Analyse der MACF1 Isoform-3 Funktionen, einem Spektraplakin Protein mit dualen Rollen im Zytoplasma und dem Nukleus

ZUSAMMENFASSUNG Das Spektraplakin Familienmitglied MACF1 (Microtubule Actin Cross-linking Factor 1) ist ein großes (620-800 kDa) Multidomänenprotein, welches Mikrofilamente und Mikrotubuli quervernetzt. Das MACF1 Gen kodiert für verschiedene alternativ gespleißte Isoformen, die in Größe, Länge und Domänenkomposition variieren. MACF1 Isoform-3 weist einen ungewöhnlichen N-Terminus auf, der auf neue Funktionen schließen lässt. Dieser ist aus einem einzigartig hoch konservierten 35 kDa N-Terminus aufgebaut, der direkt an die zweite Hälfte der Aktin-bindenden Domäne fusioniert ist. Die transiente Expression dieses N-Terminus inhibiert die Signaltransduktion der Rho-GTPasen. Durch die Verwendung des Hefe-Zwei-Hybrid Systems und Expressionsstudien konnte eine Interaktion mit dem Zytoskelettmodulator RhoA gezeigt werden. Immunfluoreszenzstudien mit einem neu generierten polyklonalen MACF1 Isoform-3 Antikörper zeigten eine ungewöhnliche Lokalisierung um und innerhalb des Nukleus in verschiedenen Zelllinien und Geweben zusätzlich zu der gut charakterisierten Mikrotubuli Lokalisation. Permeabilisationassays mit Digitonin wiesen auf die Lokalisation von MACF1 Isoform-3 an der äußeren Kernhülle hin. Dort kolokalisiert MACF1 mit Nesprinen. Nesprine sind integrale Membranproteine der äußeren Kernhülle, die den Zellkern mit dem Zytoskelett verbinden. Durch GST-Proteininteraktions-, Immunpräzipitations-, Hefe-Zwei-Hybrid- und Expressionsstudien konnte eine Interaktion von MACF1 Isoform-3 mit Nesprin-3 gezeigt werden. Weiterhin deuten die Ergebnisse an, dass durch diese Interaktion MACF1 Isoform-3 Mikrotubuli an die Kernhülle transloziert und dort stabilisiert. Während der Mitose lokalisiert MACF1 Isoform-3 entlang der mitotischen Spindel. Durch Chromatin Immunpräzipitation und Expressionsstudien konnte eine neue Assoziation mit Chromatin und Laminen für MACF1 Proteine gezeigt werden. Weiterhin konnten erste Belege für Umlagerungen von MACF1 Isoform-3 gezeigt werden. Durch Zugabe von Wachstumsfaktoren (Wnt-3, HGF und TGFß) sowie durch Serumsentzung und Zugabe von Calzium-Ionen konnte in Immunfluoreszenzstudien die subzelluläre Verteilung von MACF1 Isoform-3 beeinflusst werden, wobei MACF1 Isoform-3 nach Serumentzug mit Mitochondrien kolokalisierte. Ein möglicher Interaktionspartner an den Mitochondrien ist DISC1 (Disrupted in Schizophrenia 1), das als high risk gene für die Entstehung von Schizophrenie gilt. Durch Hefe-Zwei-Hybrid (Carmago et al., 2006) Immunpräzipitations- und Expressionsstudien wurde eine Interaktion mit MACF1 Isoform-3 nachgewiesen. Diese Daten deuten an, dass MACF1 Isoform-3 eine mögliche Funktion in der Entstehung von Schizophrenie besitzt. Wir stellen daher die Hypothese auf, dass MACF1 Isoform-3 eine wichtige Rolle in der Migration und Proliferation von Zellen ausübt und hierbei in die RhoA- und Wnt Signaltransduktion involviert ist

In vitro characterisation of the anti-intravasative properties of the marine product heteronemin

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Cytochrome P450 2B6 (CYP2B6) and constitutive androstane receptor (CAR) polymorphisms are associated with early discontinuation of efavirenz-containing regimens

Objectives Cytochrome P450 2B6 (CYP2B6) is responsible for the metabolic clearance of efavirenz and single nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with efavirenz pharmacokinetics. Since the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) correlate with CYP2B6 in liver, and a CAR polymorphism (rs2307424) and smoking correlate with efavirenz plasma concentrations, we investigated their association with early (<3 months) discontinuation of efavirenz therapy. Methods Three hundred and seventy-three patients initiating therapy with an efavirenz-based regimen were included (278 white patients and 95 black patients; 293 male). DNA was extracted from whole blood and genotyping for CYP2B6 (516G → T, rs3745274), CAR (540C → T, rs2307424) and PXR (44477T → C, rs1523130; 63396C → T, rs2472677; and 69789A → G, rs763645) was conducted. Binary logistic regression using the backwards method was employed to assess the influence of SNPs and demographics on early discontinuation. Results Of the 373 patients, 131 withdrew from therapy within the first 3 months. Black ethnicity [odds ratio (OR) = 0.27; P = 0.0001], CYP2B6 516TT (OR = 2.81; P = 0.006), CAR rs2307424 CC (OR = 1.92; P = 0.007) and smoking status (OR = 0.45; P = 0.002) were associated with discontinuation within 3 months. Conclusions These data indicate that genetic variability in CYP2B6 and CAR contributes to early treatment discontinuation for efavirenz-based antiretroviral regimens. Further studies are now required to define the clinical utility of these association

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Crowdsourced Replication Initiative: Investigating Immigration and Social Policy Preferences. Executive Report.

