Pycallingcards: an integrated environment for visualizing, analyzing, and interpreting Calling Cards data.

MOTIVATION: Unraveling the transcriptional programs that control how cells divide, differentiate, and respond to their environments requires a precise understanding of transcription factors' (TFs) DNA-binding activities. Calling cards (CC) technology uses transposons to capture transient TF binding events at one instant in time and then read them out at a later time. This methodology can also be used to simultaneously measure TF binding and mRNA expression from single-cell CC and to record and integrate TF binding events across time in any cell type of interest without the need for purification. Despite these advantages, there has been a lack of dedicated bioinformatics tools for the detailed analysis of CC data. RESULTS: We introduce Pycallingcards, a comprehensive Python module specifically designed for the analysis of single-cell and bulk CC data across multiple species. Pycallingcards introduces two innovative peak callers, CCcaller and MACCs, enhancing the accuracy and speed of pinpointing TF binding sites from CC data. Pycallingcards offers a fully integrated environment for data visualization, motif finding, and comparative analysis with RNA-seq and ChIP-seq datasets. To illustrate its practical application, we have reanalyzed previously published mouse cortex and glioblastoma datasets. This analysis revealed novel cell-type-specific binding sites and potential sex-linked TF regulators, furthering our understanding of TF binding and gene expression relationships. Thus, Pycallingcards, with its user-friendly design and seamless interface with the Python data science ecosystem, stands as a critical tool for advancing the analysis of TF functions via CC data. AVAILABILITY AND IMPLEMENTATION: Pycallingcards can be accessed on the GitHub repository: https://github.com/The-Mitra-Lab/pycallingcards.

Mecp2 deletion results in profound alterations of developmental and adult functional connectivity.

As a regressive neurodevelopmental disorder with a well-established genetic cause, Rett syndrome and its Mecp2 loss-of-function mouse model provide an excellent opportunity to define potentially translatable functional signatures of disease progression, as well as offer insight into the role of Mecp2 in functional circuit development. Thus, we applied widefield optical fluorescence imaging to assess mesoscale calcium functional connectivity (FC) in the Mecp2 cortex both at postnatal day (P)35 in development and during the disease-related decline. We found that FC between numerous cortical regions was disrupted in Mecp2 mutant males both in juvenile development and early adulthood. Female Mecp2 mice displayed an increase in homotopic contralateral FC in the motor cortex at P35 but not in adulthood, where instead more posterior parietal regions were implicated. An increase in the amplitude of connection strength, both with more positive correlations and more negative anticorrelations, was observed across the male cortex in numerous functional regions. Widespread rescue of MeCP2 protein in GABAergic neurons rescued none of these functional deficits, nor, surprisingly, the expected male lifespan. Altogether, the female results identify early signs of disease progression, while the results in males indicate MeCP2 protein is required for typical FC in the brain.

Beyond conventional bounds: Surpassing system limits for stereotactic ablative (SAbR) lung radiotherapy using CBCT-based adaptive planning system.

PURPOSE: Online adaptive radiotherapy relies on a high degree of automation to enable rapid planning procedures. The Varian Ethos intelligent optimization engine (IOE) was originally designed for conventional treatments so it is crucial to provide clear guidance for lung SAbR plans. This study investigates using the Ethos IOE together with adaptive-specific optimization tuning structures we designed and templated within Ethos to mitigate inter-planner variability in meeting RTOG metrics for both online-adaptive and offline SAbR plans. METHODS: We developed a planning strategy to automate the generation of tuning structures and optimization. This was validated by retrospective analysis of 35 lung SAbR cases (total 105 fractions) treated on Ethos. The effectiveness of our planning strategy was evaluated by comparing plan quality with-and-without auto-generated tuning structures. Internal target volume (ITV) contour was compared between that drawn from CT simulation and from cone-beam CT (CBCT) at time of treatment to verify CBCT image quality and treatment effectiveness. Planning strategy robustness for lung SAbR was quantified by frequency of plans meeting reference plan RTOG constraints. RESULTS: Our planning strategy creates a gradient within the ITV with maximum dose in the core and improves intermediate dose conformality on average by 2%. ITV size showed no significant difference between those contoured from CT simulation and first fraction, and also trended towards decreasing over course of treatment. Compared to non-adaptive plans, adaptive plans better meet reference plan goals (37% vs. 100% PTV coverage compliance, for scheduled and adapted plans) while improving plan quality (improved GI (gradient index) by 3.8%, CI (conformity index) by 1.7%). CONCLUSION: We developed a robust and readily shareable planning strategy for the treatment of adaptive lung SAbR on the Ethos system. We validated that automatic online plan re-optimization along with the formulated adaptive tuning structures can ensure consistent plan quality. With the proposed planning strategy, highly ablative treatments are feasible on Ethos.

