Converging or Crossing Curves: Untie the Gordian Knot or Cut it? Appropriate Statistics for Non-Proportional Hazards in Decitabine DACO-016 Study (AML)

Introduction: Among patients with acute myeloid leukemia (AML), the DACO-016 randomized study showed reduction in mortality for decitabine [Dacogen® (DAC), Eisai Inc., Woodcliff Lake, NJ, USA] compared with treatment choice (TC): at primary analysis the hazard ratio (HR) was 0.85 (95% confidence interval 0.69-1.04; stratified log-rank P=0.108). With two interim analyses, two-sided alpha was adjusted to 0.0462. With 1-year additional follow-up the HR reached 0.82 (nominal P=0.0373). These data resulted in approval of DAC in the European Union, though not in the United States. Though pre-specified, the log-rank test could be considered not optimal to assess the observed survival difference because of the non-proportional hazard nature of the survival curves. Methods: We applied the Wilcoxon test as a sensitivity analysis. Patients were randomized to DAC (N=242) or TC (N=243). One-hundred and eight (44.4%) patients in the TC arm and 91 (37.6%) patients in the DAC arm selectively crossed over to subsequent disease modifying therapies at progression, which might impact the survival beyond the median with resultant converging curves (and disproportional hazards). Results: The stratified Wilcoxon test showed a significant improvement in median (CI 95%) overall survival with DAC [7.7 (6.2; 9.2) months] versus TC [5.0 (4.3; 6.3) months; P=0.0458]. Conclusion: Wilcoxon test indicated significant increase in survival for DAC versus TC compared to log-rank test. Funding: Janssen-Cilag GmbH

NMR Structure of Lipoprotein YxeF from Bacillus subtilis Reveals a Calycin Fold and Distant Homology with the Lipocalin Blc from Escherichia coli

The soluble monomeric domain of lipoprotein YxeF from the Gram positive bacterium B. subtilis was selected by the Northeast Structural Genomics Consortium (NESG) as a target of a biomedical theme project focusing on the structure determination of the soluble domains of bacterial lipoproteins. The solution NMR structure of YxeF reveals a calycin fold and distant homology with the lipocalin Blc from the Gram-negative bacterium E.coli. In particular, the characteristic β-barrel, which is open to the solvent at one end, is extremely well conserved in YxeF with respect to Blc. The identification of YxeF as the first lipocalin homologue occurring in a Gram-positive bacterium suggests that lipocalins emerged before the evolutionary divergence of Gram positive and Gram negative bacteria. Since YxeF is devoid of the α-helix that packs in all lipocalins with known structure against the β-barrel to form a second hydrophobic core, we propose to introduce a new lipocalin sub-family named ‘slim lipocalins’, with YxeF and the other members of Pfam family PF11631 to which YxeF belongs constituting the first representatives. The results presented here exemplify the impact of structural genomics to enhance our understanding of biology and to generate new biological hypotheses

Characterization of amyloid-ß and other proteins related to Alzheimer's disease, their role in neurodegeneration and biomarker discovery

