Hubrecht Institute Centennial - From embryos to stem cells.

During the last 100 years, the Hubrecht Institute evolved from a small laboratory aimed at providing research material to the scientific community to a modern, fully equipped research institute with state-of-the-art infrastructure, performing research at the highest standard. The past 100 years have been eventful for the Hubrecht Institute with many glorious moments, but also threats to be shut down on several occasions. Here, we will briefly review the rich history of the Hubrecht Institute.

Efficient evaluation of Sommerfeld integrals for the optical simulation of many scattering particles in planarly layered media.

A strategy for the efficient numerical evaluation of Sommerfeld integrals in the context of electromagnetic scattering at particles embedded in a plane parallel layer system is presented. The scheme relies on a lookup-table approach in combination with an asymptotic approximation of the Bessel function in order to enable the use of fast Fourier transformation. Accuracy of the algorithm is enhanced by means of singularity extraction and a novel technique to treat the integrand at small arguments. For short particle distances, this method is accomplished by a slower but more robust direct integration along a deflected contour. As an example, we investigate enhanced light extraction from an organic light-emitting diode by optical scattering particles. The calculations are discussed with respect to accuracy and computing time. By means of the present strategy, an accurate evaluation of the scattered field for several thousand wavelength scale particles can be achieved within a few hours on a conventional workstation computer.

Advances in the management of constipation-predominant irritable bowel syndrome: the role of linaclotide.

Constipation-predominant irritable bowel syndrome (IBS-C) is a commonly prevalent and clinically challenging disorder to treat. Until recently, most therapeutic agents had limited ability to address the complexity of symptoms inherent to the syndrome. The development of linaclotide provides a physiologically sound approach to treatment of the multiple symptoms of IBS-C. Clinical trials demonstrate the efficacy of linaclotide, and a platform to better understand the symptomatology of IBS-C. Based on recent clinical evidence, linaclotide should be considered for patients with IBS-C because it improves abdominal pain and bowel symptoms. In phase III trials, linaclotide met the US Food and Drug Administration responder endpoint with a number needed to treat (NNT) of 5.1-7.9, and European Medicines Agency coprimary endpoints at 12 weeks with a NNT of 4.39-7.69, and at 26 weeks with a NNT of 4.93-5.68. It is safe and effective, with diarrhea reported as the most common adverse effect, which leads to discontinuation of the medication in approximately 5% of patients.

Sensitivity improvement in fluorescence-based particle detection.

Microfluidic flow cytometers are highly interesting candidates for biomedical point-of-care applications. However, the sensitivity, reliability, and throughput of these systems must be improved to provide the full functionality of established flow cytometric systems. One proposed method to improve fluorescence detection systems is to use spatial modulation techniques. We derive the noise-related statistics and calculate the coefficient of variation for a detection system with and without spatial modulation. We measure the noise properties of a nonmodulated microfluidic fluorescence particle detection system and analyze the possible performance gains using spatial modulation.

Anorectal physiology and pathophysiology in the elderly.

Anorectal medical disorders facing the elderly include fecal incontinence, fecal impaction with overflow fecal incontinence, chronic constipation, dyssynergic defecation, hemorrhoids, anal fissure, and pelvic floor disorders. This article discusses the latest advances in age-related changes in morphology and function of anal sphincter, changes in cellular and molecular biology, alterations in neurotransmitters and reflexes, and their impact on functional changes of the anorectum in the elderly. These biophysiologic changes have implications for the pathophysiology of anorectal disorders. A clear understanding and working knowledge of the functional anatomy and pathophysiology will enable appropriate diagnosis and treatment of these disorders.

Comorbidities in overweight children and adolescents: do we treat them effectively?

