Health-related quality of life (HRQoL) in German early benefit assessment: The importance of disease-specific instruments.

INTRODUCTION: Health-Related Quality of Life (HRQoL) data is frequently requested in early benefit assessment in Germany. To test the hypothesis that the importance of HRQoL in general and the significance of disease-specific instruments in particular has increased since the introduction of AMNOG in Germany, we analysed all early benefit assessments between 2011 and 2022. METHODS: All 793 early benefit assessments completed between 01/01/2011 and 03/11/2022 were systematically analysed. The HRQoL instruments featured in the dossier submissions were extracted for all assessments and categorized into generic and specific instruments. All G-BA resolutions were likewise assessed for consideration and acceptance of generic and specific HRQoL instruments. In addition, it was examined whether there was an association between HRQoL data and the extent of additional benefit. RESULTS: Since 2014 HRQoL data have continuously been submitted in 70% to 80% of assessments per year with the exception of 2022 (63%). Generally, disease-specific instruments are favoured, regarding submissions by industry but especially with higher acceptance by the G-BA in the resolution. Subgroup analyses revealed oncology as a major contributor to the submission and acceptance rates of disease-specific instruments. Disease-specific instruments were submitted in 81% of all oncology assessments and accepted in 53% of assessments. Overall, assessments with accepted HRQoL data tend to reach a higher overall benefit. Procedures with accepted HRQoL were most likely to receive a considerable benefit (31%), while for procedures in which HRQoL data were not accepted, a benefit was most often (65%) not proven. DISCUSSION: Industry has followed the request for submission of specific HRQoL instruments early on. Higher submission rates of specific instruments over time which at the same time meet the methodological requirements indicate that industry has learned from early assessments. A potential reason for the high submission- and acceptance rates of specific HRQoL instruments in oncology might be the particularly high relevance of HRQoL in this indication. Possible effects of changes in legislature on the future development of submission and acceptance of HRQoL data need to be monitored. CONCLUSION: In Germany, HRQoL has gained a relevant position in early benefit assessment over time. Overall specific instruments are favoured, regarding submissions by industry but especially through consideration by the G-BA in the resolution. Furthermore, HRQoL data can be supportive for benefit assessments, in particular to show that advantages in morbidity and/or mortality are reflected in HRQoL and not at the expense of HRQoL.

A drug repurposing approach for individualized cancer therapy based on transcriptome sequencing and virtual drug screening.

RNA-sequencing has been proposed as a valuable technique to develop individualized therapy concepts for cancer patients based on their tumor-specific mutational profiles. Here, we aimed to identify drugs and inhibitors in an individualized therapy-based drug repurposing approach focusing on missense mutations for 35 biopsies of cancer patients. The missense mutations belonged to 9 categories (ABC transporter, apoptosis, angiogenesis, cell cycle, DNA damage, kinase, protease, transcription factor, tumor suppressor). The highest percentages of missense mutations were observed in transcription factor genes. The mutational profiles of all 35 tumors were subjected to hierarchical heatmap clustering. All 7 leukemia biopsies clustered together and were separated from solid tumors. Based on these individual mutation profiles, two strategies for the identification of possible drug candidates were applied: Firstly, virtual screening of FDA-approved drugs based on the protein structures carrying particular missense mutations. Secondly, we mined the Drug Gene Interaction (DGI) database (https://www.dgidb.org/) to identify approved or experimental inhibitors for missense mutated proteins in our dataset of 35 tumors. In conclusion, our approach based on virtual drug screening of FDA-approved drugs and DGI-based inhibitor selection may provide new, individual treatment options for patients with otherwise refractory tumors that do not respond anymore to standard chemotherapy.

The MECP2-TRD domain interacts with the DNMT3A-ADD domain at the H3-tail binding site.

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.

CD79a promotes CNS-infiltration and leukemia engraftment in pediatric B-cell precursor acute lymphoblastic leukemia.

Central nervous system (CNS) involvement remains a challenge in the diagnosis and treatment of acute lymphoblastic leukemia (ALL). In this study, we identify CD79a (also known as Igα), a signaling component of the preB cell receptor (preBCR), to be associated with CNS-infiltration and -relapse in B-cell precursor (BCP)-ALL patients. Furthermore, we show that downregulation of CD79a hampers the engraftment of leukemia cells in different murine xenograft models, particularly in the CNS.

Sex-specific regulation of collagen I and III expression by 17ß-Estradiol in cardiac fibroblasts: role of estrogen receptors.

