Comparison of MR-soft tissue based versus biliary stent based alignment for image guidance in pancreatic SBRT.

PURPOSE: The role of biliary stents in image-guided localization for pancreatic cancer has been inconclusive. To date, stent accuracy has been largely evaluated against implanted fiducials on cone beam computed tomography. We aim to use magnetic resonance (MR) soft tissue as a direct reference to examine the geometric and dosimetric impacts of stent-based localization on the newly available MR linear accelerator. METHODS: Thirty pancreatic cancer patients (132 fractions) treated on our MR linear accelerator were identified to have a biliary stent. In our standard adaptive workflow, patients were set up to the target using soft tissue for image registration and structures were re-contoured on daily MR images. The original plan was then projected on treatment anatomy and dose predicted, followed by plan re-optimization and treatment delivery. These online predicted plans were soft tissue-based and served as reference plans. Retrospective image registration to the stent was performed offline to simulate stent-based localization and the magnitude of shifts was taken as the geometric accuracy of stent localization. New predicted plans were generated based on stent-alignment for dosimetric comparison. RESULTS: Shifts were within 3 mm for 90% of the cases (mean = 1.5 mm); however, larger shifts up to 7.2 mm were observed. Average PTV coverage dropped by 1.1% with a maximum drop of 26.8%. The mean increase in V35Gy was 0.15, 0.05, 0.02, and 0.02 cc for duodenum, stomach, small bowel and large bowel, respectively. Stent alignment was significantly worse for all metrics except for small bowel (p = 0.07). CONCLUSIONS: Overall discrepancy between stent- and soft tissue-alignment was modest; however, large discrepancies were observed for select cases. While PTV coverage loss may be compensated for by using a larger margin, the increase in dose to gastrointestinal organs at risk may limit the role of biliary stents in image-guided localization.

Low-Cost, High-Pressure-Synthesized Oxygen-Entrapping Materials to Improve Treatment of Solid Tumors.

Tumor hypoxia drives resistance to many cancer therapies, including radiotherapy and chemotherapy. Methods that increase tumor oxygen pressures, such as hyperbaric oxygen therapy and microbubble infusion, are utilized to improve the responses to current standard-of-care therapies. However, key obstacles remain, in particular delivery of oxygen at the appropriate dose and with optimal pharmacokinetics. Toward overcoming these hurdles, gas-entrapping materials (GeMs) that are capable of tunable oxygen release are formulated. It is shown that injection or implantation of these materials into tumors can mitigate tumor hypoxia by delivering oxygen locally and that these GeMs enhance responsiveness to radiation and chemotherapy in multiple tumor types. This paper also demonstrates, by comparing an oxygen (O2 )-GeM to a sham GeM, that the former generates an antitumorigenic and immunogenic tumor microenvironment in malignant peripheral nerve sheath tumors. Collectively the results indicate that the use of O2 -GeMs is promising as an adjunctive strategy for the treatment of solid tumors.

Adjuvant Radiation Field Extent and Sites of Failure in Node Positive Endometrioid Endometrial Cancer.

PURPOSE: In patients with node-positive endometrial cancer, adjuvant radiation therapy with chemotherapy decreases local-regional recurrence compared with chemotherapy alone. However, the optimal radiation field borders and extent of nodal coverage have not been well studied. In a multi-institutional cohort, survival outcomes and sites of failure were analyzed for patients with International Federation of Gynaecology and Obstetrics (FIGO) stage IIIC endometrioid endometrial cancer treated with pelvic radiation therapy (PRT) versus extended-field radiation therapy (EFRT), which encompassed high para-aortic lymph nodes. METHODS AND MATERIALS: In a multi-institutional retrospective study, 143 patients with FIGO stage IIIC1 or IIIC2 endometrioid endometrial cancer treated with adjuvant radiation therapy from 2000 to 2016 were identified. Patient subgroups were classified by substage and radiation field extent: stage IIIC1 received EFRT, stage IIIC1 received PRT, and stage IIIC2 received EFRT. Recurrence-free survival (RFS), overall survival (OS), and out-of-field recurrence were calculated by the Kaplan-Meier method. Multivariate analysis was performed using the Cox proportional hazards model. Sites of failure were categorized as within or outside the radiation field. RESULTS: The median follow-up was 59 months; 87% of patients received chemotherapy. The 5-year RFS and OS rates were 73% and 87%, respectively. By subgroup, 5-year RFS rates were 79% for stage IIIC1 EFRT, 73% for stage IIIC1 PRT, and 69% for stage IIIC2 EFRT (P = .4). On multivariate analysis, the recurrence risk was highest for stage IIIC2 EFRT, although this result was not statistically significant (adjusted hazard ratio, 2.0; P = .4). In-field vaginal and nodal recurrences were observed in 2 patients (1%) and 4 patients (3%), respectively. Of 78 patients with stage IIIC1 cancer treated with PRT, 5 (6%) had isolated para-aortic nodal relapse outside the radiation field; 3 were long-term survivors (more than 6 years after salvage therapy). For patients with para-aortic recurrence, 86% had lymphovascular invasion, 71% had myometrial invasion of ≥50%, and 57% had grade 3 disease. CONCLUSIONS: Adjuvant chemoradiation therapy resulted in excellent survival outcomes for patients with FIGO stage IIIC endometrioid endometrial cancer. For patients with positive pelvic nodes, isolated para-aortic relapse outside the PRT field was uncommon and amenable to salvage therapy.