Studies were performed to identify factors explaining the difference in the neurodegeneration seen in Alzheimer’s disease (AD) and in transgenic mice. A high level of neuronal loss is typically associated with AD, whereas in transgenic mice, this feature is only weakly displayed. Furthermore, the studies aimed at determining proteins occurring at altered levels in AD brains which could be involved in pathogenic mechanisms underlying the disease. The results of these investigations could help to identify Aβ variants and other proteins affecting the degree of neurodegeneration and would hopefully contribute to the development of biomarkers and new drug targets in AD. Using laser dissection microscopy in combination with MALDI-TOF MS and urea-based Western blotting, the Aβ composition in morphologically differentiated plaque types from three different sources could be investigated: human brains from AD patients, pathologically aging (PA) individuals – i.e. individuals who form amyloid plaques, but suffer of no cognitive impairment - and from double-transgenic PS2APP mice. Diffuse plaques are found in human AD and PA, as well as in PS2APP transgenic mice and were previously suggested to be the initially occurring plaque type. We detected almost exclusively Aβ 42 in diffuse plaques. This Aβ variant was shown to be prone to aggregation. Furthermore, no differences in the Aβ species composition between diffuse plaques from AD and PA individuals were detected. Cored plaques, the main plaque type in AD, contain also predominantly Aβ 42, whereas compact plaques, which sporadically occur in human but are found more frequently in PS2APP mice, consist of Aβ 40. This has implications on the putative evolutionary path of plaque formation: Due to the Aβ composition of the respective plaque types, a sequential maturation of diffuse plaques over compact to cored plaques can be excluded. Our data support the concept that diffuse plaques are the initially occurring plaque type and develop independently to compact or cored plaques. The Aβ composition of vascular amyloid was also investigated. Here, we detected high amounts of Aβ 40, which is consistent with previous findings using C-terminal specific antibodies. Aβ in human plaques and to a considerably lesser extent in plaques from PS2APP mice is N-terminally truncated. The investigation of different Braak stages revealed an increasing amount of N-terminally truncated Aβ variants accompanying disease progression, indicative for a successive modification by exoproteases upon deposition. Consistently low levels of Nterminally truncated Aβ forms were detected in diffuse plaques, which further support the view of diffuse plaques being at the beginning of the plaque maturation process. The major Nterminally truncated Aβ variant detected by MALDI-TOF MS in human cored plaques was pyroglutamate 3-40 / 42. To corroborate these findings, Western blotting and immunohistochemistry were applied and the existence of pyroglutamate in all types of plaques in human AD and PS2APP transgenic mice was shown. The rate of aggregation of an Aβ variant is likely to be important for initial deposition of amyloid into plaques. To test the aggregation and toxicity properties of pyroglutamate 3-42, in vitro experiments were performed on PC12 and hippocampal cell lines comparing synthetic pyroglutamate 3-42 with Aβ 1-42. These experiments showed a considerably lower rate of aggregation for synthetic pyroglutamate 3-42 when compared to Aβ 1-42. Our experiments suggest that pyroglutamate 3-40 / 42 alone is probably not a critical seed for plaque formation. The cytotoxicity of Aβ peptides is correlated with the aggregation state. Synthetic pyroglutamate 3-42 in vitro showed a significantly reduced toxicity when compared to Aβ 1-42. Cross-linking of Aβ fibrils in plaques was investigated with solubility experiments. It was found that Aβ in human plaques is cross-linked to a higher extent than Aβ in plaques from PS2APP transgenic mice. A higher degree of cross-linking in human Aβ was also indicated by our MALDI-TOF MS analysis. The high level of cross-linking may result in a hampered clearance of Aβ from the brain in AD patients. Taken together, enhanced levels of posttranslational modifications in human plaques, i.e. cross-linking and N-terminal truncation of Aβ peptides, may lead to their reduced clearance from the brain. The methodology used in our experiments allowed to discriminate between oxidized and reduced Aβ species. Diffuse plaques in PA were shown to contain a large proportion of oxidized Aβ, which could indicate a protective response in these brains and which would explain the lack of clinical symptoms in these individuals. To determine, whether the amyloid load in plaques may contribute to the high level of neuronal loss observed in AD as opposed to transgenic PS2APP mice, 15N-labeled Aβ 1-42 was used and the quantitative amyloid load in plaques from human AD and PS2APP mice was compared by MALDI-TOF MS. Roughly comparable amounts of Aβ in both species were found. Therefore, we conclude that a dose-effect of Aβ alone can not account for the higher level of neurodegeneration observed in AD patients than in PS2APP mice. Using LC/MS/MS the identification of additional proteins involved or affected in the pathogenesis of AD was aimed at. A comparison of the proteome in AD brain with control tissue was based on the analysis of over 2000 proteins. Proteins generally accepted to be related to AD like Aβ, apolipoprotein E, heat shock protein 90, glial fibrillary acidic protein (GFAP) and tau were detected at increased levels in AD. Numerous proteins previously tentatively implicated with the disease were identified at aberrant levels in AD. We found proteins indicative for an enhanced susceptibility to oxidative stress (e.g. decrease of peroxiredoxin 5), an impaired glucose metabolism (decrease of enzymes involved in citrate cycle), cytoskeletal derangement (e.g. decrease in alpha-internexin), synaptic dysfunction (e.g. decrease in presynaptic density protein-95, syntaxin-1A and synaptotagmin-1), an activation of the ubiquitin proteasome system (increase in ubiquitin), deficits in the neuritic network (decrease of various cell adhesion molecules) and membrane trafficking (e.g. decrease in AP-180), as well as altered levels of enzymes involved in the hyperphosphorylation of tau (e.g. decrease in phosphatase-2A). Most notably, we detected 18 new proteins previously not associated with the disease, which occurred at altered levels in AD. These are the synaptic proteins synaptogyrin-1 and -3, myelin associated glycoprotein, myelin oligodendrocyte glycoprotein, contactin-1, contactin-2, neurexin IV and claudin-11 involved in cell adhesion, the chaperone protein Hsp 75, plectin 1, ankyrin, α-adducin and microtubule-associated protein RP/EB family member 3 as structural cytoskeletal proteins, peroxiredoxin 5 and microsomal glutathione S-transferase 3 for the protection against oxidative stress and programmed cell death protein 8, matrin-3 and transgelin- 3, whose functions are not known. The findings obtained by LC/MS/MS were confirmed by immunohistochemistry and Western blotting for selected proteins, thereby providing a first validation of some of the proteins found at aberrant levels in AD. Interestingly, we were able to authenticate decreased levels of syntaxin-1A and reticulon-1 and increased synaptogyrin-1 levels in AD using confocal microscopy. The identification of proteins involved in the processes finally leading to neurodegeneration and cognitive deficits associated with AD could lead to the development of biological markers for the disease. Such biomarker candidates found at altered levels in AD brain tissue could possibly be reflected in the cerebrospinal fluid (CSF) and eventually in the periphery and may be explored for their value as drug targets or markers for diagnosis, disease progression or even responsiveness to treatment. The results from our studies indicate pathways which appear promising in such a quest

An automated iterative approach for protein structure refinement using pseudocontact shifts

NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing

An automated iterative approach for protein structure refinement using pseudocontact shifts

NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing

An automated iterative approach for protein structure refinement using pseudocontact shifts

NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.ISSN:0925-2738ISSN:1573-500