Aims: Sex differences in cardiac fibrosis point to the regulatory role of 17ß-Estradiol (E2) in cardiac fibroblasts (CF). We, therefore, asked whether male and female CF in rodent and human models are differentially susceptible to E2, and whether this is related to sex-specific activation of estrogen receptor alpha (ERα) and beta (ERß). Methods and results: In female rat CF (rCF), 24 h E2-treatment (10-8 M) led to a significant down-regulation of collagen I and III expression, whereas both collagens were up-regulated in male rCF. E2-induced sex-specific collagen regulation was also detected in human CF, indicating that this regulation is conserved across species. Using specific ERα- and ERß-agonists (10-7 M) for 24 h, we identified ERα as repressive and ERß as inducing factor in female and male rCF, respectively. In addition, E2-induced ERα phosphorylation at Ser118 only in female rCF, whereas Ser105 phosphorylation of ERß was exclusively found in male rCF. Further, in female rCF we found both ER bound to the collagen I and III promoters using chromatin immunoprecipitation assays. In contrast, in male rCF only ERß bound to both promoters. In engineered connective tissues (ECT) from rCF, collagen I and III mRNA were down-regulated in female ECT and up-regulated in male ECT by E2. This was accompanied by an impaired condensation of female ECT, whereas male ECT showed an increased condensation and stiffness upon E2-treatment, analysed by rheological measurements. Finally, we confirmed the E2-effect on both collagens in an in vivo mouse model with ovariectomy for E2 depletion, E2 substitution, and pressure overload by transverse aortic constriction. Conclusion: The mechanism underlying the sex-specific regulation of collagen I and III in the heart appears to involve E2-mediated differential ERα and ERß signaling in CFs.

Quantification of Stable Isotope Traces Close to Natural Enrichment in Human Plasma Metabolites Using Gas Chromatography-Mass Spectrometry.

Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger diversity of metabolites is often lacking, mainly due to the marginal percentage of fully isotopically enriched plant material in the administered food product, and hence, an even weaker 13C enrichment in downstream plasma metabolites. Therefore, we developed an analytical workflow to determine weak 13C enrichments of diverse plasma metabolites with conventional gas chromatography-mass spectrometry (GC-MS). The limit of quantification was increased by optimizing (1) the metabolite extraction from plasma, (2) the GC-MS measurement, and (3) most importantly, the computational data processing. We applied our workflow to study the catabolic dynamics of 13C-enriched wheat bread in three human subjects. For that purpose, we collected time-resolved human plasma samples at 16 timepoints after the consumption of 13C-labeled bread and quantified 13C enrichment of 12 metabolites (glucose, lactate, alanine, glycine, serine, citrate, glutamate, glutamine, valine, isoleucine, tyrosine, and threonine). Based on isotopomer specific analysis, we were able to distinguish catabolic profiles of starch and protein hydrolysis. More generally, our study highlights that conventional GC-MS equipment is sufficient to detect isotope traces below 1% if an appropriate data processing is integrated.

From powders to bulk metallic glass composites.

One way to adjust the properties of materials is by changing its microstructure. This concept is not easily applicable on bulk metallic glasses (BMGs), because they do not consist of grains or different phases and so their microstructure is very homogeneous. One obvious way to integrate inhomogeneities is to produce bulk metallic glass composites (BMGCs). Here we show how to generate BMGCs via high-pressure torsion (HPT) starting from powders (amorphous Zr-MG and crystalline Cu). Using this approach, the composition can be varied and by changing the applied shear strains, the refinement of the microstructure is adjustable. This process permits to produce amorphous/crystalline composites where the scale of the phases can be varied from the micro- to the nanometer regime. Even mixing of the two phases and the generation of new metallic glasses can be achieved. The refinement of microstructure increases the hardness and a hardness higher than the initial BMG can be obtained.

Analysis of mitochondrial metabolism in situ: Combining stable isotope labeling with selective permeabilization.

To date, it is well-established that mitochondrial dysfunction does not only play a vital role in cancer but also in other pathological conditions such as neurodegenerative diseases and inflammation. An important tool for the analysis of cellular metabolism is the application of stable isotope labeled substrates, which allow for the tracing of atoms throughout metabolic networks. While such analyses yield very detailed information about intracellular fluxes, the determination of compartment specific fluxes is far more challenging. Most approaches for the deconvolution of compartmented metabolism use computational models whereas experimental methods are rare. Here, we developed an experimental setup based on selective permeabilization of the cytosolic membrane that allows for the administration of stable isotope labeled substrates directly to mitochondria. We demonstrate how this approach can be used to infer metabolic changes in mitochondria induced by either chemical or genetic perturbations and give an outlook on its potential applications.

Pro-inflammatory Macrophages Sustain Pyruvate Oxidation through Pyruvate Dehydrogenase for the Synthesis of Itaconate and to Enable Cytokine Expression.

Upon stimulation with Th1 cytokines or bacterial lipopolysaccharides, resting macrophages shift their phenotype toward a pro-inflammatory state as part of the innate immune response. LPS-activated macrophages undergo profound metabolic changes to adapt to these new physiological requirements. One key step to mediate this metabolic adaptation is the stabilization of HIF1α, which leads to increased glycolysis and lactate release, as well as decreased oxygen consumption. HIF1 abundance can result in the induction of the gene encoding pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH) via phosphorylation. Therefore, it has been speculated that pyruvate oxidation through PDH is decreased in pro-inflammatory macrophages. However, to answer this open question, an in-depth analysis of this metabolic branching point was so far lacking. In this work, we applied stable isotope-assisted metabolomics techniques and demonstrate that pyruvate oxidation is maintained in mature pro-inflammatory macrophages. Glucose-derived pyruvate is oxidized via PDH to generate citrate in the mitochondria. Citrate is used for the synthesis of the antimicrobial metabolite itaconate and for lipogenesis. An increased demand for these metabolites decreases citrate oxidation through the tricarboxylic acid cycle, whereas increased glutamine uptake serves to replenish the TCA cycle. Furthermore, we found that the PDH flux is maintained by unchanged PDK1 abundance, despite the presence of HIF1. By pharmacological intervention, we demonstrate that the PDH flux is an important node for M(LPS) macrophage activation. Therefore, PDH represents a metabolic intervention point that might become a research target for translational medicine to treat chronic inflammatory diseases.

