Assessing the Content Validity of Preference-Based Measures in Cancer.

OBJECTIVES: This study assessed the content validity of generic and condition-specific preference-based measures (PBMs) with patients treated for cancer, evaluated against 10 Consensus-Based Standards for the Selection of Health Measurement Instruments criteria for good content validity, to best inform measurement strategies regarding the use of PBMs in oncology development programs and real-world applications. METHODS: Individual, semistructured interviews were conducted with patients who received drug treatment for cancer in the United Kingdom (n = 47) and the United States (n = 49). During the interview, patients completed 3 generic PBMs (EQ-5D-5L, EuroQol Health and Wellbeing measure-Short Form, Château Santé Base) and 2 condition-specific PBMs (Quality of Life Utility-Core 10 Dimension, Functional Assessment of Cancer Therapy Eight Dimension [FACT-8D]). Interviews were conducted via teleconference, audio recorded, and transcribed verbatim. Transcripts were coded using thematic and content analysis methods. RESULTS: Condition-specific measures were evaluated as having better relevancy than generic PBMs. Overall, the FACT-8D was evaluated as holding the best content validity in terms of relevancy, and the EuroQol Health and Wellbeing measure-Short Form received the most favorable evaluation of relevancy for generic PBMs. All measures demonstrated comparable comprehensiveness, with all suggested by patients to be missing concepts. The EQ-5D-5L was evaluated best in terms of comprehensibility. This was followed by the Quality of Life Utility-Core 10 Dimension and FACT-8D; both received similar evaluations. CONCLUSIONS: All measures were generally seen by patients as adequate in capturing appropriate aspects of health-related quality of life for measuring cancer outcomes, although together condition-specific measures were evaluated as having better relevancy than generic PBMs. Further health-related quality of life instrument development is encouraged, particularly with regard to the longer-term detrimental impacts of cancer and treatment side effects. Other developments could include new cancer-specific tools inclusive of conventional health items, treatment impacts, and psychological items.

A Methodological Study to Compare Alternative Modes of Administration With Value EQ-5D Using Preference-Elicitation Techniques.

OBJECTIVES: Time trade-off (TTO) and discrete choice experiment (DCE) preference-elicitation techniques can be administered using face-to-face interviews (F2F), unassisted online (UO) surveys, or remote-assisted (RA) interviews. The objective of this study was to explore how the mode of administration affects the quality and reliability of preference-elicitation data. METHODS: EQ-5D-5L health states were valued using composite TTO (cTTO) and DCE approaches by the UK general population. Participants were allocated to 1 of 2 study groups. Group A completed both F2F and UO surveys (n = 271), and group B completed both RA and UO surveys (n = 223). The feasibility of survey completion and the reliability and face-validity of data collected were compared across all modes of administration. RESULTS: Fewer participants reported receiving sufficient guidance on the cTTO tasks during the UO survey compared with the 2 assisted modes. Participants across all modes typically reported receiving sufficient guidance on the DCE tasks. cTTO data were less reliable from the UO survey compared with both assisted modes, but there were no differences in DCE data reliability. cTTO data from all modes demonstrated face-validity; however, the UO survey produced higher utilities for moderate and severe health states than both assisted modes. Both F2F and RA modes provided comparably reliable data. CONCLUSIONS: The reliability of DCE data is not affected by the mode of administration. Interviewer-assisted modes of administration (F2F or RA) yield more reliable cTTO data than unassisted surveys. Both F2F and RA surveys produced similar-quality data.

Mapping hippocampal glutamate in mesial temporal lobe epilepsy with glutamate weighted CEST (GluCEST) imaging.

Temporal lobe epilepsy (TLE) is one of the most common subtypes of focal epilepsy, with mesial temporal sclerosis (MTS) being a common radiological and histopathological finding. Accurate identification of MTS during presurgical evaluation confers an increased chance of good surgical outcome. Here we propose the use of glutamate-weighted chemical exchange saturation transfer (GluCEST) magnetic resonance imaging (MRI) at 7 Tesla for mapping hippocampal glutamate distribution in epilepsy, allowing to differentiate lesional from non-lesional mesial TLE. We demonstrate that a directional asymmetry index, which quantifies the relative difference between GluCEST contrast in hippocampi ipsilateral and contralateral to the seizure onset zone, can differentiate between sclerotic and non-sclerotic hippocampi, even in instances where traditional presurgical MRI assessments did not provide evidence of sclerosis. Overall, our results suggest that hippocampal glutamate mapping through GluCEST imaging is a valuable addition to the presurgical epilepsy evaluation toolbox.

