Heat-shock mediated overexpression of HNF1β mutations has differential effects on gene expression in the Xenopus pronephric kidney.

The transcription factor HNF1B, encoded by the TCF2 gene, plays an important role in the organogenesis of vertebrates. In humans, heterozygous mutations of HNF1B are associated with several diseases, such as pancreatic β-cell dysfunction leading to maturity-onset diabetes of the young (MODY5), defective kidney development, disturbed liver function, pancreas atrophy, and malformations of the genital tract. The African claw frog Xenopus laevis is an excellent model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a series of methods. In the present study, we overexpressed HNF1β mutants in the developing Xenopus embryo to assess their roles during organogenesis, particularly in the developing pronephric kidney. Towards this goal, we developed a heat-shock inducible binary Cre/loxP system with activator and effector strains. Heat-shock activation of the mutant HNF1B variants P328L329del and A263insGG resulted in malformations of various organs and the affected larvae developed large edemas. Defects in the pronephros were primarily confined to malformed proximal tubules. Furthermore, the expression of the proximal tubule marker genes tmem27 and slc3a1, both involved in amino acid transport, was affected. Both P328L329del and A263insGG downregulated expression of slc3a1. In addition, P328L329del reduced tmem27 expression while A263insGG overexpression decreased expression of the chloride channel clcnk and the transcription factor pax2. Overexpression of two mutant HNF1B derivatives resulted in distinct phenotypes reflected by either a reduction or an enlargement of pronephros size. The expression of selected pronephric marker genes was differentially affected upon overexpression of HNF1B mutations. Based on our findings, we postulate that HNF1B mutations influence gene regulation upon overexpression in specific and distinct manners. Furthermore, our study demonstrates that the newly established Cre/loxP system for Xenopus embryos is an attractive alternative to examine the gene regulatory potential of transcription factors in developing pronephric kidney as exemplified here for HNF1B

Conditional overexpression of the transcription fachtor HNF1b and its influence on kidney development in Xenopus laevis

