[Position paper on working in the intensive care unit during pregnancy : DIVI recommendations for improving the situation of pregnant employees in the ICU]. / Positionspapier zum Arbeiten in der Schwangerschaft auf der Intensivstation : DIVI-Empfehlungen zur Verbesserung der Lage von schwangeren Mitarbeiterinnen auf einer Intensivstation.

The Maternity Protection Act is intended to protect the mother and the child from hazards, excessive demands and damage to health in the workplace, and from financial disadvantages and loss of employment. However, the objectives defined by the Maternity Protection Act-the safety and health of the pregnant employee on the one hand and the prevention of disadvantages in working life on the other-are not yet adequately achieved in the intensive care unit (ICU). Implementation of the Maternity Protection Act to the benefit of all involved parties should also be promoted in the specialist areas represented by the DIVI, in particular the work of pregnant physicians and nursing staff and other working specialists (respiratory therapists, physiotherapists, speech therapists, psychotherapists, and social workers) in the ICU. The aim of this paper is to raise awareness of the need to consider each pregnant and breastfeeding staff member individually and to work together to find a personal solution for continuing to work in the ICU. Possible ways and solutions to achieve this goal are outlined and practical examples are given for implementation in everyday clinical routine. These are also based on comprehensive presentation of activities according to a traffic light color-code system for all occupational groups. Arguments against pregnant employees working in the ICU are discussed and possible solutions are presented.

[Position paper on working in the intensive care unit during pregnancy : DIVI recommendations for improving the situation of pregnant employees in the ICU]. / Positionspapier zum Arbeiten in der Schwangerschaft auf der Intensivstation : DIVI-Empfehlungen zur Verbesserung der Lage von schwangeren Mitarbeiterinnen auf einer Intensivstation.

The Maternity Protection Act is intended to protect the mother and the child from hazards, excessive demands and damage to health in the workplace, and from financial disadvantages and loss of employment. However, the objectives defined by the Maternity Protection Act-the safety and health of the pregnant employee on the one hand and the prevention of disadvantages in working life on the other-are not yet adequately achieved in the intensive care unit (ICU). Implementation of the Maternity Protection Act to the benefit of all involved parties should also be promoted in the specialist areas represented by the DIVI, in particular the work of pregnant physicians and nursing staff and other working specialists (respiratory therapists, physiotherapists, speech therapists, psychotherapists, and social workers) in the ICU. The aim of this paper is to raise awareness of the need to consider each pregnant and breastfeeding staff member individually and to work together to find a personal solution for continuing to work in the ICU. Possible ways and solutions to achieve this goal are outlined and practical examples are given for implementation in everyday clinical routine. These are also based on comprehensive presentation of activities according to a traffic light color-code system for all occupational groups. Arguments against pregnant employees working in the ICU are discussed and possible solutions are presented.

Polymorphic Variants in the GRK5 Gene Promoter Are Associated With Diastolic Dysfunction in Coronary Artery Bypass Graft Surgery Patients.