Cambrian edrioasteroid reveals new mechanism for secondary reduction of the skeleton in echinoderms.

Echinoderms are characterized by a distinctive high-magnesium calcite endoskeleton as adults, but elements of this have been drastically reduced in some groups. Herein, we describe a new pentaradial echinoderm, Yorkicystis haefneri n. gen. n. sp., which provides, to our knowledge, the oldest evidence of secondary non-mineralization of the echinoderm skeleton. This material was collected from the Cambrian Kinzers Formation in York (Pennsylvania, USA) and is dated as ca 510 Ma. Detailed morphological observations demonstrate that the ambulacra (i.e. axial region) are composed of flooring and cover plates, but the rest of the body (i.e. extraxial region) is preserved as a dark film and lacks any evidence of skeletal plating. Moreover, X-ray fluorescence analysis reveals that the axial region is elevated in iron. Based on our morphological and chemical data and on taphonomic comparisons with other fossils from the Kinzers Formation, we infer that the axial region was originally calcified, while the extraxial region was non-mineralized. Phylogenetic analyses recover Yorkicystis as an edrioasteroid, indicating that this partial absence of skeleton resulted from a secondary reduction. We hypothesize that skeletal reduction resulted from lack of expression of the skeletogenic gene regulatory network in the extraxial body wall during development. Secondary reduction of the skeleton in Yorkicystis might have allowed for greater flexibility of the body wall.

COVID-19 pandemic impacts on conservation research, management, and public engagement in US national parks.

The COVID-19 pandemic has disrupted the timing and substance of conservation research, management, and public engagement in protected areas around the world. This disruption is evident in US national parks, which play a key role in protecting natural and cultural resources and providing outdoor experiences for the public. Collectively, US national parks protect 34 million ha, host more than 300 million visits annually, and serve as one of the world's largest informal education organizations. The pandemic has altered park conditions and operations in a variety of ways. Shifts in operational conditions related to safety issues, reduced staffing, and decreased park revenues have forced managers to make difficult trade-offs among competing priorities. Long-term research and monitoring of the health of ecosystems and wildlife populations have been interrupted. Time-sensitive management practices, such as control of invasive plants and restoration of degraded habitat, have been delayed. And public engagement has largely shifted from in-person experiences to virtual engagement through social media and other online interactions. These changes pose challenges for accomplishing important science, management, and public engagement goals, but they also create opportunities for developing more flexible monitoring programs and inclusive methods of public engagement. The COVID-19 pandemic reinforces the need for strategic science, management planning, flexible operations, and online public engagement to help managers address rapid and unpredictable challenges.

Diffuse optical tomography for the detection of perinatal stroke at the cot side: a pilot study.

BACKGROUND: Perinatal stroke is a potentially debilitating injury, often under-diagnosed in the neonatal period. We conducted a pilot study investigating the role of the portable, non-invasive brain monitoring technique, diffuse optical tomography (DOT), as an early detection tool for infants with perinatal stroke. METHODS: Four stroke-affected infants were scanned with a DOT system within the first 3 days of life and compared to four healthy control subjects. Spectral power, correlation, and phase lag between interhemispheric low frequency (0.0055-0.3 Hz) hemoglobin signals were assessed. Optical data analyses were conducted with and without magnetic resonance imaging (MRI)-guided stroke localization to assess the efficacy of DOT when used without stroke anatomical information. RESULTS: Interhemispheric correlations of both oxyhemoglobin and deoxyhemoglobin concentration were significantly reduced in the stroke-affected group within the very low (0.0055-0.0095 Hz) and resting state (0.01-0.08 Hz) frequencies (p < 0.003). There were no interhemispheric differences for spectral power. These results were observed even without MRI stroke localization. CONCLUSION: This suggests that DOT and correlation-based analyses in the low-frequency range can potentially aid the early detection of perinatal stroke, prior to MRI acquisition. Additional methodological advances are required to increase the sensitivity and specificity of this technique.

