A structured proteomic approach identifies 14-3-3Sigma as a novel and reliable protein biomarker in panel based differential diagnostics of liver tumors.

Hepatocellular carcinoma (HCC) is a major lethal cancer worldwide. Despite sophisticated diagnostic algorithms, the differential diagnosis of small liver nodules still is difficult. While imaging techniques have advanced, adjuvant protein-biomarkers as glypican3 (GPC3), glutamine-synthetase (GS) and heat-shock protein 70 (HSP70) have enhanced diagnostic accuracy. The aim was to further detect useful protein-biomarkers of HCC with a structured systematic approach using differential proteome techniques, bring the results to practical application and compare the diagnostic accuracy of the candidates with the established biomarkers. After label-free and gel-based proteomics (n=18 HCC/corresponding non-tumorous liver tissue (NTLT)) biomarker candidates were tested for diagnostic accuracy in immunohistochemical analyses (n=14 HCC/NTLT). Suitable candidates were further tested for consistency in comparison to known protein-biomarkers in HCC (n=78), hepatocellular adenoma (n=25; HCA), focal nodular hyperplasia (n=28; FNH) and cirrhosis (n=28). Of all protein-biomarkers, 14-3-3Sigma (14-3-3S) exhibited the most pronounced up-regulation (58.8×) in proteomics and superior diagnostic accuracy (73.0%) in the differentiation of HCC from non-tumorous hepatocytes also compared to established biomarkers as GPC3 (64.7%) and GS (45.4%). 14-3-3S was part of the best diagnostic three-biomarker panel (GPC3, HSP70, 14-3-3S) for the differentiation of HCC and HCA which is of most important significance. Exclusion of GS and inclusion of 14-3-3S in the panel (>1 marker positive) resulted in a profound increase in specificity (+44.0%) and accuracy (+11.0%) while sensitivity remained stable (96.0%). 14-3-3S is an interesting protein biomarker with the potential to further improve the accuracy of differential diagnostic process of hepatocellular tumors. This article is part of a Special Issue entitled: Medical Proteomics.

Determinants of decreasing major amputation rates in Germany.

BACKGROUND: We analysed a potential association between the decrease in major amputations in Germany and the number of doctors, prescribed podologic foot care (PFC) and antidiabetic drugs, and performed percutaneous endoluminal angioplasties (PTA). PATIENTS AND METHODS: Data of all lower limb major amputations between 2007 and 2011, the cases hospitalised with an additional diagnosis of diabetes mellitus, and the numbers of PTAs, and the number of doctors in private practices and in hospitals were obtained from the Federal Statistical Office. Furthermore, the number of PFC treatments and prescribed antidiabetics for each of the five years were derived from the federal report of the statutory health insurance. RESULTS: Within the 5 year time period, major amputations decreased by 19.0%, from 17,846 in 2007 to 14,463 in 2011. There is an inverse relation between the number of major amputations and the increasing number of prescribed PFC, of doctors working in hospital and of below-the-knee PTA in the multiple Poisson regression analysis. The number of prescribed antidiabetics and that of all PTA showed a positive relation. In the multiple linear regression analysis with the dependent variable ratio of amputations and the cases hospitalised with an additional diagnosis of diabetes mellitus, only numbers of prescribed PFC and below-the-knee PTA still showed an inverse relation that reached a level of significance. CONCLUSIONS: While substantial improvements in patients care by doctors, endovascular interventions, prescriptions of PFC and antidiabetic drugs are under discussion to reduce major amputation rates, in this approach including comprehensive data from Germany, only prescriptions of PFC and the number of below-the-knee PTA had an independent and significant impact on the reduction of major amputations. It has to be pointed out that such a statistical association does not prove any causality.

Prescription of enoxaparin is associated with decreasing pulmonary embolism mortality rate in Germany.

