Rekombinante Hepatitis B Viren-Vektoren als Werkzeuge für Molekularbiologie und Therapie : Optimierung eines Systems

Hepatitis B Virus (HBV) - Vektoren sind vielversprechende Kandidaten für einen lebergerichteten Gentransfer, da sie bevorzugt Hepatozyten infizieren und ihre Genexpression hepatozytenspezifisch ist. Es war bekannt, dass infektiöse HBV produziert werden konnten, in denen das kleine Hüllprotein (S) durch das grün fluoreszierende Protein (GFP) ersetzt wurde. Nach Infektion primärer humaner Hepatozyten (PHH) mit diesen Viren zeigten einzelne Zellen eine grüne Fluoreszenz. Der Einsatz von HBV-Vektoren ist einerseits als Werkzeug in der Molekularbiologie und andererseits in der Therapie von Lebererkrankungen denkbar. In der vorliegenden Arbeit sollte der Nachweis eines Gentransfers verbessert sowie die Transgenkapazität exakt bestimmt und vergrößert werden. Im Hinblick auf eine mögliche Therapie von chronischer Hepatitis B oder C sollte ein lebergerichteter Interferontransfer etabliert werden. Der Gentansfers in Hepatozyten durch Infektion konnte erst reproduzierbar untersucht werden, als PHH als Testsystem für die Vektoren etabliert waren und die Virusausbeute durch die Verwendung von Lipofektion sowie serumfreiem Medium bei der Virusproduktion 10fach erhöht wurde. Durch das Ersetzen des preS2/S-Promoters durch den Transthyretin-Promoter konnte eine Infektion von lebenden PHH zu einem früheren Zeitpunkt nachgewiesen werden, ohne die Leberspezifität des Promotors zu verlieren. Mit Renilla-Luziferase als Transgen gelang es, den Nachweis einer Infektion zu vereinfachen und quantifizierbare Ergebnisse zu erhalten. Diese Viren ermöglichten es, die Transgenkapazität und die Genexpression der HBV-Vektoren genauer zu untersuchen. Da die HBV-Vektoren eine über 400 bp hinausgehende Überlänge des Genoms nicht tolerierten, konnte die Transgenkapazität von ca. 800 bp nur durch Einführung weiterer Deletionen im viralen Genom vergrößert werden. Eine andere genomische Deletion erhöhte die Genexpression 5fach. Für einen lebergerichteten Interferontransfer wurden sowohl adenovirale Vektoren als auch HBV-Vektoren benutzt. Mit beiden Systemen gelang es, Viren zu produzieren, und mit beiden Vektoren war es auch möglich, Interferon-Expression in infizierten PHH nachzuweisen. Durch diese Verbesserungen stellen die HBV-Vektoren ein Werkzeug zur mikroskopischen Identifikation infizierter, lebender PHH in Zellkultur da. Mit den Luziferase exprimierenden Viren ist es möglich, die Infektion einer gesamten Kulturschale zu quantifizieren und den Einfluss von Änderungen im viralen Genom auf Infektiösität, Transkription und Genexpression zu untersuchen. Interferon exprimierende HBV-Vektoren werden aktuell im Schimpansen Modell der chronischen Hepatitis C getestet

PBRM1 mutations might render a subtype of biliary tract cancers sensitive to drugs targeting the DNA damage repair system

Cancer genomics; OncologyGenómica del cáncer; OncologíaGenòmica del càncer; OncologiaPolybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821–1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs

Characterization of microsatellite markers for Moricandia moricandioides (Brassicaceae) and related species

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed to study population structure and mating patterns of the monocarpic herb Moricandia moricandioides (Brassicaceae). METHODS AND RESULTS: Illumina MiSeq sequencing was used to develop a panel of 15 polymorphic microsatellite markers that were tested across 77 individuals from three populations on the Iberian Peninsula. All markers were polymorphic in at least two studied populations, and the number of alleles ranged from one to 11 per locus. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.153 to 0.865, respectively. Nine and 11 loci were successfully amplified in the congeneric species M. arvensis and M. foetida, respectively. CONCLUSIONS: The 15 microsatellite markers will be useful for population genetic studies of the genus Moricandia. These markers will serve as a useful tool for exploring population structure and mating patterns of M. moricandioides.This work received funding from the European Union’s Horizon 2020 (Marie Skłodowska-Curie No. 655653), Fundación BBVA (PR17-ECO- 0021), and the Spanish Ministerio de Economia y Competitividad (CGL2017-86626- C2- 1- P)

RDML: structured language and reporting guidelines for real-time quantitative PCR data

The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results

Effect of plant-based feed ingredients on osmoregulation in the Atlantic salmon lens

Lenses of adult Atlantic salmon fed with a plant oil and plant protein-based diet (plant diet) were compared to lenses of fish fed a diet based on traditional marine ingredients (marine diet) with respect to biochemical composition and functionality ex vivo. After 12 months of feeding, plant diet-fed fish had smaller lenses with higher water contents and lower concentrations of histidine (His) and N-acetylhistidine (NAH) than fish fed with the marine diet. Cataract development in both dietary groups was minimal and no differences between the groups were observed. Lens fatty acid and lipid class composition differed minimally, although a significant increase in linoleic acid was observed. The lenses were examined for their ability to withstand osmotic disturbances ex vivo. Culture in hypoosmotic and hyperosmotic media led to increase and decrease of lens volume, respectively. Lenses from plant diet-fed fish were less resistant to swelling and shrinking, released less NAH into the culture medium, and accumulated His and NAH at higher rates than lenses from marine diet-fed fish. Culture in hypoosmotic medium resulted in higher cataract scores than in control and hyperosmotic medium. mRNA expression of selected genes, including glutathione peroxidase 4 and SPARC (secreted protein acidic and rich in cysteine), was affected by diet and osmotic treatment. It can be concluded that lenses of farmed Atlantic salmon are affected by the diet composition, both in biochemical composition and physiological functionality in relation to osmoregulation

Vasohibin inhibits angiogenic sprouting in vitro and supports vascular maturation processes in vivo

<p>Abstract</p> <p>Background</p> <p>The murine homologue of human vasohibin (mVASH1), a putative antiangiogenic protein, was investigated for its effects on <it>in vitro </it>and <it>in vivo </it>angiogenesis.</p> <p>Methods</p> <p>Cell growth and migration were analyzed in murine fibroblasts, smooth muscle cells and endothelial cells. Angiogenic sprouting was studied in human umbilical vein endothelial cells (HUVECs) in the spheroid sprouting assay. <it>In vivo </it>effects on blood vessel formation were investigated in the chorioallantoic membrane (CAM) assay and in the C57BL/6 melanoma xenograft model.</p> <p>Results</p> <p>Purified murine and human VASH1 protein induced apoptosis of murine fibroblasts <it>in vitro</it>, but not of vascular aortic smooth muscle cells (AoSMC) or endothelial cells. Adenoviral overexpression of murine and human VASH1 inhibited capillary sprouting of HUVECs in the spheroid assay. Administration of recombinant murine and human VASH1 inhibited growth of large vessels in the CAM assay and promoted the formation of a dense, fine vascular network. Murine VASH1-overexpressing B16F10 melanomas displayed a reduction in large vessels and vascular area. Moreover, tumors showed more microvessels that stained positive for the mural cell markers α-smooth muscle cell actin (ASMA) and proteoglycan (NG2).</p> <p>Conclusion</p> <p>Our data imply that murine VASH1 causes angiogenic remodelling by inhibiting angiogenic sprouting and large vessel growth, thereby supporting the formation of a vascular bed consisting predominantly of mature microvessels.</p