A Switch in p53 Dynamics Marks Cells That Escape from DSB-Induced Cell Cycle Arrest.

Cellular responses to stimuli can evolve over time, resulting in distinct early and late phases in response to a single signal. DNA damage induces a complex response that is largely orchestrated by the transcription factor p53, whose dynamics influence whether a damaged cell will arrest and repair the damage or will initiate cell death. How p53 responses and cellular outcomes evolve in the presence of continuous DNA damage remains unknown. Here, we have found that a subset of cells switches from oscillating to sustained p53 dynamics several days after undergoing damage. The switch results from cell cycle progression in the presence of damaged DNA, which activates the caspase-2-PIDDosome, a complex that stabilizes p53 by inactivating its negative regulator MDM2. This work defines a molecular pathway that is activated if the canonical checkpoints fail to halt mitosis in the presence of damaged DNA.

Cytosolic NADH-NAD(+) Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging.

AIM: Cytosolic NADH-NAD(+) redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD(+) redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. RESULTS: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD(+) ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. INNOVATION: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. CONCLUSION: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD(+) ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553-563.

Imaging GFP-based reporters in neurons with multiwavelength optogenetic control.

To study the impact of neural activity on cellular physiology, one would like to combine precise control of firing patterns with highly sensitive probes of cellular physiology. Light-gated ion channels, e.g., Channelrhodopsin-2, enable precise control of firing patterns; green fluorescent protein-based reporters, e.g., the GCaMP6f Ca(2+) reporter, enable highly sensitive probing of cellular physiology. However, for most actuator-reporter combinations, spectral overlap prevents straightforward combination within a single cell. Here we explore multiwavelength control of channelrhodopsins to circumvent this limitation. The "stoplight" technique described in this article uses channelrhodopsin variants that are opened by blue light and closed by orange light. Cells are illuminated with constant blue light to excite fluorescence of a green fluorescent protein-based reporter. Modulated illumination with orange light negatively regulates activation of the channelrhodopsin. We performed detailed photophysical characterization and kinetic modeling of four candidate stoplight channelrhodopsins. The variant with the highest contrast, sdChR(C138S,E154A), enabled all-optical measurements of activity-induced calcium transients in cultured rat hippocampal neurons, although cell-to-cell variation in expression levels presents a challenge for quantification.

Bringing bioelectricity to light.

Any bilayer lipid membrane can support a membrane voltage. The combination of optical perturbation and optical readout of membrane voltage opens the door to studies of electrophysiology in a huge variety of systems previously inaccessible to electrode-based measurements. Yet, the application of optogenetic electrophysiology requires careful reconsideration of the fundamentals of bioelectricity. Rules of thumb appropriate for neuroscience and cardiology may not apply in systems with dramatically different sizes, lipid compositions, charge carriers, or protein machinery. Optogenetic tools are not electrodes; thus, optical and electrode-based measurements have different quirks. Here we review the fundamental aspects of bioelectricity with the aim of laying a conceptual framework for all-optical electrophysiology.

Temporal dynamics of microbial rhodopsin fluorescence reports absolute membrane voltage.