Quality assurance in proton beam therapy using a plastic scintillator and a commercially available digital camera.

PURPOSE: In this article, we evaluate a plastic scintillation detector system for quality assurance in proton therapy using a BC-408 plastic scintillator, a commercial camera, and a computer. METHODS: The basic characteristics of the system were assessed in a series of proton irradiations. The reproducibility and response to changes of dose, dose-rate, and proton energy were determined. Photographs of the scintillation light distributions were acquired, and compared with Geant4 Monte Carlo simulations and with depth-dose curves measured with an ionization chamber. A quenching effect was observed at the Bragg peak of the 60 MeV proton beam where less light was produced than expected. We developed an approach using Birks equation to correct for this quenching. We simulated the linear energy transfer (LET) as a function of depth in Geant4 and found Birks constant by comparing the calculated LET and measured scintillation light distribution. We then used the derived value of Birks constant to correct the measured scintillation light distribution for quenching using Geant4. RESULTS: The corrected light output from the scintillator increased linearly with dose. The system is stable and offers short-term reproducibility to within 0.80%. No dose rate dependency was observed in this work. CONCLUSIONS: This approach offers an effective way to correct for quenching, and could provide a method for rapid, convenient, routine quality assurance for clinical proton beams. Furthermore, the system has the advantage of providing 2D visualization of individual radiation fields, with potential application for quality assurance of complex, time-varying fields.

A comparison of the dose distributions from three proton treatment planning systems in the planning of meningioma patients with single-field uniform dose pencil beam scanning.

With the number of new proton centers increasing rapidly, there is a need for an assessment of the available proton treatment planning systems (TPSs). This study compares the dose distributions of complex meningioma plans produced by three proton TPSs: Eclipse, Pinnacle3, and XiO. All three systems were commissioned with the same beam data and, as best as possible, matched configuration settings. Proton treatment plans for ten patients were produced on each system with a pencil beam scanning, single-field uniform dose approach, using a fixed horizontal beamline. All 30 plans were subjected to identical dose constraints, both for the target coverage and organ at risk (OAR) sparing, with a consistent order of priority. Beam geometry, lateral field margins, and lateral spot resolutions were made consistent across all systems. Few statistically significant differences were found between the target coverage and OAR sparing of each system, with all optimizers managing to produce plans within clinical tolerances (D2 < 107% of prescribed dose, D5 < 105%, D95 > 95%, D99 > 90%, and OAR maximum doses) despite strict constraints and overlapping structures.

A 3D in vitro cancer model as a platform for nanoparticle uptake and imaging investigations.

In order to maximize the potential of nanoparticles (NPs) in cancer imaging and therapy, their mechanisms of interaction with host tissue need to be fully understood. NP uptake is known to be dramatically influenced by the tumor microenvironment, and an imaging platform that could replicate in vivo cellular conditions would make big strides in NP uptake studies. Here, a novel NP uptake platform consisting of a tissue-engineered 3D in vitro cancer model (tumoroid), which mimics the microarchitecture of a solid cancer mass and stroma, is presented. As the tumoroid exhibits fundamental characteristics of solid cancer tissue and its cellular and biochemical parameters are controllable, it provides a real alternative to animal models. Furthermore, an X-ray fluorescence imaging system is developed to demonstrate 3D imaging of GNPs and to determine uptake efficiency within the tumoroid. This platform has implications for optimizing the targeted delivery of NPs to cells to benefit cancer diagnostics and therapy.

Determination of output factor for CyberKnife using scintillation dosimetry and deep learning.

Objective. Small-field dosimetry is an ongoing challenge in radiotherapy quality assurance (QA) especially for radiosurgery systems such as CyberKnifeTM. The objective of this work is to demonstrate the use of a plastic scintillator imaged with a commercial camera to measure the output factor of a CyberKnife system. The output factor describes the dose on the central axis as a function of collimator size, and is a fundamental part of CyberKnife QA and integral to the data used in the treatment planning system.Approach. A self-contained device consisting of a solid plastic scintillator and a camera was build in a portable Pelicase. Photographs were analysed using classical methods and with convolutional neural networks (CNN) to predict beam parameters which were then compared to measurements.Main results. Initial results using classical image processing to determine standard QA parameters such as percentage depth dose (PDD) were unsuccessful, with 34% of points failing to meet the Gamma criterion (which measures the distance between corresponding points and the relative difference in dose) of 2 mm/2%. However, when images were processed using a CNN trained on simulated data and a green scintillator sheet, 92% of PDD curves agreed with measurements with a microdiamond detector to within 2 mm/2% and 78% to 1%/1 mm. The mean difference between the output factors measured using this system and a microdiamond detector was 1.1%. Confidence in the results was enhanced by using the algorithm to predict the known collimator sizes from the photographs which it was able to do with an accuracy of less than 1 mm.Significance. With refinement, a full output factor curve could be measured in less than an hour, offering a new approach for rapid, convenient small-field dosimetry.

