Flexible Designs in klinischen Prüfungen mit binärer und ordinaler Zielvariable

In dieser Arbeit werden klinische Studien mit flexiblem Design untersucht. Hierbei handelt es sich um in mehreren voneinander unabhängigen Stufenabschnitten durchgeführten Verfahren, die basierend auf den bis zu einem bestimmten Zeitpunkt vorliegenden Information Änderungen am Studiendesign erlauben. Zur Kombination der Ergebnisse der einzelnen Sequenzen werden die gewichtete inverse Normal- sowie die verallgemeinerte inverse x^2-Methode betrachtet (vgl. Hartung (2006)). Wird die Anzahl der Sequenzen fest vorgegeben, handelt es sich um adaptiv gruppensequentielle Verfahren. Diese ermöglichen neben der Anpassung des Studiendesigns eine Überprüfung des Testproblems in jeder Zwischenauswertung. Die durch das mehrfache Testen resultierende Erhöhung des Fehlers I. Art erfordert eine Adjustierung der kritischen Schranken. Hierfür werden die Strategien von Pocock (1977), O'Brien und Fleming (1979), Wang und Tsiatis (1987) sowie Haybittle (1971) und Peto et al. (1976) vorgestellt. Die beiden letzteren werden für die Kombination der Ergebnisse mit der inversen x^2-Methode übertragen. Die zugehörigen kritische Werte, welche mittels Simulationen erhalten wurden, werden angegeben. Kommt das Self-Designing klinischer Studien zur Anwendung, ist die Anzahl der Sequenzen zu Beginn der Untersuchung offen. Zudem wird die Gewichtung der einzelnen Stufen basierend auf den vorliegenden Beobachtungen bestimmt. Bei Verwendung der inversen x^2-{Methode kann in jeder Sequenz die Überprüfung der Nullhypothese erfolgen, bei der inversen Normalmethode hingegen erst nach Vergabe des globalen Gewichts. Neben der Überprüfung von Testproblemen ist die Abschätzung des Ausma es eines Wirkungsunterschieds von Interesse. Hierfür werden für die verschiedenen Designs geeignete Methoden für die Punkt- und Intervallschätzung vorgestellt. Für den Fall, dass in den Zwischenauswertungen kein Testen der Nullhypothese sondern nur eine Anpassung der Fallzahl erfolgt, wird ein unverzerrter Schätzer hergeleitet. Die in der Literatur vorgestellten flexiblen Designs basieren häufig auf der Annahme normalverteilter Zielvariable mit bekannter Varianz. Für die Untersuchung dieser Designs bei Vorliegen diskreter Beobachtungen werden klinische Studien mit binärer und ordinaler Zielgröße betrachtet. Für beide Fälle werden Möglichkeiten zur Ermittlung des notwendigen Umfangs basierend auf den vorliegenden Daten vorgestellt. Die verschiedenen flexiblen Designs werden für diese Art von Beobachtungen ausgiebig in Simulationsstudien untersucht. Insbesondere von Interesse ist hierbei, ob Prüfgrößen, welche bereits im nicht-sequentiellen Fall das Testniveau nur asymptotisch einhalten, in adaptiven Designs Anwendung finden können

Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

Kato O, Youn J-W, Stansen KC, Matsui D, Oikawa T, Wendisch VF. Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate. BMC Microbiology. 2010;10(1): 321.Background: Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032. Results: Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer. Conclusions: Cg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer

Anti-acid therapy in idiopathic pulmonary fibrosis: insights from the INPULSIS (R) trials