BACKGROUND: The G-protein-coupled receptor kinase 5 (GRK5) is a mediator of cardiovascular homeostasis and participates in inflammation and cardiac fibrosis, both being involved in the development of diastolic dysfunction (DD). While mechanisms of transcriptional regulation of the GRK5 promoter are unclear, we tested the hypotheses, that (1) GRK5 expression varies depending on functional single nucleotide polymorphisms (SNPs) in the GRK5 promoter and (2) this is associated with DD in patients undergoing coronary artery bypass graft (CABG) surgery. METHODS: We amplified and sequenced the GRK5 promoter followed by cloning, reporter assays, and electrophoretic mobility shift assays (EMSA). GRK5 messenger ribonucleic acid (mRNA) expression was determined in right atrial tissue sampled from 50 patients undergoing CABG surgery. In another prospective study, GRK5 genotypes were associated with determinants of diastolic function using transesophageal echocardiography in 255 patients with CABG with normal systolic left ventricular (LV) function. Specifically, we measured ejection fraction (EF), transmitral Doppler early filling velocity (E), tissue Doppler early diastolic lateral mitral annular velocity (E' lateral), and calculated E/E', E' norm and the difference of E' lateral and E' norm to account for age-related changes in diastolic function. RESULTS: We identified 6 SNPs creating 3 novel haplotypes with the greatest promoter activation in haplotype tagging (ht) SNP T(-678)C T-allele constructs (P < .001). EMSAs showed allele-specific transcription factor binding proving functional activity. GRK5 mRNA expression was greatest in TT genotypes (TT: 131 fg/µg [95% CI, 108-154]; CT: 109 [95% confidence interval {CI}, 93-124]; CC: 83 [95% CI, 54-112]; P = .012). Moreover, GRK5 genotypes were significantly associated with determinants of diastolic function. Grading of DD revealed more grade 3 patients in TT compared to CT and CC genotypes (58% vs 38% vs 4%; P = .023). E´ lateral was lowest in TT genotypes (P = .007) and corresponding E/E' measurements showed 1.27-fold increased values in TT versus CC genotypes (P = .01), respectively. While E' norm values were not different between genotypes (P = .182), the difference between E' lateral and E' norm was significantly higher in TT genotypes compared to CC and CT genotypes (-1.2 [interquartile range {IQR}, 2.7], -0.5 [IQR, 3.4], and -0.4 [IQR, 4.2; P = .035], respectively). CONCLUSIONS: A functional GRK5 SNP results in allele-dependent differences in GRK5 promoter activity and mRNA expression. This is associated with altered echocardiographic determinants of diastolic function. Thus, SNPs in the GRK5 promoter are associated with altered perioperative diastolic cardiac function. In the future, preoperative testing for these and other SNPs might allow to initiate more specific diagnostic and perioperative pathways to benefit patients at risk.

Genetic variations in G-protein signal pathways influence progression of coronary artery calcification: Results from the Heinz Nixdorf Recall study.

BACKGROUND AND AIMS: Coronary artery calcification (CAC) is one of the most sensitive and specific markers of coronary atherosclerosis and believed to be heritable. We hypothesized that functionally relevant single-nucleotide polymorphisms (SNPs) in the G-protein signal pathway, which have been previously related to coronary artery disease, are associated with CAC progression. METHODS: 3108 participants from the Heinz Nixdorf Recall study with CAC measurements at both baseline (CACb) and 5-year follow-up (CAC5y) were included. We genotyped SNPs rs1042714 (ADRB2), rs6026584 and rs12481583 (GNAS), and rs5443 (GNB3) and defined a priori risk alleles derived from literature data. Regression analyses were applied to measures of 5-year CAC progression, unadjusted, adjusted for age, sex, and adjusted for age, sex, log(CACb+1) as well as for cardiovascular risk factors. RESULTS: The presence of one or more risk alleles was associated with a 26.9% (95% CI 5.5-52.4) increase in 5-year CAC progression (p = 0.011) and a 29.2% (95% CI 5.9-57.6) accelerated increase of CAC over the 5-year period compared to what was expected with respect to the baseline CAC percentile value (p = 0.012). Each of those risk alleles increased the 5-year CAC progression by 4.4% (95% CI 1.3-7.6, p = 0.006) and resulted in a 4.9% accelerated increase of CAC over the 5-year period (95% CI 1.6-8.4, p = 0.004). These unadjusted data did not change after adjustment. CONCLUSIONS: Genetic variations in the G-protein signal pathway are associated with CAC progression in a cumulative fashion, indicating the importance of the pathway for genetic heritability in CAC progression and coronary artery disease.

Genetic variability in postoperative nausea and vomiting: A systematic review.

