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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. Graphical abstract

Background and purpose Diabetes is a predictor for poor outcome after thrombolysis in stroke patients, and early post-stroke glycaemia is associated with higher rates of post-thrombolytic symptomatic intracerebral haemorrhages (SICHs). Diabetic stroke patients may nevertheless profit from thrombolysis. Here, we compared outcome data of matched thrombolysed and non-thrombolysed diabetic and non-diabetic stroke patients from a national database. Methods The outcomes of 1079 matched quadruples, each consisting of a thrombolysed diabetic, a non-thrombolysed diabetic, a thrombolysed non-diabetic and a non-thrombolysed non-diabetic case (a total of 4316 cases), enrolled in the Austrian Stroke Unit Registry (2004–2013), were compared. Patients were matched according to sex, age, stroke severity, pre-stroke disability and prior stroke. Results A regression model with improvement as depending variable found no effect of diabetes (P = 0.158) or the interaction diabetes × thrombolysis (P = 0.507), whereas the effect of thrombolysis itself was highly significant (P

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.

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-1β and IL-8 (p

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.

In the European Union the use of anabolic hormones in meat production is forbidden since 1988 and this ban of anabolic agents in animal production is strictly controlled. New hormone cocktails passing the detection systems are attractive for the practice and so new approaches to discover their illegal use have to be developed steadily. Verifying physiological effects caused by anabolic steroids will be a new way to develop potential monitoring systems. One promising matrix in female animals will be vaginal smear containing vaginal epithelial cells, because the vaginal epithelium is a primary steroid hormone responsive organ. In this study we quantified the gene expression in vaginal smear of sexually mature cattle in order to observe physiological effects. Further we aimed to establish a new screening method by testing the effect of a combination of certain anabolic steroid hormones on physiological regulations of mRNA expression of selected genes. In an animal trial Nguni heifers were treated with the anabolic combination trenbolone acetate plus estradiol. Vaginal smear samples were taken at 4 different time points. Gene expression of 27 candidate genes, selected by screening the actual literature for steroidal effects on vaginal epithelial cells, were estimated using quantitative real-time RT-PCR. There were different expression changes observed at different time points. It could be shown that the applied anabolic combination significantly influenced the expression of the steroid receptor ERα, the keratinization factor CK8, the proinflammatory interleukins IL-1α and IL-1β, the growth factors FGF7, EGF, EGFR, IGF-1R, TGFα and LTF, the oncogen c-jun and other factors like actinβ and ubiquitin 3. Using biostatistical tools like principal components analysis or hierarchical cluster analysis, the potential to develop a gene expression pattern for targeting the illegal use of growth promoters could be demonstrated.

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.

Real-time PCR has become an important tool for quantitative nucleic acids analysis. By now, a wide rang of real-time PCR instruments, PCR plastic consumables and sealing systems are available. In this study both transparent and white plates were taken to test possible position and plate effects on quantitative PCR results on different real-time platforms: ep realplex (Eppendorf, Hamburg, Germany) and the iQ5 (Bio-Rad, Palo Alto, USA). Heat and adhesive sealing was used to compare sealing systems. Heterogeneity in Ct values was calculated to show possible effects of plate type and sealing system. Heterogeneity in amplification efficiency should show possible positional effects of 96-well plates.White plates showed higher amplification efficiency because of higher fluorescence reflection. This had no significant effect on the PCR efficiency but on the sensitivity of the quantification assay. Constant positional effects concerning wells, columns or rows could not be detected.

Introduction: Multiple, complex interventions are increasingly tested in randomized, clinical trials without enabling a clear view of the strength of the intervention, especially its probability to influence outcome. Such interventions have to be strong enough to create group differences, be acceptable by patients, be realistically designed and deliverable within the health system, and ethical. If found to have a positive effect, it should have a sustainable effect after the trial is drawn to a close. It is necessary to adapt the intensity of the intervention to contrast with guideline-based treated controls and to ensure sufficient means to carry through to the end of the trial. Method: Using a multiple domain life-style based intervention in a randomized trial to prevent cognitive decline following stroke (ClinicalTrials.gov Identifier: NCT01109836), our focus was on nutrition and physical exercise in addition to risk factor management. We performed single component analysis as well as the use of a life-style summary score and a laboratory supported life-style summary score. Results: 80 participants in the intervention group and 87 in the control group completed the 12 months visit. We found a significant improvement in adherence to healthy lifestyle factors and adequately controlled physiological parameters as well as significant differences for both summary scores, all p 2 and currently non-smoking. Laboratory values included blood pressure Conclusion: Complex interventions used for secondary prevention stroke trials have to be intense enough to become effective when comparing to treatment according to current guidelines.

