Acknowledging and citing core facilities: Key contributions to data lifecycle should be recognised in the scientific literature: Key contributions to data lifecycle should be recognised in the scientific literature.

Core facilities play a central role in the life sciences by generating data and ensuring quality standards. Their contributions to research should be appropriately acknowledged or cited in research papers.

A familial disorder of altered DNA-methylation.

BACKGROUND: In a subset of imprinting disorders caused by epimutations, multiple imprinted loci are affected. Familial occurrence of multilocus imprinting disorders is rare. PURPOSE/OBJECTIVE: We have investigated the clinical and molecular features of a familial DNA-methylation disorder. METHODS: Tissues of affected individuals and blood samples of family members were investigated by conventional and molecular karyotyping. Sanger sequencing and RT-PCR of imprinting-associated genes (NLRP2, NLRP7, ZFP57, KHDC3L, DNMT1o), exome sequencing and locus-specific, array-based and genome-wide technologies to determine DNA-methylation were performed. RESULTS: In three offspring of a healthy couple, we observed prenatal onset of severe growth retardation and dysmorphism associated with altered DNA-methylation at paternally and maternally imprinted loci. Array-based analyses in various tissues of the offspring identified the DNA-methylation of 2.1% of the genes in the genome to be recurrently altered. Despite significant enrichment of imprinted genes (OR 9.49), altered DNA-methylation predominately (90.2%) affected genes not known to be imprinted. Sequencing of genes known to cause comparable conditions and exome sequencing in affected individuals and their ancestors did not unambiguously point to a causative gene. CONCLUSIONS: The family presented herein suggests the existence of a familial disorder of DNA-methylation affecting imprinted but also not imprinted gene loci potentially caused by a maternal effect mutation in a hitherto not identified gene.

Tobacco-smoking-related differential DNA methylation: 27K discovery and replication.

Tobacco smoking is responsible for substantial morbidity and mortality worldwide, in particular through cardiovascular, pulmonary, and malignant pathology. CpG methylation might plausibly play a role in a variety of smoking-related phenomena, as suggested by candidate gene promoter or global methylation studies. Arrays allowing hypothesis-free searches on a scale resembling genome-wide studies of SNPs have become available only very recently. Methylation extents in peripheral-blood DNA were assessed at 27,578 sites in more than 14,000 gene promoter regions in 177 current smokers, former smokers, and those who had never smoked, with the use of the Illumina HumanMethylation 27K BeadChip. This revealed a single locus, cg03636183, located in F2RL3, with genome-wide significance for lower methylation in smokers (p = 2.68 × 10(-31)). This was similarly significant in 316 independent replication samples analyzed by mass spectrometry and Sequenom EpiTyper (p = 6.33 × 10(-34)). Our results, which were based on a rigorous replication approach, show that the gene coding for a potential drug target of cardiovascular importance features altered methylation patterns in smokers. To date, this gene had not attracted attention in the literature on smoking.

F-box and leucine-rich repeat protein 22 is a cardiac-enriched F-box protein that regulates sarcomeric protein turnover and is essential for maintenance of contractile function in vivo.

RATIONALE: The emerging role of the ubiquitin-proteasome system in cardiomyocyte function and homeostasis implies the necessity of tight regulation of protein degradation. However, little is known about cardiac components of this machinery. OBJECTIVE: We sought to determine whether molecules exist that control turnover of cardiac-specific proteins. METHODS AND RESULTS: Using a bioinformatic approach to identify novel cardiac-enriched sarcomere proteins, we identified F-box and leucine-rich repeat protein 22 (Fbxl22). Tissue-specific expression was confirmed by multiple tissue Northern and Western Blot analyses as well as quantitative reverse-transcriptase polymerase chain reaction on a human cDNA library. Immunocolocalization experiments in neonatal and adult rat ventricular cardiomyocytes as well as murine heart tissue located Fbxl22 to the sarcomeric z-disc. To detect cardiac protein interaction partners, we performed a yeast 2-hybrid screen using Fbxl22 as bait. Coimmunoprecipitation confirmed the identified interactions of Fbxl22 with S-phase kinase-associated protein 1 and Cullin1, 2 critical components of SCF (Skp1/Cul1/F-box) E3- ligases. Moreover, we identified several potential substrates, including the z-disc proteins α-actinin and filamin C. Consistently, in vitro overexpression of Fbxl22-mediated degradation of both substrates in a dose-dependent fashion, whereas proteasome inhibition with MG-132 markedly attenuated degradation of both α-actinin and filamin C. Finally, targeted knockdown of Fbxl22 in rat cardiomyocytes as well as zebrafish embryos results in the accumulation of α-actinin associated with severely impaired contractile function and cardiomyopathy in vivo. CONCLUSIONS: These findings reveal the previously uncharacterized cardiac-specific F-box protein Fbxl22 as a component of a novel cardiac E3 ligase. Fbxl22 promotes the proteasome-dependent degradation of key sarcomeric proteins, such as α-actinin and filamin C, and is essential for maintenance of normal contractile function in vivo.

