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1.
Exp Dermatol ; 33(3): e15042, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38459626

ABSTRACT

In the context of rare genetic diseases caused by nonsense mutations, the concept of induced stop codon readthrough (SCR) represents an attractive avenue in the ongoing search for improved treatment options. Epidermolysis bullosa (EB)-exemplary for this group of diseases-describes a diverse group of rare, blistering genodermatoses. Characterized by extreme skin fragility upon minor mechanical trauma, the most severe forms often result from nonsense mutations that lead to premature translation termination and loss of function of essential proteins at the dermo-epidermal junction. Since no curative interventions are currently available, medical care is mainly limited to alleviating symptoms and preventing complications. Complementary to attempts of gene, cell and protein therapy in EB, SCR represents a promising medical alternative. While gentamicin has already been examined in several clinical trials involving EB, other potent SCR inducers, such as ataluren, may also show promise in treating the hitherto non-curative disease. In addition to the extensively studied aminoglycosides and their derivatives, several other substance classes-non-aminoglycoside antibiotics and non-aminoglycoside compounds-are currently under investigation. The extensive data gathered in numerous in vitro experiments and the perspectives they reveal in the clinical setting will be discussed in this review.


Subject(s)
Codon, Nonsense , Epidermolysis Bullosa , Humans , Codon, Terminator , Gentamicins/pharmacology , Gentamicins/therapeutic use , Aminoglycosides/pharmacology , Aminoglycosides/therapeutic use , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/pharmacology , Epidermolysis Bullosa/genetics , Epidermolysis Bullosa/therapy
2.
J Opt Soc Am A Opt Image Sci Vis ; 40(7): 1382-1391, 2023 Jul 01.
Article in English | MEDLINE | ID: mdl-37706739

ABSTRACT

Modern giant segmented mirror telescopes (GSMTs) such as the Extremely Large Telescope, which is currently under construction, depend heavily on adaptive optics (AO) systems to correct for atmospheric distortions. However, a residual blur always remains in the astronomical images corrected by single conjugate AO (SCAO) systems due to fitting and bandwidth errors, which can mathematically be described by a convolution of the true image with a point spread function (PSF). Due to the nature of the turbulent atmosphere and its correction, the PSF is spatially varying, which is known as an anisoplanatic effect. The PSF serves, e.g., as a quality measure for science images and therefore needs to be known as accurately as possible. In this paper, we present an algorithm for PSF reconstruction from pupil-plane data in directions apart from the guide star direction in an SCAO system. Our algorithm is adapted to the needs of GSMTs focused on estimating the contribution of the anisoplanatic and generalized fitting error to the PSF. Results obtained in an end-to-end simulation tool show a qualitatively good reconstruction of the PSF compared to the PSF calculated directly from the simulated incoming wavefront as well as stable performance with respect to imprecise knowledge of atmospheric parameters.

3.
Int J Mol Sci ; 24(7)2023 Mar 23.
Article in English | MEDLINE | ID: mdl-37047074

ABSTRACT

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.


Subject(s)
Codon, Nonsense , Genetic Diseases, Inborn , Peptide Chain Elongation, Translational , Precision Medicine , Rare Diseases , Suppression, Genetic , Animals , Humans , Breast Neoplasms/genetics , Breast Neoplasms/therapy , Codon, Nonsense/genetics , Cystic Fibrosis/genetics , Cystic Fibrosis/therapy , Epidermolysis Bullosa/genetics , Epidermolysis Bullosa/therapy , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/therapy , Nephritis, Hereditary/genetics , Nephritis, Hereditary/therapy , Nonsense Mediated mRNA Decay , Peptide Chain Elongation, Translational/drug effects , Precision Medicine/methods , Precision Medicine/trends , Rare Diseases/genetics , Rare Diseases/therapy , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/therapy , Shwachman-Diamond Syndrome/genetics , Shwachman-Diamond Syndrome/therapy , Suppression, Genetic/drug effects , Suppression, Genetic/genetics , Peptide Chain Termination, Translational/drug effects , Aminoglycosides/pharmacology
4.
Front Bioeng Biotechnol ; 9: 725498, 2021.
Article in English | MEDLINE | ID: mdl-34513814