A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues.

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.

Flow-Field Simulations and Hemolysis Estimates for the Food and Drug Administration Critical Path Initiative Centrifugal Blood Pump.

The design of blood pumps for use in ventricular assist devices, which provide life-saving circulatory support in patients with heart failure, require remarkable precision and attention to detail to replicate the functionality of the native heart. The United States Food and Drug Administration (FDA) initiated a Critical Path Initiative to standardize and facilitate the use of computational fluid dynamics in the study and development of these devices. As a part of the study, a simplified centrifugal blood pump model generated by computer-aided design was released to universities and laboratories nationwide. The effects of changes in fluid rheology due to temperature, hematocrit, and turbulent flow on key metrics of the FDA pump were examined in depth using results from a finite volume-based commercial computational fluid dynamics code. Differences in blood damage indices obtained using Eulerian and Lagrangian formulations were considered. These results are presented and discussed awaiting future validation using experimental results, which will be released by the FDA at a future date.

Spatial and temporal localization of caveolin-1 protein in the developing retina.

Caveolin-1 (Cav-1), the signature protein of caveolae is expressed in several cell types in the adult retina and is linked to ocular pathologies including uveitis, diabetic retinopathy, and primary open angle glaucoma. Genetic ablation of Cav-1 causes retinal functional deficits due to disruptions in environmental homeostasis. To better understand Cav-1 function in the retina, we examined its expression/localization during postnatal retinal development. From P0-P5, Cav-1 was detected only in the developing superficial retinal vessels, in hyaloid and choroidal vasculature, and in the retinal pigment epithelium (RPE). At P7, staining began to be observed centrally in radial cells in the neuroretina, and this staining increased dramatically by P9/10 in identifiable Müller glia. Prominent vascular staining continued throughout development. These results support the idea that Cav-1 is an indicator of Müller glial differentiation and suggests that it plays an important role in Müller cell function.

Optimizing Online Adaptation Timing in the Treatment of Locally Advanced Cervical Cancer.

PURPOSE: Online adaptive radiation therapy (ART) has emerged as a new treatment modality for cervical cancer. Daily online adapting improves target coverage and organ-at-risk (OAR) sparing compared with traditional image guided radiation therapy (IGRT); however, the required resources may not be feasible in a busy clinical setting. Less frequent adapting may still benefit cervical cancer patients due to large volume changes of the uterocervix of the treatment course. In this study, the dosimetry from different online adapt-on-demand schedules was compared. MATERIALS AND METHODS: A retrospective cohort of 10 patients with cervical cancer treated with 260 fractions of definitive daily online ART was included. Plans with different adaptation schedules were simulated with adaptations weekly, every other week, once during treatment, and no adaptations (IGRT). These plans were applied to the synthetic computed tomography (CT) images and contours generated during the patient's delivered daily adaptive workflow. The dosimetry of the weekly replan, every-other-week replan, once replan, and IGRT plans were compared using a paired t test. RESULTS: Compared with traditional IGRT plans, weekly and every-other-week ART plans had similar clinical target volume (CTV) coverage, but statistically significant improved sparing of OARs. Weekly and every-other-week ART had reduced bowel bag V40 by 1.57% and 1.41%, bladder V40 by 3.82% and 1.64%, rectum V40 by 8.49% and 7.50%, and bone marrow Dmean by 0.81% and 0.61%, respectively. Plans with a single adaptation had statistically significantly worse target coverage, and moderate improvements in OAR sparing. Of the 18-dose metrics evaluated, improvements were seen in 15 for weekly ART, 14 for every-other-week ART, and 10 for single ART plans compared with IGRT. When every-other-week ART was compared with weekly ART, both plans had similar CTV coverage and OAR sparing with only small improvements in bone marrow dosimetry with weekly ART. CONCLUSIONS: This retrospective work compares different adapt-on-demand treatment schedules using data collected from patients treated with daily online adaptive radiation therapy. Results suggest weekly or every-other-week online ART is beneficial for reduced OAR dose compared with IGRT by exploiting the gradual changes in the uterocervix target volume.