OBJECTIVE: The aim was to analyze the effectiveness of treatment concerning obesity-associated comorbidities in clinical practice. METHODS: A total of 11,681 overweight children with ≥ 6-month follow-up treated at 175 centers specialized in pediatric obesity care in Central Europe were included in this analysis (mean body mass index (BMI) 29.0 ± 5.6 kg m(-)(2), standard deviation score body mass index (SDS-BMI) 2.48 ± 0.54, 45% boys, age 11.4 ± 2.8 years). The changes of weight status, blood pressure, fasting lipids and glucose, and oral glucose tolerance tests were documented by standardized prospective quality documentation software (APV). RESULTS: After follow-up of in median 1.2 (interquartile range 0.9-2.2) years, a mean reduction of -0.15 SDS-BMI was achieved. The prevalence of prehypertension (37->33%) and hypertension (17->12%) decreased, while prevalences of triglycerides >150 mg dl(-1) (22->21%), low-density-lipoprotein-cholesterol >130 mg dl(-1) (15->14%), impaired fasting glucose (6->6%) and impaired glucose tolerance (9->8%) remained stable. Drug treatment according to cutoffs recommended in European obesity guidelines were not frequently indicated (hypertension: 10%; dyslipidemia: 1%, type 2 diabetes <1%). None of the children with dyslipidemia received lipid-lowering drugs and only 1.4% of the children with hypertension were treated with antihypertensive drugs. CONCLUSIONS: Achieving sufficient weight loss to improve obesity associated comorbidities was difficult in clinical practice. Drug treatment of hypertension, dyslipidemia and type 2 diabetes was rarely performed even if it was indicated only in a minority of the overweight children. Future analyses should identify reasons for this insufficient drug treatment of comorbidities and analyze whether the benchmarking processes of APV improve medical care of childhood obesity.

Hyperinsulinemia euglycemia therapy for calcium channel blocker overdose: a case report.

We report the case of a patient with calcium channel blocker toxicity who was treated successfully with hyperinsulinemia euglycemia therapy, without prior use of vasopressors. The patient was a 60-year-old man with schizoaffective disorder who presented with severe hemodynamic compromise after an intentional overdose of 5,400 mg of extended-release diltiazem. He had been admitted to the hospital twice before for attempted suicide with diltiazem and nifedipine, respectively. During the previous admissions, conventional treatments were used, and complications included hemodynamic compromise, ischemic bowel requiring ileostomy, and a prolonged hospital stay. During the current admission, the patient's clinical condition failed to improve after treatment with charcoal, fluid resuscitation, calcium, and glucagon. Eight hours after admission, hyperinsulinemia euglycemia therapy was initiated; 3 hours later, the patient's hemodynamic status showed sustained improvement. His bradycardia and hypotension resolved without cardiac pacing or vasopressors. Hyperinsulinemia euglycemia therapy is a potentially life-saving treatment for calcium channel blocker toxicity. We suggest that such therapy should be considered early, in conjunction with conventional therapy, for the treatment of calcium channel blocker overdose in patients not responding to initial treatment.

Flexible planar microfluidic chip employing a light emitting diode and a PIN-photodiode for portable flow cytometers.

Detection of fluorescence particles is a key method of flow cytometry. We evaluate the performance of a design for a microfluidic fluorescence particle detection device. Due to the planar design with low layer thicknesses, we avoid optical components such as lenses or dichroic mirrors and substitute them with a shadow mask and colored film filters. A commercially available LED is used as the light source and a PIN-photodiode as detector. This design approach reduces component cost and power consumption and enables supplying the device with power from a standard USB port. From evaluation of this design, we obtain a maximum particle detection frequency of up to 600 particles per second at a sensitivity of better than 4.7 × 10(5) MESF (molecules of equivalent soluble fluorochrome) measured with particles for FITC sensitivity calibration. Lowering the flow rate increases the instrument sensitivity by an order of magnitude enabling the detection of particles with 4.5 × 10(4) MESF.

[Treatment of extremely obese adolescents and young adults at the Insula Obesity Center in Bischofswiesen]. / Therapie der extremen juvenilen Adipositas im Adipositas-Zentrum Insula in Bischofswiesen.

The Insula Obesity Center has been treating extremely obese adolescents and young adults since 1992. Various programs ranging from 2-9 months' duration are offered. The mean BMI at admission has been increasing continuously and is presently 41.5 kg/m(2) with occasional extremes over 80 kg/m(2). Obesity comorbidities are common. A mean weight reduction of 1.3 kg/week is achieved during a mean duration of treatment of 4.7 months. Follow-up in residential support groups is offered for up to 2 years for selected patients with special challenges such as lack of family support.

Monitoring of kratom or Krypton intake in urine using GC-MS in clinical and forensic toxicology.