Development of Patient and Caregiver Conceptual Models Investigating the Health-Related Quality of Life Impacts of Malignant Pleural Mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and usually fatal malignancy frequently linked to occupational asbestos exposures and associated with poor prognosis and considerable humanistic burden. The study aimed to develop conceptual models of the health-related quality of life (HRQoL) impact on patients with and receiving treatment for MPM, and the burden on their caregivers. METHODS: This multi-country study (Australia and United Kingdom) adopted a qualitative methodology to conduct semi-structured, independent interviews with people with MPM (n = 26), current caregivers (n = 20), and caregivers of people who had recently died because of MPM (n = 4). Participants were recruited using a purposive sampling approach and interviews conducted via telephone between January 2021 and January 2022. Transcripts were analysed using thematic analysis and used to construct conceptual models. RESULTS: Patient analysis yielded four overarching themes: (1) debilitating burden of breathlessness and fatigue; (2) physical mesothelioma symptoms experienced by patients; (3) distress of MPM on the self and family; and (4) treatment is worth 'having a go' despite the potential impact on symptoms. Caregiver analysis yielded five core themes: (1) daily life limited by caregiving duties; (2) emotional well-being and the need for support; (3) the relational role shift to caregiver; (4) time spent providing care negatively impacts work and productivity; and (5) positive aspects and outcomes of caregiving. CONCLUSIONS: This study highlights the substantial daily and emotional HRQoL impact that MPM symptoms have on patients and caregivers. Both groups reduced work, productivity, and social and leisure activities. There was evidence of positive HRQoL impacts as a result of immunotherapy and radiotherapy, but less for chemotherapy. Caregiver impacts were intensified during the end-of-life period and persisted following patient death. Evident is a need for increased psychological support, information, and advice for caregivers, increased during the end-of-life period.

Medical imaging applied to heritage.

The use of imaging has transformed the study of cultural heritage artefacts in the same way that medical imaging has transformed medicine. X-ray-based techniques are common in both medical and heritage imaging. Optical imaging, including scientific photography and spectral imaging techniques, is also common in both domains. Some common medical imaging methods such as ultrasound and MRI have not yet found routine application in heritage, whereas other methods such as imaging with charged and uncharged particles and 3D surface imaging are more common in heritage. Here, we review the field of heritage imaging from the point of view of medical imaging and include some classic challenges of heritage imaging such as reading the text on burnt scrolls, identifying underdrawings in paintings, and CT scanning of mummies, an ancient calculating device and sealed documents. We show how hyperspectral imaging can offer insight into the drawing techniques of Leonardo da Vinci and explain how laparoscopy has identified the method of construction of a 500-year-old pop-up anatomical text book.

The Rat Genome Database: Genetic, Genomic, and Phenotypic Data Across Multiple Species.

The laboratory rat, Rattus norvegicus, is an important model of human health and disease, and experimental findings in the rat have relevance to human physiology and disease. The Rat Genome Database (RGD, https://rgd.mcw.edu) is a model organism database that provides access to a wide variety of curated rat data including disease associations, phenotypes, pathways, molecular functions, biological processes, cellular components, and chemical interactions for genes, quantitative trait loci, and strains. We present an overview of the database followed by specific examples that can be used to gain experience in employing RGD to explore the wealth of functional data available for the rat and other species. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Navigating the Rat Genome Database (RGD) home page Basic Protocol 2: Using the RGD search functions Basic Protocol 3: Searching for quantitative trait loci Basic Protocol 4: Using the RGD genome browser (JBrowse) to find phenotypic annotations Basic Protocol 5: Using OntoMate to find gene-disease data Basic Protocol 6: Using MOET to find gene-ontology enrichment Basic Protocol 7: Using OLGA to generate gene lists for analysis Basic Protocol 8: Using the GA tool to analyze ontology annotations for genes Basic Protocol 9: Using the RGD InterViewer tool to find protein interaction data Basic Protocol 10: Using the RGD Variant Visualizer tool to find genetic variant data Basic Protocol 11: Using the RGD Disease Portals to find disease, phenotype, and other information Basic Protocol 12: Using the RGD Phenotypes & Models Portal to find qualitative and quantitative phenotype data and other rat strain-related information Basic Protocol 13: Using the RGD Pathway Portal to find disease and phenotype data via molecular pathways.

