Comparing the EQ-5D-5L and stroke impact scale 2.0 in stroke patients: an analysis of measurement properties.

BACKGROUND: Stroke has evolved to become a chronic disease and a major public health challenge. To adequately capture the full disease burden of stroke patients, the assessment of health-related quality of life (HRQoL) and thus the performance of respective measures is increasingly relevant. The aim of this analysis was to compare the measurement properties of two self-report instruments, the EQ-5D-5L and the Stroke Impact Scale 2.0. METHODS: The data used for the analysis was derived from a quasi-experimental case management study for mildly to moderately affected incident stroke and transient ischemic attack (TIA) patients aged ≥ 18 in Germany. Data was collected patient-individually at 3, 6 and 12 months after initial stroke. The EQ-5D-5L and SIS 2.0 were compared in terms of feasibility, ceiling and floor effects, responsiveness and known-groups validity (Kruskal-Wallis H and Wilcoxon rank-sum test). RESULTS: A response for all three follow-ups is available for n = 855 patients. The feasibility of the EQ-5D-5L is determined as good (completion rate: 96.4-96.6%, ≥ one item missing: 3.2 - 3.3%), whereas the SIS 2.0 is moderately feasible (overall completion rate: 44.9-46.1%, ≥ one item missing in domains: 4.7 - 28.7%). The SIS 2.0 shows substantial ceiling effects in comparable domains (physical function: 10.4 - 13%, others: 3.5-31.3%) which are mainly larger than ceiling effects in the EQ-5D-5L index (17.1-21.5%). In terms of responsiveness, the EQ-5D-5L shows small to moderate change while the SIS 2.0 presents with moderate to large responsiveness. The EQ-5D-5L index, mobility, usual activities and Visual Analogue Scale show known-groups validity (p < 0.05). Content-related domains of the SIS 2.0 show known-groups validity as well (p < 0.05). However, it is compromised in the emotion domain in both measures (p > 0.05). CONCLUSIONS: The EQ-5D-5L seems to be slightly more suitable for this cohort. Nonetheless, the results of both measures indicate limited suitability for TIA patients. Large-scale studies concerning responsiveness and known-groups validity are encouraged. TRIAL REGISTRATION: The study was registered in the German Clinical Trials Register, retrospective registration on 21.09.2022. REGISTRATION ID: DRKS00030297.

[Digital Health Applications: A Qualitative Study of Approaches to Improve Access to Statutory Health Insurance]. / Lösungsansätze für den Zugang digitaler Gesundheitsanwendungen zur Gesetzlichen Krankenversicherung: eine qualitative Studie.

BACKGROUND: There is a lack of integration of appropriate digital health applications (DiGA) into the first healthcare market in Germany. In order to enable a valid and reliable use of previously examined digital health products, their implementation into services of the statutory health insurance (SHI) is necessary. The aim of this study was the development of strategies to modify and improve access of DiGA to SHI reimbursement. The recently introduced Digitale-Versorgung-Gesetz (DVG) is an initial step in this direction. METHODS: Using a qualitative approach, focus group interviews were conducted with key stakeholders of existing access paths. Previously elaborated problem-solving approaches were discussed. The approaches ranged between adapting existing structures and implementing an original digital pathway. Subsequently, a comparison of the project results and legislative provision of the DVG was carried out. RESULTS: The proposed approaches were discussed heterogeneously and varied depending on the position of the participants. The implementation of an Advisory Council had a greater consensus than the introduction of a digital-specific pathway. Also individual measures like administrative support for generating the necessary evidence was considered as positive and beneficial. However, a deviation from the current evidence standards should be avoided. Furthermore, the legitimacy and time expenditure for the digital-specific pathway was called into doubt. CONCLUSIONS: In principle, a better focus on existing structures on digital health applications can be endorsed. For a short-term use of DiGA potentials, adaptions of existing structures are preferable. The DVG legislation, although conforming to the project results only to some degree, can be considered as a first step. An amendment, in particular from the viewpoint of diagnostic or therapeutic DiGA, appears to be necessary.