Background: The benefits and risks of anti-acid medication in patients with idiopathic pulmonary fibrosis (IPF) remain a topic of debate. We investigated whether use of anti-acid medication at baseline was associated with differences in the natural course of disease or influenced the treatment effect of nintedanib in patients with IPF. Methods: Post-hoc analyses of outcomes in patients receiving versus not receiving anti-acid medication (proton pump or histamine-2 receptor inhibitor) at baseline using pooled data from the two Phase III randomized placebo-controlled INPULSIS (R) trials of nintedanib in patients with IPF. Results: At baseline, 406 patients were receiving anti-acid medication (244 nintedanib;162 placebo) and 655 were not (394 nintedanib;261 placebo). In an analysis of the natural course of IPF by anti-acid medication use at baseline, the adjusted annual rate of decline in FVC was -252.9 mL/year in placebo-treated patients who were receiving anti-acid medication at baseline and -205.4 mL/year in placebo-treated patients who were not (difference of -47.5 mL/year [95% CI: -105.1, 10.1];p = 0.1057). In an analysis of the potential influence of anti-acid medication use on the treatment effect of nintedanib, the adjusted annual rates of decline in FVC were -124.4 mL/year in the nintedanib group and 252.9 mL/year in the placebo group (difference of 128.6 mL/year [95% CI: 74.9, 182.2]) in patients who were receiving anti-acid medication at baseline and -107.0 mL/year in the nintedanib group and -205.3 mL/year in the placebo group (difference of 98.3 mL/year [95% CI: 54.1, 142.5]) in patients who were not (treatment-by-time-by-subgroup interaction p = 0.3869). The proportions of patients who had >= 1 investigator-reported acute exacerbation were 11.7% and 5.0% in placebo-treated patients, and 4.9% and 4.8% of nintedanib-treated patients, among patients who were and were not receiving anti-acid medication at baseline, respectively. Conclusions: In post-hoc analyses of data from the INPULSIS (R) trials, anti-acid medication use at baseline was not associated with a more favorable course of disease, and did not impact the treatment effect of nintedanib, in patients with IPF

Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in l-lactate and sugar utilization

LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-Å resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn2+ binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed

Nintedanib with add-on pirfenidone in idiopathic pulmonary fibrosis: Results of the INJOURNEY trial

Measurements and Main Results: On-treatment gastrointestinal adverse events were reported in 37 of 53 patients (69.8%) treated with nintedanib with add-on pirfenidone and 27 of 51 patients (52.9%) treated with nintedanib alone. Predose plasma trough concentrations of nintedanib were similar when it was administered alone or with add-on pirfenidone. Mean (SE) changes from baseline in FVC at Week 12 were 213.3 (17.4) ml and 240.9 (31.4) ml in patients treated with nintedanib with add-on pirfenidone (n = 48) and nintedanib alone (n = 44), respectively. Conclusions: Nintedanib with add-on pirfenidone had a manageable safety and tolerability profile in patients with IPF, in line with the adverse event profiles of each drug. These data support further research into combination regimens in the treatment of IPF. Rationale: Nintedanib and pirfenidone slow the progression of idiopathic pulmonary fibrosis (IPF), but the disease continues to progress. More data are needed on the safety and efficacy of combination therapy with nintedanib and add-on pirfenidone. Objectives: To investigate safety, tolerability, and pharmacokinetic and exploratory efficacy endpoints in patients treated with nintedanib and add-on pirfenidone versus nintedanib alone. Methods: Patients with IPF and FVC greater than or equal to 50% predicted at screening who completed a 4- to 5-week run-in with nintedanib 150 mg twice daily without dose reduction or treatment interruption were randomized to receive nintedanib 150 mg twice daily with add-on pirfenidone (titrated to 801 mg three times daily) or nintedanib 150 mg twice daily alone in an open-label manner for 12 weeks. The primary endpoint was the percentage of patients with on-treatment gastrointestinal adverse events from baseline to Week 12. Analyses were descriptive and exploratory

Analysis of body mass index, weight loss and progression of idiopathic pulmonary fibrosis

Background: Nintedanib is an approved therapy for idiopathic pulmonary fibrosis (IPF). Some patients treated with nintedanib experience weight loss. Exploratory data suggest that low body mass index or weight loss are associated with worse outcomes in patients with IPF. We investigated whether BMI at baseline or weight loss over 52 weeks was associated with FVC decline, or influenced the effect of nintedanib, in patients with IPF. Methods: Using pooled data from the two INPULSIS trials, we analysed the rate of decline in FVC (mL/yr) over 52 weeks in patients treated with nintedanib and placebo in subgroups by baseline BMI ( 5%) using random coefficient regression. Results: In the placebo group, the mean rate of FVC decline over 52 weeks was numerically greater in patients with lower baseline BMI (− 283.3 [SE 22.4], − 207.9 [20.9] and − 104.5 [21.4] in patients with BMI 5% than ≤5% weight loss over 52 weeks (− 312.7 [SE 32.2] versus − 199.5 [SE 14.4] mL/year). Nintedanib reduced the rate of FVC decline versus placebo in both subgroups by weight loss, with a greater treatment effect in patients with > 5% weight loss (interaction p = 0.0008). The adverse event profile of nintedanib was similar across subgroups. Conclusions: In patients with IPF, lower BMI and weight loss may be associated with faster decline in FVC. Nintedanib reduces the rate of FVC decline both in patients who lose weight on treatment and those who do not. Trial registration: ClinicalTrials.gov; Nos. NCT01335464 and NCT01335477; URL: www.clinicaltrials.gov

