespiratory motion induces position uncertainties resulting in decreased dose conformality. Electocardiograms (ECG) are typically used during cardiac MRI (CMR) to acquire images in a predefined cardiac phase, thus mitigating cardiac motion during image acquisition. Purpose: We demonstrate real-time cardiac physiology-based radiotherapy beam gating within a preset cardiac phase on an MR-linac. Methods: MR images were acquired in healthy volunteers (n = 5, mean age = 29.6 years, mean heart-rate (HR) = 56.2 bpm) on the 1.5 T Unity MR-linac (Elekta AB, Stockholm, Sweden) after obtaining written informed consent. The images were acquired using a single-slice balance steady-state free precession (bSSFP) sequence in the coronal or sagittal plane (TR/TE = 3/1.48 ms, flip angle = 48 (Formula presented.), SENSE = 1.5, (Formula presented.) (Formula presented.), voxel size = (Formula presented.) (Formula presented.), partial Fourier factor = 0.65, frame rate = 13.3 Hz). In parallel, a 4-lead ECG-signal was acquired using MR-compatible equipment. The feasibility of ECG-based beam gating was demonstrated with a prototype gating workflow using a Quasar MRI4D motion phantom (IBA Quasar, London, ON, Canada), which was deployed in the bore of the MR-linac. Two volunteer-derived combined ECG-motion traces (n = 2, mean age = 26 years, mean HR = 57.4 bpm, peak-to-peak amplitude = 14.7 mm) were programmed into the phantom to mimic dose delivery on a cardiac target in breath-hold. Clinical ECG-equipment was connected to the phantom for ECG-voltage-streaming in real-time using research software. Treatment beam gating was performed in the quiescent phase (end-diastole). System latencies were compensated by delay time correction. A previously developed MRI-based gating workflow was used as a benchmark in this study. A 15-beam intensity-modulated radiotherapy (IMRT) plan ((Formula presented.) Gy) was delivered for different motion scenarios onto radiochromic films. Next, cardiac motion was then estimated at the basal anterolateral myocardial wall via normalized cross-correlation-based template matching. The estimated motion signal was temporally aligned with the ECG-signal, which were then used for position- and ECG-based gating simulations in the cranial–caudal (CC), anterior–posterior (AP), and right–left (RL) directions. The effect of gating was investigated by analyzing the differences in residual motion at 30, 50, and 70% treatment beam duty cycles. Results: ECG-based (MRI-based) beam gating was performed with effective duty cycles of 60.5% (68.8%) and 47.7% (50.4%) with residual motion reductions of 62.5% (44.7%) and 43.9% (59.3%). Local gamma analyses (1%/1 mm) returned pass rates of 97.6% (94.1%) and 90.5% (98.3%) for gated scenarios, which exceed the pass rates of 70.3% and 82.0% for nongated scenarios, respectively. In average, the gating simulations returned maximum residual motion reductions of 88%, 74%, and 81% at 30%, 50%, and 70% duty cycles, respectively, in favor of MRI-based gating. Conclusions: Real-time ECG-based beam gating is a feasible alternative to MRI-based gating, resulting in improved dose delivery in terms of high (Formula presented.) rates, decreased dose deposition outside the PTV and residual motion reduction, while by-passing cardiac MRI challenges

Assessing mutual accountability to strengthen national WASH systems and achieve the SDG targets for water and sanitation

Abstract Multi-stakeholder engagement is critical for making progress towards Sustainable Development Goal (SDG) 6 ‘Ensure access to water and sanitation for all’, which is currently off track to be achieved by 2030. The aim of this paper was to investigate mutual accountability and multi-stakeholder platforms in the WASH sector in a diverse range of countries. Data were collected by Sanitation and Water for All (SWA) Research and Learning Constituency partners and collaborators in five SWA member countries: Bangladesh, Indonesia, Kenya, Peru and Somalia. Data collection involved document review, key informant interviews and workshops, and an online questionnaire. Across all the case study countries, there were no clear examples of mutual accountability mechanisms being widely used in the WASH sector. However, the findings indicate that some of the case study countries have active WASH multi-stakeholder platforms involving a range of actors from government, civil society and the private sector; however, these typically function as coordination and communication platforms rather than supporting mutual accountability. Other case study countries did not have multi-stakeholder platforms involving a diverse range of actors, and instead had platforms established for single stakeholder groups such as the private sector or civil society, leaving certain groups out of activities. Overall, the study highlights the importance of establishing strong multi-stakeholder processes and platforms that bring together a range of actors including government, civil society, private sector, research actors, and WASH external support organizations. Such platforms could provide a foundation to enable mutual accountability between these actors by providing a space to set commitments and monitor progress and have potential to strengthen WASH systems both nationally and globally.</jats:p