BACKGROUND: Postoperative nausea and vomiting (PONV) is the most frequent side effect following anaesthesia. Predisposition to developing PONV is multifactorial with patient risk factors and anaesthetic techniques both being contributory. However, there is also a genetic susceptibility to PONV, and several studies have aimed to identify polymorphisms contributing to a genetic PONV risk. OBJECTIVE: We summarised previous published studies investigating genetic contribution to PONV risk. DESIGN: Systematic review without meta-analysis. DATA SOURCE: We searched MEDLINE until June 2019. ELIGIBILITY CRITERIA: Articles were chosen for review when PONV and polymorphisms were included. Exclusion criteria were reviews/meta-analysis/comments, articles not in the English language, nonappropriate content (e.g. PONV not as primary aim of the study, study investigated opioid-induced nausea) or if articles were pharmacogenetic studies addressing treatment of PONV. RESULTS: A total of 59 studies were screened and 14 articles were reviewed including one genome-wide association study (GWAS). Seven studies were performed in East Asians, and seven in Caucasians. Seventeen polymorphisms have been positively associated with PONV in at least one study. Allele frequency of the investigated polymorphisms differs widely between the ethnicities. Furthermore, the anaesthesia regimen and the postoperative time point at which the association with PONV was reported were quite different. Only two polymorphisms, the CHRM3 rs2165870 and the KCNB2 rs349358 (both first associated with PONV in a GWAS), have been significantly associated with PONV incidence in Caucasians in independent studies. CONCLUSION: There is a genetic susceptibility to the development of PONV. Two single nucleotide polymorphisms (SNPs), the CHRM3 rs2165870 and the KCNB2 rs349358 SNP, seem to have a major influence on PONV incidence, at least in Caucasians. Both SNPs were primarily identified in a GWAS and this association may lead to a better understanding of the disease aetiology. Further high-quality studies are needed to reveal more insights in genetic PONV susceptibility, particularly so in non-Caucasian ethnicities.

Correction to: Methylation in HT22 cells and primary hippocampal neurons with and without isoflurane exposure.

Following publication of the original article [1], it was brought to our attention of an error in the article title.

Methylation in HT22 cells and primary hippocampal neurons with and without isoflurane exposurewhether isoflurane causes.

BACKGROUND: Epigenetic modulation may play a role in anesthesia related phenotypes, such as cognitive impairment or memory loss, especially with exposure to anesthetics in the vulnerable phase of brain development. While isoflurane anesthesia can evoke neuroinflammation and neuroapoptosis in young animals, we investigated in a permanent hippocampal cell line (HT22) and in primary hippocampal neurons in an a priori in vitro analysis, whether isoflurane exposure 1) evokes DNA methylation changes in genes involved in apoptosis and inflammation, and 2) results observed in a permanent hippocampal cell line are comparable to primary hippocampal neurons. In case of methylation changes in specific genes, (3) mRNA analysis was performed to assess possible effects on gene expression. METHODS: HT22 cells and primary mouse hippocampal neurons were exposed to 3% isoflurane for 4 h and DNA (each 6 single experiments) and RNA (3 single independent experiments) were extracted. Methylation analysis (EpiTect Methyl II PCR Array Systems, Qiagen) included the methylation status of 66 genes involved in apoptosis, cytokine production, inflammatory response, and autoimmunity. Quantitative Real-Time PCR was performed using the Quantitect SYBR Green Kit on a Step One Plus. RESULTS: Methylation status was markedly different between immortalized HT22 cells and cultured primary hippocampal neurons without isoflurane exposure. Of 66 genes investigated, 29 were methylated to a significantly greater degree in HT22 cells compared to primary hippocampal neurons. In cultured primary hippocampal neurons, in contrast, there was a greater methylation in several genes involved in inflammation, accompanied with significant downregulation of C-X-C motif chemokine 12 with isoflurane exposure (p = 0.023). CONCLUSIONS: We demonstrate marked differences in gene methylation between HT22 cells and cultured primary hippocampal neurons without isoflurane exposure, with a greater methylation of several genes involved in inflammation upon isoflurane exposure and significant downregulation of Cxcl12 mRNA expression in primary hippocampal neurons. Accordingly, further investigations of anesthesia related DNA methylation should be performed with special consideration being given to the choice of cells targeted for such investigations.

