Bettina Knapp

Intraindividual genome expression analysis reveals a specific molecular signature of psoriasis and eczema

Signatures from patients with both psoriasis and eczema contribute to understanding disease pathogenesis and diagnosis. Discrimination That’s Skin Deep A molecular signature may be able to aid in the differential treatment of psoriasis from eczema. Quaranta et al. examined a group of individuals affected by both psoriasis and eczema and compared molecular signatures from psoriasis and eczema lesions on the same individual. This approach limited noise from interindividual variability, allowing the authors to focus on genes involved in disease pathogenesis. They found that psoriasis-specific genes involved not only immune mediators but also regulators of metabolism. In contrast, eczema-related genes included those related to the epidermal barrier and inflammasome activation. These insights provide not only new targets for disease-specific therapies but also an independently verified classifier that can be used in difficult to diagnose patients. Previous attempts to gain insight into the pathogenesis of psoriasis and eczema by comparing their molecular signatures were hampered by the high interindividual variability of those complex diseases. In patients affected by both psoriasis and nonatopic or atopic eczema simultaneously (n = 24), an intraindividual comparison of the molecular signatures of psoriasis and eczema identified genes and signaling pathways regulated in common and exclusive for each disease across all patients. Psoriasis-specific genes were important regulators of glucose and lipid metabolism, epidermal differentiation, as well as immune mediators of T helper 17 (TH17) responses, interleukin-10 (IL-10) family cytokines, and IL-36. Genes in eczema related to epidermal barrier, reduced innate immunity, increased IL-6, and a TH2 signature. Within eczema subtypes, a mutually exclusive regulation of epidermal differentiation genes was observed. Furthermore, only contact eczema was driven by inflammasome activation, apoptosis, and cellular adhesion. On the basis of this comprehensive picture of the pathogenesis of psoriasis and eczema, a disease classifier consisting of NOS2 and CCL27 was created. In an independent cohort of eczema (n = 28) and psoriasis patients (n = 25), respectively, this classifier diagnosed all patients correctly and also identified initially misdiagnosed or clinically undifferentiated patients.

YidC occupies the lateral gate of the SecYEG translocon and is sequentially displaced by a nascent membrane protein

The immunoblot data in Fig. 4A was not obtained from a conditional secDF depletion strain Escherichia coli BL325 with a plasmid-borne copy of SecY(I91pBpa)EG as stated. The correct images representing SecD, SecF, SecY, and YidC levels in inner membrane vesicles of the conditional secDF depletion strain E. coli BL325 with a plasmid-borne copy of SecY(I91pBpa)EG are now shown. This correction does not affect the interpretation of the results or the conclusions of this work. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 23, p. 14492, June 5, 2015

RNAither, an automated pipeline for the statistical analysis of high-throughput RNAi screens

SUMMARY We present RNAither, a package for the free statistical environment R which performs an analysis of high-throughput RNA interference (RNAi) knock-down experiments, generating lists of relevant genes and pathways out of raw experimental data. The library provides a quality assessment of the signal intensities, as well as a broad range of options for data normalization, different statistical tests for the identification of significant siRNAs, and a significance analysis of the biological processes involving corresponding genes. The results of the analysis are presented as a set of HTML pages. Additionally, all values and plots are available as either text files or pdf and png files. AVAILABILITY http://bioconductor.org/

Coenzyme A dependent aerobic metabolism of benzoate via epoxide formation

In the aerobic metabolism of aromatic substrates, oxygenases use molecular oxygen to hydroxylate and finally cleave the aromatic ring. In the case of the common intermediate benzoate, the ring cleavage substrates are either catechol (in bacteria) or 3,4-dihydroxybenzoate (protocatechuate, mainly in fungi). We have shown before that many bacteria, e.g. Azoarcus evansii, the organism studied here, use a completely different mechanism. This elaborate pathway requires formation of benzoyl-CoA, followed by an oxygenase reaction and a nonoxygenolytic ring cleavage. Benzoyl-CoA transformation is catalyzed by the iron-containing benzoyl-CoA oxygenase (BoxB) in conjunction with an FAD and iron-sulfur centers containing reductase (BoxA), which donates electrons from NADPH. Here we show that benzoyl-CoA oxygenase actually does not form the 2,3-dihydrodiol of benzoyl-CoA, as formerly postulated, but the 2,3-epoxide. An enoyl-CoA hydratase (BoxC) uses two molecules of water to first hydrolytically open the ring of 2,3-epoxybenzoyl-CoA, which may proceed via its tautomeric seven-membered oxepin ring form. Then ring C2 is hydrolyzed off as formic acid, yielding 3,4-dehydroadipyl-CoA semialdehyde. The semialdehyde is oxidized by a NADP+-dependent aldehyde dehydrogenase (BoxD) to 3,4-dehydroadipyl-CoA. Final products of the pathway are formic acid, acetyl-CoA, and succinyl-CoA. This overlooked pathway occurs in 4–5% of all bacteria whose genomes have been sequenced and represents an elegant strategy to cope with the high resonance energy of aromatic substrates by forming a nonaromatic epoxide.