The application of anabolic steroids in food producing animals is forbidden in the EU since 1988, but the abuse of such drugs is a potential problem. The existing test systems are based on known compounds and can be eluded by newly emerging substances. The examination of physiological effects of anabolic hormones on different tissues to indirectly detect misuse might overcome this problem. Two studies were conducted with post-pubertal 24-months old Nguni heifers and pre-pubertal female 2–4 weeks old Holstein Friesian calves, respectively. The animals of the accordant treatment groups were administered combinations of estrogenic and androgenic compounds. The measurement of the gene expression pattern was undertaken with RT-qPCR. Target genes of different functional groups (receptors, angiogenesis, steroid synthesis, proliferation, apoptosis, nutrient metabolism and others) have been quantified. Several biochemical pathways were shown to be influenced by anabolic treatment. Both studies identified significant regulations in steroid and growth factor receptors (AR, ERβ, LHR, FSHR, Flt-1, PR, IGF-1R, Alk-6), angiogenic and tissue remodeling factors (VEGFs, FGFs, BMPs, ANGPT-2, MMPs, TIMP-2, CTSB), steroid synthesis (S5A1, HSD17, CYP19A1), proliferation (TNFα, IGF-1, IGFBPs, p53, c-fos; CEBPD, c-kit), apoptosis (CASP3, FasL, p53) and others (C7, INHA, STAR). Several genes were regulated to opposite directions in post-pubertal compared to pre-pubertal animals. PCA for Nguni heifers demonstrated a distinct separation between the control and the treatment group. In conclusion, anabolics modify hormone sensitivity and steroid synthesis, and they induce proliferative effects in the whole reproductive tract (uterus and ovary) as well as anti-angiogenic effects in the ovary. However, the extent will depend on the developmental stage of the animals.

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.

Doping with anabolic agents is a topic in sports where strength is crucial, e.g. sprinting, weight lifting and many more. Testosterone and its functional analogs are the drugs of choice taken as pills, creams, tape or injections to increase muscle mass and body performance, and to reduce body fat. Stanozolol (17β-hydroxy-17α-methyl-5α-androst-2-eno[3,2c]pyrazol) is a testosterone analogue with the same anabolic effect like testosterone but its ring structure makes it possible to take it orally. Therefore, stanozolol is one of the most frequently used anabolic steroids. Common verification methods for anabolic drugs exist, identifying the chemicals in tissues, like hair or blood samples. The idea of this feasibility study was to search for specific gene expression regulations induced by stanozolol to identify the possible influence of the synthetically hormone on different metabolic pathways. Finding biomarkers for anabolic drugs could be supportive of the existing methods and an additional proof for illegal drug abuse. In two separate cell cultures, human HFDPC (hair follicle dermal papilla cells) from a female and a male donor were treated with stanozolol. In the female cell culture treatment concentrations of 0 nM (control), 1 nM, 10 nM and 100 nM were chosen. Cells were taken 0 h, 6 h, 24 h and 48 h after stimulation and totalRNA was extracted. Learning from the results of the pilot experiment, the male cell culture was treated in 10 nM and 100 nM concentrations and taken after 0 h, 6 h, 24 h and 72 h. Using quantitative real-time RT-PCR expression of characteristics of different target genes were analysed. Totally 13 genes were selected according to their functionality by screening the actual literature and composed to functional groups: factors of apoptosis regulation were Fas Ligand (FasL), its receptor (FasR), Caspase 8 and Bcl-2. Androgen receptor (AR) and both estrogen receptors (ERα, ERβ) were summarized in the steroid receptor group. The growth factor group included the insulin like growth factor receptor (IGF1R) and growth hormone receptor (GHR). Fibroblast growth factor 2 (FGF2) and keratinocyte growth factor (FGF7) were summarized in the hair cycle factor group. 5α-Steroidreductases (SRD5A1, SRD5A2) represented the enzyme group. Three reference genes were taken for relative quantification: ubiquitin (UBQ), glycerinaldehyde-3-phsophate-dehydrogenase (GAPDH), and β-actin (ACTB). In cell culture 1 AR, FasR, FGF2 showed significant regulations within one treatment time, significant gene expressions over time were analysed for Caspase 8. In cell culture 2 AR, FasR and SRD5A2 were significantly regulated within one treatment time. In this feasibility study first biomarker for a screening pattern of anabolic agents could be identified providing the rationality to investigate modified, metabolic pathways in the whole hair follicle.

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.

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.

In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly, the biological expression variances of GAPDH, TNFα, IL-1β, TLR4 were measured by mRNA profiling experiment in single lymphocytes. The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell. Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis.

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.