Genetic contribution to PONV risk.

BACKGROUND: Clinical risk factors for postoperative nausea and vomiting (PONV) are usually stratified using the Apfel Score. While a genetic predisposition has recently been demonstrated with the muscarinic acetylcholine receptor (CHRM3) rs2165870 single nucleotide polymorphism (SNP), we investigated whether (1) other SNPs contribute to PONV risk and (2) a genetic risk score might summarise genetic PONV risk. METHODS: We retrospectively analysed data from a study with 472 patients undergoing elective surgery. We investigated the SNPs rs3218315 (IL2RB), rs349358 (KCNB2), rs703363 (intergenic variant), rs1800497 (DRD2), rs1799971 (OPRM1), and rs1176713 (HTR3A). A genetic risk score was established and association with PONV investigated. RESULTS: Early PONV occurred in 37%. There was a significant association of the KCNB2 rs349358 SNP with nausea (P = 0.021), retching (P = 0.001), and PONV (P = 0.006). The rs349358 genotype distribution was TT in 310 and TC/CC in 155 patients. The KCNB2 SNP was associated with an Odds Ratio (OR) of 1.6 for CT/CC vs. TT (95% CI 1-2.5; P = 0.031) to develop PONV and this was independent from the Apfel Score, and the CHRM3 rs2165870 SNP. A genetic risk score based on the CHRM3 rs2165870 and the KCNB2 rs349358 SNP was created and this genetic score (OR per genetic risk score point: 1.6 (1.3-2.1), P < 0.0001) was independent from the Apfel Score (OR per Apfel score point: 1.6 (1.3-1.9), P < 0.0001) associated with PONV. CONCLUSION: The KCNB2 rs349358 SNP is also an independent PONV predictor and a genetic risk score has a similar impact on PONV susceptibility compared to the Apfel Score.

The small molecule Bcl-2/Mcl-1 inhibitor TW-37 shows single-agent cytotoxicity in neuroblastoma cell lines.

BACKGROUND: High-risk neuroblastoma with N-Myc amplification remains a therapeutic challenge in paediatric oncology. Antagonism of pro-death Bcl-2 homology (BH) proteins to pro-survival BH members such as Mcl-1 and Bcl-2 has become a treatment approach, but previous studies suggest that a combined inhibition of Bcl-2 and Mcl-1 is necessary. TW-37 inhibits Mcl-1 and Bcl-2 with almost the same affinity. However, single-agent cytotoxicity of TW-37 in neuroblastoma cell lines has not been investigated. METHODS: Cell viability, apoptosis, proliferation and changes in growth properties were determined in SKNAS, IMR-5, SY5Y and Kelly cells after treatment with TW-37. After transfection with Mcl-1 or Bcl-2 siRNA, apoptosis and proliferation were investigated in Kelly cells. Mice with Kelly cell line xenografts were treated with TW-37 and tumor growth, survival and apoptosis were determined. RESULTS: Cell lines with N-Myc amplification were more sensitive to TW-37 treatment, IC50 values for IMR-5 and Kelly cells being 0.28 µM and 0.22 µM, compared to SY5Y cells and SKNAS cells (IC50 0.96 µM and 0.83 µM). Treatment with TW-37 resulted in increased apoptosis and reduced proliferation rates, especially in IMR5 and Kelly cells. Bcl-2 as well as Mcl-1 knockdown induced apoptosis in Kelly cells. TW-37 led to a decrease in tumor growth and a favorable survival (p = 0.0379) in a Kelly neuroblastoma xenografts mouse model. CONCLUSION: TW-37 has strong single-agent cytotoxicity in vitro and in vivo. Therefore, combined inhibition of Bcl-2/Mcl-1 by TW-37 in N-Myc amplified neuroblastoma may represent an interesting therapeutic strategy.