From experimental setup to bioinformatics: An RNAi screening platform to identify host factors involved in HIV-1 replication

RNA interference (RNAi) has emerged as a powerful technique for studying loss‐of‐function phenotypes by specific down‐regulation of gene expression, allowing the investigation of virus‐host interactions by large‐scale high‐throughput RNAi screens. Here we present a robust and sensitive small interfering RNA screening platform consisting of an experimental setup, single‐cell image and statistical analysis as well as bioinformatics. The workflow has been established to elucidate host gene functions exploited by viruses, monitoring both suppression and enhancement of viral replication simultaneously by fluorescence microscopy. The platform comprises a two‐stage procedure in which potential host factors are first identified in a primary screen and afterwards re‐tested in a validation screen to confirm true positive hits. Subsequent bioinformatics allows the identification of cellular genes participating in metabolic pathways and cellular networks utilised by viruses for efficient infection. Our workflow has been used to investigate host factor usage by the human immunodeficiency virus‐1 (HIV‐1), but can also be adapted to other viruses. Importantly, we expect that the description of the platform will guide further screening approaches for virus‐host interactions. The ViroQuant‐CellNetworks RNAi Screening core facility is an integral part of the recently founded BioQuant centre for systems biology at the University of Heidelberg and will provide service to external users in the near future.

miR-17-5p regulates endocytic trafficking through targeting TBC1D2/Armus.

miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.

Normalizing for individual cell population context in the analysis of high-content cellular screens

BackgroundHigh-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cells population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology.ResultsWe present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cells individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach.ConclusionsUsing a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.

Charting organellar importomes by quantitative mass spectrometry

Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelles importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles.

Allergic Contact Dermatitis in Psoriasis Patients: Typical, Delayed, and Non-Interacting

Psoriasis is characterized by an apoptosis-resistant and metabolic active epidermis, while a hallmark for allergic contact dermatitis (ACD) is T cell-induced keratinocyte apoptosis. Here, we induced ACD reactions in psoriasis patients sensitized to nickel (n = 14) to investigate underlying mechanisms of psoriasis and ACD simultaneously. All patients developed a clinically and histologically typical dermatitis upon nickel challenge even in close proximity to pre-existing psoriasis plaques. However, the ACD reaction was delayed as compared to non-psoriatic patients, with a maximum intensity after 7 days. Whole genome expression analysis revealed alterations in numerous pathways related to metabolism and proliferation in non-involved skin of psoriasis patients as compared to non-psoriatic individuals, indicating that even in clinically non-involved skin of psoriasis patients molecular events opposing contact dermatitis may occur. Immunohistochemical comparison of ACD reactions as well as in vitro secretion analysis of lesional T cells showed a higher Th17 and neutrophilic migration as well as epidermal proliferation in psoriasis, while ACD reactions were dominated by cytotoxic CD8+ T cells and a Th2 signature. Based on these findings, we hypothesized an ACD reaction directly on top of a pre-existing psoriasis plaque might influence the clinical course of psoriasis. We observed a strong clinical inflammation with a mixed psoriasis and eczema phenotype in histology. Surprisingly, the initial psoriasis plaque was unaltered after self-limitation of the ACD reaction. We conclude that sensitized psoriasis patients develop a typical, but delayed ACD reaction which might be relevant for patch test evaluation in clinical practice. Psoriasis and ACD are driven by distinct and independent immune mechanisms.

Anaerobic Metabolism of Indoleacetate

The anaerobic metabolism of indoleacetate (indole-3-acetic acid [IAA]) in the denitrifying betaproteobacterium Azoarcus evansii was studied. The strain oxidized IAA completely and grew with a generation time of 10 h. Enzyme activities that transformed IAA were present in the soluble cell fraction of IAA-grown cells but were 10-fold downregulated in cells grown on 2-aminobenzoate or benzoate. The transformation of IAA did not require molecular oxygen but required electron acceptors like NAD(+) or artificial dyes. The first products identified were the enol and keto forms of 2-oxo-IAA. Later, polar products were observed, which could not yet be identified. The first steps likely consist of the anaerobic hydroxylation of the N-heterocyclic pyrrole ring to the enol form of 2-oxo-IAA, which is catalyzed by a molybdenum cofactor-containing dehydrogenase. This step is probably followed by the hydrolytic ring opening of the keto form, which is catalyzed by a hydantoinase-like enzyme. A comparison of the proteome of IAA- and benzoate-grown cells identified IAA-induced proteins. Owing to the high similarity of A. evansii with strain EbN1, whose genome is known, we identified a cluster of 14 genes that code for IAA-induced proteins involved in the early steps of IAA metabolism. These genes include a molybdenum cofactor-dependent dehydrogenase of the xanthine oxidase/aldehyde dehydrogenase family, a hydantoinase, a coenzyme A (CoA) ligase, a CoA transferase, a coenzyme B(12)-dependent mutase, an acyl-CoA dehydrogenase, a fusion protein of an enoyl-CoA hydratase and a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, and a periplasmic substrate binding protein for ABC transport as well as a transcriptional regulator of the GntR family. Five predicted enzymes form or act on CoA thioesters, indicating that soon after the initial oxidation of IAA and possibly ring opening, CoA thioesters are formed, and the carbon skeleton is rearranged, followed by a CoA-dependent thiolytic release of another CoA thioester. We propose a scheme of an anaerobic IAA metabolic pathway that ultimately leads to 2-aminobenzoyl-CoA or benzoyl-CoA.