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In the version of this article initially published, in the Acknowledgements, the grant number for the Start Grant TWILIGHT awarded to M.A.L. by the European Research Council was omitted. The correct wording is “Supported by...the European Research Council (...Start Grant TWILIGHT (637801) to M.A.L.).” The error has not been corrected in the original article. © 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.

Tenascin-X, is an extracellular matrix glycoprotein expressed in skin, muscle, tendons, and blood vessels with an anti-adhesive function. Biallelic Tenascin-X mutations cause classical-like Ehlers-Danlos syndrome. We report a 46-year-old woman with slowly progressive weakness of the lower limbs and myalgia from age 28 years. In the past she had Raynaud's phenomenon, multiple sprains and joint dislocations, conjunctival haemorrhages and a colonic perforation during colonoscopy. Neurologic examination showed moderate asymmetric proximal and axial muscular weakness, distal amyotrophy of 4 limbs, moderate skin hyperextensibility, and hypermobility of distal joints of fingers. Whole body Magnetic Resonance Imaging showed symmetric fatty infiltration of thigh and leg muscles, with predominant atrophy of thighs. Next Generation Sequencing revealed two pathogenic TNXB variants, g.32024681C>G, c.7826-1G>C, and g.32016181dup, c.9998dupA, p.(Asn3333Lysfs* 35). Western Blot and immunofluorescence studies confirmed a marked Tenascin-X reduction in both patient's serum and muscle. Here we further detail the clinical and genetic spectrum of a patient with classical-like Ehlers-Danlos syndrome and prominent muscle involvement. (C) 2020 Elsevier B.V. All rights reserved.

Contains fulltext : 182646.pdf (Publisher’s version ) (Open Access) Terminally differentiating epidermal keratinocytes express a large number of structural and antimicrobial proteins that are involved in the physical barrier function of the stratum corneum and provide innate cutaneous host defense. Late cornified envelope (LCE) genes, located in the epidermal differentiation complex on chromosome 1, encode a family of 18 proteins of unknown function, whose expression is largely restricted to epidermis. Deletion of two members, LCE3B and LCE3C (LCE3B/C-del), is a widely-replicated psoriasis risk factor that interacts with the major psoriasis-psoriasis risk gene HLA-C*06. Here we performed quantitative trait locus analysis, utilizing RNA-seq data from human skin and found that LCE3B/C-del was associated with a markedly increased expression of LCE3A, a gene directly adjacent to LCE3B/C-del. We confirmed these findings in a 3-dimensional skin model using primary keratinocytes from LCE3B/C-del genotyped donors. Functional analysis revealed that LCE3 proteins, and LCE3A in particular, have defensin-like antimicrobial activity against a variety of bacterial taxa at low micromolar concentrations. No genotype-dependent effect was observed for the inside-out or outside-in physical skin barrier function. Our findings identify an unknown biological function for LCE3 proteins and suggest a role in epidermal host defense and LCE3B/C-del-mediated psoriasis risk.

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Background: Defensins are antimicrobial peptides that exert immunomodulatory and chemotactic functions. Based on these properties and their high expression levels in the skin, they are likely to affect skin inflammation, infection, and wound healing. This may lead to therapeutic applications in (burn) wound healing. Objective: We aimed to investigate the effects of human β-defensins (hBDs) on keratinocytes and fibroblasts, 2 major skin cell types involved in skin regeneration. Methods: Monolayer keratinocyte and fibroblast cultures were exposed to recombinant hBDs, and we overexpressed hBD2 and hBD3 in keratinocytes of reconstructed epidermal equivalents by lentiviral transduction. The effects were measured by immunohistochemistry, quantitative real-time PCR, and migration assays. Kinome analyses were performed on cultured keratinocytes to investigate the signal transduction events elicited by hBD stimulation. Results: We found that hBD3 induced the expression of cytokines and chemokines in keratinocytes, which was not observed in fibroblasts. hBD2, however, stimulated cell migration only in fibroblasts, which was not found for hBD3. Both defensins are likely to exert receptor-mediated effects in keratinocytes, as witnessed by changes in protein kinase activation following stimulation by hBD2 and hBD3. Kinome analysis suggested that protein kinase C activation was a common event for both defensins. We observed, however, considerable differences in keratinocyte responses between stimulation by exogenous recombinant defensins and endogenous defensins expressed following lentiviral transduction. Conclusion: Defensins exert modest biological effects on skin cells that are potentially beneficial in wound healing, but many questions regarding the biological mechanisms of action and relevance for the in vivo situation are still remaining.