Utilizing inherent fluorescence of therapeutics to analyze real-time uptake and multi-parametric effector kinetics.

The precise detection of pharmaceutical drug uptake and knowledge of a drug's efficacy at the single-cell level is crucial for understanding a compound's performance. Many pharmaceutical drugs, like the model substances Doxorubicin, Mitoxantrone or Irinotecan, have a distinctive natural fluorescence that can be readily exploited for research purposes. Utilizing this respective natural fluorescence, we propose a method analyzing simultaneously in real-time the efficiency, effects and the associated kinetics of compound-uptake and efflux in mammalian cells by flow cytometry. We show that real-time flow cytometric quantification of compound-uptake is reliably measured and that analyzing their respective uptake kinetic provides additional valuable information which can be used for improving drug dosage and delivery. Exploiting the native fluorescence of natural compounds is clearly advantageous compared to the usage of non-related fluorescent uptake-reporter substances, possibly yielding in unphysiological data. Flow cytometric analysis allows live-dye based multi-parametric high-throughput screening of pharmaceutical compound activity, improving cytotoxicity testing by combining several assays into a single, high resolution readout. This approach can be a useful tool identifying potential inhibitors for multiple drug resistance (MDR), representing a major challenge to the targeted treatment of various diseases.

Aging and chronic sun exposure cause distinct epigenetic changes in human skin.

Epigenetic changes are widely considered to play an important role in aging, but experimental evidence to support this hypothesis has been scarce. We have used array-based analysis to determine genome-scale DNA methylation patterns from human skin samples and to investigate the effects of aging, chronic sun exposure, and tissue variation. Our results reveal a high degree of tissue specificity in the methylation patterns and also showed very little interindividual variation within tissues. Data stratification by age revealed that DNA from older individuals was characterized by a specific hypermethylation pattern affecting less than 1% of the markers analyzed. Interestingly, stratification by sun exposure produced a fundamentally different pattern with a significant trend towards hypomethylation. Our results thus identify defined age-related DNA methylation changes and suggest that these alterations might contribute to the phenotypic changes associated with skin aging.

Myomasp/LRRC39, a heart- and muscle-specific protein, is a novel component of the sarcomeric M-band and is involved in stretch sensing.

RATIONALE AND OBJECTIVE: The M-band represents a transverse structure in the center of the sarcomeric A-band and provides an anchor for the myosin-containing thick filaments. In contrast to other sarcomeric structures, eg, the Z-disc, only few M-band-specific proteins have been identified to date, and its exact molecular composition remains unclear. METHODS AND RESULTS: Using a bioinformatic approach to identify novel heart- and muscle-specific genes, we found a leucine rich protein, myomasp (Myosin-interacting, M-band-associated stress-responsive protein)/LRRC39. RT-PCR and Northern and Western blot analyses confirmed a cardiac-enriched expression pattern, and immunolocalization of myomasp revealed a strong and specific signal at the sarcomeric M-band. Yeast 2-hybrid screens, as well as coimmunoprecipitation experiments, identified the C terminus of myosin heavy chain (MYH)7 as an interaction partner for myomasp. Knockdown of myomasp in neonatal rat ventricular myocytes (NRVCMs) led to a significant upregulation of the stretch-sensitive genes GDF-15 and BNP. Conversely, the expression of MYH7 and the M-band proteins myomesin-1 and -2 was found to be markedly reduced. Mechanistically, knockdown of myomasp in NRVCM led to a dose-dependent suppression of serum response factor-dependent gene expression, consistent with earlier observations linking the M-band to serum response factor-mediated signaling. Finally, downregulation of myomasp/LRRC39 in spontaneously beating engineered heart tissue constructs resulted in significantly lower force generation and reduced fractional shortening. Likewise, knockdown of the myomasp/LRRC39 ortholog in zebrafish resulted in severely impaired heart function and cardiomyopathy in vivo. CONCLUSIONS: These findings reveal myomasp as a previously unrecognized component of an M-band-associated signaling pathway that regulates cardiomyocyte gene expression in response to biomechanical stress.

Lentiviral vector integration profiles differ in rodent postmitotic tissues.

Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.

A community standard format for the representation of protein affinity reagents.

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.

New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling.

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

Retinal and retinol are potential regulators of gene expression in the keratinocyte cell line HaCaT.

