Characterisation of sequence-structure-function space in sensor-effector integrators of phytochrome-regulated diguanylate cyclases.

Understanding the relationship between protein sequence, structure and function is one of the fundamental challenges in biochemistry. A direct correlation, however, is often not trivial since protein dynamics also play an important functional role-especially in signal transduction processes. In a subfamily of bacterial light sensors, phytochrome-activated diguanylate cyclases (PadCs), a characteristic coiled-coil linker element connects photoreceptor and output module, playing an essential role in signal integration. Combining phylogenetic analyses with biochemical characterisations, we were able to show that length and composition of this linker determine sensor-effector function and as such are under considerable evolutionary pressure. The linker length, together with the upstream PHY-specific domain, influences the dynamic range of effector activation and can even cause light-induced enzyme inhibition. We demonstrate phylogenetic clustering according to linker length, and the development of new linker lengths as well as new protein function within linker families. The biochemical characterisation of PadC homologs revealed that the functional coupling of PHY dimer interface and linker element defines signal integration and regulation of output functionality. A small subfamily of PadCs, characterised by a linker length breaking the coiled-coil pattern, shows a markedly different behaviour from other homologs. The effect of the central helical spine on PadC function highlights its essential role in signal integration as well as direct regulation of diguanylate cyclase activity. Appreciation of sensor-effector linkers as integrator elements and their coevolution with sensory modules is a further step towards the use of functionally diverse homologs as building blocks for rationally designed optogenetic tools.

Effect of irradiation on the expression of DNA repair genes studied in human fibroblasts by real-time qPCR using three methods of reference gene validation.

The aim of this study was to determine the effects of ionizing radiation on gene expression by using for a first time a qPCR platform specifically established for the detection of 94 DNA repair genes but also to test the robustness of these results by using three analytical methods (global pattern recognition, ΔΔCq/Normfinder and ΔΔCq/Genorm). Study was focused on these genes because DNA repair is known primarily to determine the radiation response. Six strains of normal human fibroblasts were exposed to 2 Gy, and changes in gene expression were analyzed 24 h thereafter. A significant change in gene expression was found for only few genes, but the genes detected were mostly different for the three analytical methods used. For GPR, a significant change was found for four genes, in contrast to the eight or nine genes when applying ΔΔCq/Genorm or ΔΔCq/Normfinder, respectively. When using all three methods, a significant change in expression was only seen for GADD45A and PCNA. These data demonstrate that (1) the genes identified to show an altered expression upon irradiation strongly depend on the analytical method applied, and that (2) overall GADD45A and PCNA appear to play a central role in this response, while no significant change is induced for any of the other DNA repair genes tested.

Helicopter transport of stroke patients and its influence on thrombolysis rates: data from the Austrian Stroke Unit Registry.

BACKGROUND AND PURPOSE: Acute stroke management requires minimization of prehospital time. This study addresses the value of helicopter transport compared with other means of transportation to a stroke unit and compares their rates of thrombolysis on a nationwide basis. METHODS: Prospective data collection and prespecified evaluation of data from 32 stroke units between 2003 and 2009 were used. We distinguished between patients transported either directly to a stroke unit or transferred indirectly via a peripheral hospital. Thus, there were 6 transport groups: helicopter emergency service (HEMS) direct and indirect, ambulance accompanied by an emergency physician direct and indirect, and ambulance without physician direct and indirect. Demographic and clinical factors, time delays, and rates of thrombolysis of patients transported by helicopter were compared with factors of patients transported otherwise. RESULTS: Of 21 712 ischemic stroke patients, 905 patients (4.1%) were transported by helicopter. Of these, 752 patients (3.4%) were transported by direct HEMS, and 153 patients (0.7%) were transported by indirect HEMS. Thrombolysis rates were highest for HEMS (24% direct, 29% indirect) transport, followed by ambulance accompanied by an emergency physician (18% direct, 15% indirect). The probability of receiving thrombolysis was highest for indirect HEMS transport (OR 3.6, 2.2-6.0), followed by indirect ambulance accompanied by an emergency physician transport (OR 1.5, 1.1-1.9). The shortest times, 90 minutes or less from stroke onset to hospital arrival, were achieved with direct AMBP and direct HEMS transport. CONCLUSIONS: The shortest hospital arrival times and highest thrombolysis rates were seen in ischemic stroke patients transported by helicopter.

