Computational and experimental prediction of human C-type lectin receptor druggability

Mammalian C-type lectin receptors (CTLRS) are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies, and lymphocyte homing. Despite a great interest in modulating CTLR recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 CTLRs using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable. To further explore these findings, we employed a fluorine-based nuclear magnetic resonance screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rate = 13.5%). A surface plasmon resonance-based follow-up assay confirmed 18 of these fragments (47%) and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7 and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular, with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of CTLRs can be developed

NFAT5 expression in bone marrow-derived cells enhances atherosclerosis and drives macrophage migration

Objective: We have previously shown that the transcription factor, nuclear factor of activated T-cells 5 (NFAT5), regulates vascular smooth muscle cell phenotypic modulation, but the role of NFAT5 in atherosclerosis is unknown. Our main objective was to determine if NFAT5 expression in bone marrow (BM)-derived cells altered atherosclerotic development and macrophage function. Methods and Results: NFAT5+/-ApoE-/- mice were generated for in vivo atherosclerosis studies. Following high fat diet feeding, en face analysis of the thoracic aorta established that genome-wide NFAT5 haploinsufficiency reduced atherosclerotic lesion formation by 73%. BM transplant studies revealed that transplantation of NFAT5+/-ApoE-/- marrow into NFAT5+/+ApoE-/- mice resulted in a similar 86% reduction in lesion formation. In vitro functional analysis of BM-derived macrophages demonstrated that NFAT5 is required for macrophage migration, which is a key event in the propagation of atherosclerosis. Conclusion: We have identified NFAT5 in BM-derived cells as a positive regulator of atherosclerotic lesion formation and macrophage function in the vasculature.open

The Theatre of Ambiguity

The increased diversity of beliefs surrounding the definition of gender identity and gender roles has enhanced the need for research on historical societies\u27 beliefs and practices. By examining the private and professional theatre of Elizabethan England through contemporary scholarship, primary sources, and plays of the time, I found a contrast between companies comprised entirely of boys and those comprised of adult men. Boy players were able to play female and male characters across a wide age range. My analysis shows that the Elizabethan society\u27s acceptance of this theatrical convention depended upon the widespread view of adolescents as ambiguous in age and gender identity. Playwrights, including Shakespeare, exploited this ambiguity of boys who were no longer children and not yet adults by using conventions such as double cross dressing and gender switching onstage to appeal to both male and female members of the audience. Although Puritan critics of the time like John Rainold claimed such conventions were detrimental to audiences, the backlash failed to stop or hamper the boy companies from producing art that took advantage of a culturally ambiguous understanding of gender identity

Molecular analysis and visualisation of adventitious root formation in rose (Rosa L.)