The low-frequency contribution to the systematic and random sampling errors in single-tower eddy-covariance flux measurements is investigated using large-eddy simulation (LES). We use a continuous LES integration that covers a full year of realistic weather conditions over Cabauw, the Netherlands, and emulate eddy-covariance measurements. We focus on the daytime flux imbalance, when the turbulent flux is sufficiently resolved. Averaged over the year, daytime single-tower eddy-covariance flux measurements lead to a significant systematic underestimation of the turbulent flux. This underestimation depends on the averaging period and measurement height. For a 3600-s averaging period at 16-m height, the systematic underestimation reduces to a few percent, but for 900-s averaged tall-tower measurements at 100-m height, the fluxes are systematically underestimated by over 20 %. The year-long dataset facilitates an investigation into the environmental conditions that influence the eddy-covariance flux imbalance. The imbalance problem is found to vary widely from day to day, strongly dependent on the flow regime. In general, the imbalance problem reduces with increased mean wind speed, but days having the largest imbalance (over twice the average) are characterized by roll vortices that occur for average wind speeds, typically having a boundary-layer height (zi) to Obukhov length (L) ratio of 10zi/L<100.

Contains fulltext : 147170.pdf (Publisher’s version ) (Open Access) The present invention concerns novel pantothenone compounds having pantetheinase inhibitory activity as well as antiplasmodial activity. These compounds can suitably be used in therapeutic and or prophylactic treatment of malaria. Furthermore, the present invention provides combinations of pantothenone compounds and pantothenamides. Combining a pantothenone with a pantothenamide increases the antimalarial potency by an order of magnitude. It is hypothesized that inhibition of pantetheinase activity could protect pantothenamides against degradation by serum-derived pantetheinases, thereby revealing the hitherto unknown antimalaria activity of pantothenamides. The present invention thus, for the first time, makes available compounds and combinations of compounds for use in therapeutic and or prophylactic treatment of malaria infection in a human or animal subject in need thereof, relying on interference with host or pathogen-derived pantetheinase dependent pathways. 08 december 2011