MYT1L deficiency impairs excitatory neuron trajectory during cortical development.

Mutations that reduce the function of MYT1L, a neuron-specific transcription factor, are associated with a syndromic neurodevelopmental disorder. Furthermore, MYT1L is routinely used as a proneural factor in fibroblast-to-neuron transdifferentiation. MYT1L has been hypothesized to play a role in the trajectory of neuronal specification and subtype specific maturation, but this hypothesis has not been directly tested, nor is it clear which neuron types are most impacted by MYT1L loss. In this study, we profiled 313,335 nuclei from the forebrains of wild-type and MYT1L-deficient mice at two developmental stages: E14 at the peak of neurogenesis and P21, when neurogenesis is complete, to examine the role of MYT1L levels in the trajectory of neuronal development. We found that MYT1L deficiency significantly disrupted the relative proportion of cortical excitatory neurons at E14 and P21. Significant changes in gene expression were largely concentrated in excitatory neurons, suggesting that transcriptional effects of MYT1L deficiency are largely due to disruption of neuronal maturation programs. Most effects on gene expression were cell autonomous and persistent through development. In addition, while MYT1L can both activate and repress gene expression, the repressive effects were most sensitive to haploinsufficiency, and thus more likely mediate MYT1L syndrome. These findings illuminate the intricate role of MYT1L in orchestrating gene expression dynamics during neuronal development, providing insights into the molecular underpinnings of MYT1L syndrome.

The accuracy of artificial intelligence deformed nodal structures in cervical online cone-beam-based adaptive radiotherapy.

Background and Purpose: Online cone-beam-based adaptive radiotherapy (ART) adjusts for anatomical changes during external beam radiotherapy. However, limited cone-beam image quality complicates nodal contouring. Despite this challenge, artificial-intelligence guided deformation (AID) can auto-generate nodal contours. Our study investigated the optimal use of such contours in cervical online cone-beam-based ART. Materials and Methods: From 136 adaptive fractions across 21 cervical cancer patients with nodal disease, we extracted 649 clinically-delivered and AID clinical target volume (CTV) lymph node boost structures. We assessed geometric alignment between AID and clinical CTVs via dice similarity coefficient, and 95% Hausdorff distance, and geometric coverage of clinical CTVs by AID planning target volumes by false positive dice. Coverage of clinical CTVs by AID contour-based plans was evaluated using D100, D95, V100%, and V95%. Results: Between AID and clinical CTVs, the median dice similarity coefficient was 0.66 and the median 95 % Hausdorff distance was 4.0 mm. The median false positive dice of clinical CTV coverage by AID planning target volumes was 0. The median D100 was 1.00, the median D95 was 1.01, the median V100% was 1.00, and the median V95% was 1.00. Increased nodal volume, fraction number, and daily adaptation were associated with reduced clinical CTV coverage by AID-based plans. Conclusion: In one of the first reports on pelvic nodal ART, AID-based plans could adequately cover nodal targets. However, physician review is required due to performance variation. Greater attention is needed for larger, daily-adapted nodes further into treatment.

Norepinephrine Signals Through Astrocytes To Modulate Synapses.

