Plastizität von schnell teilenden humanen mesenchymalen Stammzellen auf Einzelzellebene

Zellkulturen humaner mesenchymaler Stammzellen (hMSC) enthalten überwiegend drei Subpopulationen: spindelige fibroblastenähnliche Zellen, große abflachte Zellen und kleine hoch proliferative Zellen, die sogenannten rapidly self-renewing cells (RS-Zellen). Ziel dieser Studie war zunächst die Isolation dieser RS-Zellen auf Einzelzellniveau und ihre anschließende klonale Expansion auf eine für Folgeexperimente hohe Zellzahl. Das Hauptziel war das Stammzellkriterium der Plastizität für eine RS-Zelle durch Differenzierung in die adipogene, osteogene und chondrogene Richtung ausgehend von einer Zelle nachzuweisen. HMSCs der Fa. Cambrex (USA) wurden entsprechend den Herstellerangaben kultiviert. Einzelne Zellen wurden mittels single cell picking isoliert und klonal expandiert sowie anschließend entweder nach Standardprotokollen adipogen, osteogen und chondrogen differenziert, oder als Kontrolle unstimuliert kultiviert. Die histologische Auswertung der Differenzierung erfolgte mit Oil Red-O- (Fettzellen), von Kossa- (Knochenzellen) und Toluidin Blau- (Knorpelzellen) Färbung. Für die chondrogene Differenzierung wurde zudem eine spezifische Immunfluoreszenzfärbung gegen Kollagen Typ-II durchgeführt. Nach Optimierung des Isolationsverfahrens mittels Einzelzellpickens konnte ausgehend von einer einzelnen Zelle die Zellzahl innerhalb von 5 Wochen auf ca. 1 Mio. Zellen expandiert werden. Die adiopogene, osteogene und chondrogene Differenzierung konnte bei den stimulierten RS-Zellen durch die oben beschriebenen histologisch Färbemethoden nachgewiesen werden. Die unstimulierten Kontrollen veränderten sich nicht. Die Versuche wurden stets mit einer heterogenen Kontrollgruppe durchgeführt. In dieser Studie ist es gelungen, ausgehend von einer einzelnen RS-Zelle, die Differenzierung in drei verschiedene Richtungen nachzuweisen. Somit konnten für die RS-Zellen erstmals die Stammzellkriterien einer hohen Replikationsrate sowie die Plastizität durch Differenzierung in drei mesenchymale Gewebetypen nachgewiesen werden. Zudem konnten für die Klassifizierung der RS-Zellen in Bezug auf Morphologie und Wachstumskinetik wichtige Erkenntnisse erbracht werden. Aufgrund ihrer Vermehrungsfähigkeit in vitro sind RS-Zellen für das tissue engineering besonders von Bedeutung. Jedoch bedarf es weiterer Studien, um das Verhalten der RS-Zellen als Subpopulation der humanen mesenchymalen Stammzellen besser zu verstehen

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1- like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in overreplication and leads to chromosome breaks and chromosome-wide, strandbiased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select \~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors

Transcription Factor MAFF (MAF Basic Leucine Zipper Transcription Factor F) Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism

BACKGROUND: Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue-specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterize central regulators and networks leading to atherosclerosis. METHODS: Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knockout models) and human (as shown by genome-wide association studies), liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models, as well as experimental studies including chromatin immunoprecipitation DNA-sequencing, chromatin immunoprecipitation mass spectrometry, overexpression, small interfering RNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. RESULTS: The transcription factor MAFF (MAF basic leucine zipper transcription factor F) interacted as a key driver of a liver network with 3 human genes at CAD genome-wide association studies loci and 11 atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor (LDLR) gene correlated with MAFF in 600 CAD patients undergoing bypass surgery (STARNET [Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task]) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of MAFF were tested in noninflammatory conditions and showed positive correlation between MAFF and LDLR in vitro and in vivo. Interestingly, after lipopolysaccharide stimulation (inflammatory conditions), an inverse correlation between MAFF and LDLR in vitro and in vivo was observed. Chromatin immunoprecipitation mass spectrometry revealed that the human CAD genome-wide association studies candidate BACH1 (BTB domain and CNC homolog 1) assists MAFF in the presence of lipopolysaccharide stimulation with respective heterodimers binding at the MAF recognition element of the LDLR promoter to transcriptionally downregulate LDLR expression. CONCLUSIONS: The transcription factor MAFF was identified as a novel central regulator of an atherosclerosis/CAD-relevant liver network. MAFF triggered context-specific expression of LDLR and other genes known to affect CAD risk. Our results suggest that MAFF is a missing link between inflammation, lipid and lipoprotein metabolism, and a possible treatment target

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins. We quantified the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA indicating head-to-tail replication fork collisions as key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins. We quantified the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA indicating head-to-tail replication fork collisions as key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation

África como mercado potencialmente atractivo

El mercado de plástico en África parece ser atractivo para la compañía X la cual opera en diversas zonas del mundo y en parte también en esta. Este mercado cuenta con alrededor de 1.300 millones de africanos y un consumo de plástico de 16 kg per cápita, siendo este un lugar ideal en el cual operar. (Babayemi, Nnorom, Osibanjo, & Weber, 2019). La Compañía X espera contar con información sobre el mercado africano de moldes de inyección de plástico, estando interesada especialmente en el tamaño de mercado actual, los clientes potenciales, en la forma de hacer negocios y en factores externos e internos que afectan la operación en estos países. Con el objetivo de tomar una decisión sobre si entrar o no a este mercado, la empresa delegó a un grupo de estudiantes de la University of Applied Sciences of Upper para llevar a cabo una investigación y formular recomendaciones sobre este. Este informe ofrece una perspectiva detallada de los resultados de la investigación realizada sobre el mercado objetivo, los clientes potenciales, los aspectos comerciales y el análisis PESTEL para seis países africanos seleccionados: Egipto, Etiopía, Kenia, Nigeria, Sudáfrica y Uganda (en sustitución de Marruecos). Estos países fueron elegidos con base a un estudio inicial sobre su número de clientes potenciales identificados y su estabilidad política y económica, a cada uno de estos aspectos se les dio una ponderación específica según la importancia para la Compañía X. En la investigación principal al clasificar los países en función de sus resultados de cada aspecto evaluado, Egipto resultó ser el más prometedor seguido de Sudáfrica y Nigeria, principalmente por su gran mercado de productos de plásticos y la cantidad de clientes potenciales que la Compañía X podría encontrar en este. Adicionalmente, aunque fue sustituido por Uganda en el transcurso de la investigación, Marruecos resultó ser muy atractivo. Por tanto, no hay que dejar pasar esta oportunidad y encontrar la manera de superar la barrera del idiomaThe African plastic market seems to be utterly appealing to Company X which is operative in many areas of the world, partly also in this one. With around 1,3 billion Africans and a plastic consumption of 16kg per capita, the market is highly promising (Babayemi, Nnorom, Osibanjo, & Weber, 2019). Company X is looking forward to having information about the African market for plastic injection moulds. They are especially concerned about the actual market size, potential customers, doing business aspects as well as external and internal factors affecting countries’ performances. To make an informed decision about whether to enter this market, students at the University of Applied Sciences of Upper Austria were delegated to conduct research and formulate recommendations. The start point was a kick-off meeting with Company X. The purpose was to research if there is a profitable and sustainable market for a specific selection of rigid plastic products in the African continent. This report gives a profound overview by providing findings of conducted research about the target market, potential clients, business aspects, and PESTEL analysis for six selected African countries: Egypt, Ethiopia, Kenya, Nigeria, South Africa, and Uganda (substitute for Morocco). These countries were chosen based on initial research about their number of identified potential clients, and political and economic stability. The aspects were given different weightings according to the importance to Company X. In the main research, by ranking the countries based on their performance for each category chosen in the country ranking (namely PESTEL, target market, potential clients, and cultural proximity). Egypt turned out to be the most promising one followed by South Africa and Nigeria. Mainly because of its huge market for plastic products and a lot of potential customers, Egypt outperforms other countries. Furthermore, although substituted by Uganda during the course of the research, Morocco turned out to be highly appealing. Thus, one should not miss out on this opportunity and find ways to overcome the language barrie

Whole-body CT in haemodynamically unstable severely injured patients--a retrospective, multicentre study.

The current common and dogmatic opinion is that whole-body computed tomography (WBCT) should not be performed in major trauma patients in shock. We aimed to assess whether WBCT during trauma-room treatment has any effect on the mortality of severely injured patients in shock.In a retrospective multicenter cohort study involving 16719 adult blunt major trauma patients we compared the survival of patients who were in moderate, severe or no shock (systolic blood pressure 90-110,<90 or >110 mmHg) at hospital admission and who received WBCT during resuscitation to those who did not. Using data derived from the 2002-2009 version of TraumaRegister®, we determined the observed and predicted mortality and calculated the standardized mortality ratio (SMR) as well as logistic regressions.9233 (55.2%) of the 16719 patients received WBCT. The mean injury severity score was 28.8±12.1. The overall mortality rate was 17.4% (SMR  = 0.85, 95%CI 0.81-0.89) for patients with WBCT and 21.4% (SMR = 0.98, 95%CI 0.94-1.02) for those without WBCT (p<0.001). 4280 (25.6%) patients were in moderate shock and 1821 (10.9%) in severe shock. The mortality rate for patients in moderate shock with WBCT was 18.1% (SMR 0.85, CI95% 0.78-0.93) compared to 22.6% (SMR 1.03, CI95% 0.94-1.12) to those without WBCT (p<0.001, p = 0.002 for the SMRs). The mortality rate for patients in severe shock with WBCT was 42.1% (SMR 0.99, CI95% 0.92-1.06) compared to 54.9% (SMR 1.10, CI95% 1.02-1.16) to those without WBCT (p<0.001, p = 0.049 for the SMRs). Adjusted logistic regression analyses showed that WBCT is an independent predictor for survival that significantly increases the chance of survival in patients in moderate shock (OR = 0.73; 95%CI 0.60-0.90, p = 0.002) as well as in severe shock (OR = 0.67; 95%CI 0.52-0.88, p = 0.004). The number needed to scan related to survival was 35 for all patients, 26 for those in moderate shock and 20 for those in severe shock.WBCT during trauma resuscitation significantly increased the survival in haemodynamically stable as well as in haemodynamically unstable major trauma patients. Thus, the application of WBCT in haemodynamically unstable severely injured patients seems to be safe, feasible and justified if performed quickly within a well-structured environment and by a well-organized trauma team

Adjusted logistic regression models 2,3 and 4; shock-subgroups.

<p>RISC  =  revised injury severity classification, CI 95%  =  confidence interval; * Inverse logistic transformation of the predicted outcome probability of RISC (mortality).</p