Dynamic light- and acetate-dependent regulation of the proteome and lysine acetylome of Chlamydomonas.

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

Detection of Novel Poxvirus from Gray Seal (Halichoerus grypus), Germany.

We detected a novel poxvirus from a gray seal (Halichoerus grypus) from the North Sea, Germany. The juvenile animal showed pox-like lesions and deteriorating overall health condition and was finally euthanized. Histology, electron microscopy, sequencing, and PCR confirmed a previously undescribed poxvirus of the Chordopoxvirinae subfamily, tentatively named Wadden Sea poxvirus.

The individual relationship between atrial fibrillation sources from CARTOFINDER mapping and atrial cardiomyopathy: The catch me if you can trial.

BACKGROUND: Targeting individual sources identified during atrial fibrillation (AF) has been used as an ablation strategy with varying results. OBJECTIVE: Aim of this study was to evaluate the relationship between regions of interest (ROIs) from CARTOFINDER (CF) mapping and atrial cardiomyopathy from late gadolinium enhancement (LGE) cardiovascular magnetic resonance imaging (CMR). METHODS: Twenty consecutive patients underwent index catheter ablation for persistent AF (PERS AF). Pre-processed LGE CMR images were merged with the results from CF mapping to visualize harboring regions for focal and rotational activities. Atrial cardiomyopathy was classified based on the four Utah stages. RESULTS: Procedural success was achieved in all patients (n = 20, 100%). LGE CMR revealed an intermediate amount of 21.41% ± 6.32% for LA fibrosis. ROIs were identified in all patients (mean no ROIs per patient n = 416.45 ± 204.57). A tendency towards a positive correlation between the total amount of atrial cardiomyopathy and the total number of ROIs per patient (regression coefficient, ß = 10.86, p = .15) was observed. The degree of fibrosis and the presence of ROIs per segment showed no consistent spatial correlation (posterior: ß = 0.36, p-value (p) = .24; anterior: ß = -0.08, p = .54; lateral: ß = 0.31, p = 39; septal: ß = -0.12; p = .66; right PVs: ß = 0.34, p = .27; left PVs: ß = 0.07, p = .79; LAA: ß = -0.91, p = .12). 12 months AF-free survival was 70% (n = 14) after ablation. CONCLUSION: The presence of ROIs from CF mapping was not directly associated with the extent and location of fibrosis. Further studies evaluating the relationship between focal and rotational activity and atrial cardiomyopathy are mandatory.

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.

The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed ß propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

A Golgi-Released Subpopulation of the Trans-Golgi Network Mediates Protein Secretion in Arabidopsis.

Spatiotemporal coordination of protein trafficking among organelles is essential for eukaryotic cells. The post-Golgi interface, including the trans-Golgi network (TGN), is a pivotal hub for multiple trafficking pathways. The Golgi-released independent TGN (GI-TGN) is a compartment described only in plant cells, and its cellular and physiological roles remain elusive. In Arabidopsis (Arabidopsis thaliana), the SYNTAXIN OF PLANTS (SYP) 4 group Qa-SNARE (soluble N-ethylmaleimide) membrane fusion proteins are shared components of TGN and GI-TGN and regulate secretory and vacuolar transport. Here we reveal that GI-TGNs mediate the transport of the R-SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 to the plasma membrane. In interactions with a nonadapted powdery mildew pathogen, the SYP4 group of SNAREs is required for the dynamic relocation of VAMP721 to plant-fungus contact sites via GI-TGNs, thereby facilitating complex formation with its cognate SNARE partner PENETRATION1 to restrict pathogen entry. Furthermore, quantitative proteomic analysis of leaf apoplastic fluid revealed constitutive and pathogen-inducible secretion of cell wall-modification enzymes in a SYP4- and VAMP721-dependent manner. Hence, the GI-TGN acts as a transit compartment between the Golgi apparatus and the plasma membrane. We propose a model in which the GA-TGN matures into the GI-TGN and then into secretory vesicles by increasing the abundance of VAMP721-dependent secretory pathway components.

Principles and characteristics of the Arabidopsis WRKY regulatory network during early MAMP-triggered immunity.