The Thai medicinal plant Mitragyna speciosa (kratom) is misused as a herbal drug. Besides this, a new herbal blend has appeared on the drugs of abuse market, named Krypton, a mixture of O-demethyltramadol (ODT) and kratom. Therefore, urine drug screenings should include ODT and focus on the metabolites of the kratom alkaloids mitragynine (MG), paynantheine (PAY), speciogynine (SG), and speciociliatine (SC). The aim of this study was to develop a full-scan gas chromatography-mass spectrometry procedure for monitoring kratom or Krypton intake in urine after enzymatic cleavage of conjugates, solid-phase extraction, and trimethylsilylation. With use of reconstructed mass chromatography with the ions m/z 271, 286, 329, 344, 470, 526, 528, and 586, the presence of MG, 16-carboxy-MG, 9-O-demethyl-MG, and/or 9-O-demethyl-16-carboxy-MG could be indicated, and in case of Krypton, with m/z 58, 84, 116, 142, 303, 361, 393, and 451, the additional presence of ODT and its nor metabolite could be indicated. Compounds were identified by comparison with their respective reference spectra. Depending on the plant type, dose, administration route, and/or sampling time, further metabolites of MG, PAY, SG, and SC could be detected. The limits of detection (signal-to-noise ratio of 3) were 100 ng/ml for the parent alkaloids and 50 ng/ml for ODT. As mainly metabolites of the kratom alkaloids were detected in urine, the detectability of kratom was tested successfully using rat urine after administration of a common user's dose of MG. As the metabolism in humans was similar, this procedure should be suitable to prove an intake of kratom or Krypton.

Use of liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry for studying the metabolism of paynantheine, an alkaloid of the herbal drug Kratom in rat and human urine.

The Thai medicinal plant Mitragyna speciosa (Kratom in Thai) is misused as a herbal drug of abuse. During studies on the main Kratom alkaloid mitragynine (MG) in rats and humans, several dehydro analogs could be detected in urine of Kratom users, which were not found in rat urine after administration of pure MG. Questions arose as to whether these compounds are formed from MG only by humans or whether they are metabolites formed from the second abundant Kratom alkaloid paynantheine (PAY), the dehydro analog of MG. Therefore, the aim of the presented study was to identify the phase I and II metabolites of PAY in rat urine after administration of the pure alkaloid. This was first isolated from Kratom leaves. Liquid chromatography-linear ion trap mass spectrometry provided detailed structure information of the metabolites in the MS(n) mode particularly with high resolution. Besides PAY, the following phase I metabolites could be identified: 9-O-demethyl PAY, 16-carboxy PAY, 9-O-demethyl-16-carboxy PAY, 17-O-demethyl PAY, 17-O-demethyl-16,17-dihydro PAY, 9,17-O-bisdemethyl PAY, 9,17-O-bisdemethyl-16,17-dihydro PAY, 17-carboxy-16,17-dihydro PAY, and 9-O-demethyl-17-carboxy-16,17-dihydro PAY. These metabolites indicated that PAY was metabolized via the same pathways as MG. Several metabolites were excreted as glucuronides or sulfates. The metabolism studies in rats showed that PAY and its metabolites corresponded to the MG-related dehydro compounds detected in urine of the Kratom users. In conclusion, PAY and its metabolites may be further markers for a Kratom abuse in addition of MG and its metabolites.

Studies on the metabolism of mitragynine, the main alkaloid of the herbal drug Kratom, in rat and human urine using liquid chromatography-linear ion trap mass spectrometry.

Mitragynine (MG) is an indole alkaloid of the Thai medicinal plant Mitragyna speciosa (Kratom in Thai) and reported to have opioid agonistic properties. Because of its stimulant and euphoric effects, Kratom is used as a herbal drug of abuse. The aim of the presented study is to identify the phase I and II metabolites of MG in rat and human urine after solid-phase extraction (SPE) using liquid chromatography-linear ion trap mass spectrometry providing detailed structure information in the MSn mode particularly with high resolution. The seven identified phase I metabolites indicated that MG was metabolized by hydrolysis of the methylester in position 16, O-demethylation of the 9-methoxy group and of the 17-methoxy group, followed, via the intermediate aldehydes, by oxidation to carboxylic acids or reduction to alcohols and combinations of some steps. In rats, four metabolites were additionally conjugated to glucuronides and one to sulfate, but in humans, three metabolites to glucuronides and three to sulfates.