Vitamin A is a pivotal regulator of differentiation and growth of developing and adult skin. Retinoic acid is the major physiologically active form of vitamin A regulating the expression of different genes through retinoic acid nuclear receptors. Here, we present evidence that other vitamin A derivates - retinol and retinal - are also capable of functioning as regulators of gene expression in the keratinocyte cell line HaCaT. We have shown that all-trans retinol (ATRol) and all-trans retinal (ATRal) are capable of modulating gene expression in keratinocytes, which is not because of vitamin A metabolism in the cells, and retinol and retinal modulate gene expression through nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Based on the data, we propose that ATRol and all-trans retinal, in addition to all-trans retinoic acid, can function as important regulators of gene expression manifesting their effect through the nuclear receptors RARs and RXRs.

Transporter molecules influence the gene expression in HeLa cells.

Progresses in biology and pharmacology led to highly specific bioactive substances, but their poor bioavailability at the site of action is a result of their physico-chemical properties. Various design approaches for transport carrier molecules facilitating the cellular entry of bioactive substances could help to reach their molecular target in cells and tissues. The transfer efficacy and the subsequent pharmacological effects of the cargo molecules are well investigated, but the investigations of effects of the carrier molecules themselves on the target cells or tissues remain necessary. A special attention should be paid to the differential gene expression, particularly in the interpretation of the data achieved by highly specific active pharmaceutical products. After application of transmembrane transport peptides, particularly the pAnt and also the HIV-1 Tat, cells respond with a conspicuous altered gene expression of at least three genes. The PKN1 gene was induced and two genes (ZCD1 and BSG) were slightly repressed. The genes and the chromosomes are described, the moderate differential gene expression graphed, and the ontology is listed.

Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation.

Constitutive and induced protein SUMOylation is involved in the regulation of a variety of cellular processes, such as regulation of gene expression and protein transport, and proceeds mainly in the nucleus of the cell. So far, several hundred SUMOylation targets have been identified, but presumably they represent only a part of the total of proteins which are regulated by SUMOylation. Here, we used the Ubc9 fusion-dependent SUMOylation system (UFDS) to screen for constitutive and induced SUMOylation of 46 randomly chosen proteins with proven or potential nuclear localization. Fourteen new UFDS-substrate proteins were identified of which eight could be demonstrated to be SUMOylated in a UFDS-independent manner in vivo. Of these, three were constitutively SUMOylated (FOS, CRSP9 and CDC37) while the remaining five substrates (CSNK2B, TAF10, HSF2BP, PSMC3 and DRG1) showed a stimulation-dependent SUMOylation induced by the MAP3 kinase MEKK1. Hence, UFDS is appropriate for the identification and characterization of constitutive and, more importantly, induced protein SUMOylation in vivo.

Effect of artemisinins and other endoperoxides on nitric oxide-related signaling pathway in RAW 264.7 mouse macrophage cells.

Artemisinin is the active principle of the Chinese herb Artemisia annua L. In addition to its anti-malarial activity, artemisinin and its derivatives have been shown to exert profound anti-cancer activity. The endoperoxide moiety in the chemical structure of artemisinin is thought to be responsible for the bioactivity. Here, we analyzed the cytotoxicity and the ability of artemisinin, five of its derivatives, and two other endoperoxides to inhibit generation of nitric oxide (NO). In the RAW 264.7 mouse macrophage cell line, the well-established model cell line to analyze NO generation, artesunate revealed the highest ability to inhibit NO production among all compounds tested. In cytotoxicity assays (XTT assay), the IC(50) value of RAW 264.7 cells for artesunate was determined to be 3.1+/-0.7 microM. In order to associate the cytotoxic effects with specific alteration in gene expression related to NO metabolism and signaling, whole genome mRNA microarray analyses were conducted. RAW 264.7 cells were treated with artesunate using DMSO as vehicle control followed by microarray analysis. A total of 36 genes related to NO metabolism and signaling were found to be differentially expressed upon exposure to artesunate. Apart from NO-related genes, the expression of genes associated with other functional groups was also analyzed. Out of 24 functional groups, differential expression was most prominent in genes involved in cell-to-cell signaling and interactions. Further refinement of this analysis showed that the pathways for cAMP-mediated signaling and Wnt/beta-catenin signaling were most closely related to changes in mRNA expression. In conclusion, NO generation and signaling play a role in exhibiting cytotoxic activity of artesunate. In addition, other signaling pathways also contribute to the inhibitory effect of artesunate towards RAW 264.7 cells pointing to a multi-factorial mode of action of artesunate.

A systematic approach for testing expression of human full-length proteins in cell-free expression systems.

BACKGROUND: The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. RESULTS: In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. CONCLUSION: We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield.