In an era of mass migration, social scientists, populist parties and social movements raise concerns over the future of immigration-destination societies. What impacts does this have on policy and social solidarity? Comparative cross-national research, relying mostly on secondary data, has findings in different directions. There is a threat of selective model reporting and lack of replicability. The heterogeneity of countries obscures attempts to clearly define data-generating models. P-hacking and HARKing lurk among standard research practices in this area.This project employs crowdsourcing to address these issues. It draws on replication, deliberation, meta-analysis and harnessing the power of many minds at once. The Crowdsourced Replication Initiative carries two main goals, (a) to better investigate the linkage between immigration and social policy preferences across countries, and (b) to develop crowdsourcing as a social science method. The Executive Report provides short reviews of the area of social policy preferences and immigration, and the methods and impetus behind crowdsourcing plus a description of the entire project. Three main areas of findings will appear in three papers, that are registered as PAPs or in process

From niche to mainstream : the dilemmas of scaling up sustainable alternatives

At the heart of transition research lies the question of how to "scale up" sustainable alternatives from a protected niche to the creation of mainstream practices. While upscaling processes are often seen as an essential element that contributes to societal transformation, upscaling itself remains a fuzzy concept. We argue that some fundamental dilemmas of upscaling can be identified, for example, the different understanding of the concept by researchers and practitioners. The dilemmas should be addressed in a more reflexive way by those from the worlds of science and practice who are involved in collaborative research settings

MR Fingerprinting and ADC Mapping for Characterization of Lesions in the Transition Zone of the Prostate Gland.

Background: Preliminary studies have shown that MR fingerprinting-based relaxometry combined with apparent diffusion coefficient (ADC) mapping can be used to differentiate normal peripheral zone from prostate cancer and prostatitis. The utility of relaxometry and ADC mapping for the transition zone (TZ) is unknown. Purpose: To evaluate the utility of MR fingerprinting combined with ADC mapping for characterizing TZ lesions. Materials and Methods: TZ lesions that were suspicious for cancer in men who underwent MRI with T2-weighted imaging and ADC mapping ( values, 50-1400 sec/mm), MR fingerprinting with steady-state free precession, and targeted biopsy (60 in-gantry and 15 cognitive targeting) between September 2014 and August 2018 in a single university hospital were retrospectively analyzed. Two radiologists blinded to Prostate Imaging Reporting and Data System (PI-RADS) scores and pathologic diagnosis drew regions of interest on cancer-suspicious lesions and contralateral visually normal TZs (NTZs) on MR fingerprinting and ADC maps. Linear mixed models compared two-reader means of T1, T2, and ADC. Generalized estimating equations logistic regression analysis was used to evaluate both MR fingerprinting and ADC in differentiating NTZ, cancers and noncancers, clinically significant (Gleason score ≥ 7) cancers from clinically insignificant lesions (noncancers and Gleason 6 cancers), and characterizing PI-RADS version 2 category 3 lesions. Results: In 67 men (mean age, 66 years ± 8 [standard deviation]) with 75 lesions, targeted biopsy revealed 37 cancers (six PI-RADS category 3 cancers and 31 PI-RADS category 4 or 5 cancers) and 38 noncancers (31 PI-RADS category 3 lesions and seven PI-RADS category 4 or 5 lesions). The T1, T2, and ADC of NTZ (1800 msec ± 150, 65 msec ± 22, and [1.13 ± 0.19] × 10 mm/sec, respectively) were higher than those in cancers (1450 msec ± 110, 36 msec ± 11, and [0.57 ± 0.13] × 10 mm/sec, respectively; < .001 for all). The T1, T2, and ADC in cancers were lower than those in noncancers (1620 msec ± 120, 47 msec ± 16, and [0.82 ± 0.13] × 10 mm/sec, respectively; = .001 for T1 and ADC and = .03 for T2). The area under the receiver operating characteristic curve (AUC) for T1 plus ADC was 0.94 for separation. T1 and ADC in clinically significant cancers (1440 msec ± 140 and [0.58 ± 0.14] × 10 mm/sec, respectively) were lower than those in clinically insignificant lesions (1580 msec ± 120 and [0.75 ± 0.17] × 10 mm/sec, respectively; = .001 for all). The AUC for T1 plus ADC was 0.81 for separation. Within PI-RADS category 3 lesions, T1 and ADC of cancers (1430 msec ± 220 and [0.60 ± 0.17] × 10 mm/sec, respectively) were lower than those of noncancers (1630 msec ± 120 and [0.81 ± 0.13] × 10 mm/sec, respectively; = .006 for T1 and = .004 for ADC). The AUC for T1 was 0.79 for differentiating category 3 lesions. Conclusion: MR fingerprinting-based relaxometry combined with apparent diffusion coefficient mapping may improve transition zone lesion characterization