Locus coeruleus (LC)-derived norepinephrine (NE) drives network and behavioral adaptations to environmental saliencies by reconfiguring circuit connectivity, but the underlying synapse-level mechanisms are elusive. Here, we show that NE remodeling of synaptic function is independent from its binding on neuronal receptors. Instead, astrocytic adrenergic receptors and Ca2+ dynamics fully gate the effect of NE on synapses as the astrocyte-specific deletion of adrenergic receptors and three independent astrocyte-silencing approaches all render synapses insensitive to NE. Additionally, we find that NE suppression of synaptic strength results from an ATP-derived and adenosine A1 receptor-mediated control of presynaptic efficacy. An accompanying study from Chen et al. reveals the existence of an analogous pathway in the larval zebrafish and highlights its importance to behavioral state transitions. Together, these findings fuel a new model wherein astrocytes are a core component of neuromodulatory systems and the circuit effector through which norepinephrine produces network and behavioral adaptations, challenging an 80-year-old status quo.

The New Kid on the Block: Online Adaptive Radiotherapy in the Treatment of Gynecologic Cancers.

Online adaptive radiation is a new and exciting modality of treatment for gynecologic cancers. Traditional radiation treatments deliver the same radiation plan to cancers with large margins. Improvements in imaging, technology, and artificial intelligence have made it possible to account for changes between treatments and improve the delivery of radiation. These advances can potentially lead to significant benefits in tumor coverage and normal tissue sparing. Gynecologic cancers can uniquely benefit from this technology due to the significant changes in bladder, bowel, and rectum between treatments as well as the changes in tumors commonly seen between treatments. Preliminary studies have shown that online adaptive radiation can maintain coverage of the tumor while sparing nearby organs. Given these potential benefits, numerous clinical trials are ongoing to investigate the clinical benefits of online adaptive radiotherapy. Despite the benefits, implementation of online adaptive radiotherapy requires significant clinical resources. Additionally, the timing and workflow for online adaptive radiotherapy is being optimized. In this review, we discuss the history and evolution of radiation techniques, the logistics and implementation of online adaptive radiation, and the potential benefits of online adaptive radiotherapy for gynecologic cancers.

Spare the Bowel, Don't Spoil the Target: Optimal Margin Assessment for Online Cone Beam Adaptive Radiation Therapy (OnC-ART) of the Cervix.

PURPOSE: The standard treatment for locally advanced cervical cancer involves pelvic chemoradiation. Intensity modulated radiation therapy planning requires expansion of the cervix and uterus clinical target volume (CTV) by 1.5 to 2 cm to account for motion. With online cone beam adaptive radiation therapy (OnC-ART), interfractional movement is accounted for, which can potentially lead to smaller CTV to planned target volume (PTV) margins. In this study, we attempted to determine the optimal CTV-to-PTV margin for adequate coverage with OnC-ART and factors that can affect CTV coverage. METHODS AND MATERIALS: A retrospective cohort of 21 patients with cervical cancer treated with definitive chemoradiation was included. Nine patients treated with nonadaptive radiation had the uterocervix contoured on pretreatment cone beam computed tomography (CBCT) and end-treatment CBCTs. Anterior-posterior, lateral, and superior-inferior shifts and the average shift in all directions were calculated. A CTV-to-PTV expansion was determined and verified on a validation cohort of 12 patients treated with OnC-ART. RESULTS: The average anterior-posterior, lateral, and superior-inferior shifts with standard deviation were 0.32 ± 1.55 cm, 0.12 ± 2.31 cm, and 1.67 ± 3.41 cm, respectively. A uniform 5-mm expansion around the pretreatment CTV covered 98.85% ± 1.23% of the end-treatment CTV. This 5-mm expansion was applied to our validation cohort treated with OnC-ART, and 98.39% ± 3.0% of the end-treatment CTV was covered. Time between CBCTs >30 minutes and change in bladder volume were significantly correlated to CTV coverage. CONCLUSIONS: Based on our analysis, a CTV-to-PTV margin of 5 mm is adequate to encompass 98% of the CTV. A significantly reduced margin could potentially decrease the toxicities associated with radiation for patients with cervical cancer and lead to improved patient reported toxicity outcomes. We recommend physicians begin with a 5-mm margin and assess adequate coverage with image guidance during daily adaptation.