[Long-term inpatient treatment of extreme juvenile obesity: an 18-month catamnestic study]. / Studie zur stationären Langzeittherapie der extremen juvenilen Adipositas. Jeder Zweite nimmt langfristig ab.

Eighteen months after completion of long-term treatment of 98 extremely overweight juveniles in the rehabilitation center Insula, this study revealed an improvement in the age-specific body mass index (BMI-SDS) in 55.1% of the cases, when all the non-responders (approx. 22%) were evaluated as failures. An improvement of at least 0.2 or 0.5 BMI SDS points was achieved in 41.8% and 21.4%, respectively.

Mutation screen of the brain derived neurotrophic factor gene (BDNF): identification of several genetic variants and association studies in patients with obesity, eating disorders, and attention-deficit/hyperactivity disorder.

Several lines of evidence indicate an involvement of brain derived neurotrophic factor (BDNF) in body weight regulation and activity: heterozygous Bdnf knockout mice (Bdnf(+/-)) are hyperphagic, obese, and hyperactive; furthermore, central infusion of BDNF leads to severe, dose-dependent appetite suppression and weight loss in rats. We searched for the role of BDNF variants in obesity, eating disorders, and attention-deficit/hyperactivity disorder (ADHD). A mutation screen (SSCP and DHPLC) of the translated region of BDNF in 183 extremely obese children and adolescents and 187 underweight students was performed. Additionally, we genotyped two common polymorphisms (rs6265: p.V66M; c.-46C > T) in 118 patients with anorexia nervosa, 80 patients with bulimia nervosa, 88 patients with ADHD, and 96 normal weight controls. Three rare variants (c.5C > T: p.T2I; c.273G > A; c.*137A > G) and the known polymorphism (p.V66M) were identified. A role of the I2 allele in the etiology of obesity cannot be excluded. We found no association between p.V66M or the additionally genotyped variant c.-46C > T and obesity, ADHD or eating disorders. This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148-7299:1/suppmat/index.html.

No evidence for involvement of the promoter polymorphism -866 G/A of the UCP2 gene in childhood-onset obesity in humans.

Recently, an association between obesity and the G-allele of the - 866 G/A polymorphism in the promoter region of uncoupling protein-2 gene (UCP2) was reported. Both allele frequencies and genotype distributions for this polymorphism differed between obese individuals and never-obese controls. We attempted to confirm this finding. Genotyping was performed by polymerase chain reaction with subsequent restriction fragment length polymorphism analysis (PCR-RFLP). We analysed transmission disequilibrium of the (wild type) G-allele for 200 extremely obese children and adolescents from 93 concordant sib pair families using the pedigree disequilibrium test. Additionally, using a one-sided asymptotic Pearson's chi 2-test, we tested whether the G-allele occurs more frequently in 277 extremely obese children and adolescents (including the 93 index patients of the concordant sib pairs) than in 188 never-obese controls. The one-sided asymptotic Cochran Armitage trend test was used to determine differences in genotype frequencies between extremely obese and healthy underweight individuals. The PDT analysis revealed no evidence for transmission disequilibrium in obesity. Allele and genotype frequencies did not differ between the extremely obese and never-obese subjects. In conclusion, we cannot confirm the results of ) in our young sample.

No evidence for involvement of alleles of the 825-C/T polymorphism of the G-protein subunit beta 3 in body weight regulation.

The 825-C/T polymorphism of the beta 3 subunit of the heterotrimeric G protein gene (GNB3) has been shown to be associated with essential hypertension in humans. Recently, it was also reported that the 825-T allele has a higher frequency in obese than non-obese hypertensives suggesting that the primary effect of this allele is on body weight. The association to hypertension might merely be a secondary effect of the higher weight of the respective allele carriers. To investigate an involvement of the 825-T allele in body weight regulation in young individuals, we evaluated allele frequencies in 440 extremely obese children and adolescents (82.9% had a body mass index [BMI] > or = 99th percentile), 51 obese students (BMI > or = 90th percentile), 110 normal weight students (BMI between 40th and 60th percentile) and 144 underweight students (BMI < or = 15th percentile). The study groups were genotyped by polymerase chain reaction with subsequent restriction fragment length polymorphism analysis (PCR-RFLP). The one-sided Yates-corrected chi(2)-test and the Cochran-Armitage trend test for association were performed. Tests for association were negative. The 825-T allele frequencies were similar in the four study groups belonging to different weight ranges (extreme early onset obesity: 0.29; obesity: 0.28; normal weight: 0.35; underweight: 0.32). Similarly, genotype frequencies did not differ between the groups. We concluded that the 825-T allele of the GNB3 does not play a major role in weight regulation in German children, adolescents and young adults.