Roses (Rosa L.) are among the most important ornamental crops and of great importance in the market segments of cut flowers, garden and pot plants. Due to their highly heterozygous nature, roses must be propagated vegeta-tively to achieve true-to-type multiplication. Most cut and garden rose cultivars are still propagated by xenovegeta-tive propagation, which is labour-intensive and costly. Therefore, propagation by cuttings is of increasing interest to rose breeders. However, prerequisite for this is adventitious root (AR) formation, a complex multiphasic process that is under highly quantitative genetic control. In roses, huge genotypic differences between individual cultivars can be observed and some genotypes even do not form roots at all. Factors that limit AR formation are difficult to identify due to the complexity of the overall process, particularly in the early stages. Therefore, a better under-standing of the process of AR formation in roses on the molecular level and knowledge about the anatomical and biochemical characteristics in the different developmental stages is desirable. The aims of this study were to unravel possible constraints of AR formation in different developmental phases and to achieve a better understanding of the genetic control of this quantitative trait in rose. To characterise the genetic regulation of AR formation, existing genome-wide association studies (GWAS) in a population of 96 garden roses were followed up by analysing a larger number of genotypes and of cuttings per genotype to increase the power of the GWAS. Genotypic single nucleotide polymorphism (SNP) information generated with the 68k WagRhSNP Axiom® SNP array was available for the population of the 96 garden roses and a population of 96 cut roses. First, the popu-lations were characterised for their AR formation in vivo. The experiments were performed with stem cuttings in a hydroponic rooting system under greenhouse conditions for 6 weeks. Analyses of rooting percentage after 6 weeks, root number and root fresh mass revealed no significant associations, but several SNPs with strong allele dosage effects for these traits were identified, that were located in distinct peak regions. Of these, ten SNPs were located in the coding region of the putative phosphoinositide phosphatase SUPRESSOR OF ACTIN 9 (SAC9). This is the first di-rect evidence that the phosphoinositide (PI) signalling pathway may also be involved in the regulation of AR for-mation. For one of these SNPs - namely RhK5_69_1627P - an allele-specific PCR marker was derived and verified in an independent group of rose genotypes homozygous for this SNP. Thus, this allele-specific PCR marker can be used in rose breeding programs to improve AR formation by marker-assisted selection. In further GWAS analyses the early events in AR formation in vitro were investigated for 106 rose genotypes. The formation of root primordia (RP), AR formation and anatomical characteristics of the shoot base were correlated. Significant correlations were found for several rooting-associated traits with vasculature dimensions after 1 week of culture. In addition, it was observed that some genotypes showed RP in high numbers after 1 week, but low AR formation percentages after 3 weeks. GWAS analyses for traits at different stages of AR development showed a higher contribution of distinct genomic regions to phenotypic variation for traits at earlier developmental stages and identified nine SNPs to be significantly associated with different traits. The combination of early RP and later AR formation data as an index enabled to identify genomic regions and factors that potentially prevent RP from emer-gence. In addition, two SNPs located in homologues of genes that are involved in the PI signalling pathway showed strong allele dosage effects for RP/AR formation traits. The visualisation of the early stages in AR formation is of particular interest for genotypes with low rooting capacity, but challenging as most methods are destructive and do not allow observations over time. Magnetic resonance imaging was used to non-destructively visualise microstructures in AR formation already 3 days post excision. Both, the difficult-to-root genotype ᶦMariatheresiaᶦ (MT) and the easy-to-root genotype ᶦHerzogin Friederikeᶦ (HF) showed a pronounced formation of RP. Conspicuous differences in the MRI signal in the region of the shoot cortex in front of the RP referred to further investigation of the biochemical composition in this region of the shoot base. Spatially-resolved Fourier-transformed infrared spectroscopy revealed distinct differences in cell wall composition. In particular, absorbance for lignin and hemicelluloses were significantly higher for MT compared to HF. Thus, not the RP development limited the AR formation capacity in MT, but the emergence of RP was hindered by the altered composition and mechanical strength of the cell walls in regions of the cortex. In conclusion, by genetically dissecting AR formation using GWAS, this study contributed to a better understanding and potential improvement of AR formation in rose in breeding programmes and for the first time provided evi-dence of a possible role of the PI signalling pathway in AR formation. The combination of imaging techniques made it possible to identify anatomical and biochemical factors that limit AR formation, particularly in the early events of AR formation.Die Rose (Rosa L.) ist eine der wichtigsten Zierpflanzen und von großer Bedeutung für die Marktsegmente Schnittblumen, Garten- und Topfpflanzen. Aufgrund ausgeprägter Heterozygotie müssen Rosen für eine sortenechte Vermehrung vegetativ vermehrt werden. Die meisten Schnitt- und Gartenrosensorten werden derzeit noch xenove-getativ vermehrt, was sehr zeit- und kostenintensiv ist. Daher ist die Vermehrung durch Stecklinge von zunehmen-dem Interesse. Die dafür erforderliche Bildung von Adventivwurzeln („adventitious roots“ = ARs) ist jedoch ein kom-plexes, mehrphasiges und hochgradig quantitativ vererbtes Merkmal. Rosen weisen große genotypische Unter-schiede für dieses Merkmal auf und einige Genotypen bewurzeln gar nicht. Die Faktoren, die die Bildung von ARs limitieren, sind aufgrund der Komplexität des Prozesses, insbesondere in den frühen Entwicklungsstadien schwer zu identifizieren. Ein besseres Verständnis der Regulation der Bildung von ARs auf molekularer Ebene und Einblicke in die phänotypischen Merkmale in den verschiedenen Entwicklungsstadien sind daher wünschenswert. Ziele dieser Arbeit waren, ein besseres Verständnis der genetischen Kontrolle des quantitativen Merkmals der AR-Bildung bei Rosen zu erlangen und potentielle Limitationen zu identifizieren. Um die genetische Regulation zu charakterisieren, wurden bestehende genomweite Assoziationsstudien (GWAS) zur AR-Bildung in einer Population von 96 Gartenrosen weiterverfolgt. Dabei wurden eine größere Anzahl Genotypen und Stecklinge je Genotyp analy-siert, um die Aussagekraft der GWAS-Ergebnisse zu erhöhen. Genotypische Informationen in Form von Single Nucle-otide Polymorphismen (SNPs), ermittelt mit dem 68k WagRhSNP Axiom® SNP-Array, standen bereits für 96 Garten-rosen und 96 Schnittrosen zur Verfügung. Die Genotypen wurden auf ihre Bewurzelungsfähigkeit in vivo charakteri-siert. Die Versuche wurden 6 Wochen lang mit Stecklingen in einem hydroponischen Bewurzelungssystem unter Gewächshausbedingungen durchgeführt. GWAS zur Bewurzelung nach 6 Wochen, Wurzelanzahl und Wurzelfrisch-masse brachten keine signifikant assoziierten SNPs hervor. Allerdings zeigten mehrere SNPs starke Alleldosiseffekte für diese Merkmale. Von diesen lagen zehn SNPs in der kodierenden Region des Gens SUPRESSOR OF ACTIN 9 (SAC9). Dies ist ein erster direkter Hinweis, dass der Phosphoinositid (PI) Signalweg auch in der Regulation der Aus-bildung von ARs involviert sein kann. Für den SNP RhK5_69_1627P wurden allelspezifische PCR-Primer abgeleitet und in einer unabhängigen Gruppe selektierter, für den SNP homozygoter Genotypen verifiziert. Dieser allelspezifi-sche PCR-Marker kann somit zur Selektion zur Verbesserung der Adventivwurzelbildung in Rosenpopulationen an-gewendet werden. In weiteren GWAS-Analysen wurden frühe Ereignisse der AR-Bildung in vitro für 106 Rosen-Genotypen untersucht. Die Bildung von Wurzelprimordien („root primordia“ = RP), die Bewurzelung und anatomische Merkmale der Sprossbasis wurden korreliert. Signifikante Korrelationen zwischen mehreren Merkmalen der RP- und AR-Bildung mit der räumlichen Ausdehnung der Leitgefäße nach einwöchiger Bewurzelung wurden gefunden. Einige Genotypen bildeten eine hohe Anzahl RP nach einer Woche aus, wiesen jedoch geringe Bewurzelungsprozente nach 3 Wochen auf. GWAS-Analysen für Merkmale verschiedener Entwicklungsstadien zeigten einen höheren Beitrag genomischer Regionen zur phänotypischen Variation für Merkmale früherer Entwicklungsstadien. Insgesamt neun SNPs assoziier-ten signifikant mit verschiedenen, teils mehreren Merkmalen. Die Kombination von Daten zur frühen RP- und späte-ren AR-Bildung in Form eines Index ermöglichte es, genomische Regionen und Faktoren zu identifizieren, die mög-licherweise das Auswachsen von RP verhindern. Darüber hinaus zeigten zwei SNPs, die in Homologen von Genen liegen, welche am PI-Signalweg beteiligt sind, starke Alleldosiseffekte für Merkmale der RP/AR-Bildung. Die Visualisierung früher Stadien der AR-Bildung ist besonders für Genotypen mit geringer Bewurzelungsfähigkeit von Interesse. Aufgrund der Destruktivität der meisten Methoden waren Beobachtungen an identischen Explantaten über die Zeit bisher nicht möglich. Mittels Magnetresonanztomographie konnten mikrostrukturelle Veränderungen während der AR-Bildung bereits nach 3 Tagen an In-vitro-Stecklingen zerstörungsfrei visualisiert werden. Sowohl für den schwer zu bewurzelnden Genotyp ᶦMariatheresiaᶦ (MT) als auch den leicht zu bewurzelnden Genotyp ᶦHerzogin Friederikeᶦ (HF) wurde die Entwicklung zahlreicher RP in der Sprossbasis gezeigt. Auffällige Unterschiede im MRT-Signal im Sprosscortex initiierten weitere Untersuchungen zur biochemischen Zusammensetzung in dieser Region der Sprossbasis. Die ortsaufgelöste Fourier-transformierte Infrarotspektroskopie zeigte Unterschiede in der Zell-wandzusammensetzung. Insbesondere die Absorptionswerte für Lignin und Hemicellulosen waren bei MT im Ver-gleich zu HF deutlich höher. Somit begrenzt nicht die RP-Entwicklung die Bewurzelung bei MT, sondern das Aus-wachsen von RP war wahrscheinlich durch die veränderte Zellwandzusammensetzung in bestimmten Regionen der Sprossbasis behindert. Zusammenfassend trug diese Studie durch die genetische Analyse mittels GWAS zu einem besseren Verständnis und zur Verbesserung der AR-Bildung bei Rosen in Zuchtprogrammen bei und lieferte erstmals Hinweise auf eine mögliche Rolle des PI-Signalweges in der AR-Bildung. Darüber hinaus ermöglichte die Kombination moderner bild-gebender Verfahren die zerstörungsfreie Identifizierung anatomischer und biochemischer Faktoren, die zur Limita-tion der AR-Bildung beitragen können und das insbesondere in den frühen Stadien

Chromatin Immunoprecipitation (ChIP): Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Sheared DNA, and Analysis via PCR

The “quantitative” ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically

N,N′-Bis(4-chloro­phen­yl)urea

The carbonyl unit of the title compound, C13H10Cl2N2O, lies on a twofold rotation axis. The ring is aligned at 51.6 (1)° with respect to the N—C(=O)—N fragment. The two –NH– fragments of one mol­ecule form hydrogen bonds [2.845 (2) Å] to the C=O fragment of an adjacent mol­ecule, giving rise to the formation of a linear hydrogen-bonded chain

Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection

Apple replant disease (ARD) negatively affects growth and yield of apple plants worldwide. Fungi belonging to the Nectriaceae have often been isolated from roots grown in replant soils and thus are proposed among others as one biotic cause of the disease complex. Microscopic analyses of ARD-affected roots revealed characteristic symptoms associated with fungal infection sites. Here, two extraction methods of such tissue sites were applied to directly identify an unknown fungus that forms typical cauliflower-like structures in diseased root cortex cells. Punching small tissue samples of about 0.5 mm3 volume with the Harris Uni-Core is a quick and easy method to harvest symptomatic material. Secondly, a laser microdissection (LMD) protocol for apple roots was established. This technique allows the extraction of defined cell or tissue fractions from thin cryo-sections. Tissue harvesting was followed by the identification of fungi via PCR amplification of two gene fragments and Sanger sequencing. For Harris samples, Chelex was used for DNA stabilization, while LMD samples were directly submitted to PCR. In Harris samples, mainly the Nectriaceae species Dactylonectria torresensis, Ilyonectria robusta and Rugonectria rugulosa were identified. In addition to these, in LMD samples Cylindrocladiella sp. and Ilyonectria europaea were detected. Thus, the intracellular CF structures contained different species of Nectriaceae in the ARD-affected cortex cells. These results contribute considerably to the etiology of the ARD. Both protocols offer the possibility to identify fungi from selected symptomatic small root sections by molecular tools avoiding isolation and subsequent axenic pure cultures of single fungal isolates

GWAS of adventitious root formation in roses identifies a putative phosphoinositide phosphatase (SAC9) for marker-assisted selection

Rose propagation by cuttings is limited by substantial genotypic differences in adventitious root formation. To identify possible genetic factors causing these differences and to develop a marker for marker-assisted selection for high rooting ability, we phenotyped 95 cut and 95 garden rose genotypes in a hydroponic rooting system over 6 weeks. Data on rooting percentage after 3 to 6 weeks, root number, and root fresh mass were highly variable among genotypes and used in association mappings performed on genotypic information from the WagRhSNP 68 K Axiom SNP array for roses. GWAS analyses revealed only one significantly associated SNP for rooting percentage after 3 weeks. Nevertheless, prominent genomic regions/peaks were observed and further analysed for rooting percentage after 6 weeks, root number and root fresh mass. Some of the SNPs in these peak regions were associated with large effects on adventitious root formation traits. Very prominent were ten SNPs, which were all located in a putative phosphoinositide phosphatase SAC9 on chromosome 2 and showed very high effects on rooting percentage after 6 weeks of more than 40% difference between nulliplex and quadruplex genotypes. SAC9 was reported to be involved in the regulation of endocytosis and in combination with other members of the SAC gene family to regulate the translocation of auxin-efflux PIN proteins via the dephosphorylation of phosphoinositides. For one SNP within SAC9, a KASP marker was successfully derived and used to select genotypes with a homozygous allele configuration. Phenotyping these homozygous genotypes for adventitious root formation verified the SNP allele dosage effect on rooting. Hence, the presented KASP derived from a SNP located in SAC9 can be used for marker-assisted selection in breeding programs for high rooting ability in the future