Artificial intelligence guided physician directive improves head and neck planning quality and practice Uniformity: A prospective study.

•AI dose predictor was fully integrated with treatment planning system and used as a physicain decision support tool to improve uniformity of practice.•Model was trained based on our standard of practice, but implemented at the time of expansion with 3 new physicians join the practice.•Phase 1 retrospective evaluation demonstrated the non-uniform practice among 3 MDs and only 52.9% frequency planner can achieve physicians' directives.•Significant improvement in practice uniformity of practice was observed after utilizing AI as DST and 80.4% frequency clinical plan can achieve AI-guided physician directives.

Aspirin Use Is Associated With Improved Outcomes in Inflammatory Breast Cancer Patients.

PURPOSE: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer and has a high propensity for distant metastases. Our previous data suggested that aspirin (acetylsalicylic acid, ASA) use may be associated with reduced risk of distant metastases in aggressive breast cancer; however, there are no reported studies on the potential benefit of ASA use in patients with IBC. METHODS: Data from patients with non-metastatic IBC treated between 2000-2017 at two institutions, were reviewed. Overall survival (OS), disease-free survival (DFS), and distant metastasis-free survival (DMFS) were performed using Kaplan-Meier analysis. Univariate and multivariable logistic regression models were used to identify significant associated factors. RESULTS: Of 59 patients meeting the criteria for analysis and available for review, 14 ASA users were identified. ASA users demonstrated increased OS (p = 0.03) and DMFS (p = 0.02), with 5-year OS and DMFS of 92% (p = 0.01) and 85% (p = 0.01) compared to 51% and 43%, respectively, for non-ASA users. In univariate analysis, pT stage, pN stage, and ASA use were significantly correlated (p < 0.05) with OS and DFS. On multivariable analysis, ASA use (hazard ratio [HR], 0.11; 95% confidence interval [CI], 0.01-0.8) and lymph node stage (HR, 5.9; 95% CI, 1.4-25.9) remained significant for OS and DFS ASA use (HR, 0.13; 95% CI, 0.03-0.56) and lymph node stage (HR, 5.6; 95% CI, 1.9-16.4). CONCLUSION: ASA use during remission was associated with significantly improved OS and DMFS in patients with IBC. These results suggest that ASA may provide survival benefits to patients with IBC. Prospective clinical trials of ASA use in patients with high-risk IBC in remission should be considered.

Calling Cards: a customizable platform to longitudinally record protein-DNA interactions over time in cells and tissues.

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon (SRT) "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile specific transcription factor binding with custom transcription factor (TF)-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Card reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 1-2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. Basic Protocol 1: Preparation and delivery of Calling Cards reagentsBasic Protocol 2: Sample preparationBasic Protocol 3: Sequencing library preparationBasic Protocol 4: Library pooling and sequencingBasic Protocol 5: Data analysis.

Bony structure enhanced synthetic CT generation using Dixon sequences for pelvis MR-only radiotherapy.

BACKGROUND: MRI-only radiotherapy planning (MROP) is beneficial to patients by avoiding MRI/CT registration errors, simplifying the radiation treatment simulation workflow and reducing exposure to ionizing radiation. MRI is the primary imaging modality for soft tissue delineation. Treatment planning CTs (i.e., CT simulation scan) are redundant if a synthetic CT (sCT) can be generated from the MRI to provide the patient positioning and electron density information. Unsupervised deep learning (DL) models like CycleGAN are widely used in MR-to-sCT conversion, when paired patient CT and MR image datasets are not available for model training. However, compared to supervised DL models, they cannot guarantee anatomic consistency, especially around bone. PURPOSE: The purpose of this work was to improve the sCT accuracy generated from MRI around bone for MROP. METHODS: To generate more reliable bony structures on sCT images, we proposed to add bony structure constraints in the unsupervised CycleGAN model's loss function and leverage Dixon constructed fat and in-phase (IP) MR images. Dixon images provide better bone contrast than T2-weighted images as inputs to a modified multi-channel CycleGAN. A private dataset with a total of 31 prostate cancer patients were used for training (20) and testing (11). RESULTS: We compared model performance with and without bony structure constraints using single- and multi-channel inputs. Among all the models, multi-channel CycleGAN with bony structure constraints had the lowest mean absolute error, both inside the bone and whole body (50.7 and 145.2 HU). This approach also resulted in the highest Dice similarity coefficient (0.88) of all bony structures compared with the planning CT. CONCLUSION: Modified multi-channel CycleGAN with bony structure constraints, taking Dixon-constructed fat and IP images as inputs, can generate clinically suitable sCT images in both bone and soft tissue. The generated sCT images have the potential to be used for accurate dose calculation and patient positioning in MROP radiation therapy.