De vorige eeuw was getuige van de opkomst van numerieke weer- en klimaatmodellen. Weersvoorspelling, voorheen een empirische procedure gebaseerd op de persoonlijke ervaring van de meteoroloog, ontwikkelde zich tot een wetenschappelijke methode om de toekomstige toestand van de atmosfeer te voorspellen op basis van de huidige toestand en tendens. Tegelijkertijd werden klimaatmodellen ontwikkeld om inzicht te krijgen in het gedrag van ons klimaat over tijdspannes van meer dan honderden jaren. Als gevolg van de enorme rekenkracht die vereist is om deze weer- of klimaatmodellen te kunnen draaien, ontstond een nauwe verwikkeling tussen atmosferische en computerwetenschap. Door de combinatie van toenemende computerkracht, beschikbaarheid van globale observaties als invoer en de onophoudelijk doorontwikkeling van weer- en klimaatmodellen, kan de wereldwijde atmosferische stroming nu worden berekend met een ongeloo?ijke nauwkeurigheid. De resterende achilleshiel van weer- en klimaatmodellen blijft echter de representatie van fenomenen op de schaal van turbulentie, zoals turbulent transport, wolken en neerslag. Deze fenomenen kunnen (nog) niet opgelost worden door hedendaagse weer- en klimaatmodellen (daar hebben deze modellen te weinig detail voor), waardoor ze vertegenwoordigd moeten worden door parametrische schema’s. Om de kwaliteit van dit soort schema’s te testen zijn specialistische modellen ontwikkeld, zogenaamde Large-Eddy Simulations (LES), die een beperkt oppervlak betrekken waarop wolken wel realistisch kunnen worden opgelost. In dit proefschrift worden LES modellen ingezet met het doel weer en klimaatvoorspellingen te verbeteren. In hoofdstuk twee worden LES resultaten gebruikt om een analytisch ’geraamte’ op te zetten met betrekking tot de evenwichtsoplossingen van cumulus-bewolking (stapelwolken) voor verschillende grootschalige weersituaties. Zo’n opzet kan ons begrip verbeteren van de manier waarop wolken reageren als de grootschalige situatie veranderd. Gezien weer- en klimaatmodellen projecties kunnen maken van de grootschalige situatie, kunnen we zo beter voorspellen hoe wolken zich zullen gedragen. De ’tradionele’ manier om kennis te combineren van LES en van weer- en klimaatstudies, zoals hierboven beschreven, leunt op de mogelijkheid om de grootschalige situatie te onderscheiden van de kleinschalige situatie. Dit is tegenwoordig echter helemaal niet meer vanzelfsprekend. Computerkracht neemt dermate snel toe dat we binnen twintig jaar in staat zullen zijn om de ontwikkeling van de globale atmosfeer voor de komende week te berekenen op een turbulentie-oplossende manier: dus op een detailniveau dat het mogelijk maakt turbulentie en individuele (stapel)wolken te beschrijven. Traditionele weer- en klimaatmodellen zijn echter niet ontworpen voor dit detailniveau, en kunnen dit niet aan zonder grote ingrepen. LES modellen zijn wel ontworpen voor dit detailniveau, maar niet voor grote oppervlaktes. Het ziet er dus naar uit dat toekomstige weer- en klimaatmodellen kenmerken van beide specializaties zullen krijgen. De rode draad van dit proefschrift is de studie in hoeverre we LES kunnen gebruiken om het ’werkelijke’ weer of klimaat te modelleren. Door de grote hoeveelheid benodigde rekenkracht worden LES modellen typisch alleen toegepast voor geidealiseerde casussen. In hoofdstuk drie wordt echter de mogelijkheid onderzocht om een nieuwe computertechniek toe te passen bij LES modellering: het gebruik van gra?sche co-processor chips (graphics processing unit, GPU). Het blijkt dat als we ons LES model aanpassen op het gebruik van deze chips, het model zodanig versneld kan worden dat nieuwe toepassingen, bijvoorbeeld in weersvoorspelling, mogelijk worden. Het GPU-LES model wordt daarom uitgerust met procedures om op een realistische manier de grond-atmosfeer interactie en de grootschalige invloeden te behandelen. In hoofdstuk vier testen we het model door middel van simulaties van het dagelijkse weer rondom Cabauw voor het gehele jaar 2012. Deze simulaties laten zien dat het LES model waarde kan toevoegen door zijn gedetailleerde representatie van turbulentie en wolkenprocessen. Daarnaast wordt in hoofdstuk vijf deze simulatie gebruikt om inzicht te geven in de kwaliteit van metingen van turbulent transport van warmte en vocht. Deze metingen zijn van groot belang om een goede weerspiegeling van de atmosferische grenslaag te kunnen maken in weer- en klimaatmodellen. Tenslotte wordt in hoofdstuk zes de grootste GPU-supercomputer van Europa ingezet om een ’proof of concept’ te bewerkstelligen van turbulentie-oplossende weersvoorspelling over Nederland met behulp van LES. Dit is een eerste kleine stap richting turbulentie-oplossende (dus met een detailniveau van individuele stapelwolken) weersvoorspelling vanuit een LES invalshoek. Het onderzoek in dit proefschrift kan zo een weg banen voor een nadere fusie van wolkensimulaties en globale weer- en klimaatmodellen.