We analysed time trends in the pulmonary embolism (PE) mortality rates in Germany from 2004 and assessed for an association between the use of anticoagulants and PE caused mortality. We extracted age-specific number of deaths due to PE (ICD-10 I26) from 2004 to 2011 as available from the WHO mortality databases. In addition we derived defined daily dosage (DDD) of prescribed anticoagulants and the low molecular heparin Enoxaparin for the years 2004-2011 from the statutory health insurance-drug-information system reports. Age-standardized PE mortality per 100,000 decreased from 5.9283 in year 2004 to 4.4876 in 2011 (-24.3 %). Amounts of prescribed anticoagulants increased in this period from 271,810.7 × 1000 DDD to 416,611.8 × 1000 DDD (+53.3 %), that of Enoxaparin increased from 27,071.1 × 1000 DDD in 2004 97,276.5 × 1000 DDD in 2011. The PE mortality is negatively correlated with anticoagulants (-0.9463, p = 0.0004) as well as with enoxaparin (-0.9740, p < 0.0001) and of DDD of Enoxaparin per 1000 insured (-0.9682, p < 0.0001). In univariate linear regression model, anticoagulants, Enoxaparin and Enoxaparin per 1000 insured all reach significance (p = 0.0004, p = 4.31 × 10(-5) and p = 0.0001 respectively). Multiple regression models show that Enoxaparin has the most robust effect. Including the time trend in the model does not alter the results. Our study shows that increasing number of prescribed Enoxaparin in an outpatient setting might be one determinant of decreasing PE mortality rate in Germany since 2004.

Repetitive exposure of mice to strong static magnetic fields in utero does not impair fertility in adulthood but may affect placental weight of offspring.

PURPOSE: To investigate the effect of daily exposure in utero to static magnetic fields during prenatal development on germ cell development and fertility of exposed offspring in adulthood. MATERIALS AND METHODS: Mice were exposed daily in utero to different static magnetic field strengths at the bore entrance or in the isocenter of 1.5 T and 7 T MRI systems during the entire course of prenatal development. RESULTS: In utero-exposed male mice revealed no effect of magnetic field strength on weight of testes and epididymis or on sperm count, sperm morphology, or fertility. Exposed pregnant female mice showed no reduced fertility in terms of pregnancy rates and litter size, pointing to a normal ovarian function. However, a reduced placental weight of offspring of intrauterine exposed female mice was observed that correlated with a decrease in embryonic weight in those animals exposed at the strongest magnetic field. This effect seemed to be parent-dependent, since it was not observed in those embryos fathered by in utero-exposed male mice. CONCLUSION: Repetitive in utero exposure to strong static magnetic fields does not impair fertility but may have a parental-dependent effect on fetal programming with regard to placental development and fetal growth.

Impact of repetitive exposure to strong static magnetic fields on pregnancy and embryonic development of mice.

PURPOSE: To evaluate possible risks of strong static magnetic fields for embryo implantation, gestation, organogenesis, and embryonic development. MATERIALS AND METHODS: Pregnant mice were exposed for 75 minutes daily during the entire course of pregnancy at the bore entrance, representing the position of medical staff, and at the isocenter, representing the position of patients, of a 1.5 T and a 7 T human MRI scanner. RESULTS: No effect of static magnetic field strength was observed with regard to pregnancy rate, duration of pregnancy, litter size, still births, malformations, sex distribution, or postpartum death of offspring. During the first 8 weeks postnatal, mice exposed in utero to a magnetic field strength of 1.5 T or stronger showed a slight delay in weight gain and in time to eye opening compared to controls. CONCLUSION: Daily exposure to strong magnetic fields during pregnancy had no deleterious effect on offspring; however, a developmental retardation could be observed postnatally with regard to weight gain and eye opening.

Relationship between carotid intima-media thickness and metabolic syndrome in adolescents.