["Alternative study designs" for the evaluation of digital health applications - a real alternative?] / "Alternative Studiendesigns" zur Bewertung digitaler Gesundheitsanwendungen ­ eine echte Alternative?

INTRODUCTION: After the Digital Healthcare Act (Digitale-Versorgung-Gesetz, DVG) reformed digital health applications' (Digitale Gesundheitsanwendungen, DiGAs) access to German Statutory Health Insurance (SHI) reimbursement, the discussion concerning necessary evidence requirements has intensified. In the past, different "alternative study designs" have been proposed to replace randomized controlled trials (RCTs) in the DiGA efficacy and benefit assessments. The present paper examines the suitability of these alternative designs for informing SHI reimbursement decisions. METHODS: The four alternative study designs primarily discussed in the context of DiGA - "Continuous Evaluation of Evolving Behavioral Intervention Technologies" (CEEBIT), "Multiphase Optimization Strategy" (MOST), "Sequential Multiple Assignment Randomized Trial" (SMART) and "Micro-Randomized Trial" (MRT) - are characterized and compared on the basis of relevant primary and secondary sources. Subsequently, their suitability for effectiveness and benefit evaluation in the context of SHI reimbursement decisions is discussed. RESULTS: None of the study designs examined aims primarily at conclusively demonstrating efficacy and benefit. Three of the four designs (MOST, SMART, MRT) focus on the development and optimization of interventions. In order to reduce resource requirements, the approaches presented sometimes deviate considerably from the methodological approach in traditional RCTs. This is especially true for their applied statistical error tolerance and their underlying randomization logic. Three of the four concepts (MOST, SMART, MRT) therefore still require RCTs after the development phase in order to demonstrate the effectiveness and benefit of the optimized intervention. DISCUSSION: The methodological differences of the alternative study designs compared to classical RCTs are accompanied by serious potentials for bias and uncertainties with regard to the identified intervention effects. These may be acceptable in the context of intervention development, but do not appear to be appropriate for use in collective SHI reimbursement decisions. CONCLUSION: The alternative study designs presented cannot be regarded as a suitable RCT alternative for efficacy and benefit assessments. A pragmatic study design, which continues to meet high methodological standards, and better utilization of real-world data could, in the future, contribute to a compromise between the justified claims to sufficient certainty of results on the one hand and appropriate procedural effort on the other.

Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility.

The c-di-GMP-binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re-annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ-mediated downregulation of motility is fine-tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c-di-GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c-di-GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c-di-GMP-dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella-driven swimming and swarming motility.

glnA Truncation in Salmonella enterica Results in a Small Colony Variant Phenotype, Attenuated Host Cell Entry, and Reduced Expression of Flagellin and SPI-1-Associated Effector Genes.