Remote ischaemic preconditioning increases serum extracellular vesicle concentrations with altered micro-RNA signature in CABG patients.

BACKGROUND: Remote ischaemic preconditioning (RIPC) can attenuate myocardial ischaemia/reperfusion injury but its underlying mechanisms remain largely unknown. Recently, extracellular vesicles (EVs) containing microRNAs (miRNAs) were shown to mediate distant intercellular communication that may be involved in cardioprotection. We tested the hypothesis that RIPC in anaesthetized patients undergoing coronary artery bypass (CABG) surgery results in the release of EVs from the ischaemic/reperfused arm into the blood stream harbouring cardioprotective miRNAs. METHODS: In 58 patients randomised to RIPC (three 5/5 minutes episodes of left arm ischaemia/reperfusion by suprasystolic blood pressure cuff inflations/deflations) or Sham, a subprotocol comprising of parallel right radial artery and regional (left subclavian) venous blood sampling before (awake) and 5 and 60 minutes after RIPC/Sham during isoflurane/sufentanil anaesthesia could be completed. EVs were extracted by polymer-based precipitation methods, their concentrations measured, and their miRNA signature analysed. RESULTS: Five minutes after RIPC, regional venous EV concentrations downstream from the cuff increased and arterial concentrations increased after 60 minutes (fold change [fc]: RIPC: 1.33 ± 0.5, Sham: 0.91 ± 0.31; P = 0.003 for interaction). Already 5 minutes after RIPC, expression of 26 miRNAs (threshold fc: 3.0, P < 0.05) isolated from EVs including the cardioprotective miR-21 had increased. RIPC also decreased postoperative Troponin I concentrations (AUC RIPC: 336 ng/mL × 72 hours ± 306 vs Sham: 713 ± 1013; P  = â€Š0.041). CONCLUSIONS: Remote ischaemic preconditioning increases serum EV concentrations, most likely by early EV release from the patients' left (RIPC) arm, alters their miRNA signature, and is associated with myocardial protection. Thus, an increased EV concentration with an altered miR-signature may mediate the RIPC effect.

Circulating miR-192 is a prognostic marker in patients with ischemic cardiomyopathy.

Ischemic cardiomyopathy (ICM) is characterized by accumulation of p53 causing apoptosis of cardiomyocytes and resulting in upregulation of miRNA (miR)-192, which plays an important role in the development of heart failure after acute myocardial infarction. However, for other cardiomyopathies, miR-192 seems to have minor relevance. We tested in a prospective, observational study comprising 91 patients with diagnosed heart failure (59.3% ICM and 40.7% non-ICM), the hypothesis that miR-192 expression predicts survival in patients with ICM. Median follow-up was 59 months (range: 1-118). While miR-192 expression was significantly associated with age (p = 0.028), log-rank analysis revealed significant association with survival in ICM (p = 0.003) but not in non-ICM (p = 0.6). In ICM, median age at time of death was 84 years in patients with low miR-192 expression but 67 years with high miR-192 expression. Thus, miR-192 expression is associated with survival in ICM and represents a prognostic marker in ischemic heart failure.

The GRK2 Promoter Is Regulated by Early-Growth Response Transcription Factor EGR-1.

