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1.  Functional capacity of reconstituted blood in 1:1:1 versus 3:1:1 ratios: A thrombelastometry study 
Introduction
Different transfusion ratio concepts of packed red blood cells (pRBCs), fresh frozen plasma (FFP) and platelets (PLTs) have been implemented in trauma care, but the optimal ratios are still discussed. In this study the hemostatic potential of two predefined ratios was assessed by using an in vitro thrombelastometric approach. Furthermore, age effects of reconstituted blood were analyzed.
Methods
Whole blood (WB) of voluntary donors was separated into pRBCs, FFP and PLTs and reconstituted into the ratios 1:1:1 and 3:1:1 at day 1, 4, 14, and 24. Standard blood count, electrolytes and coagulation proteins were quantified. The functional coagulation in ratio- and age-specific groups was evaluated using rotational thromboelastometry (ROTEM).
Results
Several coagulation factors reduced significantly in the 3:1:1 ratio and were consistent with increased INR, decelerated clot formation times and A10 (amplitude 10 minutes after clotting time (CT)), flattened α-angle during the EXTEM and diminished MCF for distinct time points during the INTEM, FIBTEM and APTEM assays. With rising age of pRBCs the pH, sodium and potassium reached non-physiological levels.
Conclusion
Under standardized in vitro conditions the higher amount of pRBCs in the 3:1:1 ratio diluted coagulation factors significantly on the expense of its functional coagulation capacity as revealed by ROTEM results. Thus, the coagulation functionality of the 1:1:1 ratio predominated.
doi:10.1186/s13049-014-0080-0
PMCID: PMC4296528  PMID: 25571924
Transfusion practices; Acute bleeding injury; Coagulopathy; Thrombelastometry in trauma; Ratio of components
2.  Dynamics of mRNA and polysomal abundance in early 3T3-L1 adipogenesis 
BMC Genomics  2014;15(1):381.
Background
Adipogenesis is a complex process, in which immature pre-adipocytes change morphology, micro-anatomy and physiology to become mature adipocytes. These store and accumulate fat and release diverse hormones. Massive changes in protein content and protein composition of the transforming cell take place within a short time-frame.
In a previous study we analyzed changes in the abundance of free and polysomal, i.e. ribosome bound, RNAs in the first hours of adipogenesis in the murine cell line 3T3-L1. Here we analyze changes of mRNA levels and their potential contribution to the changing protein pool by determination of mRNA levels and ribosome binding to mRNAs in 3T3-L1 cells stimulated for adipogenesis. We grouped mRNA species into categories with respect to up- or down-regulated transcription and translation and analyzed the groups regarding specific functionalities based on Gene Ontology (GO).
Results
A shift towards up-regulation of gene expression in early adipogenesis was detected. Genes up-regulated at the transcriptional (TC:up) and translational (TL:up) level (TC:up/TL:up) are very likely involved in control and logistics of translation. Many of them are known to contain a TOP motif. In the TC:up/TL:unchanged group we detected most of the metal binding proteins and metal transporters. In the TC:unchanged/TL:up group several factors of the olfactory receptor family were identified, while in TC:unchanged/TL:down methylation and repair genes are represented. In the TC:down/TL:up group we detected many signaling factors. The TC:down/TL:unchanged group mainly consists of regulatory factors.
Conclusions
Within the first hours of adipogenesis, changes in transcriptional and translational regulation take place. Notably, genes with a specific biological or molecular function tend to cluster in groups according to their transcriptional and translational regulation.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-381) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-381
PMCID: PMC4039748  PMID: 24886538
Adipogenesis; Transcription; Translation; 3T3-L1 pre-adipocytes; Gene ontology; Gene enrichment; Differential expression; TOP motif
3.  Extrinsic and intrinsic regulation of DOR/TP53INP2 expression in mice: effects of dietary fat content, tissue type and sex in adipose and muscle tissues 
Background
DOR/TP53INP2 acts both at the chromosomal level as a nuclear co-factor e.g. for the thyroid hormone receptor and at the extrachromosomal level as an organizing factor of the autophagosome. In a previous study, DOR was shown to be down-regulated in skeletal muscle of obese diabetic Zucker fa/fa rats.
Methods
To identify sites of differential DOR expression in metabolically active tissues, we measured differences in DOR expression in white adipose tissue (WAT), brown adipose tissue (BAT), skeletal muscle (SM) and heart muscle (HM) by qPCR. To assess whether DOR expression is influenced in the short term by nutritional factors, NMRI mice were fed different fat rich diets (fat diet, FD: 18% or high fat diet, HFD: 80% fat) for one week and DOR expression was compared to NMRI mice fed a control diet (normal diet, ND: 3.3% fat). Additionally, DOR expression was measured in young (45 days old) and adult (100 days old) genetically obese (DU6/DU6i) mice and compared to control (DUKs/DUKsi) animals.
Results
ANOVA results demonstrate a significant influence of diet, tissue type and sex on DOR expression in adipose and muscle tissues of FD and HFD mice. In SM, DOR expression was higher in HFD than in FD male mice. In WAT, DOR expression was increased compared to BAT in male FD and HFD mice. In contrast, expression levels in female mice were higher in BAT for both dietary conditions.
DOR expression levels in all tissues of 100 days old genetically obese animals were mainly influenced by sex. In HM, DOR expression was higher in male than female animals.
Conclusions
DOR expression varies under the influence of dietary fat content, tissue type and sex. We identified target tissues for further studies to analyze the specific function of DOR in obesity. DOR might be part of a defense mechanism against fat storage in high fat diets or obesity.
doi:10.1186/1743-7075-9-86
PMCID: PMC3497704  PMID: 22995226
DOR/TP53INP2; High fat diet; Genetically induced obesity; Fat tissue; Muscle tissue
4.  Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice 
PLoS ONE  2012;7(6):e39711.
We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.
doi:10.1371/journal.pone.0039711
PMCID: PMC3387210  PMID: 22768110
5.  Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells 
Background
Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting.
Results
We identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes.
Conclusions
We conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis.
doi:10.1186/1471-2199-13-9
PMCID: PMC3347988  PMID: 22436005

Results 1-5 (5)