Der südafrikanische Krallenfrosch Xenopus laevis ist ein attraktives Modell, um die frühe Entwicklung von Vertebraten auf molekularer und zellularer Ebene zu untersuchen. Die Transparenz der Larven ermöglicht eine effiziente Beobachtung der zahlreichen morphologischen Veränderungen während der Organogenese. In der vorliegenden Arbeit wurde die Funktion des gewebespezifischen Transkriptionsfaktors HNF1β insbesondere in der Nierenentwicklung von Xenopus laevis näher charakterisiert. Zu diesem Zweck wurde das binäre Cre/loxP-System mit Aktivator- und Effektorstämmen verwendet. In diesem System kann die Cre-Rekombinase konditional durch einen Hitzeschock aktiviert werden. Es konnte zunächst gezeigt werden, dass die Transkription der Cre-Rekombinase sowohl im HSPCre1- und im HSPCre13-Aktivatorstamm durch einen einstündigen Hitzeschock effizient aktiviert wird und beide Stämme als Aktivatorstämme für das Cre/loxP-System verwendet werden können. Durch Kreuzung des C5-Stammes mit dem HSPCre1-Stamm wurde gezeigt, dass bereits eine Stunde nach Beginn des Hitzeschocks Transkripte eines induzierbaren Reportergens detektierbar waren. Die HSPCre13 vermittelte Überexpression der P328L329del-Mutation durch einen einstündigen Hitzeschock führte zu Nierendefekten und Fehlentwicklungen anderer Organe. Es wiesen jedoch nicht alle mutanten Larven Nierendefekte auf. Dieses Ergebnis konnte in weiteren Kreuzungen mit unabhängigen Aktivator- und Effektorstämmen bestätigt werden. Nach der Optimierung des binären Cre/loxP-Systems durch einen zweistündigen Hitzeschock konnte gezeigt werden, dass die ubiquitäre Überexpression der P328L329del- Mutation in allen mutanten Larven zu einem Nierenphänotyp führte. Insgesamt war die Niere signifikant verkleinert. Zusätzlich interferierte die Überexpression mit der Entwicklung des Magen-Darm-Traktes, des Schwanzes und des Auges. Durch die Analyse ausgewählter Marker für die einzelnen Segmente des sich entwickelnden Pronephros konnte gezeigt werden, dass die Überexpression des mutanten Derivates P328L329del die Expression der Gene slc3a1 und tmem27, welche spezifisch in den proximalen Tubuli exprimiert werden, beeinflusste. Beide Gene spielen eine Rolle bei der renalen Reabsorption von Aminosäuren. Dieses Resultat enthüllt eine neue Rolle von HNF1β in der Nierenphysiologie. Larven in denen das mutante HNF1β-Derivat A263insGG ubiquitär aktiviert war, wiesen ebenfalls Defekte der Niere, des Magen-Darm-Traktes, des Schwanzes und des Auges auf. Im Gegensatz zu dem durch die Überexpression von P328L329del verursachten Nierenphänotyp, war die Niere hier signifikant vergrößert. Die Überexpression von HNF1β hatte hingegen keinen Einfluss auf die Larvenentwicklung. Desweiteren konnten zwei neue Effektorstämme etabliert werden, mit denen HNF1β und das mutante Derivat P328L329del im Gegensatz zu den vorherigen Experimenten ausschließlich im Pronephros überexprimiert werden können. Es konnte gezeigt werden, dass die pronephrosspezifische Überexpression von HNF1β mit der Entwicklung der Niere und des Magen-Darm-Traktes interferierte. Im Schwanzbereich und im Auge wurden keine Defekte beobachtet. Weiterhin wurde eine neue Methode in Xenopus laevis getestet und optimiert, bei der die lokale Hitzeschockaktivierung eines Gens mittels Laserbestrahlung erfolgt. Mit dieser Methode war es möglich, das EYFP-Reportergen des C5-Stammes lokal in einzelnen Schwanzmuskelzellen und im Pronephros zu aktivieren. Diese Methode wurde in dieser Arbeit erstmalig in Xenopus laevis angewandt und ermöglicht eine gewebespezifische Überexpression von Genen ohne die Verwendung gewebespezifischer Promotoren.The frog Xenopus laevis is an excellent model system to analyze the molecular processes involved in early embryogenesis and organogenesis. The advantages include a large number of embryos and the development that takes place outside the maternal body allowing for a continuous observation of morphogenetic events in the highly transparent larvae. In this work the function of the tissue specific transcription factor HNF1β was examined particularly in kidney development of Xenopus laevis. For this purpose it was made use of the binary Cre/loxP-system with activator and effector strains. In this system the Cre recombinase can be conditionally activated by a heat-shock. Transcription of the cre recombinase is efficiently activated by a one hour heat-shock in both the HSPCre1 and HSPCre13 strain. Therefore both strains can be used as activator strains in the binary Cre/loxP-system. A crossing of the C5 strain with the HSPCre1 strain revealed that transcripts of an inducible reporter gene could be detected already one hour after heat-shock-activation of the cre recombinase. Ubiquitous overexpression of the HNF1β derivative P328L329del by a one-hour heat-shock caused kidney defects and malformations of other organs in the mutant larvae, but this was not the case in all of these animals. This result was validated with additional crossings of independent activator and effector strains. The binary system was optimized by application of a two hour heat-shock and upon ubiquitous overexpression of P328L329del 100 % of the mutant larvae showed a kidney phenotype, which became manifested in defects particularly in the proximal tubules and in a significantly reduced kidney size. Additionally the overexpression of P328L329del interfered with the development of the gut, the tail and the eye. Furthermore the expression of the two proximal tubule markers slc3a1 and tmem27 was affected. Both genes play a crucial role in the renal absorption of amino acids. This finding reveals an unknown function of HNF1β in kidney development. The overexpression of the HNF1β derivative A263insGG also caused malformations of the kidney, gut, tail and eye in mutant larvae. In comparison to animals with activated P328L329del the kidney size was significantly increased. The overexpression of HNF1β had no effect on development. Furthermore two new effector strains for pronephros specific overexpression of HNF1β and the mutant derivative P328L329del were established. The pronephros specific overexpression of HNF1β led to kidney defects and gut malformations. In contrast to the ubiquitous overexpression no malformations of tail and eye were observed. In addition a new method was established where a local heat-shock is induced by laser beams. With this approach it was possible to activate the inducible EYFP reporter gene in single cells of the tail and in the pronephros. This method was applied in Xenopus laevis for the first time and allows for a tissue specific gene activation without the need of tissue specific promoters

Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2

International audienceInnate immune recognition of the major human-specific Gram-positive pathogen Strepto-coccus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2-and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon in-ternalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13 −/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms

天然型エストロゲン及び抗エストロゲン剤トレミフェンによるマウス子宮内膜発癌に対する影響

Modern communication technology facilitates communication from anywhere to anywhere. As a result, low speech intelligibility has become a common problem, which is exacerbated by the lack of feedback to the talker about the rendering environment. In recent years, a range of algorithms has been developed to enhance the intelligibility of speech rendered in a noisy environment. We describe methods for intelligibility enhancement from a unified vantage point. Before one defines a measure of intelligibility, the level of abstraction of the representation must be selected. For example, intelligibility can be measured on the message, the sequence of words spoken, the sequence of sounds, or a sequence of states of the auditory system. Natural measures of intelligibility defined at the message level are mutual information and the hit-or-miss criterion. The direct evaluation of high-level measures requires quantitative knowledge of human cognitive processing. Lower-level measures can be derived from higher-level measures by making restrictive assumptions. We discuss the implementation and performance of some specific enhancement systems in detail, including speech intelligibility index (SII)-based systems and systems aimed at enhancing the sound-field where it is perceived by the listener. We conclude with a discussion of the current state of the field and open problems. © 2015 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works

Studies on the Coordination of Ribosomal Protein Assembly Events Involved in Prosessing and Stabiliz

Cellular production of ribosomes involves the formation of highly defined interactions between ribosomal proteins (r-proteins) and ribosomal RNAs (rRNAs). Moreover in eukaryotic cells, efficient ribosome maturation requires the transient association of a large number of ribosome biogenesis factors (RBFs) with newly forming ribosomal subunits. Here, we investigated how r-protein assembly events in the large ribosomal subunit (LSU) rRNA domain II are coordinated with each other and with the association of RBFs in early LSU precursors of the yeast Saccharomyces cerevisiae. Specific effects on the pre-ribosomal association of RBFs could be observed in yeast mutants blocked in LSU rRNA domain II assembly. Moreover, formation of a cluster of r-proteins was identified as a downstream event in LSU rRNA domain II assembly. We analyzed in more detail the functional relevance of eukaryote specific bridges established by this r-protein cluster between LSU rRNA domain II and VI and discuss how they can support the stabilization and efficient processing of yeast early LSU precursor RNAs

A metric for predicting binaural speech intelligibility in stationary noise and competing speech maskers

One criterion in the design of binaural sound scenes in audio production is the extent to which the intended speech message is correctly understood. Object-based audio broadcasting systems have permitted sound editors to gain more access to the metadata (e.g., intensity and location) of each sound source, providing better control over speech intelligibility. The current study describes and evaluates a binaural distortion-weighted glimpse proportion metric -- BiDWGP -- which is motivated by better-ear glimpsing and binaural masking level differences. BiDWGP predicts intelligibility from two alternative input forms: either binaural recordings or monophonic recordings from each sound source along with their locations. Two listening experiments were performed with stationary noise and competing speech, one in the presence of a single masker, the other with multiple maskers, for a variety of spatial configurations. Overall, BiDWGP with both input forms predicts listener keyword scores with correlations of 0.95 and 0.91 for single- and multi-masker conditions, respectively. When considering masker type separately, correlations rise to 0.95 and above for both types of maskers. Predictions using the two input forms are very similar, suggesting that BiDWGP can be applied to the design of sound scenes where only individual sound sources and their locations are available

The listening talker: A review of human and algorithmic context-induced modifications of speech

International audienceSpeech output technology is finding widespread application, including in scenarios where intelligibility might be compromised - at least for some listeners - by adverse conditions. Unlike most current algorithms, talkers continually adapt their speech patterns as a response to the immediate context of spoken communication, where the type of interlocutor and the environment are the dominant situational factors influencing speech production. Observations of talker behaviour can motivate the design of more robust speech output algorithms. Starting with a listener-oriented categorisation of possible goals for speech modification, this review article summarises the extensive set of behavioural findings related to human speech modification, identifies which factors appear to be beneficial, and goes on to examine previous computational attempts to improve intelligibility in noise. The review concludes by tabulating 46 speech modifications, many of which have yet to be perceptually or algorithmically evaluated. Consequently, the review provides a roadmap for future work in improving the robustness of speech output

Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs

Mammalian cells are an indispensable tool for the production of recombinant proteins in contexts where function depends on post-translational modifications. Among them, Chinese Hamster Ovary (CHO) cells are the primary factories for the production of therapeutic proteins, including monoclonal antibodies (MAbs). To improve expression and stability, several methodologies have been adopted, including methods based on media formulation, selective pressure and cell- or vector engineering. This review presents current approaches aimed at improving mammalian cell factories that are based on the enhancement of translation. Among well-established techniques (codon optimization and improvement of mRNA secondary structure), we describe SINEUPs, a family of antisense long non-coding RNAs that are able to increase translation of partially overlapping protein-coding mRNAs. By exploiting their modular structure, SINEUP molecules can be designed to target virtually any mRNA of interest, and thus to increase the production of secreted proteins. Thus, synthetic SINEUPs represent a new versatile tool to improve the production of secreted proteins in biomanufacturing processes. \ua9 2016 The Author