The G-protein-coupled receptor kinase 2 (GRK2) plays a major role in cardiovascular diseases, and its expression is increased in heart failure. However, only little is known about factors being involved in up-regulation of GRK2 expression through transcriptional regulation of its promoter. Since the transcription factor early-growth response 1 (EGR-1) is also up-regulated in patients with heart failure, we tested the hypothesis that EGR-1 regulates GRK2 transcription. Stimulation of immortalized rat cardiomyocytes (H9c2) with phorbol 12-myristate 13-acetate (PMA) resulted in up-regulation of Egr-1 and subsequently of Grk2 mRNA expression, with maximum Grk2 expression (p = 0.008) 5 hr after PMA stimulation and being abolished by actinomycin D, indicating a transcriptional mechanism. To identify naturally occurring variants affecting promoter transcriptional activity, we identified a novel G(-43)A polymorphism (rs182084609), which surrounded a putative EGR-1-binding site. While the minor A allele frequency was rare (0.02), this variant was used to explore regulation by EGR-1 and promoter construct with altered alleles at nt-43 were subjected of reporter assays in human embryonic kidney cells (Hek293). Here, EGR-1 over-expression resulted in a more than twofold increase in GRK2 promoter activity but only in the presence of the G-allele (p = 0.04). In electrophoretic mobility shift assays, EGR-1 over-expression resulted in a specific binding of transcription factors only to the G oligonucleotide. Finally, EGR-1 over-expression resulted in increased GRK2 mRNA expression (p = 0.03). We identified EGR-1 as a regulator of GRK2 transcription. Suppression of GRK2 expression by inhibition of EGR-1 binding to GRK2 might be a promising approach to mitigate adrenergic desensitization.

Relationship between GNAS1 T393C polymorphism and aseptic loosening after total hip arthroplasty.

BACKGROUND: Aseptic loosening is a main cause for revision surgery after total hip arthroplasty (THA) and there is no reliable marker for the early detection of patients at high risk. This study has been performed to validate association of the T393C polymorphism (rs7121) in the GNAS1 gene, encoding for the alpha-subunit of heterotrimeric G-protein Gs, with risk for and time to aseptic loosening after THA, which has been demonstrated in our previous study. METHODS: 231 patients with primary THA and 234 patients suffering from aseptic loosening were genotyped for dependency on GNAS1 genotypes and analyzed. RESULTS: Genotyping revealed almost similar minor allele frequencies of 0.49 and 0.46, respectively. Consistently, genotype distributions of both groups were not significantly different (p = 0.572). Neither gender nor GNAS1 genotype showed a statistically significant association with time to loosening (p = 0.501 and p = 0.840). Stratification by gender, as performed in our previous study, was not able to show a significant genotype-dependent difference in time (female p = 0.313; male p = 0.584) as well as median time to aseptic loosening (female p = 0.353; male p = 0.868). CONCLUSION: This study was not able to confirm the results of our preliminary study. An association of the GNAS1 T393C polymorphisms with risk for and time to aseptic loosening after THA is unlikely.

GNAQ TT(-695/-694)GC Polymorphism Is Associated with Increased Gq Expression, Vascular Reactivity, and Myocardial Injury after Coronary Artery Bypass Surgery.

BACKGROUND: Angiotensin II receptor type 1-mediated activation of the α-subunit of the heterotrimeric Gq protein evokes increased vasoconstriction and may promote hypertrophy-induced myocardial damage. The authors recently identified a TT(-695/-694)GC polymorphism in the human Gq promoter, the GC allele being associated with an increased prevalence of cardiac hypertrophy. In this article, the authors tested whether the TT(-695/-694)GC polymorphism is associated with differences in (1) myocardial Gq protein expression, (2) vascular reactivity, and (3) myocardial damage after coronary artery bypass grafting. METHODS: Gq protein expression was measured in right atrial muscle from 55 patients undergoing coronary artery bypass grafting as were skin perfusion changes (n = 18; laser Doppler imaging), saphenous vein ring vascular reactivity (n = 50, organ bath) in response to angiotensin II, and myocardial damage (227 patients undergoing coronary artery bypass grafting), as assessed by postoperative cardiac troponin I concentration. RESULTS: Myocardial Gq expression was greater in GC/GC genotypes (GC/GC vs. TT/TT: 1.27-fold change; P = 0.006). Skin perfusion after intradermal angiotensin II injection decreased only in GC/GC genotypes (P = 0.0002). Saphenous vein rings exposed to increasing angiotensin II concentrations showed an almost doubled maximum contraction in GC/GC compared with individuals with the TT/TT genotype (P = 0.022). In patients undergoing coronary artery bypass grafting, baseline cardiac ejection fraction was different (GC/GC: 55 ± 13%; GC/TT: 54 ± 14%; TT/TT: 48 ± 15%; P = 0.037) and postoperative peak cardiac troponin I was greater in patients with the GC/GC (11.5 ± 13.8 ng/ml) than in patients with the GC/TT (9.2 ± 9.2 ng/ml) or patients with the TT/TT genotype (6.6 ± 4.8 ng/ml, P = 0.015). CONCLUSIONS: The GC/GC genotype of the TT(-695/-694)GC polymorphism is associated with increased Gq protein expression, augmented angiotensin II receptor type 1-related vasoconstriction, and increased myocardial injury after coronary artery bypass grafting, highlighting the impact of Gq genotype variation.

Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiments.

Real-time PCR is an indispensable technique for mRNA expression analysis but conclusions depend on appropriate reference gene selection. However, while reference gene selection has been a topic of publications, this issue is often disregarded when measuring target mRNA expression. Therefore, we (1) evaluated the frequency of appropriate reference gene selection, (2) suggest an easy-to-use tool for least variability reference gene selection, (3) demonstrate application of this tool, and (4) show effects on target gene expression profiles. All 2015 published articles in Naunyn-Schmiedeberg's Archives of Pharmacology were screened for the use of quantitative real-time PCR analysis and selection of reference genes. Target gene expression (Vegfa, Grk2, Sirt4, and Timp3) in H9c2 cells was analyzed following various interventions (hypoxia, hyperglycemia, and/or isoflurane exposure with and without subsequent hypoxia) in relation to putative reference genes (Actb, Gapdh, B2m, Sdha, and Rplp1) using the least variability method vs. an arbitrarily selected but established reference gene. In the vast majority (18 of 21) of papers, no information was provided regarding selection of an appropriate reference gene. In only 1 of 21 papers, a method of appropriate reference gene selection was described and in 2 papers reference gene selection remains unclear. The method of reference gene selection had major impact on interpretation of target gene expression. With hypoxia, for instance, the least variability gene was Rplp1 and target gene expression (Vefga) heavily showed a 2-fold up-regulation (p = 0.022) but no change (p = 0.3) when arbitrarily using Gapdh. Frequency of appropriate reference gene selection in this journal is low, and we propose our strategy for reference gene selection as an easy tool for proper target gene expression.

The BCL2 -938C>A Promoter Polymorphism Is Associated with Risk for and Time to Aseptic Loosening of Total Hip Arthroplasty.

Aseptic loosening is a major cause of revision surgery of total hip arthroplasty (THA). Only few host factors affecting aseptic loosening have been identified until now, although they are urgently needed to identify and possibly treat those patients at higher risk for aseptic loosening. To determine whether the functional single nucleotide polymorphism (SNP) c.-938C>A (rs2279115), located in the promoter region of the BCL2 gene has an impact on aseptic loosening of THA we genotyped and analyzed 234 patients suffering from aseptic loosening and 231 patients after primary THA. The polymorphism is associated with risk for aseptic loosening with the CC genotype at highest risk for aseptic loosening, Odds Ratio CC vs. AA 1.93, 95%CI 1.15-3.25, p = 0.013. In contrast, low risk AA genotype carriers that still developed aseptic loosening showed a significantly shorter time to aseptic loosening than patients carrying the C allele (p = 0.004). These results indicate that the BCL2 -938C>A polymorphism influences the occurrence and course of aseptic loosening and suggests this polymorphism as an interesting candidate for prospective studies and analyses in THA registers.

Characterization of the PLCB1 promoter and regulation by early growth response transcription factor EGR-1.