During microbe-associated molecular pattern-triggered immunity more than 5000 Arabidopsis genes are significantly altered in their expression, and the question arises, how such an enormous reprogramming of the transcriptome can be regulated in a safe and robust manner? For the WRKY transcription factors (TFs), which are important regulators of numerous defense responses, it appears that they act in a complex regulatory sub-network rather than in a linear fashion, which would be much more vulnerable to gene function loss either by pathogen-derived effectors or by mutations. In this study we employed RNA-seq, mass spectrometry and chromatin immunoprecipitation-seq to find evidence for and uncover principles and characteristics of this network. Upon flg22-treatment, one can distinguish between two sets of WRKY genes: constitutively expressed and induced WRKY genes. Prior to elicitation the induced WRKY genes appear to be maintained in a repressed state mainly by the constitutively expressed WRKY factors, which themselves appear to be regulated by non-WRKY TFs. Upon elicitation, induced WRKYs rapidly bind to induced WRKY gene promoters and by auto- and cross-regulation build up the regulatory network. Maintenance of this flg22-induced network appears highly robust as removal of three key WRKY factors can be physically and functionally compensated for by other WRKY family members.

A Multi-Omics Analysis of Glycine max Leaves Reveals Alteration in Flavonoid and Isoflavonoid Metabolism Upon Ethylene and Abscisic Acid Treatment.

Phytohormones are central to plant growth and development. Despite the advancement in our knowledge of hormone signaling, downstream targets, and their interactions upon hormones action remain largely fragmented, especially at the protein and metabolite levels. With an aim to get new insight into the effects of two hormones, ethylene (ET) and abscisic acid (ABA), this study utilizes an integrated proteomics and metabolomics approach to investigate their individual and combined (ABA+ET) signaling in soybean leaves. Targeting low-abundance proteins, our previously established protamine sulfate precipitation method was applied, followed by label-free quantification of identified proteins. A total of 4129 unique protein groups including 1083 differentially modulated in one (individual) or other (combined) treatments were discerned. Functional annotation of the identified proteins showed an increased abundance of proteins related to the flavonoid and isoflavonoid biosynthesis and MAPK signaling pathway in response to ET treatment. HPLC analysis showed an accumulation of isoflavones (genistin, daidzein, and genistein) upon ET treatment, in agreement with the proteomics results. A metabolome analysis assigned 79 metabolites and further confirmed the accumulation of flavonoids and isoflavonoids in response to ET. A potential cross-talk between ET and MAPK signaling, leading to the accumulation of flavonoids and isoflavonoids in soybean leaves is suggested.

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis.

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome-wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis Relative quantification of the changes in the lysine acetylation levels was determined on a proteome-wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1-like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar-localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss-of-function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low-light conditions.

Genes for seed longevity in barley identified by genomic analysis on near isogenic lines.

Genes controlling differences in seed longevity between 2 barley (Hordeum vulgare) accessions were identified by combining quantitative genetics "omics" technologies in near isogenic lines (NILs). The NILs were derived from crosses between the spring barley landraces L94 from Ethiopia and Cebada Capa from Argentina. A combined transcriptome and proteome analysis on mature, nonaged seeds of the 2 parental lines and the L94 NILs by RNA-sequencing and total seed proteomic profiling identified the UDP-glycosyltransferase MLOC_11661.1 as candidate gene for the quantitative trait loci on 2H, and the NADP-dependent malic enzyme (NADP-ME) MLOC_35785.1 as possible downstream target gene. To validate these candidates, they were expressed in Arabidopsis under the control of constitutive promoters to attempt complementing the T-DNA knockout line nadp-me1. Both the NADP-ME MLOC_35785.1 and the UDP-glycosyltransferase MLOC_11661.1 were able to rescue the nadp-me1 seed longevity phenotype. In the case of the UDP-glycosyltransferase, with high accumulation in NILs, only the coding sequence of Cebada Capa had a rescue effect.

The SAGA complex in the rice pathogen Fusarium fujikuroi: structure and functional characterization.

Post-translational modification of histones is a crucial mode of transcriptional regulation in eukaryotes. A well-described acetylation modifier of certain lysine residues is the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex assembled around the histone acetyltransferase Gcn5 in Saccharomyces cerevisiae. We identified and characterized the SAGA complex in the rice pathogen Fusarium fujikuroi, well-known for producing a large variety of secondary metabolites (SMs). By using a co-immunoprecipitation approach, almost all of the S. cerevisiae SAGA complex components have been identified, except for the ubiquitinating DUBm module and the chromodomain containing Chd1. Deletion of GCN5 led to impaired growth, loss of conidiation and alteration of SM biosynthesis. Furthermore, we show that Gcn5 is essential for the acetylation of several histone 3 lysines in F. fujikuroi, that is, H3K4, H3K9, H3K18 and H3K27. A genome-wide microarray analysis revealed differential expression of about 30% of the genome with an enrichment of genes involved in primary and secondary metabolism, transport and histone modification. HPLC-based analysis of known SMs revealed significant alterations in the Δgcn5 mutant. While most SM genes were activated by Gcn5 activity, the biosynthesis of the pigment bikaverin was strongly increased upon GCN5 deletion underlining the diverse roles of the SAGA complex in F. fujikuroi.

Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins.

RNA-protein complexes play pivotal roles in many central biological processes. Although methods based on high-throughput sequencing have advanced our ability to identify the specific RNAs bound by a particular protein, there is a need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced cross-linking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked RNA oligonucleotide moieties of such RNA-binding proteins. The workflow can be applied to any RNA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mRNA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct RNA-binding proteins. The open-source software pipeline developed for this purpose, RNP(xl), is available as part of the OpenMS project.

Sequence Polymorphisms at the REDUCED DORMANCY5 Pseudophosphatase Underlie Natural Variation in Arabidopsis Dormancy.

Seed dormancy controls the timing of germination, which regulates the adaptation of plants to their environment and influences agricultural production. The time of germination is under strong natural selection and shows variation within species due to local adaptation. The identification of genes underlying dormancy quantitative trait loci is a major scientific challenge, which is relevant for agricultural and ecological goals. In this study, we describe the identification of the DELAY OF GERMINATION18 (DOG18) quantitative trait locus, which was identified as a factor in natural variation for seed dormancy in Arabidopsis (Arabidopsis thaliana). DOG18 encodes a member of the clade A of the type 2C protein phosphatases family, which we previously identified as the REDUCED DORMANCY5 (RDO5) gene. DOG18/RDO5 shows a relatively high frequency of loss-of-function alleles in natural accessions restricted to northwestern Europe. The loss of dormancy in these loss-of-function alleles can be compensated for by genetic factors like DOG1 and DOG6, and by environmental factors such as low temperature. RDO5 does not have detectable phosphatase activity. Analysis of the phosphoproteome in dry and imbibed seeds revealed a general decrease in protein phosphorylation during seed imbibition that is enhanced in the rdo5 mutant. We conclude that RDO5 acts as a pseudophosphatase that inhibits dephosphorylation during seed imbibition.

Doxycycline in early CJD: a double-blinded randomised phase II and observational study.

OBJECTIVES: The main objective of the present study is to study the therapeutic efficiency of doxycycline in a double-blinded randomised phase II study in a cohort of patients with sporadic Creutzfeldt-Jakob disease (sCJD). METHODS: From the National Reference Center of TSE Surveillance in Germany, patients with probable or definite sCJD were recruited for a double-blinded randomised study with oral doxycycline (EudraCT 2006-003934-14). In addition, we analysed the data from patients with CJD who received compassionate treatment with doxycycline in a separate group. Potential factors which influence survival such as age at onset, gender, codon 129 polymorphism and cognitive functions were evaluated. The primary outcome measure was survival. RESULTS: Group 1: in the double-blinded randomised phase II study, 7 patients in the treatment group were compared with 5 controls. Group 2: 55 patients with sCJD treated with oral doxycycline were analysed and compared with 33 controls by a stratified propensity score applied to a Cox proportional hazard analysis. The results of both studies were combined by means of a random-effects meta-analysis. A slight increase in survival time in the doxycycline treatment group was observed (p=0.049, HR=0.63 (95% CI 0.402 to 0.999)). CONCLUSIONS: On the basis of our studies, a larger trial of doxycycline should be performed in persons in the earliest stages of CJD. TRIAL REGISTRATION NUMBER: EudraCT 2006-003934-14; Results.

Dithiothreitol (DTT) Acts as a Specific, UV-inducible Cross-linker in Elucidation of Protein-RNA Interactions.

Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein-RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks, which is observable as a mass increment of 151.9966 Da (C(4)H(8)S(2)O(2)) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive sites as the most probable structure of the cross-linking products.

Crystal structure of Cwc2 reveals a novel architecture of a multipartite RNA-binding protein.