ABSTRACT

Cultivations of mammalian cells are routinely conducted in shake flasks. In contrast to instrumented bioreactors, reliable options for non-invasive, time-resolved monitoring of the culture status in shake flasks are lacking. The Respiration Activity Monitoring Respiration Activity Monitoring System system was used to determine the oxygen transfer rate (OTR) in shake flasks. It was proven that the OTR could be regarded as equal to the oxygen uptake rate as the change of the dissolved oxygen concentration in the liquid phase over time was negligibly small. Thus, monitoring the oxygen transfer rate (OTR) was used to increase the information content from shake flask experiments. The OTR of a Chinese hamster ovary cell line was monitored by applying electrochemical sensors. Glass flasks stoppered with cotton plugs and polycarbonate flasks stoppered with vent-caps were compared in terms of mass transfer characteristics and culture behavior. Similar mass transfer resistances were determined for both sterile closures. The OTR was found to be well reproducible within one experiment (standard deviation <10%). It correlated with changes in cell viability and depletion of carbon sources, thus, giving more profound insights into the cultivation process. Culture behavior in glass and polycarbonate flasks was identical. Monitoring of the OTR was applied to a second culture medium. Media differed in the maximum OTR reached during cultivation and in the time when all carbon sources were depleted. By applying non-invasive, parallelized, time-resolved monitoring of the OTR, the information content and amount of data from shake flask experiments was significantly increased compared to manual sampling and offline analysis. The potential of the technology for early-stage process development was demonstrated.

5.
Exp Dermatol ; 30(8): 1009-1022, 2021 08.
Article in English | MEDLINE | ID: mdl-33600038

ABSTRACT

Continuous exposure of the skin to environmental, mechanical and chemical stress necessitates constant self-renewal of the epidermis to maintain its barrier function. This self-renewal ability is attributed to epidermal stem cells (EPSCs), which are long-lived, multipotent cells located in the basal layer of the epidermis. Epidermal homeostasis - coordinated proliferation and differentiation of EPSCs - relies on fine-tuned adaptations in gene expression which in turn are tightly associated with specific epigenetic signatures and metabolic requirements. In this review, we will briefly summarize basic concepts of EPSC biology and epigenetic regulation with relevance to epidermal homeostasis. We will highlight the intricate interplay between mitochondrial energy metabolism and epigenetic events - including miRNA-mediated mechanisms - and discuss how the loss of epigenetic regulation and epidermal homeostasis manifests in skin disease. Discussion of inherited epidermolysis bullosa (EB) and disorders of cornification will focus on evidence for epigenetic deregulation and failure in epidermal homeostasis, including stem cell exhaustion and signs of premature ageing. We reason that the epigenetic and metabolic component of epidermal homeostasis is significant and warrants close attention. Charting epigenetic and metabolic complexities also represents an important step in the development of future systemic interventions aimed at restoring epidermal homeostasis and ameliorating disease burden in severe skin conditions.


Subject(s)
Epidermis/metabolism , Epigenesis, Genetic , Homeostasis , Skin Diseases/genetics , Cell Differentiation/genetics , Humans , Skin Diseases/metabolism
6.
Mol Ther Nucleic Acids ; 18: 496-507, 2019 Dec 06.
Article in English | MEDLINE | ID: mdl-31670199

ABSTRACT

Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.