3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies.

Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between experimental in vitro and in vivo data. Using 3D HepaRG organotypic cultures we tested four drugs (aflatoxin B1, amiodarone, valproic acid and chlorpromazine) for toxic effects and compared the results with 2D HepaRG and HepG2 cultures. We show that 3D HepaRG cultures are more sensitive than the other tested cultures to aflatoxin B1 which is only toxic upon metabolic activation in the liver. We observed that CYP3A4 activity is higher in the 3D HepaRG cultures compared to the 2D HepaRG cultures. Furthermore, we investigated repeated dose toxicity of chlorpromazine and assessed its effects on glucose and lactate metabolism. Sub-toxic concentrations of chlorpromazine induced significant metabolic changes in both 2D and 3D HepaRG cultures upon acute and repeated dose (3 doses) exposure. In summary, our data support the hypothesis that 3D cell culture models better mimic the in vivo tissue and improve cellular functionality. The 3D HepaRG organotypic cultures represent a high throughput system for drug toxicity screening. This system is therefore a promising tool in preclinical testing of human relevance which can allow reducing and/or replacing animal testing for drug adverse effects.

Effects of daylight-saving time changes on stock market returns and stock market volatility: rebuttal.

In a 2011 reply to our 2010 comment in this journal, Berument and Dogen maintained their challenge to the existence of the negative daylight-saving effect in stock returns reported by Kamstra, Kramer, and Levi in 2000. Unfortunately, in their reply, Berument and Dogen ignored all of the points raised in the comment, failing even to cite the Kamstra, et al. comment. Berument and Dogen continued to use inappropriate estimation techniques, over-parameterized models, and low-power tests and perhaps most surprisingly even failed to replicate results they themselves reported in their previous paper, written by Berument, Dogen, and Onar in 2010. The findings reported by Berument and Dogen, as well as by Berument, Dogen, and Onar, are neither well-supported nor well-reasoned. We maintain our original objections to their analysis, highlight new serious empirical and theoretical problems, and emphasize that there remains statistically significant evidence of an economically large negative daylight-saving effect in U.S. stock returns. The issues raised in this rebuttal extend beyond the daylight-saving effect itself, touching on methodological points that arise more generally when deciding how to model financial returns data.

Effects of daylight-saving time changes on stock market volatility: a comment.

In a recent article in this journal, Berument, Dogan, and Onar (2010) challenged the existence of the previously documented daylight-saving effect. Kamstra, Kramer, and Levi's original finding (2000) was that average stock market returns on Mondays following time changes are economically and statistically significantly lower than typical Monday returns. Kamstra, et al. hypothesized that the effect may arise due to heightened anxiety or risk aversion on the part of market participants after they experience a 1-hr. disruption in their sleep habits, in accordance with prior findings in the psychology literature linking sleep desynchronosis with anxiety. Berument, et al. replicated the original findings using ordinary least squares estimation, but when they modeled the mean of returns using a method prone to producing biased estimates, they obtained puzzling results. The analysis here, based on standard, unbiased modeling techniques, shows that the daylight-saving effect remains intact in the U.S.

Amino acid 49 polymorphisms of the human beta1-adrenergic receptor affect agonist-promoted trafficking.

The signaling impact of a human beta1-adrenergic receptor (beta1 AR) polymorphism at residue 49 of the aminoterminus (Ser-to-Gly substitution) was studied by recombinantly expressing each receptor. The two receptors displayed identical agonist and antagonist binding affinities. Furthermore, basal and agonist-stimulated adenylyl cyclase activities were the same for these receptors as assessed in both cell types. Although short-term agonist exposure resulted in similar degrees of receptor internalization, long-term agonist-promoted downregulation was greater for Gly49 compared with Ser49. The Gly49 receptor underwent a 24 +/- 3% loss of receptor density after exposure to isoproterenol for 18 h, whereas Ser49 underwent no such loss. In studies in which receptor synthesis was inhibited, agonist-promoted downregulation for Gly49 was 55 +/- 3% compared with 36 +/- 5% for Ser49. In the absence of agonist, degradative turnover of each receptor was the same. Immunoblotting revealed that some of the Ser49 receptor exists as a highly N-glycosylated form (approximately 105-kD molecular mass), which is not present with Gly49. Thus the phenotype of the Gly49 polymorphic receptor is that of wild-type coupling with enhanced agonist-promoted downregulation, which is associated with altered N-glycosylation. Based on this cellular phenotype, the beta1 AR Gly49 polymorphism may impart a beneficial effect in chronic heart failure.