The yeast splicing factor Cwc2 contacts several catalytically important RNA elements in the active spliceosome, suggesting that Cwc2 is involved in determining their spatial arrangement at the spliceosome's catalytic centre. We have determined the crystal structure of the Cwc2 functional core, revealing how a previously uncharacterized Torus domain, an RNA recognition motif (RRM) and a zinc finger (ZnF) are tightly integrated in a compact folding unit. The ZnF plays a pivotal role in the architecture of the whole assembly. UV-induced crosslinking of Cwc2-U6 snRNA allowed the identification by mass spectrometry of six RNA-contacting sites: four in or close to the RRM domain, one in the ZnF and one on a protruding element connecting the Torus and RRM domains. The three distinct regions contacting RNA are connected by a contiguous and conserved positively charged surface, suggesting an expanded interface for RNA accommodation. Cwc2 mutations confirmed that the connector element plays a crucial role in splicing. We conclude that Cwc2 acts as a multipartite RNA-binding platform to bring RNA elements of the spliceosome's catalytic centre into an active conformation.

Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1.

BACKGROUND: Modulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many - if not all - molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase. RESULTS: Here we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates. CONCLUSION: The here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology.

Probing the structure-activity relationship of endogenous histone deacetylase complexes with immobilized peptide-inhibitors.

Histone deacetylases (HDACs) are key regulators of numerous cellular proteins by removing acetylation marks from modified lysine residues. Peptide-based HDAC probes containing α-aminosuberic acid ω-hydroxamate have been established as useful tools for investigating substrate selectivity and composition of endogenous HDAC complexes in cellular lysates. Here we report a structure-activity study of potential HDAC-probes containing derivatives of the hydroxamate moieties. While most of these probes did not recruit significant amounts of endogenous HDACs from cellular lysates, peptides containing Nε-acetyl-Nε-hydroxy-L-lysine served as HDAC probe. The recruitment efficiency varied between HDACs and was generally lower than that of α-aminosuberic acid ω-hydroxamate probes, but showed a similar global interaction profile. These findings indicate that Nε-acetyl-Nε-hydroxy-L-lysine might be a useful tool for investigations on HDAC complexes and the development of HDAC inhibitors. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry.

Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different single- and multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 - RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.

The influence of the utilization time of brush heads from different types of power toothbrushes on oral hygiene assessed over a 6-month observation period: A randomized clinical trial.

PURPOSE: This randomized clinical trial investigated the influence of the utilization time of brush heads from different types of power toothbrushes [oscillating rotating (OR) and sonic action (SA)93; on oral hygiene (plaque accumulation and gingival inflammation) over a 6-month observation period. METHODS: 49 participants were randomly allocated into two groups: use of the same brush head over 6 months (NR: non-replacement) or replacement of brush head every 4 weeks over 6 months (R: replacement). Each group was subdivided into two subgroups according to kind of toothbrush (TB) used (OR and SA). Modified Quigley-Hein plaque index (QHI), papilla bleeding index (PBI), and gingival index (GI) were recorded at baseline and 2, 8, 12, 16, and 24 weeks after baseline. After 24 weeks, participants of both groups (R and NR) received a new brush head. At week 26, final QHI, PBI, and GI were recorded. RESULTS: QHI decreased between baseline and follow-up visits in R groups (P< 0.05), with the exception of week 12 (P= 0.26). In NR groups, no significant decrease was detected (P> 0.05). There was no significant effect of time on PBI or GI in any of R subgroups (P> 0.05). In NR oscillating/rotating TB: significant increase in PBI and GI was detected 24 weeks after baseline (PBI: P= 0.02, GI: P= 0.03); sonic action TBs showed significant decrease in PBI at every follow-up visit (P< 0.05), except at 24 weeks after baseline (P= 0.73). GI was significantly decreased at 2 weeks after baseline only (P< 0.01). CLINICAL SIGNIFICANCE: Six-month use of the same brush head reduced effectiveness in removing plaque, and gingival inflammation appeared to increase after a utilization time of over 4 months. Replacing brush heads is advised after 4 months.

Nonparametric meta-analysis for diagnostic accuracy studies.

Summarizing the information of many studies using a meta-analysis becomes more and more important, also in the field of diagnostic studies. The special challenge in meta-analysis of diagnostic accuracy studies is that in general sensitivity and specificity are co-primary endpoints. Across the studies both endpoints are correlated, and this correlation has to be considered in the analysis. The standard approach for such a meta-analysis is the bivariate logistic random effects model. An alternative approach is to use marginal beta-binomial distributions for the true positives and the true negatives, linked by copula distributions. In this article, we propose a new, nonparametric approach of analysis, which has greater flexibility with respect to the correlation structure, and always converges. In a simulation study, it becomes apparent that the empirical coverage of all three approaches is in general below the nominal level. Regarding bias, empirical coverage, and mean squared error the nonparametric model is often superior to the standard model, and comparable with the copula model. The three approaches are also applied to two example meta-analyses.