2022 updates to the Rat Genome Database: a Findable, Accessible, Interoperable, and Reusable (FAIR) resource.

The Rat Genome Database (RGD, https://rgd.mcw.edu) has evolved from simply a resource for rat genetic markers, maps, and genes, by adding multiple genomic data types and extensive disease and phenotype annotations and developing tools to effectively mine, analyze, and visualize the available data, to empower investigators in their hypothesis-driven research. Leveraging its robust and flexible infrastructure, RGD has added data for human and eight other model organisms (mouse, 13-lined ground squirrel, chinchilla, naked mole-rat, dog, pig, African green monkey/vervet, and bonobo) besides rat to enhance its translational aspect. This article presents an overview of the database with the most recent additions to RGD's genome, variant, and quantitative phenotype data. We also briefly introduce Virtual Comparative Map (VCMap), an updated tool that explores synteny between species as an improvement to RGD's suite of tools, followed by a discussion regarding the refinements to the existing PhenoMiner tool that assists researchers in finding and comparing quantitative data across rat strains. Collectively, RGD focuses on providing a continuously improving, consistent, and high-quality data resource for researchers while advancing data reproducibility and fulfilling Findable, Accessible, Interoperable, and Reusable (FAIR) data principles.

Rare disease research resources at the Rat Genome Database.

Rare diseases individually affect relatively few people, but as a group they impact considerable numbers of people. The Rat Genome Database (https://rgd.mcw.edu) is a knowledgebase that offers resources for rare disease research. This includes disease definitions, genes, quantitative trail loci (QTLs), genetic variants, annotations to published literature, links to external resources, and more. One important resource is identifying relevant cell lines and rat strains that serve as models for disease research. Diseases, genes, and strains have report pages with consolidated data, and links to analysis tools. Utilizing these globally accessible resources for rare disease research, potentiating discovery of mechanisms and new treatments, can point researchers toward solutions to alleviate the suffering of those afflicted with these diseases.

Recommendations for research design and reporting in computer-assisted diagnosis to facilitate meta-analysis.

Computer-assisted diagnosis (CAD) describes a diverse, heterogeneous range of applications rather than a single entity. The aims and functions of CAD systems vary considerably and comparing studies and systems is challenging due to methodological and design differences. In addition, poor study quality and reporting can reduce the value of some publications. Meta-analyses of CAD are therefore difficult and may not provide reliable conclusions. Aiming to determine the major sources of heterogeneity and thereby what CAD researchers could change to allow this sort of assessment, this study reviews a sample of 147 papers concerning CAD used with imaging for cancer diagnosis. It discusses sources of variability, including the goal of the CAD system, learning methodology, study population, design, outcome measures, inclusion of radiologists, and study quality. Based upon this evidence, recommendations are made to help researchers optimize the quality and comparability of their trial design and reporting.

Farewell editorial.

Biomedical Optics Express Editor-in-Chief Christoph K. Hitzenberger and Deputy Editor Adam Gibson share some final remarks as they prepare to end their editorial terms on 31 December 2021.

Deep learning-based automated segmentation of resection cavities on postsurgical epilepsy MRI.