A Brief, Daily, Online Mental Health and Well-being Intervention for University Staff During the COVID-19 Pandemic: Program Description and Outcomes Using a Mixed Methods Design

BACKGROUND: The unprecedented changes and isolation measures to contain COVID-19 have had multiple psychological and social impacts, with implications for professional and personal functioning. Evidence-informed interventions that can be rapidly implemented under pandemic conditions to support mental health during such times are urgently needed. OBJECTIVE: The aim of this study was to determine the acceptability and preliminary outcomes of a daily online mental health promotion program for tertiary education staff during the COVID-19 pandemic. METHODS: The “Victoria University (VU) Elevenses” program was delivered as an uncontrolled intervention at Victoria University (VU) in the western metropolitan region of Melbourne, Australia. In April 2020, an email invitation was sent to all academic and professional staff inviting them to: (1) participate in the program and (2) opt-in to the research component. The “VU Elevenses” program provided 10-15–minute microinterventions comprising lifestyle and well-being strategies to promote mental health via an online meeting platform at 11 AM each weekday. A mixed methods approach was used to evaluate the program, combining structured questionnaires with semistructured interviews to investigate the experiences of staff who participated in the program. RESULTS: Between 16 and 90 participants provided weekly program feedback. A total of 106 university staff opted into the longitudinal research component and 10 staff participated in the interviews. Participants reported high levels of satisfaction with sessions and perceived benefits for mental health. Approximately one quarter of participants reported moderate to severe symptoms of depression, anxiety, and stress at baseline, with significant reductions in these symptoms in the first 7 weeks of the program, corresponding with easing in mandatory isolation (“lockdown”) restrictions. Symptoms of depression, anxiety, and stress all increased when lockdown measures were reintroduced, but not to the same levels as found during the initial lockdown period. Overall changes in depression and anxiety from baseline to the end of the program were explained by changes in COVID-19–related distress, whereas changes in self-compassion explained changes in stress. CONCLUSIONS: We show that it is feasible and acceptable to develop and deliver a program of brief interventions in a timely manner, using a simple and accessible online platform. Although participation in the program was initially associated with reduced symptoms of depression, anxiety, and stress, participants’ mental health worsened with the reintroduction of a “lockdown” period. However, as symptoms of depression, anxiety, and stress did not return to levels observed at the start of the VU Elevenses program, participation in the uncontrolled intervention may have offered a protective benefit against the impact of the second significant lockdown period

NAD-Independent L-Lactate Dehydrogenase Is Required for L-Lactate Utilization in Pseudomonas stutzeri SDM

BACKGROUND: Various Pseudomonas strains can use L-lactate as their sole carbon source for growth. However, the L-lactate-utilizing enzymes in Pseudomonas have never been identified and further studied. METHODOLOGY/PRINCIPAL FINDINGS: An NAD-independent L-lactate dehydrogenase (L-iLDH) was purified from the membrane fraction of Pseudomonas stutzeri SDM. The enzyme catalyzes the oxidation of L-lactate to pyruvate by using FMN as cofactor. After cloning its encoding gene (lldD), L-iLDH was successfully expressed, purified from a recombinant Escherichia coli strain, and characterized. An lldD mutant of P. stutzeri SDM was constructed by gene knockout technology. This mutant was unable to grow on L-lactate, but retained the ability to grow on pyruvate. CONCLUSIONS/SIGNIFICANCE: It is proposed that L-iLDH plays an indispensable function in Pseudomonas L-lactate utilization by catalyzing the conversion of L-lactate into pyruvate

Charakterisierung der Ausscheidung von L-Glutamat bei Corynebacterium glutamicum\textit{Corynebacterium glutamicum}

Mit Hilfe von $\textit{Corynebacterium glutamicum}$ werden jährlich 1,5 Millionen Tonnen L-Glutamat produziert. Dennoch konnte bislang weder ein L-Glutamat-Exporter identifiziert werden, noch ist genau verstanden, warum die Exkretion von L-Glutamat immer erst nach Induktion durch verschiedenste Auslöser erfolgt. Aus diesem Grund wurden in dieser Arbeit fünf Methoden der Glutamatausscheidung etabliert und die genomweiten Genexpressionsmuster unter diesen Induktionsbedingungen quantifiziert. Durch die Auslöser der Glutamatausscheidung Ethambutol und Tween40 wurden 74 bzw. 137 Gene differentiell exprimiert, durch Biotin-Mangel und Penicillin aber nur 19 bzw. 18 Gene. Überraschenderweise zeigte sich kein Expressionsmuster, welches unabhängig von der Art der Induktion stets bei der Ausscheidung von L-Glutamat auftritt. Ebenso überraschend war, dass trotz stark verändertem Zentralstoffwechsel während der Ausscheidung von L-Glutamat keine veränderte Expression von Genen z.B. der Enzyme des Pentosephosphatwegs oder der α-Ketoglutarat-Dehydrogenase auftritt, was eher auf eine Regulation der Stoffflüsse auf Protein-, bzw. Aktivitätsebene schließen lässt. Es gab jedoch spezifische und teilweise sogar extreme Expressionsveränderungen. So führten Ethambutol und Tween40 zu einem bis zu 31-fach erhöhten mRNA-Spiegel von $\textit{mepA}$, das vermutlich für eine Metalloendopeptidase kodiert, und zu einem zweifach reduzierten mRNA-Spiegel von $\textit{accBC}$, welches für die α-Untereinheit der Acetyl-CoA-Carboxylase kodiert. Dies ist ein deutlicher Hinweis auf die Beeinflussung der Zellwandstruktur unter diesen Induktionsbedingungen. Auch das für einen mechanosensitiven Kanal der Osmoregulation kodierende $\textit{yggB}$ wurde durch Ethambutol und Tween40 bis zu vierfach verstärkt exprimiert. Durch zusätzliche Untersuchungen konnte gezeigt werden, dass unabhängig von der Art der Induktion hyperosmotischer Schock die Exkretion drastisch reduziert, während hypoosmotischer Stress die Ausscheidung steigert. Aufgrund dessen ist anzunehmen, dass der noch zu identifizierende L-Glutamat-Exporter durch die Membranspannung in seiner Aktivität beeinflusst wird, worüber auch ein Zusammenhang mit der Zellwand als bekannter Wirkort der verschiedenen Auslöser der Glutamatausscheidung hergestellt werden könnte. Die genomweiten Expressionsanalysen ergaben, dass 13 Gene, die für putative Transporter und Membranproteine kodieren, nach Induktion der Glutamatausscheidung verstärkt exprimiert werden. Durch Inaktivierung konnte für die meisten dieser Gene gezeigt werden, dass sie weder die Ausscheidung von L-Glutamat noch das Wachstum beeinflussen. Diese Gene sind somit als nicht-essentiell zu betrachten. Die Inaktivierung des Gens $\textit{NCgl0944}$ führte zu einer deutlichen Reduktion der L-Glutamat-Exkretion nach Induktion durch Biotin- Mangel, Ethambutol und Penicillin. Eine weitere Mutante, in der das Gen $\textit{NCgl2566}$ deletiert war, exkretierte dagegen nach Induktion durch Tween40 kaum noch L-Glutamat. Aus den Ergebnissen kann geschlossen werden, dass diese zwei Transporter direkt oder indirekt in die Glutamatausscheidung involviert sind. Während der Temperatur-induzierten Glutamatausscheidung wurden die zwei benachbart liegenden Gene $\textit{NCgl2816}$ und $\textit{NCgl2817}$ verstärkt exprimiert. Es konnte gezeigt werden, dass diese Gene durch L-Lactat induziert werden und als Operon vorliegen. Enzymtests zeigten, dass $\textit{NCgl2817}$ für eine L-Lactat-Dehydrogenase kodiert. Das Enzym oxidiert L-Lactat Quinon-abhängig zu Pyruvat und weist einen K$_{m}$-Wert von 0,5 mM für das einzige Substrat L-Lactat auf. Die Analyse einer Inaktivierungsmutante ergab, dass dieses $\textit{lldD}$ genannte Gen die Verwertung von L-Lactat essentiell ist, wogegen das kotranskribierte Gen sehr wahrscheinlich die L-Lactat-Aufnahme katalysiert

Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum

Schneider J, Peters-Wendisch P, Stansen KC, et al. Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum. BMC Microbiology. 2012;12(6): 6.Background: The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. Results: By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. Conclusions: The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation