Mechanism of actin polymerization with yeast formin Bni1p

Formin proteins are actin nucleators and elongators which can be found in most eukaryotic cells. In this work, structure-function relationships between yeast formin Bni1p and actin polymerization were studied. In the first part of this work, it was attempted to clone and express formin constructs derived from yeast Bni1p (Saccharomyces cerevisiae), including the key FH2 domain and a modified FH1 domain. Biomathematical models involving both diffusion and concentration-limited actin recruitment kinetics could be tested with such proteins. Cloning was mostly successful, but only the FH2 domain alone was expressed. In the second part of this work, a salt effect on FH2 mediated actin nucleation was discovered by means of pyrene assays and epifluorescence microscopy. Potassium chloride (KCl) is a downregulator of FH2 nucleation activity: a higher KCl concentration leads to a significantly lower actin polymerization speed (kp, m), to a bigger lag time (tlag) and to a bigger t1/2, with the respective actin filament length distributions. The salt effect was shown to be significant in a KCl concentration range from 10 mM to 90 mM at two different FH2 concentration, but not in absence of FH2. The critical KCl concentration is lowered in the presence of FH2. Some initial experiments with sodium chloride point to a non-specific nature of this salt. This is in agreement with the electrostatic nature of the salt effect, which was studied further by computational means: A decrease of the KCl concentration leads to lower binding free energies of the protein-protein interactions in the crystallographically characterized actin-FH2 complex 1Y64. This is especially the case for the electrostatic Coulomb interaction of a specific area ("lasso" site). ANCHOR calculation results of solvent accessible surface areas (SASAs) corroborate the importance of this site. The experimentally found downregulation of FH2 mediated actin nucleation by KCl can therefore be explained by reduced actin recruitment by the FH2 dimer: KCl diminishes the surface charge of FH2 and actin and thus weakens electrostatic Coulomb interactions. In future, this newly discovered salt effect should be considered in experiments on formins, for example when performing in vitro screens for FH2 inhibitors. The relevance of this new salt effect in vivo remains to be demonstrated.Forminproteine sind Aktinnukleatoren und -elongatoren, die in fast allen eukaryotischen Zellen vorkommen. Es wurden in dieser Arbeit Struktur-Funktionsbeziehungen zwischen dem Hefeformin Bni1p und der Aktinpolymerisation untersucht. Im ersten Teil der Arbeit wurde versucht, verschiedene Forminkonstrukte des Hefeformins bni1p (Saccharomyces cerevisiae) mit der Schlüsseldomäne FH2 und gegebenenfalls modifizierter FH1-Domänen zu exprimieren, um damit biomathematische Modelle testen zu können, die sowohl eine diffusions- als auch eine konzentrationslimitierte Kinetik der Aktinrekrutierung annehmen. Die meisten Klonierungen gelangen, exprimiert werden konnte jedoch nur die FH2-Domäne allein. Pyrene-Assays und epifluoreszenzmikroskopische Aufnahmen konnten im zweiten Teil der Arbeit einen bisher nicht bekannten Salzeffekt auf die FH2-vermittelte Aktinnukleation nachweisen. Der Salzeffekt wurde für Kaliumchlorid (KCl) im Konzentrationsbereich 10 mM < c(KCl) < 90 mM untersucht und war signifikant für zwei Versuchsserien mit unterschiedlichen FH2-Konzentrationen, jedoch nicht in Abwesenheit von FH2. KCl reguliert im genannten Konzentrationsbereich die Aktinnukleationsaktivität von FH2 herunter: Eine höhere KCl-Konzentration führt zu einer signifikant niedrigeren Polymerisationsgeschwindigkeit (kp, m), zu einer größeren lag time (tlag) und zu einer größeren t1/2, mit einer dazu passenden Längenverteilung der Aktinfilamente. Die kritische KCl-Konzentration sinkt in Anwesenheit von KCl. Erste Experimente mit Natriumchlorid deuten an, dass der Effekt unspezifisch ist. Das passt zu der elektrostatischen Natur dieses Salzeffektes, die auch bioinformatisch untersucht wurde: Für eine niedrigere KCl-Konzentration wurden niedrigere freie Bindungsenergien für die Protein-Protein-Interaktion eines kristallographisch beschriebenen Aktin-FH2-Komplexes (1Y64) berechnet. Das gilt insbesondere für die elektrostatischen Coulomb-Wechselwirkungen eines bestimmten Bereiches (Lasso). ANCHOR-Berechnungen der der exponierten Proteinoberflächen (SASAs) weisen ebenfalls auf die Bedeutung dieses Bereiches hin. Die Herunterregulierung der FH2-vermittelten Aktinnukleation durch Kaliumchlorid kann daher mit einer reduzierten Aktinrekrutierung durch das FH2-Dimer erklärt werden: KCl vermindert die Oberflächenladung von FH2 und Aktin und schwächt so elektrostatische Coulomb-Wechselwirkungen. In der Zukunft sollte dieser neu gefundene Salzeffekt bei in vitro - Experimenten mit Forminen berücksichtigt werden, zum Beispiel beim Screening nach FH2-Inhibitoren. Es ist künftig ferner von Interesse, welche Folgen dieser neu gefundene Salzeffekt auf die FH2-vermittelte Aktinnukleation in vivo hat

MicroRNA Expression Profile in the Vitreous of Proliferative Diabetic Retinopathy Patients and Differences from Patients Treated with Anti-VEGF

Purpose: microRNAs (miRNAs) mediate the pathological mechanisms of diabetic retinopathy. In this study, we compared miRNA expression profiles in the vitreous between patients with proliferative diabetic retinopathy (PDR) and patients with a macular hole as non-diabetic controls, and between PDR patients treated with antivascular endothelial growth factor (VEGF) therapy and untreated PDR patients. Methods: Vitreous samples of non-diabetic and PDR patients were screened for miRNAs with quantitative polymerase chain reaction (qPCR) panels. miRNA candidates were validated in vitreous samples of a second, independent cohort. In addition, the effect of anti-VEGF therapy was investigated in the vitreous of a third study population consisting of PDR patients who had not received anti-VEGF therapy and PDR patients who had received preoperative anti-VEGF therapy. Results: During screening, seven miRNAs were found to be significantly higher in the vitreous of PDR patients, whereas two miRNAs were found to be significantly lower compared with non-diabetic controls. Validating the expression of these miRNAs in a second cohort resulted in the identification of six miRNAs that were expressed at significantly higher rates in the vitreous of PDR patients: hsa-miR-20a-5p, hsa-miR-23b3p, hsa-miR-142-3p, hsa-miR-185-5p, hsa-miR-326, and hsa-miR-362-5p. Among these six miRNAs, hsa-miR-23b-3p levels were lower in the anti-VEGF-treated group of PDR patients compared with untreated PDR patients. Conclusions: In this study, we identified six miRNAs that are expressed more highly in PDR patients and one miRNA that is expressed at a lower levels in anti-VEGF-treated PDR patients. Translational Relevance: miRNAs identified in the vitreous of PDR patients may improve our understanding of the mechanisms leading to PDR

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors

Gastric Emphysema: An Etiologic Classification

I Gas within the wall of the stomach is a rare radiologic finding. The stomach has been the least often reported site of intramural gas in the hollow viscera. Based on etiology, gas in the wall of the stomach can be classified as either gastric emphysema or emphysematous gastritis. Gastric emphysema may be classified into traumatic, pulmonary or obstructive types depending upon the mechanism and pathogenesis. Three cases of gastric emphysema, each of different etiology, are presented to emphasize the subclassification of gastric emphysema. The clinical and prognostic significance of this classification is emphasized.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72543/1/j.1440-1673.1984.tb02363.x.pd

2- and 3-substituted imidazo [1,2-a] pyrazines as inhibitors of bacterial type IV secretion

A novel series of 8-amino imidazo[1,2-a]pyrazine derivatives has been developed as inhibitors of the VirB11 ATPase HP0525, a key component of the bacterial type IV secretion system. A flexible synthetic route to both 2- and 3-aryl substituted regioisomers has been developed. The resulting series of imidazo[1,2-a]pyrazines has been used to probe the structure–activity relationships of these inhibitors, which show potential as antibacterial agents

New insights from deep VLA data on the potentially recoiling black hole CID-42 in the COSMOS field

We present deep 3-GHz Karl G. Jansky Very Large Array (VLA) observations of the potentially recoiling black hole CID-42 in the Cosmic Evolution Survey (COSMOS) field. This galaxy shows two optical nuclei in the Hubble Space Telescope/Advanced Camera for Surveys (HST/ACS) image and a large velocity offset of ˜1300 km s-1 between the broad and narrow Hß emission line although the spectrum is not spacially resolved (Civano et al. 2010). The new 3 GHz VLA data have a bandwidth of 2 GHz and to correctly interpret the flux densities imaging was done with two different methods: multiscale multifrequency (MSMF) synthesis and spectral windows (SPWs) stacking. The final resolutions and sensitivities of these maps are 0.7 arcsec with rms = 4.6 µJy beam-1 and 0.9 arcsec with rms = 4.8 µJy beam-1, respectively. With a 7s detection, we find that the entire observed 3-GHz radio emission can be associated with the south-eastern component of CID-42, coincident with the detected X-ray emission. We use our 3 GHz data combined with other radio data from the literature ranging from 320 MHz to 9 GHz, which include the VLA, Very Long Baseline Array (VLBA) and Giant Metrewave Radio Telescope (GMRT) data, to construct a radio synchrotron spectrum of CID-42. The radio spectrum suggests a type I unobscured radio-quiet flat-spectrum active galactic nucleus (AGN) in the south-eastern component which may be surrounded by a more extended region of old synchrotron electron population or shocks generated by the outflow from the supermassive black hole (SMBH). Our data are consistent with the recoiling black hole picture but cannot rule out the presence of an obscured and radio-quiet SMBH in the north-western component

Corticosteroids and regional variations in thickness of the human cerebral cortex across the lifespan

International audienceExposures to life stressors accumulate across the lifespan, with possible impact on brain health. Little is known, however, about the mechanisms mediating age-related changes in brain structure. We use a lifespan sample of participants (n = 21 251; 4–97 years) to investigate the relationship between the thickness of cerebral cortex and the expression of the glucocorticoid- and the mineralocorticoid-receptor genes (NR3C1 and NR3C2, respectively), obtained from the Allen Human Brain Atlas. In all participants, cortical thickness correlated negatively with the expression of both NR3C1 and NR3C2 across 34 cortical regions. The magnitude of this correlation varied across the lifespan. From childhood through early adulthood, the profile similarity (between NR3C1/NR3C2 expression and thickness) increased with age. Conversely, both profile similarities decreased with age in late life. These variations do not reflect age-related changes in NR3C1 and NR3C2 expression, as observed in 5 databases of gene expression in the human cerebral cortex (502 donors). Based on the co-expression of NR3C1 (and NR3C2) with genes specific to neural cell types, we determine the potential involvement of microglia, astrocytes, and CA1 pyramidal cells in mediating the relationship between corticosteroid exposure and cortical thickness. Therefore, corticosteroids may influence brain structure to a variable degree throughout life

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response