Plasma membrane voltage is a fundamentally important property of a living cell; its value is tightly coupled to membrane transport, the dynamics of transmembrane proteins, and to intercellular communication. Accurate measurement of the membrane voltage could elucidate subtle changes in cellular physiology, but existing genetically encoded fluorescent voltage reporters are better at reporting relative changes than absolute numbers. We developed an Archaerhodopsin-based fluorescent voltage sensor whose time-domain response to a stepwise change in illumination encodes the absolute membrane voltage. We validated this sensor in human embryonic kidney cells. Measurements were robust to variation in imaging parameters and in gene expression levels, and reported voltage with an absolute accuracy of 10 mV. With further improvements in membrane trafficking and signal amplitude, time-domain encoding of absolute voltage could be applied to investigate many important and previously intractable bioelectric phenomena.

Flash memory: photochemical imprinting of neuronal action potentials onto a microbial rhodopsin.

We developed a technique, "flash memory", to record a photochemical imprint of the activity state--firing or not firing--of a neuron at a user-selected moment in time. The key element is an engineered microbial rhodopsin protein with three states. Two nonfluorescent states, D1 and D2, exist in a voltage-dependent equilibrium. A stable fluorescent state, F, is reached by a photochemical conversion from D2. When exposed to light of a wavelength λ(write), population transfers from D2 to F, at a rate determined by the D1 ⇌ D2 equilibrium. The population of F maintains a record of membrane voltage which persists in the dark. Illumination at a later time at a wavelength λ(read) excites fluorescence of F, probing this record. An optional third flash at a wavelength λ(reset) converts F back to D2, for a subsequent write-read cycle. The flash memory method offers the promise to decouple the recording of neural activity from its readout. In principle, the technique may enable one to generate snapshots of neural activity in a large volume of neural tissue, e.g., a complete mouse brain, by circumventing the challenge of imaging a large volume with simultaneous high spatial and high temporal resolution. The proof-of-principle flash memory sensors presented here will need improvements in sensitivity, speed, brightness, and membrane trafficking before this goal can be realized.

Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin.

Microbial rhodopsins were recently introduced as genetically encoded fluorescent indicators of membrane voltage. An understanding of the mechanism underlying this function would aid in the design of improved voltage indicators. We asked, what states can the protein adopt, and which states are fluorescent? How does membrane voltage affect the photostationary distribution of states? Here, we present a detailed spectroscopic characterization of Archaerhodopsin 3 (Arch). We performed fluorescence spectroscopy on Arch and its photogenerated intermediates in Escherichia coli and in single HEK293 cells under voltage-clamp conditions. These experiments probed the effects of time-dependent illumination and membrane voltage on absorption, fluorescence, membrane current, and membrane capacitance. The fluorescence of Arch arises through a sequential three-photon process. Membrane voltage modulates protonation of the Schiff base in a 13-cis photocycle intermediate (M ⇌ N equilibrium), not in the ground state as previously hypothesized. We present experimental protocols for optimized voltage imaging with Arch, and we discuss strategies for engineering improved rhodopsin-based voltage indicators.

Screening fluorescent voltage indicators with spontaneously spiking HEK cells.

Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators.

Residual structure within the disordered C-terminal segment of p21(Waf1/Cip1/Sdi1) and its implications for molecular recognition.

Probably the most unusual class of proteins in nature is the intrinsically unstructured proteins (IUPs), because they are not structured yet play essential roles in protein-protein signaling. Many IUPs can bind different proteins, and in many cases, adopt different bound conformations. The p21 protein is a small IUP (164 residues) that is ubiquitous in cellular signaling, for example, cell cycle control, apoptosis, transcription, differentiation, and so forth; it binds to approximately 25 targets. How does this small, unstructured protein recognize each of these targets with high affinity? Here, we characterize residual structural elements of the C-terminal segment of p21 encompassing residues 145-164 using a combination of NMR measurements and molecular dynamics simulations. The N-terminal half of the peptide has a significant helical propensity which is recognized by calmodulin while the C-terminal half of the peptide prefers extended conformations that facilitate binding to the proliferating cell nuclear antigen (PCNA). Our results suggest that the final bound conformations of p21 (145-164) pre-exist in the free peptide even without its binding partners. While the conformational flexibility of the p21 peptide is essential for adapting to diverse binding environments, the intrinsic structural preferences of the free peptide enable promiscuous yet high affinity binding to a diverse array of molecular targets.