First experimental exploration of real-time cardiorespiratory motion management for future stereotactic arrhythmia radioablation treatments on the MR-linac

Objective.Stereotactic arrhythmia radioablation (STAR) is a novel, non-invasive treatment for refractory ventricular tachycardia (VT). The VT isthmus is subject to both respiratory and cardiac motion. Rapid cardiac motion presents a unique challenge. In this study, we provide first experimental evidence for real-time cardiorespiratory motion-mitigated MRI-guided STAR on the 1.5 T Unity MR-linac (Elekta AB, Stockholm, Sweden) aimed at simultaneously compensating cardiac and respiratory motions. Approach.A real-time cardiorespiratory motion-mitigated radiotherapy workflow was developed on the Unity MR-linac in research mode. A 15-beam intensity-modulated radiation therapy treatment plan (1 × 25 Gy) was created in Monaco v.5.40.01 (Elekta AB) for the Quasar MRI 4Dphantom (ModusQA, London, ON). A film dosimetry insert was moved by combining either artificial (cos 4, 70 bpm, 10 mm peak-to-peak) or subject-derived (59 average bpm, 15.3 mm peak-to-peak) cardiac motion with respiratory (sin, 12 bpm, 20 mm peak-to-peak) motion. A balanced 2D cine MRI sequence (13 Hz, field-of-view = 400 × 207 mm 2, resolution = 3 × 3 × 15 mm 3) was developed to estimate cardiorespiratory motion. Cardiorespiratory motion was estimated by rigid registration and then deconvoluted into cardiac and respiratory components. For beam gating, the cardiac component was used, whereas the respiratory component was used for MLC-tracking. In-silico dose accumulation experiments were performed on three patient data sets to simulate the dosimetric effect of cardiac motion on VT targets. Main results.Experimentally, a duty cycle of 57% was achieved when simultaneously applying respiratory MLC-tracking and cardiac gating. Using film, excellent agreement was observed compared to a static reference delivery, resulting in a 1%/1 mm gamma pass rate of 99%. The end-to-end gating latency was 126 ms on the Unity MR-linac. Simulations showed that cardiac motion decreased the target's D98% dose between 0.1 and 1.3 Gy, with gating providing effective mitigation. Significance.Real-time MRI-guided cardiorespiratory motion management greatly reduces motion-induced dosimetric uncertainty and warrants further research and development for potential future use in STAR

Identification of alleles of carotenoid pathway genes important for zeaxanthin accumulation in potato tubers

We have investigated the genetics and molecular biology of orange flesh colour in potato (Solanum tuberosum L.). To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (Chy2) alleles only one dominant allele has a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (Lcye) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (Zep) alleles showed that all (diploid) genotypes with orange tuber flesh were homozygous for one specific Zep allele. This Zep allele showed a reduced level of expression. The complete genomic sequence of the recessive Zep allele, including the promoter, was determined, and compared with the sequence of other Zep alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive Zep allele, which was absent in all other Zep alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced Zep activity and accumulation of zeaxanthin. Only genotypes combining presence of the dominant Chy2 allele with homozygosity for the recessive Zep allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin

First VMAT delivery with MLC-tracking for single and multi fraction lung SBRT on a Unity MR-linac