Changes in the miRNA profile under the influence of anabolic steroids in bovine liver.

miRNAs are regulatory RNA molecules. The analytical interest rose over the past 10 years especially in clinical diagnostics as miRNAs show specific expression patterns in several human diseases like diabetes or cancer. Therefore, it is expected that miRNA profiles might be used as biomarkers in early diagnosis. The idea of establishing biomarkers is also present in veterinary drug analysis, e.g. in the surveillance of illegal use of anabolics. Transcriptomics is a promising approach in the detection of anabolics misuse. However, miRNA expression patterns have shown their superiority over mRNA patterns in clinical diagnostics. Thus, the influence of anabolic steroids on miRNA expression in bovine liver should be investigated and an expression pattern should be validated, which might be used as a treatment biomarker. An animal experiment was conducted with 18 heifers equally allocated to a control and a treatment group, which was implanted with TBA plus E2. Liver samples were screened for miRNA expression using PCR arrays. Expression of 11 prominent miRNAs was validated via single assay qPCR. Herein, the following expression pattern could be found with an up-regulation of miR-29c and miR-103 and a down-regulation of miR-34a, miR-181c, miR-20a and miR-15a (p<0.05 each). Using principal components analysis (PCA), the control group could clearly be distinguished from the treatment group, when integrating gene expression results from both miRNA and mRNA. So, the combination of different transcribed targets (mRNA plus miRNA) might be a promising approach to find a valid expression pattern to be used for anabolic treatment screening.

Expression of immune relevant genes in pigs under the influence of low doses of deoxynivalenol (DON).

Deoxynivalenol (DON) is one of the most common Fusarium toxins in animal feed and poses a potential risk especially for monogastric animals like pigs. DON is known to modulate the immune system, dependent on dose and frequency of exposure. The aim of the study was to investigate the effects of chronic exposure to low levels of DON on the expression of immune relevant genes. In a feeding trial (84 days), 20 pigs were assigned equally to a control and a treatment group. The DON-content of the contaminated diet was 1.2 mg/kg from day 1 to 41, from day 42 it was elevated to 2.0 mg/kg. The control group (n = 10) was fed a diet with a DON concentration lower than 0.05 mg/kg. Blood samples were taken over the course of the study and ileum samples were taken at slaughter. Gene expression measurement was done using real-time RT-qPCR. For target genes, those cytokines were chosen, which were estimated to be implicated in the modulation of the immune system induced by DON ingestion. In ileum, significant down-regulations could be observed for IL-1ß and IL-8 (p < 0.05). Most significant regulations in blood could be detected on day 45 after increasing the dietary DON content in the experimental diet. Herein, down-regulations of IL-1ß, IL-8 and TNFα were demonstrated. In conclusion, the present study provides data concerning chronic application of DON in low doses, as little is known in this area. Down-regulations of immune-related transcription factors and pro-inflammatory immune factors could be demonstrated.

Effect of trenbolone acetate plus estradiol on transcriptional regulation of metabolism pathways in bovine liver.