The Gαq/-Gα11-PLCß1 pathway is important for intracellular signalling and associated with pathological conditions, such as cardiac hypertrophy. The GNAQ and GNA11 promoters (encoding for Gαq and Gα11) have already been characterized and are both regulated by the transcription factor early growth response 1 (Egr-1). In contrast, the PLCB1 promoter (encoding for the direct downstream effector PLCß1) has neither been cloned nor characterized. Therefore, the purpose of this study was to 1) characterize the PLCB1 promoter, and 2) assess its potential regulation by Egr-1. By means of 5'- Rapid Amplification of 5'-cDNA ends analysis in human heart tissue we found an initiation of transcription from multiple starting points, the main transcription starting point being located at nt-235 relative to the translation start point. The PLCB1 promoter was cloned and deletion constructs were generated. Luciferase assays were performed in three different cell lines and regulatory regions were identified between nt-595/nt-313 (Hek293: P=0.013; HASMC: P=0.019; H9c2: P=0.005). In electrophoretic mobility shift assays one specific Egr-1 binding site was identified at nt-451/-419 and PLCB1 promoter activity was increased more than 5-fold (Hek293: P=0.0008) and 1,6- fold (H9c2: P=0.0499) following overexpression of Egr-1. Thus, the PLCB1 promoter was characterized for the first time and a specific interaction with the transcription factor Egr-1 was shown. Our data provide a potential molecular mechanism relating to pathophysiological conditions such as cardiac hypertrophy where activation by Egr-1 of Gαq/Gα11-PLCß1 plays an important role.

Cloning and characterization of the GNA11 promoter and its regulation by early growth response 1.

GNAQ and GNA11, encoding the G-proteins Gα(q) and Gα11, are members of the Gα(q)/Gα11 subfamily, which transmits signals from the cell surface to intracellular signalling cascades. The GNAQ promoter was already characterized, and regulation by the transcription factor early growth response 1 (Egr-1) was demonstrated. Interestingly, in silico analysis revealed putative Egr-1 binding sites in sequences potentially representing the GNA11 promoter. However, the GNA11 promoter has not been characterized so far. Therefore, the purpose of the study was the characterization of the GNA11 promoter and investigation of its potential regulation by Egr-1. The putative GNA11 promoter was cloned, and deletion constructs were generated. Luciferase assays were performed, and essential regulatory regions identified between nt-805/-177. In electrophoretic mobility shift assays (EMSAs), one specific Egr-1 binding site at nt-475/-445 was identified. An Egr-1 expression plasmid was generated, which evoked increased Egr-1 content in nuclear extracts and a > 2-fold increase in GNA11 promoter activity in construct nt-805/+54 (p = 0.035). Finally, real-time PCR analysis was performed, and an increased Gα11 mRNA (p = 0.035) expression induced by Egr-1 was found. Here, we characterize for the first time the GNA11 promoter and its specific interaction with Egr-1. Both the GNAQ and the GNA11 promoter appear to be regulated by the same transcription factor, Egr-1, which may be a molecular mechanism leading to Gα(q)-/Gα11-associated phenotypes.

The GNB3 C825T polymorphism as a pharmacogenetic marker in the treatment of hypertension, obesity, and depression.

Heterotrimeric guanine-binding proteins (G proteins) transmit signals from the cell surface to intracellular signal cascades. The ß3-subunit encoded by the gene GNB3 is widely expressed and, therefore, involved in various physiological and pathophysiological processes. A C825T polymorphism located in exon 10 of GNB3 was described in 1998 and the T allele was associated with alternative splicing and with increased signal transduction in human cells and tissues. In several disease-association studies, the 825T allele could be linked to hypertension, obesity, and depression. Meta-analysis available for hypertension and depression confirmed association with these phenotypes. On the basis of these findings, subsequent studies investigated whether the C825T polymorphism serves as a pharmacogenetic marker. Most pharmacogenetic investigations have focused on the treatment of hypertension, obesity, and depression. In this study, we will comprehensively describe and discuss these studies.