Item does not contain fulltext PlsX is an acyl-acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram-positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The DeltaplsX S. pneumoniae strain was refractory to myristic acid-dependent growth arrest, and unlike the wild-type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The DeltaplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18- and 20-carbon saturated fatty acids was detected in the DeltaplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl-ACP in vitro, and the DeltatesS DeltaplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl-ACP in the DeltaplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the DeltaplsX strains of S. aureus and S. pneumoniae.

Contains fulltext : 147172.pdf (Publisher’s version ) (Open Access) 08 december 2011

Contains fulltext : 50224.pdf (Publisher’s version ) (Open Access) Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine proteases, no high affinity binding for any member of this family has been demonstrated so far. We report that human cathepsin V (CTSV) and human cathepsin L (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that Ki values of cystatin M/E for the interaction with CTSV and CTSL are 0.47 and 1.78 nM, respectively. On the basis of the analogous sites in cystatin C, we used site-directed mutagenesis to identify the binding sites of these proteases in cystatin M/E. We found that the W135A mutant was rendered inactive against CTSV and CTSL but retained legumain-inhibiting activity. Conversely, the N64A mutant lost legumain-inhibiting activity but remained active against the papain-like cysteine proteases. We conclude that legumain and papain-like cysteine proteases are inhibited by two distinct non-overlapping sites. Using immunohistochemistry on normal human skin, we found that cystatin M/E co-localizes with CTSV and CTSL. In addition, we show that CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3. The identification of CTSV and CTSL as novel targets for cystatin M/E, their (co)-expression in the stratum granulosum of human skin, and the activity of CTSL toward transglutaminase 3 strongly imply an important role for these enzymes in the differentiation process of human epidermis.

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Contains fulltext : 57333.pdf (Publisher’s version ) (Open Access) Contains fulltext : 57333schalkwijk.pdf (Publisher’s version ) (Closed access) Contains fulltext : 57333timmermans.pdf (Publisher’s version ) (Closed access) BACKGROUND: We recently described a new autosomal recessive type of Ehlers-Danlos syndrome (EDS) based on a deficiency of the extracellular matrix protein tenascin-X (TNX). TNX-deficient patients have hypermobile joints, hyperextensible skin and show easy bruising. Because of the reported cardiovascular abnormalities in other EDS types and the excessive haematoma formation after mild trauma in TNX-deficient individuals, we investigated whether cardiovascular or coagulation abnormalities occur in these patients. METHODS: We examined seven TNX-deficient patients. One of them had a mitral valve prolapse and died postoperatively after valve replacement, before the study was completed. RESULTS: Bleeding time and coagulation factors (INR, APTT, PT and fibrinogen) were all within the normal range. Ultrasonographic examination of the carotid and femoral arteries showed normal vessel wall compliance and distensibility. Echocardiography showed a slight billowing of the mitral valve in two patients from one family. All patients had normal diameters of aortic root and ascending aorta. CONCLUSION: Although the patient group is small, there are no indications of generalised cardiovascular abnormalities in this type of EDS. We would recommend echocardiography for all these patients at the first evaluation and when a cardiac murmur appears.

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Item does not contain fulltext Psoriasis was until recently regarded as a T-cell-driven disease with presumed (auto)immune mechanisms as its primary cause. This view was supported by clinical data and genetic studies that identified risk factors functioning in adaptive and innate immunity, such as HLA-C*06, ERAP1, the IL-23 pathway, and NF-kappaB signaling. Candidate gene approaches and genome-wide association studies, however, have identified copy number polymorphisms of the beta-defensin cluster and deletion of late cornified envelope (LCE) 3B and 3C genes (LCE3C_LCE3B-del) as psoriasis risk factors. As these genes are expressed in epithelial cells and not by the immune system, these findings may cause a change of paradigm for psoriasis, not unlike the reported filaggrin association that has profoundly changed the views on atopic dermatitis. In addition to genetic polymorphisms of the immune system, genetic variations affecting the skin barrier are likely to contribute to psoriasis. Recent studies have shown epistatic interactions involving HLA-C*06, ERAP1, and LCE3C_LCE3B-del, which makes psoriasis a unique model to investigate genetic and biological interactions of associated genes in a complex disease. We present a model for disease initiation and perpetuation, which integrates the available genetic, immunobiological, and clinical data.Journal of Investigative Dermatology advance online publication, 24 May 2012; doi:10.1038/jid.2012.167.