BACKGROUND: The use of anabolic steroids is forbidden for food producing animals in the EU. Owing to the advantages of anabolics for production profitability, illegal application is appealing. Anabolics are known to influence gene expression of several tissues. We focused on the liver because of its important role in nutrient and hormone metabolism. The aim of the present study was to find differentially regulated metabolic pathways, which might be used as treatment biomarkers. MATERIAL AND METHODS: A total of 18 Nguni heifers were allocated equally to a control group and a treatment group and were implanted with Revalor H. Expression of 34 target genes was measured using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). RESULTS: Upregulation of androgen receptor and insulin-like growth factor 1 (IGF-1) and downregulation of IGF-2, insulin-like growth factor binding protein 2, steroid hormone binding globulin, insulin receptor α, insulin receptor ß, tyrosine aminotransferase, 17ß-hydroxy steroid dehydrogenase 2,3-hydroxy-methylglutaryl-coenzym-A-synthase, cathepsin B, hepatocyte growth factor, steroidogenic acute regulatory protein, apolipoprotein 2 and tumor necrosis factor α was demonstrated. CONCLUSION: Several biochemical pathways showed different regulations on mRNA level under the influence of trenbolone acetate plus estradiol. The inhibition of nutrient metabolism and protein breakdown seems to support growth processes. IGF-1 plays an important role in growth and development and thus the upregulation of IGF-1 could be responsible for the stimulation of growth in treated animals. The upregulation of IGF-1 could also be revealed as a possible risk factor for the generation of artherosclerotic plaques, which are known as long-term side effects following the use of anabolic steroids. Principal components analysis of RT-qPCR results showed that both groups arrange together and can be clearly separated. Therefore, these might be used as possible biomarkers in bovine liver.

Monitoring gene expression in muscle tissue of macaca fascicularis under the influence of testosterone and SARM.

The focus of this study was to evaluate data on the gene expression profiles induced by testosterone and a selective androgen receptor modulator (SARM, TAP Pharmaceutical Products Inc., Lake Forest, IL, USA) in androgen sensitive muscle tissue to obtain a better understanding on the molecular mechanisms of action and to identify biomarkers for SARM function in primate organs. A total of 24 male cyomolgus monkeys were divided into four groups: testosterone group, SARM1 group, SARM10 group, and control group, each consisting of six animals. The testosterone group was treated i.m. with 3.0 mg/kg Testostoviron®-depot-250 (Schering, Berlin, Germany) every 2 weeks, the SARM1 and SARM10 groups with 1 mg/kg or 10 mg/kg SARM LGD2941 daily, and the control group was not treated. Muscle biopsies from musculus quadriceps and musculus triceps were collected at three time points: baseline time point before SARM application (control), on day 16, and on day 90 of treatment. A total of 30 candidate genes were selected according to their functionality by screening the actual literature and were composed to the following functional groups: cell cycle, endocrine factors, energy metabolism, muscle fiber proteins, muscle specific transcription factors, protein metabolism, and satellite cell biology. Biomarkers were identified as genes regulated from baseline in any of the three treatment groups at day 16 or day 90 using analysis of variance with baseline defined as the contrast group. Out of 23 tested candidate genes, 3 were significantly regulated in m. quadriceps after 90 days treatment; in m. triceps no significant differences were identified. Cathepsin L, calpain 3, and insulin like growth factor binding protein 3 could be identified as first biomarkers, and first physiological differences between control and treatment samples were determined. Both testosterone and SARM LGD2941 appear to have similar effects after 90 days treatment, and thus a longer-term therapy with these substances can be recommended.

Influence of testosterone and a novel SARM on gene expression in whole blood of Macaca fascicularis.

Anabolic hormones, including testosterone, have been suggested as a therapy for aging-related conditions, such as osteoporosis and sarcopenia. These therapies are sometimes associated with severe androgenic side effects. A promising alternative to testosterone replacement therapy are selective androgen receptor modulators (SARMs). SARMs have the potential to mimic the desirable central and peripheral androgenic anabolic effects of testosterone without having its side effects. In this study we evaluated the effects of LGD2941, in comparison to testosterone, on mRNA expression of selected target genes in whole blood in an non-human model. The regulated genes can act as potential blood biomarker candidates in future studies with AR ligands. Cynomolgus monkeys (Macaca fascicularis) were treated either with testosterone or LGD2941 for 90 days in order to compare their effects on mRNA expression in blood. Blood samples were taken before SARM application, on day 16 and on day 90 of treatment. Gene expression of 37 candidate genes was measured using quantitative real-time RT-PCR (qRT-PCR) technology. Our study shows that both testosterone and LGD2941 influence mRNA expression of 6 selected genes out of 37 in whole blood. The apoptosis regulators CD30L, Fas, TNFR1 and TNFR2 and the interleukins IL-12B and IL-15 showed significant changes in gene expression between control and the treatment groups and represent potential biomarkers for androgen receptor ligands in whole blood.