7.
Biotechnol J ; 14(3): e1800219, 2019 Mar.
Article in English | MEDLINE | ID: mdl-29989353

ABSTRACT

With the ability to affect multiple genes and fundamental pathways simultaneously, miRNA engineering of Chinese Hamster Ovary (CHO) cells has significant advantages over single gene expression or repression. Tight control of these molecular triggers is desirable as it could in theory allow on/off or even tunable regulation of desirable cellular phenotypes. The present study investigated the potential of employing a tetracycline inducible (TET-On) system for conditional knockdown of specific miRNAs but encountered several challenges. The authors show a significant reduction in cell proliferation and culture viability when maintained in media supplemented with the TET-On induction agent Doxycycline at concentrations commonly reported. Calculation of a mature miRNA and miRNA sponge mRNA copy number demonstrates that leaky basal transgene expression in the un-induced state, is sufficient for significant miRNA knockdown. This work highlights challenges of the TET-On inducible expression system for controlled manipulation of endogenous miRNAs with two examples; miR-378 and miR-455. The authors suggest a solution involving isolation of highly inducible clones and use a single cell analysis platform to demonstrate the heterogeneity of basal expression and inducibility. Finally, the authors describe numerous strategies to minimize leaky transgene expression and alterations to current miRNA sponge design.


Subject(s)
Gene Expression/genetics , MicroRNAs/genetics , Tetracycline/pharmacology , Animals , CHO Cells , Cell Line , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cricetulus , Doxycycline/pharmacology , Gene Expression/drug effects , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Genetic Vectors/genetics , Transgenes/genetics
8.
Hepatology ; 69(1): 222-236, 2019 01.
Article in English | MEDLINE | ID: mdl-30014484

ABSTRACT

Transforming growth factor (TGF)-ß suppresses early hepatocellular carcinoma (HCC) development but triggers pro-oncogenic abilities at later stages. Recent data suggest that the receptor tyrosine kinase Axl causes a TGF-ß switch toward dedifferentiation and invasion of HCC cells. Here, we analyzed two human cellular HCC models with opposing phenotypes in response to TGF-ß. Both HCC models showed reduced proliferation and clonogenic growth behavior following TGF-ß stimulation, although they exhibited differences in chemosensitivity and migratory abilities, suggesting that HCC cells evade traits of anti-oncogenic TGF-ß. Transcriptome profiling revealed differential regulation of the chemokine CXCL5, which positively correlated with TGF-ß expression in HCC patients. The expression and secretion of CXCL5 was dependent on Axl expression, suggesting that CXCL5 is a TGF-ß target gene collaborating with Axl signaling. Loss of either TGF-ß or Axl signaling abrogated CXCL5-dependent attraction of neutrophils. In mice, tumor formation of transplanted HCC cells relied on CXCL5 expression. In HCC patients, high levels of Axl and CXCL5 correlated with advanced tumor stages, recruitment of neutrophils into HCC tissue, and reduced survival. Conclusion: The synergy of TGF-ß and Axl induces CXCL5 secretion, causing the infiltration of neutrophils into HCC tissue. Intervention with TGF-ß/Axl/CXCL5 signaling may be an effective therapeutic strategy to combat HCC progression in TGF-ß-positive patients.


Subject(s)
Carcinoma, Hepatocellular/immunology , Chemokine CXCL5/physiology , Liver Neoplasms/immunology , Neutrophil Infiltration , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/physiology , Transforming Growth Factor beta/physiology , Animals , Humans , Mice , Tumor Cells, Cultured , Axl Receptor Tyrosine Kinase
9.
Appl Opt ; 55(6): 1421-9, 2016 Feb 20.
Article in English | MEDLINE | ID: mdl-26906596

ABSTRACT

The imaging quality of modern ground-based telescopes such as the planned European Extremely Large Telescope is affected by atmospheric turbulence. In consequence, they heavily depend on stable and high-performance adaptive optics (AO) systems. Using measurements of incoming light from guide stars, an AO system compensates for the effects of turbulence by adjusting so-called deformable mirror(s) (DMs) in real time. In this paper, we introduce a novel reconstruction method for ground layer adaptive optics. In the literature, a common approach to this problem is to use Bayesian inference in order to model the specific noise structure appearing due to spot elongation. This approach leads to large coupled systems with high computational effort. Recently, fast solvers of linear order, i.e., with computational complexity O(n), where n is the number of DM actuators, have emerged. However, the quality of such methods typically degrades in low flux conditions. Our key contribution is to achieve the high quality of the standard Bayesian approach while at the same time maintaining the linear order speed of the recent solvers. Our method is based on performing a separate preprocessing step before applying the cumulative reconstructor (CuReD). The efficiency and performance of the new reconstructor are demonstrated using the OCTOPUS, the official end-to-end simulation environment of the ESO for extremely large telescopes. For more specific simulations we also use the MOST toolbox.

