App-based assessment of patient-reported outcomes in the Molecular Tumor Board in the Center for Personalized Medicine-(TRACE).

Background: Biomarker-based therapies are increasingly used in cancer patients outside clinical trials. Systematic assessment of patient-reported outcomes (PRO) is warranted to take patients' perspectives during biomarker-based therapies into consideration. We assessed the feasibility of an electronic PRO assessment via a smartphone application. Methods: An interdisciplinary expert panel developed a smartphone application based on symptom burden and health-related quality of life (HRQoL) metrics reported in a retrospective analysis of 292 neuro-oncological patients. The app included validated assessments of health-related quality of life (HRQoL), the burden of symptoms, and psychological stress. Feasibility and usability were tested in a pilot study. Semi-structured interviews with patients and health care professionals (HCP) were conducted, transcribed, and analyzed according to Mayring´s qualitative content analysis. Furthermore, we assessed compliance and descriptive data of ePROs. Results: A total of 14 patients have been enrolled, (9 female, 5 male). A total of 4 HCPs, 9 patients, and 1 caregiver were interviewed regarding usability/feasibility. The main advantages were the possibility to complete questionnaires at home and comfortable implementation in daily life. Compliance was high, for example, 82% of the weekly distributed NCCN distress thermometer questionnaires were answered on time, however, with interindividual variability. We observed a median distress score of 5 (range 0-10, 197 results, n = 12, weekly assessed) and a median Global health score of 58.3 according to the EORTC QLQ-C30 instrument (range 16.7-100, 77 results, n = 12, monthly assessed). Conclusions: This pilot study proved the feasibility and acceptance of the app. We will therefore expand its application during biomarker-guided therapies to enable systematic PRO assessments.

Usability and User Experience of an mHealth App for Therapy Support of Patients With Breast Cancer: Mixed Methods Study Using Eye Tracking.

BACKGROUND: Early identification of quality of life (QoL) loss and side effects is a key challenge in breast cancer therapy. Digital tools can be helpful components of therapeutic support. Enable, a smartphone app, was used in a multicenter, prospective randomized controlled trial in 3 breast cancer centers. The app simultaneously serves as a therapy companion (eg, by displaying appointments), a tool for documenting QoL (eg, by enabling data collection for QoL questionnaires), and documentation of patient-reported side effects. The need for digital tools is continually rising. However, evidence of the effects of long-term use of mobile health (mHealth) apps in aftercare for patients with breast cancer is limited. Therefore, evaluating the usability and understanding the user experience of this mHealth app could potentially contribute valuable insights in this field. OBJECTIVE: A usability study was conducted to explore how patients with breast cancer receiving neoadjuvant, adjuvant, or palliative outpatient treatment rated their engagement with the app , the user experience, and the benefits of using the app. METHODS: A mixed methods approach was chosen to combine subjective and objective measures, including an eye-tracking procedure, a standardized usability questionnaire (mHealth App Usability Questionnaire), and semistructured interviews. Participants were surveyed twice during the study period. Interviews were transcribed verbatim and analyzed using thematic analysis. Analysis of the eye-tracking data was carried out using the tracker-integrated software. Descriptive analysis was conducted for the quantitative data. RESULTS: The mHealth App Usability Questionnaire results (n=105) indicated good overall usability for 2 different time points (4 wk: mean 89.15, SD 9.65; 20 wk: mean 85.57, SD 12.88). The qualitative analysis of the eye-tracking recordings (n=10) and interviews (n=16) showed that users found the Enable app easy to use. The design of the app, information about therapies and side effects, and usefulness of the app as a therapy companion were rated positively. Additionally, participants contributed requests for additional app features and suggestions for improving the content and usability of the app. Relevant themes included optimization of the appointment feature, updating the app's content regularly, and self-administration. In contrast to the app's current passive method of operation, participants expressed a desire for more active engagement through messaging, alarms, or emails. CONCLUSIONS: The results of this study demonstrate the good usability of the Enable app as well as the potential for further development. We concluded from patients' feedback and requests that mHealth apps could benefit from giving patients a more active role (eg, being able to actively document side effects as they occur). Additionally, regular updates of app content could further contribute to encouraging continued use of mHealth apps. Our findings may also assist other researchers in tailoring their mHealth apps to the actual needs of patients undergoing breast cancer therapy.

German Version of the mHealth App Usability Questionnaire in a Cohort of Patients With Cancer: Translation and Validation Study.

