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

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

A chemokine network of T cell exhaustion and metabolic reprogramming in renal cell carcinoma

Renal cell carcinoma (RCC) is frequently infiltrated by immune cells, a process which is governed by chemokines. CD8+ T cells in the RCC tumor microenvironment (TME) may be exhausted which most likely influence therapy response and survival. The aim of this study was to evaluate chemokine-driven T cell recruitment, T cell exhaustion in the RCC TME, as well as metabolic processes leading to their functional anergy in RCC. Eight publicly available bulk RCC transcriptome collectives (n=1819) and a single cell RNAseq dataset (n=12) were analyzed. Immunodeconvolution, semi-supervised clustering, gene set variation analysis and Monte Carlo-based modeling of metabolic reaction activity were employed. Among 28 chemokine genes available, CXCL9/10/11/CXCR3, CXCL13/CXCR5 and XCL1/XCR1 mRNA expression were significantly increased in RCC compared to normal kidney tissue and also strongly associated with tumor-infiltrating effector memory and central memory CD8+ T cells in all investigated collectives. M1 TAMs, T cells, NK cells as well as tumor cells were identified as the major sources of these chemokines, whereas T cells, B cells and dendritic cells were found to predominantly express the cognate receptors. The cluster of RCCs characterized by high chemokine expression and high CD8+ T cell infiltration displayed a strong activation of IFN/JAK/STAT signaling with elevated expression of multiple T cell exhaustion-associated transcripts. Chemokinehigh RCCs were characterized by metabolic reprogramming, in particular by downregulated OXPHOS and increased IDO1-mediated tryptophan degradation. None of the investigated chemokine genes was significantly associated with survival or response to immunotherapy. We propose a chemokine network that mediates CD8+ T cell recruitment and identify T cell exhaustion, altered energy metabolism and high IDO1 activity as key mechanisms of their suppression. Concomitant targeting of exhaustion pathways and metabolism may pose an effective approach to RCC therapy

RDML-Ninja and RDMLdb for standardized exchange of qPCR data

Background: The universal qPCR data exchange file format RDML is today well accepted by the scientific community, part of the MIQE guidelines and implemented in many qPCR instruments. With the increased use of RDML new challenges emerge. The flexibility of the RDML format resulted in some implementations that did not meet the expectations of the consortium in the level of support or the use of elements. Results: In the current RDML version 1.2 the description of the elements was sharpened. The open source editor RDML-Ninja was released (http://sourceforge.net/projects/qpcr-ninja/). RDML-Ninja allows to visualize, edit and validate RDML files and thus clarifies the use of RDML elements. Furthermore RDML-Ninja serves as reference implementation for RDML and enables migration between RDML versions independent of the instrument software. The database RDMLdb will serve as an online repository for RDML files and facilitate the exchange of RDML data (http://www.rdmldb.org). Authors can upload their RDML files and reference them in publications by the unique identifier provided by RDMLdb. The MIQE guidelines propose a rich set of information required to document each qPCR run. RDML provides the vehicle to store and maintain this information and current development aims at further integration of MIQE requirements into the RDML format. Conclusions: The editor RDML-Ninja and the database RDMLdb enable scientists to evaluate and exchange qPCR data in the instrument-independent RDML format. We are confident that this infrastructure will build the foundation for standardized qPCR data exchange among scientists, research groups, and during publication

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/

Meeting Report from the Second 'Minimum Information for Biological and Biomedical Investigations (MIBBI) workshop

Face-to-face meetings play a central role in the birth and maturation of communities. Intensive workshops filled with presentations, discussions and working group meetings have always been at the heart of the activities of the Genomic Standards Consortium (GSC). Such work-driven meetings are a key way in which the GSC fulfils its mission. Similarly, meeting reports provide a key mechanism for preserving and disseminating the consensus built at such meetings as they describe the range of speakers and participants present, topics covered and key outcomes and priorities agreed upon by the community.This issue contains a total of nine meeting reports, from workshops held between April and October 2010 that are presented to the reader to provide a broad overview of ongoing GSC activities and initiatives

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

Doubly heterozygous LMNA and TTN mutations revealed by exome sequencing in a severe form of dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is a heterogeneous disease; although 30 disease genes have been discovered, they explain only no more than half of all cases; in addition, the causes of intra-familial variability in DCM have remained largely unknown. In this study, we exploited the use of whole-exome sequencing (WES) to investigate the causes of clinical variability in an extended family with 14 affected subjects, four of whom showed particular severe manifestations of cardiomyopathy requiring heart transplantation in early adulthood. This analysis, followed by confirmative conventional sequencing, identified the mutation p.K219T in the lamin A/C gene in all 14 affected patients. An additional variant in the gene for titin, p.L4855F, was identified in the severely affected patients. The age for heart transplantation was substantially less for LMNA:p.K219T/TTN:p.L4855F double heterozygotes than that for LMNA:p.K219T single heterozygotes. Myocardial specimens of doubly heterozygote individuals showed increased nuclear length, sarcomeric disorganization, and myonuclear clustering compared with samples from single heterozygotes. In conclusion, our results show that WES can be used for the identification of causal and modifier variants in families with variable manifestations of DCM. In addition, they not only indicate that LMNA and TTN mutational status may be useful in this family for risk stratification in individuals at risk for DCM but also suggest titin as a modifier for DCM

Cleavage modification did not alter blastomere fates during bryozoan evolution

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The study was funded by the core budget of the Sars Centre and by The European Research Council Community’s Framework Program Horizon 2020 (2014–2020) ERC grant agreement 648861 to A