Contains fulltext : 107850.pdf (Publisher’s version ) (Closed access) BACKGROUND: Recent studies have emphasized the importance of heritable and acquired skin barrier abnormalities in common inflammatory diseases such as psoriasis and atopic dermatitis (AD). To date, no comprehensive studies on the effect of experimental barrier disruption on cornified envelope protein expression have been performed. OBJECTIVES: To analyse the effect of experimental skin barrier disruption on the expression of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins. METHODS: We examined mRNA (day 1, 3 and 7) and protein (day 1, 2, 4 and 9) expression levels of structural proteins and regulatory molecules after sodium dodecyl sulphate (SDS) application on normal skin, and tape stripping of uninvolved epidermis of patients with psoriasis and AD and healthy controls. RESULTS: Upon tape stripping, several structural molecules were significantly downregulated (at the mRNA level as well as the protein level), including LCE5A, LCE2B, FLG, FLG2 and LOR, whereas others were upregulated: IVL, SPRR1, SPRR2, HRNR and most notably LCE3A. The epidermal crosslinking enzymes TGM1, TGM3 and TGM5 were all upregulated, whereas proteases involved in the desquamation process (CTSV, KLK5 and KLK7) were downregulated or unaffected. Most results were similar in SDS-instigated irritant contact dermatitis. There was no significant difference in response between normal epidermis and nonlesional skin of patients with psoriasis and AD. CONCLUSIONS: Skin barrier disruption induces a temporary barrier repair response composed of increased expression of several cornification-related proteins, and decreased expression of some structural and desquamation-related proteins. 01 juni 2012

Contains fulltext : 89038schalkwijk.pdf (Publisher’s version ) (Closed access) Tenascin-X is a large extracellular matrix glycoprotein that is widely distributed within connective tissues and is associated with an autosomal recessive type of Ehlers-Danlos syndrome (EDS). Tenascin-X represents the first EDS susceptibility gene that does not code for a fibrillar collagen or collagen-processing enzyme. We describe a paediatric case of tenascin-X deficiency and review the literature. 01 december 2010

Contains fulltext : 51741.pdf (Publisher’s version ) (Open Access) Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.

Contains fulltext : 47358schalkwijk.pdf (Publisher’s version ) (Closed access) Tenascin-X is an extracellular matrix protein initially identified because the gene encoding it overlaps with the human CYP21B gene. Because studies of gene and protein function of other tenascins had been poorly predictive of essential functions in vivo, we used a genetic approach that critically relied on an understanding of the genomic locus to uncover an association between inactivating tenascin-X mutations and novel recessive and dominant forms of Ehlers-Danlos syndrome (EDS). Tenascin-X provides the first example of a gene outside of the fibrillar collagens and their processing enzymes that causes EDS. Tenascin-X null mice recapitulate the skin findings of the human disease, confirming a causative role for this gene in EDS. Further evaluation of these mice showed that tenascin-X is an important regulator of collagen deposition in vivo, suggesting a novel mechanism of disease in this form of EDS. Further studies suggest that tenascin-X may do this through both direct and indirect interactions with the collagen fibril. Recent studies show that TNX effects on matrix extend beyond the collagen to the elastogenic pathway and matrix remodeling enzymes. Tenascin-X serves as a compelling example of how human "experiments of nature" can guide us to an understanding of genes whose function may not be evident from their sequence or in vitro studies of their encoded proteins. (c) 2005 Wiley-Liss, Inc.