10.
Retrovirology ; 12: 74, 2015 Aug 22.
Article in English | MEDLINE | ID: mdl-26297639

ABSTRACT

BACKGROUND: HIV-1 protease (PR) is essential for viral infectivity as it cleaves Gag and Gag-Pol polyprotein precursors during viral maturation. Recent evidence suggests that cellular proteins can also be cleaved by PR, perhaps representing an important viral strategy to counter host defense mechanisms. Receptor-interacting protein kinase 1 (RIPK1) and RIPK2 belong to a family of serine/threonine kinases with conserved domain architecture and important functions in apoptosis, necrosis and innate immunity. RESULTS: We found that RIPK1 and RIPK2 but not other members of the RIP kinase family are cleaved by HIV-1 PR. In RIPK1, we identified a putative PR cleavage site; a mutation at this site rendered RIPK1 resistant to PR cleavage. RIPK1 and RIPK2 were cleaved during HIV-1 infection of T cell lines or primary activated CD4(+) T cells. Interfering with the viral life cycle at different stages by the addition of specific inhibitors against RT, integrase, or PR, completely prevented RIPK1 and RIPK2 cleavage. Cleavage of RIPK1 disrupted RIPK1/RIPK3 complex formation and RIPK1-mediated induction of NF-kB. CONCLUSIONS: These findings indicate that RIPK1 and RIPK2 are targets of HIV-1 PR activity during infection, and their inactivation may contribute to modulation of cell death and host defense pathways by HIV-1.


Subject(s)
HIV Protease/metabolism , HIV-1/enzymology , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , CD4-Positive T-Lymphocytes/virology , Cell Death , Cells, Cultured , HEK293 Cells , HIV-1/drug effects , HIV-1/growth & development , HIV-1/physiology , Humans , Jurkat Cells , NF-kappa B/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/genetics
11.
Cytotechnology ; 66(3): 493-514, 2014 May.
Article in English | MEDLINE | ID: mdl-23775287

ABSTRACT

Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 µg mL(-1) 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These 'nucleotide ratios' changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.

12.
BMC Surg ; 13: 29, 2013 Jul 26.
Article in English | MEDLINE | ID: mdl-23890488

ABSTRACT

BACKGROUND: Bilateral pneumothoraces after cosmetic breast surgery are rare and sporadically reported in the literature. CASE PRESENTATION: A 65-year-old female patient developed bilateral pneumothoraces after bilateral breast reduction surgery. Emergent chest tube thoracostomy was performed on both sides. The chest drains were removed on the fourth day (left side) and sixth day (right side), and the patient was discharged after 7 days of hospitalization without any further complications. CONCLUSION: To our knowledge, the English-language literature contains no other reports of bilateral pneumothoraces after reduction mammoplasty.


Subject(s)
Mammaplasty/adverse effects , Pneumothorax/etiology , Aged , Female , Humans
13.
Tissue Barriers ; 1(2): e25039, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-24665396

ABSTRACT

Zonula occludens proteins (ZO-1, ZO-2, ZO-3), which belong to the family of membrane-associated guanylate kinase (MAGUK) homologs, serve as molecular hubs for the assembly of multi-protein networks at the cytoplasmic surface of intercellular contacts in epithelial and endothelial cells. These multi-PDZ proteins exert crucial functions in the structural organization of intercellular contacts and in transducing intracellular signals from the plasma membrane to the nucleus. The junctional MAGUK protein ZO-2 not only associates with the C-terminal PDZ-binding motif of various transmembrane junctional proteins but also transiently targets to the nucleus and interacts with a number of nuclear proteins, thereby modulating gene expression and cell proliferation. Recent evidence suggests that ZO-2 is also involved in stress response and cytoprotective mechanisms, which further highlights the multi-faceted nature of this PDZ domain-containing protein. This review focuses on ZO-2 acting as a molecular scaffold at the cytoplasmic aspect of tight junctions and within the nucleus and discusses additional aspects of its cellular activities. The multitude of proteins interacting with ZO-2 and the heterogeneity of proteins either influencing or being influenced by ZO-2 suggests an exceptional functional capacity of this protein far beyond merely serving as a structural component of cellular junctions.

14.
Biochemistry ; 51(37): 7330-41, 2012 Sep 18.
Article in English | MEDLINE | ID: mdl-22928810

ABSTRACT

NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2-α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD-PYD interaction specificity.


Subject(s)
Models, Molecular , Protein Folding , Repressor Proteins/chemistry , Adaptor Proteins, Signal Transducing , Crystallography, X-Ray , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Protein Structure, Tertiary , Repetitive Sequences, Amino Acid , Repressor Proteins/genetics , Structure-Activity Relationship
15.
Innate Immun ; 18(1): 100-11, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21310790

ABSTRACT

Nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are intracellular pattern recognition receptors (PRRs) of the nucleotide-binding domain and leucine-rich repeat containing (NLR) gene family involved in innate immune responses. Their centrally located NACHT domain displays ATPase activity and is necessary for activation and oligomerization leading to inflammatory signaling responses. Mutations affecting key residues of the ATPase domain of NOD2 are linked to severe auto-inflammatory diseases, such as Blau syndrome and early-onset sarcoidosis. By mutational dissection of the ATPase domain function, we show that the NLR-specific extended Walker B box (DGhDE) can functionally replace the canonical Walker B sequence (DDhWD) found in other ATPases. A requirement for an intact Walker A box and the magnesium-co-ordinating aspartate of the classical Walker B box suggest that an initial ATP hydrolysis step is necessary for activation of both NOD1 and NOD2. In contrast, a Blau-syndrome associated mutation located in the extended Walker B box of NOD2 that results in higher autoactivation and ligand-induced signaling does not affect NOD1 function. Moreover, mutation of a conserved histidine in the NACHT domain also has contrasting effects on NOD1 and NOD2 mediated NF-κB activation. We conclude that these two NLRs employ different modes of activation and propose distinct models for activation of NOD1 and NOD2.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Cranial Nerve Diseases/genetics , Nod1 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/genetics , Sarcoidosis/genetics , Synovitis/genetics , Uveitis/genetics , Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Arthritis , Cranial Nerve Diseases/immunology , DNA Mutational Analysis , HeLa Cells , Humans , Immunity, Innate , Mutation/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , NLR Proteins , Nod1 Signaling Adaptor Protein/immunology , Nod1 Signaling Adaptor Protein/metabolism , Nod2 Signaling Adaptor Protein/immunology , Nod2 Signaling Adaptor Protein/metabolism , Polymorphism, Genetic , Protein Structure, Tertiary/genetics , Sarcoidosis/immunology , Signal Transduction/genetics , Synovitis/immunology , Transcriptional Activation/genetics , Uveitis/immunology
16.
PLoS One ; 6(11): e27396, 2011.
Article in English | MEDLINE | ID: mdl-22087307