Calling Cards: A Customizable Platform to Longitudinally Record Protein-DNA Interactions Over Time in Cells and Tissues.

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next-generation sequencing. Compared with other genomic assays, readouts of which provide a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile-specific transcription factor (TF) binding with custom TF-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Cards reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation and delivery of Calling Cards reagents Support Protocol 1: Next-generation sequencing quantification of barcode distribution within self-reporting transposon plasmid pool and adeno-associated virus genome Basic Protocol 2: Sample collection and RNA purification Support Protocol 2: Library density quantitative PCR Basic Protocol 3: Sequencing library preparation Basic Protocol 4: Library pooling and sequencing Basic Protocol 5: Data analysis.

Case Report: Resolution of radiation pneumonitis with androgens and growth hormone.

Radiation pneumonitis (RP) occurs in some patients treated with thoracic radiation therapy. RP often self-resolves, but when severe it is most commonly treated with corticosteroids because of their anti-inflammatory properties. Androgens and human growth hormone (HGH) also have anti-inflammatory and healing properties in the lung, but have not been studied as a remedy for RP. Here we present a case of corticosteroid-refractory RP that resolved with androgen and HGH-based therapy. Case Presentation: A 62 year old male body builder with excellent performance status presented with locally advanced non-small cell lung cancer characterized by a 7 cm mass in the right lower lobe and associated right hilar and subcarinal lymph node involvement. He was treated with chemoradiation and an excellent tumor response was observed. However, 2 months post-treatment he developed severe shortness of breath and imaging was consistent with RP. His RP was refractory to prednisone and antibiotic therapy, despite various regimens over a 9 month period. The patient self-treated with an androgen and HGH-based regimen and the RP promptly resolved. Conclusion: The anti-inflammatory properties of androgens and HGH have prompted an exploration of their potential role in therapeutic strategies to treat pro-inflammatory conditions such as sepsis, infections and interstitial lung disease. This case study suggests a potential role for the use of androgens for the treatment of steroid-refractory RP after radiation therapy. However, the applicability of this strategy to general populations should be weighed carefully against secondary effects of these agents, especially in the setting of cancer survivorship.

MYT1L in the making: emerging insights on functions of a neurodevelopmental disorder gene.

Large scale human genetic studies have shown that loss of function (LoF) mutations in MYT1L are implicated in neurodevelopmental disorders (NDDs). Here, we provide an overview of the growing number of published MYT1L patient cases, and summarize prior studies in cells, zebrafish, and mice, both to understand MYT1L's molecular and cellular role during brain development and consider how its dysfunction can lead to NDDs. We integrate the conclusions from these studies and highlight conflicting findings to reassess the current model of the role of MYT1L as a transcriptional activator and/or repressor based on the biological context. Finally, we highlight additional functional studies that are needed to understand the molecular mechanisms underlying pathophysiology and propose key questions to guide future preclinical studies.

Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes.

Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing. Although immensely powerful, current implementations of calling cards in bulk experiments on populations of cells are technically cumbersome and require many replicates to identify independent insertions into the same genomic locus. Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2 and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. Lastly, we demonstrated barcoded calling cards can record binding in vivo in the mouse brain. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells.