Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

Populationsgenetische Parameter der Gewebemerkmale der deutschen Bevölkerung und ihre Anwendung bei der Suche nach nicht-verwandten Blutstammzellspendern

Zahlreiche Anwendungen, vor allem in der klinischen Medizin und den forensischen Wissenschaften, setzen eine genaue Kenntnis der HLA-Allel- und Haplotypfrequenzen voraus. Unter der Annahme eines Hardy-Weinberg-Gleichgewichtes können diese mittels eines Gene-Counting genannten Expectation-Maximisation-Algorithmus aus Stichproben geschätzt werden, in denen nur die Phänotypen der Probanden bekannt sind. Diese Arbeit vergleicht systematisch dieses mit anderen vorgeschlagenen Verfahren, belegt schlüssig seine Überlegenheit und liefert Beiträge zu seiner Verbesserung. Eine effiziente Implementierung, die Rohdaten unterschiedlicher Auflösung mit mehrdeutigen Merkmalsbezeichnern verarbeiten kann, wird mit Algorithmen zur Bereichsschätzung wie dem Bootstrapping kombiniert. Die Analyse der HLA-Genorte A, B und DR einer über 25.000 Probanden umfassenden Stichprobe der deutschen Bevölkerung wird mit den derzeit meist verwendeten Tabellenwerken aus den International Histocompatibility Workshops verglichen, die Diskrepanzen werden untersucht und die Bedeutung von Bereichsschätzungen für die Anwendungen wird herausgearbeitet. Es wird nachgewiesen, dass mit Hilfe der umfangreichen Frequenztabellen zuverlässige Prognosen für die individuelle Suche nach nicht-verwandten Blutstammzellspendern erstellt und damit Such- und Behandlungsstrategien auf einer soliden Basis festgelegt werden können. Weiter zeigt sich, dass die Versorgung der Patienten mit identischen Spendern einer Sättigungskinetik in Abhängigkeit von der Registergröße unterliegt und durch Erhöhung der Spenderzahlen nur mit mehr als exponentiell wachsenden Kosten pro versorgtem Patient weiter zu verbessern wäre. Damit liefert diese Arbeit wesentliche Grundlagen für Entscheidungen von erheblicher ökonomischer Bedeutung

The impact of HLA-C matching on donor identification rates in a European Caucasian population

The degree of HLA concordance with the patient has long been known to be the major donor-related prediction factor for the success of hematopoietic stem cell transplantations and, with the progress of HLA typing technology, selection criteria became more stringent with regard to the recommended loci and resolution. A late refinement was HLA-C matching which gained broader acceptance only after the turn of the millennium.The enormous HLA polymorphism has always necessitated registries with a large number of donors in order to be able to provide well-matched donors to a substantial fraction of patients. Using a biostatistical approach, we investigated the impact of adding HLA-C at low or high resolution as a supplementary matching criterion on some key parameters in donor provision for a European Caucasian population. Starting point is donor selection based on allele level matching for HLA A, B, -DRB1 and, optionally, HLA-DQB1.Without typing for HLA-C, 68 % of the donors selected based on matching for HLA A, B, DRB1 and -DQB1 at high resolution will also match for HLA-C, 29 % will have a single and only 3 % will have two HLA-C alleles different from the patient. In order to provide the same fraction of patients with a fully-matched donor, a registry would have to be about twice the size if HLA-C is considered in addition to the four other loci, with the exact factor increasing with the registry’s size. If the provision of donors with up to a single allele mismatch is considered, this factor doubles due to the strong linkage between HLA-B and -C. These figures only change slightly when HLA-DQB1 is completely ignored or HLA-C matching is only considered at low resolution.Our results contribute to quantifying the medical and economic impact of the progress in donor selection algorithms

Human leukocyte antigen-E mismatch is associated with better hematopoietic stem cell transplantation outcome in acute leukemia patients

The immunomodulatory role of human leukocyte antigen (HLA)-E in hematopoietic stem cell transplantation (HSCT) has not been extensively investigated. To this end, we genotyped 509 10/10 HLA unrelated transplant pairs for HLA-E, in order to study the effect of HLA-E as a natural killer (NK)-alloreactivity mediator on HSCT outcome in an acute leukemia (AL) setting. Overall survival (OS), disease free survival (DFS), relapse incidence (RI) and non-relapse mortality (NRM) were set as endpoints. Analysis of our data revealed a significant correlation between HLA-E mismatch and improved HSCT outcome, as shown by both univariate (53% vs. 38%, P=0.002, 5-year OS) and multivariate (hazard ratio (HR)=0.63, confidence interval (CI) 95%=0.48–0.83, P=0.001) analyses. Further subgroup analysis demonstrated that the positive effect of HLA-E mismatch was significant and pronounced in advanced disease patients (n=120) (5-year OS: 50% vs. 18%, P=0.005; HR=0.40, CI 95%=0.22–0.72, P=0.002; results from univariate and multivariate analyses, respectively). The study herein is the first to report an association between HLA-E incompatibility and improved post–transplant prognosis in AL patients who have undergone matched unrelated HSCT. Combined NK and T cell HLA-E-mediated mechanisms may account for the better outcomes observed. Notwithstanding the necessity for in vitro and confirmational studies, our findings highlight the clinical relevance of HLA-E matching and strongly support prospective HLA-E screening upon donor selection for matched AL unrelated HSCTs

Human leukocyte antigen-E mismatch is associated with better hematopoietic stem cell transplantation outcome in acute leukemia patients

The immunomodulatory role of human leukocyte antigen (HLA)-E in hematopoietic stem cell transplantation (HSCT) has not been extensively investigated. To this end, we genotyped 509 10/10 HLA unrelated transplant pairs for HLA-E, in order to study the effect of HLA-E as a natural killer (NK)-alloreactivity mediator on HSCT outcome in an acute leukemia (AL) setting. Overall survival (OS), disease free survival (DFS), relapse incidence (RI) and non-relapse mortality (NRM) were set as endpoints. Analysis of our data revealed a significant correlation between HLA-E mismatch and improved HSCT outcome, as shown by both univariate (53% vs. 38%, P=0.002, 5-year OS) and multivariate (hazard ratio (HR)=0.63, confidence interval (CI) 95%=0.48–0.83, P=0.001) analyses. Further subgroup analysis demonstrated that the positive effect of HLA-E mismatch was significant and pronounced in advanced disease patients (n=120) (5-year OS: 50% vs. 18%, P=0.005; HR=0.40, CI 95%=0.22–0.72, P=0.002; results from univariate and multivariate analyses, respectively). The study herein is the first to report an association between HLA-E incompatibility and improved post–transplant prognosis in AL patients who have undergone matched unrelated HSCT. Combined NK and T cell HLA-E-mediated mechanisms may account for the better outcomes observed. Notwithstanding the necessity for in vitro and confirmational studies, our findings highlight the clinical relevance of HLA-E matching and strongly support prospective HLA-E screening upon donor selection for matched AL unrelated HSCTs