OBJECTIVE: To test the hypothesis that metabolic syndrome (MetS) is more predictive of carotid intima-media thickness (IMT) than the sum of the individual components of MetS. STUDY DESIGN: We analyzed the relationships between 2 definitions of the MetS and IMT in 461 overweight adolescents aged 10-18 years (median body mass index, 28.6 kg/m(2)). We used regression models and receiver operating characteristics (ROCs) for increased IMT (defined as ≥0.7 mm). RESULTS: The prevalence of MetS was 15.0% and 26.9% according to the 2 definitions applied. At the group level, quantitative IMT was associated with body mass index, blood pressure, glucose levels at 2 hours in an oral glucose tolerance test, and with each of the MetS components (all P < .05). At an individual level, using the MetS definitions alone as a diagnostic test for the presence of increased IMT (area under the ROC curve, 0.60-0.66) was inferior when compared with the sum of all individual components (area under the ROC curve, 0.65-0.85). Adding the presence or absence of MetS to the components did not improve the accuracy. CONCLUSION: Overweight adolescents with MetS demonstrated increased IMT values compared with overweight adolescents without MetS. The best model for diagnosing increased IMT was the sum of the quantitative components of MetS. The use of dichotomized variables reduced the diagnostic accuracy. Thus, in clinical practice, treatment of overweight adolescents should be based on weighing cardiovascular risk factors themselves, rather than on the dichotomous variable MetS.

Common obesity risk alleles in childhood attention-deficit/hyperactivity disorder.

Children with attention-deficit/hyperactivity disorder (ADHD) have a higher rate of obesity than children without ADHD. Obesity risk alleles may overlap with those relevant for ADHD. We examined whether risk alleles for an increased body mass index (BMI) are associated with ADHD and related quantitative traits (inattention and hyperactivity/impulsivity). We screened 32 obesity risk alleles of single nucleotide polymorphisms (SNPs) in a genome-wide association study (GWAS) for ADHD based on 495 patients and 1,300 population-based controls and performed in silico analyses of the SNPs in an ADHD meta-analysis comprising 2,064 trios, 896 independent cases, and 2,455 controls. In the German sample rs206936 in the NUDT3 gene (nudix; nucleoside diphosphate linked moiety X-type motif 3) was associated with ADHD risk (OR: 1.39; P = 3.4 × 10(-4) ; Pcorr = 0.01). In the meta-analysis data we found rs6497416 in the intronic region of the GPRC5B gene (G protein-coupled receptor, family C, group 5, member B; P = 7.2 × 10(-4) ; Pcorr = 0.02) as a risk allele for ADHD. GPRC5B belongs to the metabotropic glutamate receptor family, which has been implicated in the etiology of ADHD. In the German sample rs206936 (NUDT3) and rs10938397 in the glucosamine-6-phosphate deaminase 2 gene (GNPDA2) were associated with inattention, whereas markers in the mitogen-activated protein kinase 5 gene (MAP2K5) and in the cell adhesion molecule 2 gene (CADM2) were associated with hyperactivity. In the meta-analysis data, MAP2K5 was associated with inattention, GPRC5B with hyperactivity/impulsivity and inattention and CADM2 with hyperactivity/impulsivity. Our results justify further research on the elucidation of the common genetic background of ADHD and obesity.

Missing heritability in the tails of quantitative traits? A simulation study on the impact of slightly altered true genetic models.

OBJECTIVE: Genome-wide association studies have identified robust associations between single nucleotide polymorphisms and complex traits. As the proportion of phenotypic variance explained is still limited for most of the traits, larger and larger meta-analyses are being conducted to detect additional associations. Here we investigate the impact of the study design and the underlying assumption about the true genetic effect in a bimodal mixture situation on the power to detect associations. METHODS: We performed simulations of quantitative phenotypes analysed by standard linear regression and dichotomized case-control data sets from the extremes of the quantitative trait analysed by standard logistic regression. RESULTS: Using linear regression, markers with an effect in the extremes of the traits were almost undetectable, whereas analysing extremes by case-control design had superior power even for much smaller sample sizes. Two real data examples are provided to support our theoretical findings and to explore our mixture and parameter assumption. CONCLUSIONS: Our findings support the idea to re-analyse the available meta-analysis data sets to detect new loci in the extremes. Moreover, our investigation offers an explanation for discrepant findings when analysing quantitative traits in the general population and in the extremes.

Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile in head and neck squamous cell carcinomas.