ABSTRACT

The "Function to Find Domain" (FIIND)-containing proteins CARD8 (Cardinal; Tucan) and NLRP1 (NALP1; NAC) are well known components of inflammasomes, multiprotein complexes responsible for activation of caspase-1, a regulator of inflammation and innate immunity. Although identified many years ago, the role of the FIIND is unknown. Here, we report that CARD8 and NLRP1 undergo autoproteolytic cleavage at a conserved SF/S motif within the FIIND. Using bioinformatics and computational modeling approaches, we detected striking structural similarity between the FIIND and the ZU5-UPA domain present in the autoproteolytic protein PIDD. This allowed us to generate a three-dimensional model and to gain insights in the molecular mechanism of the cleavage. Site-directed mutagenesis experiments revealed that the second serine of the SF/S motif is required for CARD8 and NLRP1 autoproteolysis. Furthermore, we discovered an important function for conserved glutamic acid and histidine residues, located in proximity of the cleavage site in regulating the autoprocessing efficiency. Altogether, these results identify a function for the FIIND and show that CARD8 and NLRP1 are ZU5-UPA domain-containing autoproteolytic proteins, thus suggesting a novel mechanism for regulating innate immune responses.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , CARD Signaling Adaptor Proteins/metabolism , Neoplasm Proteins/metabolism , Peptide Hydrolases/metabolism , Binding Sites , Cell Line , Humans , Immunity, Innate , Inflammasomes , Multiprotein Complexes/immunology , NLR Proteins , Protein Structure, Tertiary
18.
Science ; 327(5964): 435-9, 2010 Jan 22.
Article in English | MEDLINE | ID: mdl-20007863

ABSTRACT

Since 2004, Saturn's moon Iapetus has been observed repeatedly with the Imaging Science Subsystem of the Cassini spacecraft. The images show numerous impact craters down to the resolution limit of approximately 10 meters per pixel. Small, bright craters within the dark hemisphere indicate a dark blanket thickness on the order of meters or less. Dark, equator-facing and bright, poleward-facing crater walls suggest temperature-driven water-ice sublimation as the process responsible for local albedo patterns. Imaging data also reveal a global color dichotomy, wherein both dark and bright materials on the leading side have a substantially redder color than the respective trailing-side materials. This global pattern indicates an exogenic origin for the redder leading-side parts and suggests that the global color dichotomy initiated the thermal formation of the global albedo dichotomy.


Subject(s)
Ice , Saturn , Water , Color , Extraterrestrial Environment , Spacecraft , Temperature
19.
PLoS One ; 4(4): e4931, 2009.
Article in English | MEDLINE | ID: mdl-19337385

ABSTRACT

Members of the Nod-like receptor (NLR) family recognize intracellular pathogens and recruit a variety of effector molecules, including pro-caspases and kinases, which in turn are implicated in cytokine processing and NF-kappaB activation.In order to elucidate the intricate network of NLR signaling, which is still fragmentary in molecular terms, we applied comprehensive yeast two-hybrid analysis for unbiased evaluation of physical interactions between NLRs and their adaptors (ASC, CARD8) as well as kinase RIPK2 and inflammatory caspases (C1, C2, C4, C5) under identical conditions. Our results confirmed the interaction of NOD1 and NOD2 with RIPK2, and between NLRP3 and ASC, but most importantly, our studies revealed hitherto unrecognized interactions of NOD2 with members of the NLRP subfamily. We found that NOD2 specifically and directly interacts with NLRP1, NLRP3 and NLRP12. Furthermore, we observed homodimerization of the RIPK2 CARD domains and identified residues in NOD2 critical for interaction with RIPK2.In conclusion, our work provides further evidence for the complex network of protein-protein interactions underlying NLR function.


Subject(s)
Nod Signaling Adaptor Proteins/metabolism , Binding Sites , Cell Line , Humans , Protein Binding , Two-Hybrid System Techniques
20.
Ger Med Sci ; 5: Doc06, 2007 Sep 03.
Article in English | MEDLINE | ID: mdl-19675714

ABSTRACT

METHODS: From 1 July 1995 to 31 June 2006 we implanted 3498 intravenous port systems. In nearly all cases the indication was vascular access for chemotherapy. RESULTS: We registered 199 complications (5.7%), mostly infections (n=85 i.e. 2.4%) and thromboses (n=63 i.e. 1.8%). CONCLUSIONS: Permanent central venous catheters have become standard in the management of patients with malignancies. Because of the improvement of material and design during the past twenty years technical complications have been reduced significantly. The most frequent occurring medical complications are infection and thromboses. In order to further minimize these disadvantages we developed a "best practices" standard for port implantation combining own data with recent studies.

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