The DT40 web site: sampling and connecting the genes of a B cell line.

Thousands of new vertebrate genes have been discovered and genetic systems are needed to address their functions at the cellular level. The chicken B cell line DT40 allows efficient gene disruptions due to its high homologous recombination activity. However, cloning the gene of interest is often cumbersome, since relatively few chicken cDNA sequences are present in the public databases. In addition, the accumulation of multiple mutations within the same cell clone is limited by the consumption of one drug-resistance marker for each transfection. Here, we present the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40.html), which includes a comprehensive database of chicken bursal ESTs to identify disruption candidate genes and recyclable marker cassettes based on the loxP system. These freely available resources greatly facilitate the analysis of genes and genetic networks.

The oxidized subunit B8 from human complex I adopts a thioredoxin fold.

Subunit B8 from ubiquinone oxidoreductase (complex I) (CI-B8) is one of several nuclear-encoded supernumerary subunits that are not present in bacterial complex I. Its solution structure shows a thioredoxin fold with highest similarities to the human thioredoxin mutant C73S and thioredoxin 2 from Anabeana sp. Interestingly, these proteins contain active sites in the same area, where the disulfide bond of oxidized CI-B8 is located. The redox potential of this disulfide bond is -251.6 mV, comparing well to that of disulfides in other thioredoxin-like proteins. Analysis of the structure reveals a surface area that is exclusively composed of highly conserved residues and thus most likely a subunit interaction site within complex I.

Candidate genes for cross-resistance against DNA-damaging drugs.

Drug resistance of tumor cells leads to major drawbacks in the treatment of cancer. To identify candidate genes for drug resistance, we compared the expression patterns of the drug-sensitive human malignant melanoma cell line MeWo and three derived sublines with acquired resistance to the DNA-damaging agents cisplatin, etoposide, and fotemustine. Subarray analyses confirmed 57 candidate genes recovered from a genome-wide scan for differential expression. By specifically addressing cancer genes we retrieved another set of 209 candidates. Exemplary Northern blot studies indicated qualitative concordance for 110 of 135 (81.4%) data points. Whereas the etoposide-resistant line showed constant expression patterns over a period of approximately 2.5 years, the fotemustine- and cisplatin-resistant sublines exhibited considerable variability. Initially representing distinct entities, these two sublines finally converged in their expression patterns. A total of 110 genes was transiently or permanently deregulated in at least two resistant sublines. Fourteen genes displayed differential expression in all three of the sublines. We hypothesize that the variations in fotemustine and cisplatin resistance are based on progressive optimization and/or polyclonality. This, in addition to genomic alterations investigated by comparative genomic hybridization and evaluation of short-term response genes, can be used as a criterion for the selection of promising candidates. Among these are CYR61, AHCYL1, and MPP1, as well as several apoptosis-related genes, in particular STK17A and CRYAB. As MPP1 and CRYAB are also among the 14 genes differentially expressed in all three of the drug-resistant sublines, they represent the strongest candidates for resistance against DNA-damaging drugs.

Strengths and weaknesses of EST-based prediction of tissue-specific alternative splicing.

BACKGROUND: Alternative splicing contributes significantly to the complexity of the human transcriptome and proteome. Computational prediction of alternative splice isoforms are usually based on EST sequences that also allow to approximate the expression pattern of the related transcripts. However, the limited number of tissues represented in the EST data as well as the different cDNA construction protocols may influence the predictive capacity of ESTs to unravel tissue-specifically expressed transcripts. METHODS: We predict tissue and tumor specific splice isoforms based on the genomic mapping (SpliceNest) of the EST consensus sequences and library annotation provided in the GeneNest database. We further ascertain the potentially rare tissue specific transcripts as the ones represented only by ESTs derived from normalized libraries. A subset of the predicted tissue and tumor specific isoforms are then validated via RT-PCR experiments over a spectrum of 40 tissue types. RESULTS: Our strategy revealed 427 genes with at least one tissue specific transcript as well as 1120 genes showing tumor specific isoforms. While our experimental evaluation of computationally predicted tissue-specific isoforms revealed a high success rate in confirming the expression of these isoforms in the respective tissue, the strategy frequently failed to detect the expected restricted expression pattern. The analysis of putative lowly expressed transcripts using normalized cDNA libraries suggests that our ability to detect tissue-specific isoforms strongly depends on the expression level of the respective transcript as well as on the sensitivity of the experimental methods. Especially splice isoforms predicted to be disease-specific tend to represent transcripts that are expressed in a set of healthy tissues rather than novel isoforms. CONCLUSIONS: We propose to combine the computational prediction of alternative splice isoforms with experimental validation for efficient delineation of an accurate set of tissue-specific transcripts.