BACKGROUND: Chronic inflammation plays an important role in head and neck squamous cell carcinomas (HNSCC). This study addresses the impact of two single nucleotide polymorphisms (SNP) Asp299Gly and Thr399Ile of the toll-like receptor (TLR) 4 gene on the clinical outcome while accounting for the influence of adjuvant systemic therapy in a large cohort of HNSCC patients. METHODS: Genotype analysis was done using DNA from tissue samples from 188 patients with HNSCC; TLR4 protein expression was assessed immunohistochemically in tissue microarrays. Classical survival models were used for statistical analyses. RESULTS: Ten percent of patients with HNSCC presented with the TLR4 299Gly and 17% with the TLR4 399Ile allele. Patients with the heterozygous genotype TLR4 Asp299Gly had a significantly reduced disease-free and overall survival. Also, patients with the heterozygous genotype TLR4 Thr399Ile had a reduced disease-free survival. Notably, these associations seem to be attributable to relatively poor therapy response as e.g. reflected in a significantly shorter DFS among HNSCC patients carrying the Asp299Gly variant and receiving adjuvant systemic therapy. CONCLUSION: According to this study, TLR4 299Gly und 399Ile alleles may serve as markers for prognosis of head and neck cancer in patients with adjuvant systemic therapy, particularly chemotherapy, and might indicate therapy resistance.

Genome-wide association study in German patients with attention deficit/hyperactivity disorder.

The heritability of attention deficit hyperactivity disorder (ADHD) is approximately 0.8. Despite several larger scale attempts, genome-wide association studies (GWAS) have not led to the identification of significant results. We performed a GWAS based on 495 German young patients with ADHD (according to DSM-IV criteria; Human660W-Quadv1; Illumina, San Diego, CA) and on 1,300 population-based adult controls (HumanHap550v3; Illumina). Some genes neighboring the single nucleotide polymorphisms (SNPs) with the lowest P-values (best P-value: 8.38 × 10(-7)) have potential relevance for ADHD (e.g., glutamate receptor, metabotropic 5 gene, GRM5). After quality control, the 30 independent SNPs with the lowest P-values (P-values ≤ 7.57 × 10(-5) ) were chosen for confirmation. Genotyping of these SNPs in up to 320 independent German families comprising at least one child with ADHD revealed directionally consistent effect-size point estimates for 19 (10 not consistent) of the SNPs. In silico analyses of the 30 SNPs in the largest meta-analysis so far (2,064 trios, 896 cases, and 2,455 controls) revealed directionally consistent effect-size point estimates for 16 SNPs (11 not consistent). None of the combined analyses revealed a genome-wide significant result. SNPs in previously described autosomal candidate genes did not show significantly lower P-values compared to SNPs within random sets of genes of the same size. We did not find genome-wide significant results in a GWAS of German children with ADHD compared to controls. The second best SNP is located in an intron of GRM5, a gene located within a recently described region with an infrequent copy number variation in patients with ADHD.

High-throughput DNA methylation analysis in anorexia nervosa confirms TNXB hypermethylation.

OBJECTIVES: Patients with anorexia nervosa (AN) are ideally suited to identify differentially methylated genes in response to starvation. METHODS: We examined high-throughput DNA methylation derived from whole blood of 47 females with AN, 47 lean females without AN and 100 population-based females to compare AN with both controls. To account for different cell type compositions, we applied two reference-free methods (FastLMM-EWASher, RefFreeEWAS) and searched for consensus CpG sites identified by both methods. We used a validation sample of five monozygotic AN-discordant twin pairs. RESULTS: Fifty-one consensus sites were identified in AN vs. lean and 81 in AN vs. population-based comparisons. These sites have not been reported in AN methylation analyses, but for the latter comparison 54/81 sites showed directionally consistent differential methylation effects in the AN-discordant twins. For a single nucleotide polymorphism rs923768 in CSGALNACT1 a nearby site was nominally associated with AN. At the gene level, we confirmed hypermethylated sites at TNXB. We found support for a locus at NR1H3 in the AN vs. lean control comparison, but the methylation direction was opposite to the one previously reported. CONCLUSIONS: We confirm genes like TNXB previously described to comprise differentially methylated sites, and highlight further sites that might be specifically involved in AN starvation processes.

Neoadjuvant, anthracycline-free chemotherapy with carboplatin and docetaxel in triple-negative, early-stage breast cancer: a multicentric analysis of rates of pathologic complete response and survival.

