Molecular mechanisms of TIS7 action

Myogenese wird von myogenen regulatorischen Faktoren (MRFs) gesteuert, die zur Familie der basischen Helix-Loop-Helix (bHLH) Transkriptionsfaktoren (MyoD, Myf5, Myogenin und MRF4) gehören. TPA-induzierte Sequenz 7 (TIS7) ist ein Transkription-Co-regulator der eine essenzielle Rolle in der Differenzierung von Muskelzellen spielt. TIS7 steuert die Myogenese über MyoD und Myogenin, da deren Expression in TIS7 Knockout Mäusen herunter reguliert ist (Vadivelu et al., 2004). In dieser Arbeit stellen wir die TIS7-spezifischen molekularen Mechanismen vor, welche die Differenzierung der Skelettmuskulatur steuern. Darüber hinaus zeigen wir, dass TIS7 die Transkription von ICln reguliert welches als ein neuartiger Regulator der Myogenese über MyoD funktioniert. ICln steuert über die Interaktion mit PRMT5 die MyoD Expression und zugleich auch die folgende epigenetische Modifikation: die symmetrische Dimethylierung des Histons H3 auf Arginin 8. Zusammenfassend haben wir eine regulatorische Kaskade identifiziert in der die TIS7 transkriptionelle Regulation von ICln für die MyoD abhängige Skelettmyogenese notwendig ist.The skeletal muscle developmental program is controlled by myogenic regulatory factors (MRFs), members of the family of basic helix-loop-helix (bHLH) transcription factors (MyoD, Myf5, myogenin, and MRF4). TPA Induced Sequence 7 (TIS7) is a transcriptional co-regulator and plays a role in adult muscle differentiation. TIS7 regulates myogenesis, affecting MyoD and downstream myogenin, as expression levels are down-regulated in TIS7 knockout mice (Vadivelu et al., 2004). In this study we present the molecular mechanism underlying TIS7-specific control of skeletal muscle differentiation. We show that TIS7 transcriptionally regulates ICln expression, which in turn functions as a novel myogenesis regulatory factor upstream of MyoD. ICln regulates MyoD expression through interaction with PRMT5 by epigenetic modification that utilizes symmetrical di-methylation of histone H3 on arginine 8. In summary, we identified a regulatory cascade in which TIS7 transcriptional control of ICln is required for the MyoD-dependent skeletal myogenesis.Andrea LammiratoAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheInnsbruck, Med. Univ., Diss., 2015OeBB(VLID)79333

FlgM as a Secretion Moiety for the Development of an Inducible Type III Secretion System

<div><p>Regulation and assembly of the flagellar type III secretion system is one of the most investigated and best understood regulational cascades in molecular biology. Depending on the host organism, flagellar morphogenesis requires the interplay of more than 50 genes. Direct secretion of heterologous proteins to the supernatant is appealing due to protection against cellular proteases and simplified downstream processing. As <i>Escherichia coli</i> currently remains the predominant host organism used for recombinant prokaryotic protein expression, the generation of a strain that exhibits inducible flagellar secretion would be highly desirable for biotechnological applications.</p> <p>Here, we report the first engineered <i>Escherichia coli</i> mutant strain featuring flagellar morphogenesis upon addition of an external inducer. Using FlgM as a sensor for direct secretion in combination with this novel strain may represent a potent tool for significant improvements in future engineering of an inducible type III secretion for heterologous proteins.</p> </div

Comparison of the secretion competency via plasmid-encoded overexpression of FlgM.

<p>(A) FlgM was secreted to the supernatant in the HMS174(DE3) strain. <b>(B)</b> In contrast, in the newly generated HMS174(DE3)Δ<i>insAB</i> araBAD-<i>flhDC</i> no FlgM could be detected in the supernatant. <b>(C)</b> The ability to secrete FlgM was recovered in the HMS174(DE3)Δ<i>insAB</i> araBAD-<i>flhDC</i> strain when plasmid-encoded <i>flhDC</i> was co-expressed. SDS-PAGE, M Fermentas PageRuler Prestained, -I whole cell sample without induction of recombinant protein expression, +I whole cell samples with induction of recombinant protein expression, C cytoplasmic fraction, Sn supernatant;</p

Inducible secretion of FlgM.

<p>In the newly generated strain HMS174(DE3)LT7 the functional assembly of the hook basal body led to the secretion of plasmid-encoded FlgM into the supernatant. No secretion of FlgM occurred in the strain HMS174(DE3)LL. <b>(A)</b>, HMS174(DE3)LL, pET30-lacUV5-<i>flgM-6His </i><b>(B)</b>, HMS174(DE3)LT7, pET30-lacUV5-<i>flgM-6His </i><b>(C)</b> HMS174(DE3)LT7, pET30-lacUV5-<i>flgM-SOD </i><b>(D)</b> HMS(174)LT7, pET30-lacUV5-<i>flgM-GFPmut3.1-6H</i>, M Fermentas PageRuler Prestained, -I whole cell sample without induction of recombinant protein expression, +I whole cell samples with induction of recombinant protein expression, C cytoplasmic fraction, Sn supernatant;</p

Quantitative Real Time PCR of generated HMS174(DE3) mutant strains.