Accurate segmentation of surgical resection sites is critical for clinical assessments and neuroimaging research applications, including resection extent determination, predictive modeling of surgery outcome, and masking image processing near resection sites. In this study, an automated resection cavity segmentation algorithm is developed for analyzing postoperative MRI of epilepsy patients and deployed in an easy-to-use graphical user interface (GUI) that estimates remnant brain volumes, including postsurgical hippocampal remnant tissue. This retrospective study included postoperative T1-weighted MRI from 62 temporal lobe epilepsy (TLE) patients who underwent resective surgery. The resection site was manually segmented and reviewed by a neuroradiologist (JMS). A majority vote ensemble algorithm was used to segment surgical resections, using 3 U-Net convolutional neural networks trained on axial, coronal, and sagittal slices, respectively. The algorithm was trained using 5-fold cross validation, with data partitioned into training (N = 27) testing (N = 9), and validation (N = 9) sets, and evaluated on a separate held-out test set (N = 17). Algorithm performance was assessed using Dice-Sørensen coefficient (DSC), Hausdorff distance, and volume estimates. Additionally, we deploy a fully-automated, GUI-based pipeline that compares resection segmentations with preoperative imaging and reports estimates of resected brain structures. The cross-validation and held-out test median DSCs were 0.84 ± 0.08 and 0.74 ± 0.22 (median ± interquartile range) respectively, which approach inter-rater reliability between radiologists (0.84-0.86) as reported in the literature. Median 95 % Hausdorff distances were 3.6 mm and 4.0 mm respectively, indicating high segmentation boundary confidence. Automated and manual resection volume estimates were highly correlated for both cross-validation (r = 0.94, p < 0.0001) and held-out test subjects (r = 0.87, p < 0.0001). Automated and manual segmentations overlapped in all 62 subjects, indicating a low false negative rate. In control subjects (N = 40), the classifier segmented no voxels (N = 33), <50 voxels (N = 5), or a small volumes<0.5 cm3 (N = 2), indicating a low false positive rate that can be controlled via thresholding. There was strong agreement between postoperative hippocampal remnant volumes determined using automated and manual resection segmentations (r = 0.90, p < 0.0001, mean absolute error = 6.3 %), indicating that automated resection segmentations can permit quantification of postoperative brain volumes after epilepsy surgery. Applications include quantification of postoperative remnant brain volumes, correction of deformable registration, and localization of removed brain regions for network modeling.

Technology readiness levels for machine learning systems.

The development and deployment of machine learning systems can be executed easily with modern tools, but the process is typically rushed and means-to-an-end. Lack of diligence can lead to technical debt, scope creep and misaligned objectives, model misuse and failures, and expensive consequences. Engineering systems, on the other hand, follow well-defined processes and testing standards to streamline development for high-quality, reliable results. The extreme is spacecraft systems, with mission critical measures and robustness throughout the process. Drawing on experience in both spacecraft engineering and machine learning (research through product across domain areas), we've developed a proven systems engineering approach for machine learning and artificial intelligence: the Machine Learning Technology Readiness Levels framework defines a principled process to ensure robust, reliable, and responsible systems while being streamlined for machine learning workflows, including key distinctions from traditional software engineering, and a lingua franca for people across teams and organizations to work collaboratively on machine learning and artificial intelligence technologies. Here we describe the framework and elucidate with use-cases from physics research to computer vision apps to medical diagnostics.

MOET: a web-based gene set enrichment tool at the Rat Genome Database for multiontology and multispecies analyses.

Biological interpretation of a large amount of gene or protein data is complex. Ontology analysis tools are imperative in finding functional similarities through overrepresentation or enrichment of terms associated with the input gene or protein lists. However, most tools are limited by their ability to do ontology-specific and species-limited analyses. Furthermore, some enrichment tools are not updated frequently with recent information from databases, thus giving users inaccurate, outdated or uninformative data. Here, we present MOET or the Multi-Ontology Enrichment Tool (v.1 released in April 2019 and v.2 released in May 2021), an ontology analysis tool leveraging data that the Rat Genome Database (RGD) integrated from in-house expert curation and external databases including the National Center for Biotechnology Information (NCBI), Mouse Genome Informatics (MGI), The Kyoto Encyclopedia of Genes and Genomes (KEGG), The Gene Ontology Resource, UniProt-GOA, and others. Given a gene or protein list, MOET analysis identifies significantly overrepresented ontology terms using a hypergeometric test and provides nominal and Bonferroni corrected P-values and odds ratios for the overrepresented terms. The results are shown as a downloadable list of terms with and without Bonferroni correction, and a graph of the P-values and number of annotated genes for each term in the list. MOET can be accessed freely from https://rgd.mcw.edu/rgdweb/enrichment/start.html.