Many pathogenic bacteria use sophisticated survival strategies to overcome harsh environmental conditions. One strategy is the formation of slow-growing subpopulations termed small colony variants (SCVs). Here we characterize an SCV that spontaneously emerged from an axenic Salmonella enterica serovar Typhimurium water culture. We found that the SCV harbored a frameshift mutation in the glutamine synthetase gene glnA, leading to an ∼90% truncation of the corresponding protein. Glutamine synthetase, a central enzyme in nitrogen assimilation, converts glutamate and ammonia to glutamine. Glutamine is an important nitrogen donor that is required for the synthesis of cellular compounds. The internal glutamine pool serves as an indicator of nitrogen availability in Salmonella In our study, the SCV and a constructed glnA knockout mutant showed reduced growth rates, compared to the wild type. Moreover, the SCV and the glnA mutant displayed attenuated entry into host cells and severely reduced levels of exoproteins, including flagellin and several Salmonella pathogenicity island 1 (SPI-1)-dependent secreted virulence factors. We found that these proteins were also depleted in cell lysates, indicating their diminished synthesis. Accordingly, the SCV and the glnA mutant had severely decreased expression of flagellin genes, several SPI-1 effector genes, and a class 2 motility gene (flgB). However, the expression of a class 1 motility gene (flhD) was not affected. Supplementation with glutamine or genetic reversion of the glnA truncation restored growth, cell entry, gene expression, and protein abundance. In summary, our data show that glnA is essential for the growth of S. enterica and controls important motility- and virulence-related traits in response to glutamine availability.IMPORTANCESalmonella enterica serovar Typhimurium is a significant pathogen causing foodborne infections. Here we describe an S Typhimurium small colony variant (SCV) that spontaneously emerged from a long-term starvation experiment in water. It is important to study SCVs because (i) SCVs may arise spontaneously upon exposure to stresses, including environmental and host defense stresses, (ii) SCVs are slow growing and difficult to eradicate, and (iii) only a few descriptions of S. enterica SCVs are available. We clarify the genetic basis of the SCV described here as a frameshift mutation in the glutamine synthetase gene glnA, leading to glutamine auxotrophy. In Salmonella, internal glutamine limitation serves as a sign of external nitrogen deficiency and is thought to regulate cell growth. In addition to exhibiting impaired growth, the SCV showed reduced host cell entry and reduced expression of SPI-1 virulence and flagellin genes.

Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs.

UNLABELLED: High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein. IMPORTANCE: In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.

Constitutive production of c-di-GMP is associated with mutations in a variant of Pseudomonas aeruginosa with altered membrane composition.

Most bacteria can form multicellular communities called biofilms on biotic and abiotic surfaces. This multicellular response to surface contact correlates with an increased resistance to various adverse environmental conditions, including those encountered during infections of the human host and exposure to antimicrobial compounds. Biofilm formation occurs when freely swimming (planktonic) cells encounter a surface, which stimulates the chemosensory-like, surface-sensing system Wsp and leads to generation of the intracellular second messenger 3',5'-cyclic-di-guanosine monophosphate (c-di-GMP). We identified adaptive mutations in a clinical small colony variant (SCV) of Pseudomonas aeruginosa and correlated their presence with self-aggregating growth behavior and an enhanced capacity to form biofilms. We present evidence that a point mutation in the 5' untranslated region of the accBC gene cluster, which encodes components of an enzyme responsible for fatty acid biosynthesis, was responsible for a stabilized mRNA structure that resulted in reduced translational efficiency and an increase in the proportion of short-chain fatty acids in the plasma membrane. We propose a model in which these changes in P. aeruginosa serve as a signal for the Wsp system to constitutively produce increased amounts of c-di-GMP and thus play a role in the regulation of adhesion-stimulated bacterial responses.

Elucidation of sigma factor-associated networks in Pseudomonas aeruginosa reveals a modular architecture with limited and function-specific crosstalk.

Sigma factors are essential global regulators of transcription initiation in bacteria which confer promoter recognition specificity to the RNA polymerase core enzyme. They provide effective mechanisms for simultaneously regulating expression of large numbers of genes in response to challenging conditions, and their presence has been linked to bacterial virulence and pathogenicity. In this study, we constructed nine his-tagged sigma factor expressing and/or deletion mutant strains in the opportunistic pathogen Pseudomonas aeruginosa. To uncover the direct and indirect sigma factor regulons, we performed mRNA profiling, as well as chromatin immunoprecipitation coupled to high-throughput sequencing. We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS). Our integrated approach revealed a highly modular network architecture which is composed of insulated functional sigma factor modules. Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes. Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.

Identification of the alternative sigma factor SigX regulon and its implications for Pseudomonas aeruginosa pathogenicity.

Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma (σ) factors. The largest group of alternative σ factors is that of the extracytoplasmic function (ECF) σ factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative σ factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF σ factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative σ factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa.

A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa.

In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2'-aminohexylcarbamoyl-c-di-GMP (2'-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2'-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.