<p>(A) The generated mutant strains HMS174(DE3)Δ<i>insAB</i> lacUV5-<i>flhDC</i>, HMS174(DE3)Δ<i>insAB</i> lacUV5-<i>flhDC</i>, att7 lacUV5-<i>flhDC</i>, HMS174(DE3) Δ<i>insAB</i> T7-<i>flhDC</i> and HMS174(DE3)LT7 were incubated for 1 h after induction with 1 mM IPTG at 37°C/225 rpm. Quantification of flhD mRNA levels was accomplished with the comparative ΔΔCt method using rpoD for normalization. Resulting data were analyzed via 2-tailed type 2 Student's <i>t</i>-test, ***p<0.001, n = number of replicates.</p

Analysis of the novel host strain HMS174(DE3)LT7.

<p>(A) Scanning Electron Microscopy (SEM) of the HMS174(DE3)LT7 strain without addition of the inducer IPTG. <b>(B)</b> Scanning Electron Microscopy (SEM) of the HMS174(DE3)LT7 strain with addition of the inducer IPTG. Samples were prepared after 90 min cultivation. <b>(C)</b><b>Cell culture homogeneity.</b> The homogeneity regarding <i>fliC</i> promoter activity of three parallel HMS174(DE3)LT7 strain cultivations containing the plasmid p5′UTR-f<i>liC20-GFPmut-Flag-Linker-StrepII</i>-3′UTR with and without addition of the inducer IPTG were analyzed via Fluorescence Activated Cell Sorting (FACS). After incubation for 2 h at 220 rpm at 37 °C all samples were normalized to OD 600: 1.0 and the percentage of GFP expressing cells indicating flagellar assembly was determined. <b>(D)</b><b>FACS analysis of GFP expressing cells</b>. A sample of the HMS174(DE3)LT7 strain cultivation containing the plasmid p5′UTR-<i>fliC20-GFPmut-Flag-Linker-StrepII</i>-3′UTR (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059034#pone-0059034-g006" target="_blank">Figure 6C</a>) with and without addition of the inducer IPTG.</p

Used host strains.

a<p>These names are used to describe each mutant strain in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059034#s2" target="_blank">Results</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059034#s3" target="_blank">Discussion</a>.</p

Transcription of the master operon <i>flhDC</i>.

<p>(<b>A</b>) To verify the transcription of the plasmid-encoded master operon <i>flhDC</i> Reverse Transcriptase PCR Reaction of the isolated RNA was performed. It was assumed that within the overnight culture cells exhibiting flagellar assembly were found and therefore this culture was used as a positive control. Cells before addition of the inducer IPTG were used as reference compared to cells after induction of the plasmid-encoded <i>flhDC</i>. To evaluate genomic DNA contamination the isolated RNA samples were also subjected to subsequent PCR. Plasmid-encoded <i>flhDC</i> was used as a positive control for the PCR reaction. Fermentas 1 KB GeneRuler, ON overnight culture sample, -I whole cell sample without induction of plasmid-encoded <i>flhDC</i>, +I whole cell samples with induction of plasmid-encoded <i>flhDC</i>, RT Reverse Transcriptase Reaction, + plasmid-encoded <i>flhDC</i>; (<b>B</b>) Scheme of the plasmid-encoded master operon under the control of the lacUV5 promoter</p

Modification of the <i>flhDC</i> master operon.

<p>The expression strain HMS174(DE3) was used for modifications of the bacterial genome. Upstream regulator elements of the master operon <i>flhDC</i> were deleted and replaced by the artificial araBAD promoter resulting in the HMS174(DE3)Δ<i>insAB</i> araBAD-<i>flhDC</i> strain. Activator elements are depicted in green, whereas repressor elements are depicted in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059034#pone.0059034-Liu1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059034#pone.0059034-Claret1" target="_blank">[40]</a>.</p

Experimental Study on a Coiled Tube Solar Receiver under Variable Solar Radiation Condition

K. Wang, H. Wu, D. Wang, Y. Wang, Z. Tong, F. Lin, and A. G. Olabi, 'Experimental study on a coiled tube solar receiver under variable solar radiation condition', Solar Energy, Vol. 122, pp.. 1080-1090, December 2015, doi: https://doi.org/10.1016/j.solener.2015.10.004.This article presents an experimental investigation of the heat transfer characteristics as well as energy and exergy performance for a coil type solar dish receiver under variable solar radiation conditions. During a 4.5 h period of continuous operation in the morning, the solar irradiance fluctuates in the range between 160 W/m2 and 650 W/m2, and the concentrated solar flux is approximately 1000 kW/m2 at aperture. Experimental results show that: (1) the efficiency of the solar receiver is found to be over 58% and the highest efficiency can be reached to 64%. Generally, the efficiency is maintained at around 60% under steady state condition. (2) A very low value of the heat loss factor (0.035 kW/K) could be achieved during steady state operation. (3) The highest value of the exergy rate is approximately 7.3 kW, whereas the maximum energy rate can reach 16.2 kW. (4) The highest exergy efficiency is approximately 23%, whereas the highest energy efficiency is around 64%.Peer reviewe