BACKGROUND: Good usability is important for the adoption and continued use of mobile health (mHealth) apps. In particular, high usability can support intuitive use by patients, which improves compliance and increases the app's effectiveness. However, many usability studies do not use adequate tools to measure perceived usability. The mHealth App Usability Questionnaire (MAUQ) was developed specifically for end users in a medical context. MAUQ is a relatively new but increasingly used questionnaire to evaluate mHealth apps, but it is not yet available in German. OBJECTIVE: This study aims to translate MAUQ into German and determine its internal consistency, reliability, and construct validity. METHODS: This validation study was conducted as part of a usability evaluation project for an mHealth app used as a therapy support tool during breast cancer chemotherapy. MAUQ was translated into German through a rigorous forward-backward translation process, ensuring semantic and conceptual equivalence. Patient responses to MAUQ and System Usability Scale (SUS) were analyzed for validation. Descriptive analysis was performed for the MAUQ subscales and SUS standard scores. Significance tests and correlation coefficients assessed the relationship between the SUS and MAUQ results, confirming construct validity. Internal consistency was assessed for item reliability and consistency in measuring the target construct. Free-text questions assessed translation comprehensibility, with responses analyzed descriptively and qualitatively using content analysis. RESULTS: In this study, 133 participants responded to the questionnaire, and the validation analysis showed substantially positive correlations between the overall MAUQ score and its subscales: ease of use (r=0.56), interface and satisfaction (r=0.75), and usefulness (r=0.83). These findings support the construct validity of MAUQ and emphasize the importance of these subscales in assessing the usability of the Enable app. The correlation coefficients ranging from 0.39 to 0.68 for the items further validate the questionnaire by aligning with the overall score and capturing the intended concept. The high internal consistency reliability of MAUQ (Cronbach α=.81) and its subscales further enhances the instrument's robustness in accurately evaluating the usability of mHealth apps. CONCLUSIONS: We successfully validated the German translation of the MAUQ for stand-alone apps using a standardized approach in a cohort of patients with breast cancer. In our validation study, MAUQ exhibited strong internal consistency reliability (Cronbach α=.81) across its subscales, indicating reliable and consistent measurement. Furthermore, a significant positive correlation (P<.001) was found between the subscales and the overall score, supporting their consistent measurement of the intended construct. Therefore, MAUQ can be considered a reliable instrument for assessing the usability of mHealth apps among German-speaking adults. The availability of the German version of MAUQ will help other researchers in conducting usability studies of mHealth apps in German-speaking cohorts and allow for international comparability of their results.

Proposing a Novel Hybrid Short-Term Exchange Program in Biomedical and Health Informatics Education.

International student exchange is a valuable opportunity for Biomedical and Health Informatics students to gain new perspectives and experiences. In the past, such exchanges have been made possible through international partnerships between universities. Unfortunately, numerous obstacles such as housing, financial concerns, and environmental implications related to travel, have made it difficult to continue international exchange. Experiences with hybrid and online education during covid-19 paved the way for a new approach that allows for short international exchange with a hybrid online-offline supervision model. This will be initiated with an exploration project between two international universities , each related to their respective institute's research focus.

Exploring healthcare provider and patient perspectives on current outpatient care of venous leg ulcers and potential interventions to improve their treatment: a mixed methods study in the ulcus cruris care project.

BACKGROUND: The project "Ulcus Cruris Care" aims to improve primary care for patients with venous leg ulcer (VLU) in General Practitioner (GP) practices using a complex intervention comprised of educational components, standardized treatment recommendations, computer-assisted documentation, and case management by non-physician medical assistants (MAs). Prior to implementing and testing the intervention components in general practices, in-depth exploration of current outpatient treatment of VLU patients and relevant implementation determinants was pursued. METHODS: A mixed-methods study explored views of GPs, MAs, and patients regarding current VLU outpatient care and the planned intervention components to identify potential implementation determinants. Data were collected through semi-structured guide-based telephone interviews (n = 29) and a survey questionnaire (n = 28). Interviews were transcribed verbatim. Analysis was inductive initially and finalized in a deductive-inductive approach based on domains of the Theoretical Domains Framework to support structuring of relevant implementation determinants. Survey data were analyzed descriptively. RESULTS: Current VLU outpatient care was described as frequently tailored to individual wounds and gradient. In general, workload was shared by GPs (diagnostics, counselling) and MAs (wound care). All care providers were aware of compression therapy, yet not all of them considered it essential for VLU care. Standardized operating procedures and educational components including e-learning were considered supportive. Stronger involvement of non-physician assistants was seen as opportunity to optimize VLU care. Concerns were identified regarding integration of software-supported case management into daily practice routines and regarding potential limitations in decision-making autonomy when using standard operating procedures. CONCLUSIONS: Findings in this study emphasize a need for educational interventions addressing VLU care providers as well as patients, particularly with regards to compression therapy. The conception of the planned intervention appears to be adequate and a structured guideline-based case management might be a promising approach for optimization of VLU treatment.