INTRODUCTION: Triple-negative breast cancer (TNBC) has the highest mortality rates of all subtypes. Anthracycline and taxane regimens yield unsatisfactorily low rates of pathologic complete response (pCR) and are often not feasible in cardiac comorbidity. This study seeks to increase pCR and survival by introducing platin agents. PATIENTS AND METHODS: In this multicentric, open-label study with six cycles of docetaxel (75 mg/m(2)) and carboplatin AUC 6 q3w, patients were unwilling or unsuitable for anthracycline-based regimens. Primary endpoint was pCR (ypT0/ypTis ypN0) and survival. RESULTS: pCR rate was 50%. After 2 and 5 years, overall survival (OS) was 96.7 and 89.7%, disease-free-survival (DFS) 96.7 and 85.7%, DDFS 96.7 and 89.6%. Grade 3/4 toxicities were rare. Ninety-three per cent of patients completed six cycles. No toxicity-related treatment discontinuation or febrile neutropaenia was recorded. CONCLUSION: This regimen is highly effective and feasible in TNBC and may be combined with anthracyclines.

Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing.

INTRODUCTION: Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing. METHODS: We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99th percentile and 176 lean adults (BMI ≤ 15th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults. RESULTS AND CONCLUSION: We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (pTDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.

Long-Term Results From Tonsillectomy in Adults.

BACKGROUND: Tonsillectomy is performed more than 400 000 times in the European Union each year, making it one of the most common operations. Nonetheless, there have been only a few long-term studies of quality of life after tonsillectomy. METHODS: In 2004, data on the quality of life after tonsillectomy were obtained from adult German-speaking tonsillectomy patients by means of the Glasgow Benefit Inventory and a questionnaire specifically designed for that study. The present study concerns the further followup of these patients, sometimes many years later. 114 patients with recurrent tonsillitis were included in this descriptive study. RESULTS: Of the 114 patients, 97 (85%) provided further data at 14 months, and 71 (62%) at ca. 7 years. The Glasgow Benefit Inventory revealed postoperative improvement of quality of life at 14 months and at 7 years, with median values of 16.67 points (quartile 11.11/25) and 13.89 points (quartile 8.33/25) (p=0.168). The mean number of annual episodes of sore throat fell from 10 preoperatively to 2 postoperatively (p=0.0001). The number of visits to the doctor, the intake of analgesic drugs and antibiotics, and the number of medical absences from work also declined significantly over the period of observation. CONCLUSION: Tonsillectomy was associated with a longlasting improvement of health and quality of life, and with lower utilization of medical resources. The 62% response rate at 7 years leaves the question open whether patients with a favorable postoperative course may have been more likely than others to participate in the study.

Fine Mapping of a GWAS-Derived Obesity Candidate Region on Chromosome 16p11.2.

INTRODUCTION: Large-scale genome-wide association studies (GWASs) have identified 97 chromosomal loci associated with increased body mass index in population-based studies on adults. One of these SNPs, rs7359397, tags a large region (approx. 1MB) with high linkage disequilibrium (r2>0.7), which comprises five genes (SH2B1, APOBR, sulfotransferases: SULT1A1 and SULT1A2, TUFM). We had previously described a rare mutation in SH2B1 solely identified in extremely obese individuals but not in lean controls. METHODS: The coding regions of the genes APOBR, SULT1A1, SULT1A2, and TUFM were screened for mutations (dHPLC, SSCP, Sanger re-sequencing) in 95 extremely obese children and adolescents. Detected non-synonymous variants were genotyped (TaqMan SNP Genotyping, MALDI TOF, PCR-RFLP) in independent large study groups (up to 3,210 extremely obese/overweight cases, 485 lean controls and 615 obesity trios). In silico tools were used for the prediction of potential functional effects of detected variants. RESULTS: Except for TUFM we detected non-synonymous variants in all screened genes. Two polymorphisms rs180743 (APOBR p.Pro428Ala) and rs3833080 (APOBR p.Gly369_Asp370del9) showed nominal association to (extreme) obesity (uncorrected p = 0.003 and p = 0.002, respectively). In silico analyses predicted a functional implication for rs180743 (APOBR p.Pro428Ala). Both APOBR variants are located in the repetitive region with unknown function. CONCLUSION: Variants in APOBR contributed as strongly as variants in SH2B1 to the association with extreme obesity in the chromosomal region chr16p11.2. In silico analyses implied no functional effect of several of the detected variants. Further in vitro or in vivo analyses on the functional implications of the obesity associated variants are warranted.