Systematic screening for mutations in the human necdin gene (NDN): identification of two naturally occurring polymorphisms and association analysis in body weight regulation.

BACKGROUND: NDN, which codes for the human necdin protein, is a candidate gene for Prader-Willi syndrome (PWS). One feature of this neurogenetic disorder is hyperphagia resulting in extreme obesity observed later in development. OBJECTIVE AND DESIGN: In this study we have used single-strand conformation polymorphism (SSCP) analysis to identify sequence variants at the human necdin gene. Furthermore we tested whether these variants were associated with obesity in extremely obese German children and adolescents. RESULTS: Two gene variants could be identified: a g.1352T-->C polymorphism in the putative promotor region and a silent g.2311C-->T polymorphism in the coding region. Genotype and allele frequency distribution of both of the polymorphisms were not significantly different between lower and higher body mass index (BMI) subjects. CONCLUSIONS: Hence, it is unlikely that these polymorphisms play a major role in the emergence of juvenile onset human obesity.

Efficacy of 'functional relaxation' in comparison to terbutaline and a 'placebo relaxation' method in patients with acute asthma. A randomized, prospective, placebo-controlled, crossover experimental investigation.

BACKGROUND: 'Functional relaxation' (FR) according to Marianne Fuchs is a body-oriented psychotherapy that involves teaching the patient a type of relaxation techniques aimed at maintaining equilibrium of the nervous system. METHODS: In order to determine whether the practice of elementary parts of this therapy has an immediate beneficial effect on pulmonary function, a randomized, single-blind, prospective crossover study was done with 21 asthmatics with acute bronchoconstriction. On 3 consecutive days they were given either (1) a 5-min verbal standard instruction in elementary exercises of FR (eFR), which they were to practice during subsequent bodyplethysmographic measurement or (2) inhalative terbutaline (IT), a beta(2)-sympathomimetic drug, or (3) an unspecific 'placebo relaxation' technique (PRT), so that all subjects tried all 3 treatments in random order. Spirometric variables were assessed. RESULTS: There was a significant decrease in specific airway resistance with eFR, which, though not as pronounced as with IT, was significantly greater than with PRT. This study shows that clinically relevant effects can be achieved for patients with asthma through mind-body interaction, which can be triggered by reproducible procedures. CONCLUSION: Further development of the FR approach could lead to a nonpharmacological and effective supplementary treatment for asthma, which is in high demand by many patients.

Identification of a deletion variant in the gene encoding the human alpha(2A)-adrenergic receptor.

OBJECTIVE: The alpha(2)-adrenergic receptors are involved in the effects of catecholamines on energy metabolism. Of three known subtypes with differential expression, alpha(2A)-adrenergic receptors are also localized in adipose tissue where they counteract the lipolytic activity of beta-adrenergic receptors. This study was undertaken to assess whether variants in the alpha(2A)-adrenergic receptor gene are associated with body weight. DESIGN AND METHODS: Single strand conformation polymorphism (SSCP) screening and subsequent sequencing were applied to determine genetic variants in DNA samples from individuals with obesity, those of normal weight and those underweight. RESULTS: Analysis of the coding region resulted in the identification of an 18 bp deletion, with no other mutation found. Of 429 genotyped subjects, 7 carried the deletion, with no significant differences between lean and obese subjects. A previously identified polymorphism in the promoter of the alpha(2A)-adrenergic receptor gene also did not show an association with any of the tested body weight categories. CONCLUSION: Our data suggest that variants in the alpha(2A)-adrenergic receptor gene are unlikely to contribute to the predisposition for the lean or obese state.