[Utilization and reporting of qualitative research methods in health services research in Germany: A scoping review on original research publications (2010-2019)]. / Anwendung und Darstellung qualitativer Methoden in der Versorgungsforschung in Deutschland: ein Scoping Review zu Primärstudien (2010­2019).

BACKGROUND: Qualitative research methods offer a unique perspective on health care services. However, little is known about the actual application of qualitative methods in health services research. Therefore, the aim of this study was to gain an overview of volume and variety of the use of qualitative research methods in health services research in Germany. METHODS: By means of a scoping review, a systematic literature search of the database PubMed was conducted in September 2020. We included (1) qualitative studies in (2) a health services setting (3) in Germany, (4) published in either German or English as (5) original research in a journal (6) between 2010 and 2019. After removing duplicates, tandem teams of researchers first performed a title and abstract screening, followed by a full text screening. Data was extracted by using a category grid considering research focus, study design and reporting. RESULTS: In total, 759 articles were included in the title and abstract screening. After applying the exclusion criteria, 97 articles were included in the data extraction. The studies investigating mainly subjective perspectives of different stakeholders, especially physicians and patients, covered 13 areas of health care. Interviews were the dominant form of data collection (n=64). Data analysis was mainly conducted using content analysis (n=65). CONCLUSION: A clear absolute increase in publications since the mid-2010s can be observed. At the same time, there has been a strong tendency towards certain methods being used for data collection and analysis. Compared to reporting standards and guidelines (e.g., COREQ), incomplete reporting of research methods has been noted. The results show that both an extension of the range of methods and the quality of reporting need to be discussed.

Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor.

Single-molecule measurements provide detailed mechanistic insights into molecular processes, for example in genome regulation where DNA access is controlled by nucleosomes and the chromatin machinery. However, real-time single-molecule observations of nuclear factors acting on defined chromatin substrates are challenging to perform quantitatively and reproducibly. Here we present XSCAN (multiplexed single-molecule detection of chromatin association), a method to parallelize single-molecule experiments by simultaneous imaging of a nucleosome library, where each nucleosome type carries an identifiable DNA sequence within its nucleosomal DNA. Parallel experiments are subsequently spatially decoded, via the detection of specific binding of dye-labeled DNA probes. We use this method to reveal how the Cas9 nuclease overcomes the nucleosome barrier when invading chromatinized DNA as a function of PAM position.

Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes.

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

Data-collection strategy for challenging native SAD phasing.

Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.

Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.

The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools.

In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target.

The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a ternary complex with a guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes.

In vitro enzymology of Cas9.

Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms. Here, we describe protocols for the heterologous expression and purification of recombinant Cas9 protein and for in vitro transcription of guide RNAs. We describe in vitro reconstitution of the Cas9-guide RNA ribonucleoprotein complex and its use in endonuclease activity assays. The methods outlined here enable mechanistic characterization of the RNA-guided DNA cleavage activity of Cas9 and may assist in further development of the enzyme for genetic engineering applications.

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.

Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

A semisynthetic fusicoccane stabilizes a protein-protein interaction and enhances the expression of K+ channels at the cell surface.

Small-molecule stabilization of protein-protein interactions is an emerging field in chemical biology. We show how fusicoccanes, originally identified as fungal toxins acting on plants, promote the interaction of 14-3-3 proteins with the human potassium channel TASK-3 and present a semisynthetic fusicoccane derivative (FC-THF) that targets the 14-3-3 recognition motif (mode 3) in TASK-3. In the presence of FC-THF, the binding of 14-3-3 proteins to TASK-3 was increased 19-fold and protein crystallography provided the atomic details of the effects of FC-THF on this interaction. We also tested the functional effects of FC-THF on TASK channels heterologously expressed in Xenopus oocytes. Incubation with 10 µM FC-THF was found to promote the transport of TASK channels to the cell membrane, leading to a significantly higher density of channels at the surface membrane and increased potassium current.