Differential proteomic and tissue expression analyses identify valuable diagnostic biomarkers of hepatocellular differentiation and hepatoid adenocarcinomas.

The exact discrimination of lesions with true hepatocellular differentiation from secondary tumours and neoplasms with hepatocellular histomorphology like hepatoid adenocarcinomas (HAC) is crucial. Therefore, we aimed to identify ancillary protein biomarkers by using complementary proteomic techniques (2D-DIGE, label-free MS). The identified candidates were immunohistochemically validated in 14 paired samples of hepatocellular carcinoma (HCC) and non-tumourous liver tissue (NT). The candidates and HepPar1/Arginase1 were afterwards tested for consistency in a large cohort of hepatocellular lesions and NT (n = 290), non-hepatocellular malignancies (n = 383) and HAC (n = 13). Eight non-redundant, differentially expressed proteins were suitable for further immunohistochemical validation and four (ABAT, BHMT, FABP1, HAOX1) for further evaluation. Sensitivity and specificity rates for HCC/HAC were as follows: HepPar1 80.2%, 94.3% / 80.2%, 46.2%; Arginase1 82%, 99.4% / 82%, 69.2%; BHMT 61.4%, 93.8% / 61.4%, 100%; ABAT 84.4%, 33.7% / 84.4%, 30.8%; FABP1 87.2%, 95% / 87.2%, 69.2%; HAOX1 95.5%, 36.3% / 95.5%, 46.2%. The best 2×/3× biomarker panels for the diagnosis of HCC consisted of Arginase1/HAOX1 and BHMT/Arginase1/HAOX1 and for HAC consisted of Arginase1/FABP1 and BHMT/Arginase1/FABP1. In summary, we successfully identified, validated and benchmarked protein biomarker candidates of hepatocellular differentiation. BHMT in particular exhibited superior diagnostic characteristics in hepatocellular lesions and specifically in HAC. BHMT is therefore a promising (panel based) biomarker candidate in the differential diagnostic process of lesions with hepatocellular aspect.

Severe blunt muscle trauma in rats: only marginal hypoxia in the injured area.

BACKGROUND: After severe muscle trauma, hypoxia due to microvascular perfusion failure is generally believed to further increase local injury and to impair healing. However, detailed analysis of hypoxia at the cellular level is missing. Therefore, in the present work, spectroscopic measurements of microvascular blood flow and O2 supply were combined with immunological detection of hypoxic cells to estimate O2 conditions within the injured muscle area. MATERIALS AND METHODS: Severe blunt muscle trauma was induced in the right Musculus gastrocnemius of male Wistar rats by a standardized "weight-drop" device. Microvascular blood flow, relative hemoglobin amount, and hemoglobin O2 saturation were determined by laser Doppler and white-light spectroscopy. Hypoxic cells were detected by histologic evaluation of covalent binding of pimonidazole and expression of HIF-1α. RESULTS: Directly after trauma and until the end of experiment (480 minutes), microvascular blood flow and relative hemoglobin amount were clearly increased. In contrast to blood flow and relative hemoglobin amount, there was no immediate but a delayed increase of microvascular hemoglobin O2 saturation. Pimonidazole immunostaining revealed a hypoxic fraction (percentage area of pimonidazole-labelled muscle cells within the injured area) between 8 to 3%. There was almost no HIF-1α expression detectable in the muscle cells under each condition studied. CONCLUSIONS: In the early phase (up to 8 hours) after severe blunt muscle trauma, the overall microvascular perfusion of the injured area and thus its O2 supply is clearly increased. This increased O2 supply is obviously sufficient to ensure normoxic (or even hyperoxic) conditions in the vast majority of the cells.

No impact of obesity susceptibility loci on weight regain after a lifestyle intervention in overweight children.