Purpose or Objective Conventional lung SBRT requires large treatment margins to cover tumor motion resulting from respiration. This may avoid underdosage but increases toxicity risks. To maximize healthy tissue sparing, we previously developed MRI-guided MLC tumor tracking for the 1.5 T Unity MR-linac (Elekta AB, Stockholm, SE) in combination with IMRT. Recently, we also piloted VMAT deliveries on Unity to further maximize plan conformality and delivery efficiency. In this study, we demonstrate the feasibility of a first experimental setup on an MR-linac that combines VMAT with MLC-tracking for a range of lung SBRT indications. Materials and Methods All experiments were performed on a 1.5 T Unity MR-linac in research mode. A Quasar MRI4D phantom (ModusQA, London, CA) was used to generate: no motion (static reference), Lujan motion (cos4, peak-to-peak amplitude A = 20 mm, f = 0.25 Hz), and subject-derived real respiratory motion (average A = 11 mm, average f = 0.33 Hz) with an average baseline drift of 0.6 mm/min. The phantom contained a film insert with a 3 cm spherical target (GTV) that could be positioned centrally or 10 cm off-center (peripheral) in a water-filled body oval. Target positions were continuously estimated from 2D cine-MR (4 Hz). A linear regression prediction filter compensated for system latency. Predicted positions were used continuously to realign the MLC with the target position. We created three VMAT treatment plans with 3 mm GTV-to-PTV margins following the clinical planning template for lung SBRT: a central plan (8x7.5 Gy) and two peripheral plans (3x18 Gy and 1x34 Gy).EBT3 or EBTXD films were used to measure the delivered dose. A 1%/1mm local Gamma-analysis quantified dose differences between the static reference and tracking cases. Additionally, the dose area histogram (DAH) was determined for the target. Results The VMAT plans had a conformity index (prescribed isodose volume/ PTV) of 1.4-1.5 and an MU-weighted mean-field area of 13-16 cm2. Treatment delivery times were: 6.7 min, 13.1 min, and 24.2 min, for the 8x7.5 Gy, 3x18 Gy, 1x34 Gy lung SBRT plans respectively. The plans required an RMS leaf speed of 0.5-0.7 cm/s. Tracking required a maximal additional 2.4 cm/s leaf speed. Each plan was delivered in respectively 2, 4, and 6 arcs. The local gamma analysis for the central delivery shows that MLC-tracking improved the gamma pass-rate from 67.5% to 98.3% for Lujan motion and to 94.2% for the real respiratory trace. For peripheral deliveries with real respiratory motion, the 3x18 Gy delivery had a 97.3% pass-rate and the 1x34 Gy delivery had a 96.8% pass-rate (Fig.1). The DAH (Fig.2) shows that the target dose agrees well between static and tracking deliveries with real respiratory motion. The figure also shows that the minimum dose in the target is well above the prescribed dose. Conclusion We provided a first experimental demonstration of the technical feasibility of VMAT combined with MR-guided MLC-tracking for central and peripheral lung SBRT

Using SuperClomeleon to measure changes in intracellular chloride during development and after early life stress

Intraneuronal chloride concentrations ([Cl- ]i) decrease during development resulting in a shift from depolarizing to hyperpolarizing γ-aminobutyric acid (GABA) responses via chloride-permeable GABAA receptors. This GABA shift plays a pivotal role in postnatal brain development, and can be strongly influenced by early life experience. Here, we assessed the applicability of the recently developed fluorescent SuperClomeleon (SClm) sensor to examine changes in [Cl- ]i using two-photon microscopy in brain slices. We used SClm mice of both sexes to monitor the developmental decrease in neuronal chloride levels in organotypic hippocampal cultures. We could discern a clear reduction in [Cl-]i between DIV3 and DIV9 (equivalent to the second postnatal week in vivo) and a further decrease in some cells until DIV22. In addition, we assessed alterations in [Cl- ]i in the medial prefrontal cortex (mPFC) of P9 male SClm mouse pups after early life stress (ELS). ELS was induced by limiting nesting material between P2 and P9. ELS induced a shift towards higher (i.e. immature) chloride levels in layer 2/3 cells in the mPFC. Although conversion from SClm fluorescence to absolute chloride concentrations proved difficult, our study underscores that the SClm sensor is a powerful tool to measure physiological changes in [Cl- ]i in brain slices