Identification of potential gene expression biomarkers for the surveillance of anabolic agents in bovine blood cells.

In the EU, the use of anabolic steroids in food producing animals has been forbidden since 1988. The routine methods used in practice are based on the detection of hormonal residues. To overcome these routine methods, growth-promoting agents are sometimes administered at concentrations below the detection limit and new anabolic substances are designed. Therefore, new monitoring systems are needed to overcome the misuse of anabolic agents in meat production. In this study, a new monitoring system was applied: the quantification of mRNA gene expression changes by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). Blood was selected as ideal tissue for biomarker screening. From the literature, it is known that steroid hormones affect mRNA gene expression of the different blood cells, which can easily be taken from the living animal. In an animal trial, 18 Nguni heifers were separated to two groups of nine animals. One group served as untreated control and the other group was treated with a combination of trenbolone acetate plus estradiol for 39 days in order to allow the detection of the effect on mRNA expression in blood at three time points. Candidate genes used for developing a biomarker pattern were chosen by screening the actual literature for anabolic effects on blood cells. It could be demonstrated that the combination of trenbolone acetate plus estradiol significantly influences mRNA expression of the steroid receptors (ER-alpha and GR-alpha), the apoptosis regulator Fas, the proinflammatory interleukins IL-1alpha, IL-1beta and IL-6 and of MHCII, CK, MTPN, RBM5 and Actin-beta. Advanced statistical analysis by Principal Components Analysis (PCA) indicated that these genes represent potential biomarkers for this hormone combination in whole blood.

Modification of mRNA expression after treatment with anabolic agents and the usefulness for gene expression-biomarkers.

With this feasibility study a first step towards a new monitoring system for hormonal treatments was done. Screening of regulation and function of anabolic sex steroids via modified gene expression of mRNA in various tissues could be a new approach to trace treatments with unknown drugs or newly combined cocktails. In the study, uterus, liver and muscle tissue from 24 cycling heifers were taken after the animals were treated either with Melengestrol Acetate (MGA), Finaplix-H (200 mg Trenbolone Acetate) or Ralgro (36 mg Zeranol) for 56 days. In every treatment group always two heifers were given 1-fold, 3-fold and 10-fold doses of the standard preparation, the control group without any treatment consisted of two animals. The different tissue gene expression profiles were investigated via the candidate gene approach. Totally 57 candidate genes were selected according to their functionality by screening the actual literature and composed to functional groups: angiogenesis, apoptosis, cell cycle, endocrine factors, energy metabolism, inflammatory factors, muscle function, oncogenes, protein metabolism and transcription factors. Gene expression was measured using quantitative real-time RT-PCR (qRT-PCR) technology. From 24 tested candidate genes in the liver, 17 showed a significant regulation. Eight genes were influenced by MGA, 9 by Finaplix-H, and 4 by Ralgro. For the muscle tissue 19 genes were tested with the result that in the neck muscle 11 genes were regulated and in the hind limb muscle 8 genes. In the neck 5 genes were affected by MGA, 6 by Finaplix-H and 3 by Ralgro. Only 2 genes were influenced by MGA in the hind limb muscle. Finaplix-H affected 6 and Ralgro 4 genes. In the uterus 29 target genes were tested and 13 were significantly influenced by the anabolic sex steroids. Under Finaplix-H treatment eight target genes were regulated and Ralgro and MGA showed a significant regulation in four target genes. The highest gene expression changes under anabolic treatment were observed in the uterus. The analyzed genes showed significant regulations but further studies, testing different animal husbandry conditions will be needed to identify meaningful expression patterns for the different tissues. With the investigation of the regulation and possible function of anabolic sex steroids via gene expression, a preparatory work for the development of an expression pattern for drug screening was made.