OBJECTIVE: An obesity risk allele at the NEGR1 locus was shown to be associated with weight regain after a lifestyle intervention in obese adults. Independent confirmation and studies in children are lacking. Therefore, we analyzed the impact of this and 11 additional obesity susceptibility loci on weight regain after a lifestyle intervention in overweight children. DESIGN AND METHODS: We longitudinally analyzed the changes in weight status as body mass index standard deviation score (BMI-SDS) in 282 overweight children (10.6 ± 2.5 years, 47% male, BMI 27.1 ± 3.9 kg/m2) both at the end of a 1-year lifestyle intervention and at 1 year after the end of intervention. We genotyped obesity risk single nucleotide polymorphisms (SNPs) derived from genome-wide association studies in or in proximity to the following genes: NEGR1, TNKS, SDCCAG8, FTO, MC4R, TMEM18, PTER, MTCH2, SH2B1, MAF, NPC1, and KCTD15. RESULTS: The children reduced their BMI-SDS (-0.28 ± 0.35; p<0.001) during intervention and increased their BMI-SDS between the end of intervention and 1 year later (+0.05 ± 0.36; p=0.027). None of the SNPs including NEGR1 was related significantly to weight regain. CONCLUSIONS: We found no evidence for effects of any of the GWAS-based obesity marker alleles on weight regain in the course of 1 year after an intervention.

Significantly reducing post-tonsillectomy haemorrhage requiring surgery by suturing the faucial pillars: a retrospective analysis.

BACKGROUND: The tonsillectomy is one of the most frequently performed surgical procedures. Given the comparatively frequent postsurgical bleeding associated with this procedure, particular attention has been paid to reduction of the postoperative bleeding rate. In 2006, we introduced routine suturing of the faucial pillars at our clinic to reduce postoperative haemorrhage. METHODS: Two groups from the years 2003-2005 (n = 1000) and 2007-2009 (n = 1000) have been compared. We included all patients who had an elective tonsillectomy due to a benign, non-acute inflammatory tonsil illness. In the years 2007-2009, we additionally sutured the faucial pillars after completing haemostasis. For primary haemostasis we used suture ligation and bipolar diathermy. RESULTS: The rate of bleeding requiring second surgery for haemostasis was 3.6% in 2003-2005 but only 2.0% in 2007-2009 (absolute risk reduction 1.6% (95% CI 0.22%-2.45%, p = 0.04)). The median surgery time-including adenoidectomy and paracentesis surgery-increased from 25 to 31 minutes (p<0.01). CONCLUSIONS: We have been able to substantiate that suturing of the faucial pillars nearly halves the rate of postoperative haemorrhage. Surgery takes 8 minutes longer on average. Bleeding occurs later, mostly after 24 h. The limitations of this study relate to its retrospective character and all the potential biases related to observational studies.

Do common variants separate between obese melanocortin-4 receptor gene mutation carriers and non-carriers? The impact of cryptic relatedness.

BACKGROUND/AIMS: Genome-wide association studies revealed associations of single nucleotide polymorphisms (SNPs) flanking MC4R with body mass index variability and obesity. We genotyped 28 SNPs, covering MC4R, and searched for haplotypes discriminating between obese mutation carriers and non-carriers. METHODS: We analyzed all three-marker haplotype combinations of the 28 SNPs to discriminate between obese mutation carriers and non-carriers - overall and in functional categories for 25 different MC4R mutations: (a) 'like wild type', (b) 'partial loss of function', and (c) 'complete loss of function'. We checked for the possible impact of 'cryptic relatedness' by sensitivity analyses including only 1 randomly selected patient per mutation. RESULTS: Overall analyses revealed a haplotype of 3 SNPs downstream of the MC4R discriminating between obese mutation carriers and obese non-carriers. However, sensitivity analyses showed that the finding is most likely due to cryptic relatedness. CONCLUSION: Given a mutation prevalence of 1-5%, the sample size of 62 obese mutation carriers with overall 25 different MC4R mutations represents a unique feature of our study. Taking MC4R as an example, we demonstrate the impact of cryptic relatedness when trying to link non-coding SNPs to functionally relevant mutations. Hence, a thorough mutation screen can currently not be guided by SNP genotyping.