Oxidation of lysergic acid diethylamide (LSD) by peroxidases: a new metabolic pathway

Lysergic acid diethylamide (LSD) is a potent hallucinogen that is primarily metabolized to 2-oxo-3-hydroxy-LSD (O-H-LSD) and N-desmethyl-LSD (nor-LSD) by cytochrome P450 complex liver enzymes. Due to its extensive metabolism, there still is an interest in the identification of new metabolites and new routes of its metabolism in humans. In the present study, we investigated whether LSD could be a substrate for horseradish peroxidase or myeloperoxidase (MPO). Using liquid chromatography coupled to UV detection and electrospray ionization mass spectrometry (LC-UV-ESI-MS), we found that both peroxidases were capable of metabolizing LSD to the same compounds that have been observed in vivo (i.e., O-H-LSD and nor-LSD). In addition, we found another major metabolite, N,N-diethyl-7-formamido-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide (FOMBK), which is an opened indolic ring compound. Hydrolysis of FOMBK led to the deformylated compound 7-amino-N,N-diethyl-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide. The reactions of LSD with the peroxidases were chemiluminescent and sensitive to inhibition by reactive oxygen scavengers, which indicated that the classic peroxidase cycle is involved in this new alternative metabolic pathway. Considering that MPO is abundant in immune cells and also present in the central nervous system, the degradation pathway described in this study suggests a possible route of LSD metabolism that may occur concurrently with the in vivo reaction catalyzed by the cytochrome P450 system.

Structured antibody surfaces for bio-recognition and a label-free detection of bacteria

Antibody microarrays are of great research interest because of their potential application as biosensors for high-throughput protein and pathogen screening technologies. In this active area, there is still a need for novel structures and assemblies providing insight in binding interactions such as spherical and annulus-shaped protein structures, e.g. for the utilization of curved surfaces for the enhanced protein-protein interactions and detection of antigens. Therefore, the goal of the presented work was to establish a new technique for the label-free detection of bio-molecules and bacteria on topographically structured surfaces, suitable for antibody binding.rnIn the first part of the presented thesis, the fabrication of monolayers of inverse opals with 10 μm diameter and the immobilization of antibodies on their interior surface is described. For this purpose, several established methods for the linking of antibodies to glass, including Schiff bases, EDC/S-NHS chemistry and the biotin-streptavidin affinity system, were tested. The employed methods included immunofluorescence and image analysis by phase contrast microscopy. It could be shown that these methods were not successful in terms of antibody immobilization and adjacent bacteria binding. Hence, a method based on the application of an active-ester-silane was introduced. It showed promising results but also the need for further analysis. Especially the search for alternative antibodies addressing other antigens on the exterior of bacteria will be sought-after in the future.rnAs a consequence of the ability to control antibody-functionalized surfaces, a new technique employing colloidal templating to yield large scale (~cm2) 2D arrays of antibodies against E. coli K12, eGFP and human integrin αvβ3 on a versatile useful glass surface is presented. The antibodies were swept to reside around the templating microspheres during solution drying, and physisorbed on the glass. After removing the microspheres, the formation of annuli-shaped antibody structures was observed. The preserved antibody structure and functionality is shown by binding the specific antigens and secondary antibodies. The improved detection of specific bacteria from a crude solution compared to conventional “flat” antibody surfaces and the setting up of an integrin-binding platform for targeted recognition and surface interactions of eukaryotic cells is demonstrated. The structures were investigated by atomic force, confocal and fluorescence microscopy. Operational parameters like drying time, temperature, humidity and surfactants were optimized to obtain a stable antibody structure.

Regulation of placental growth by aldosterone and cortisol

During pregnancy, trophoblasts grow to adapt the feto-maternal unit to fetal requirements. Aldosterone and cortisol levels increase, the latter being inactivated by a healthy placenta. By contrast, preeclamptic placental growth is reduced while aldosterone levels are low and placental cortisol tissue levels are high due to improper deactivation. Aldosterone acts as a growth factor in many tissues, whereas cortisol inhibits growth. We hypothesized that in preeclampsia low aldosterone and enhanced cortisol availability might mutually affect placental growth and function. Proliferation of cultured human trophoblasts was time- and dose-dependently increased with aldosterone (P < 0.04 to P < 0.0001) and inhibited by spironolactone and glucocorticoids (P < 0.01). Mineralo- and glucocorticoid receptor expression and activation upon agonist stimulation was verified by visualization of nuclear translocation of the receptors. Functional aldosterone deficiency simulated in pregnant mice by spironolactone treatment (15 μg/g body weight/day) led to a reduced fetal umbilical blood flow (P < 0.05). In rat (P < 0.05; R(2) = 0.2055) and human (X(2) = 3.85; P = 0.0249) pregnancy, placental size was positively related to plasma aldosterone. Autocrine production of these steroid hormones was excluded functionally and via the absence of specific enzymatic transcripts for CYP11B2 and CYP11B1. In conclusion, activation of mineralocorticoid receptors by maternal aldosterone appears to be required for trophoblast growth and a normal feto-placental function. Thus, low aldosterone levels and enhanced cortisol availability may be one explanation for the reduced placental size in preeclampsia and related disorders.

Targeting PI3KC2β impairs proliferation and survival in acute leukemia, brain tumours and neuroendocrine tumours

Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks.

Therapeutic hypothermia in term infants after perinatal encephalopathy: The last 5 years in Switzerland

Background: Therapeutic hypothermia (TH) following perinatal asphyxial encephalopathy in term infants improves mortality and neurodevelopmental outcome. In Europe, most neonatal units perform active cooling whereas in Switzerland passive cooling is predominantly used. Aims: (i) To determine how many infants were cooled within the last 5 years in Switzerland, (ii) to assess the cooling methods, (iii) to evaluate the variation of temperature of different cooling methods, and (iv) to evaluate the use of neuromonitoring. Study design: Retrospective cohort study. Patients: Notes of all cooled term infants between March 2005 and December 2010 in 9 perinatal and two paediatric intensive care centres were retrospectively reviewed. Active cooling was compared to passive cooling alone and to passive cooling in combination with gel packs. Results: 150 infants were cooled. Twenty-seven (18.2%) were cooled actively, 34 (23%) passively and 87 (58.8%) passively in combination with gel packs. Variation of temperature was significantly different between the three methods. Passive cooling had a significant higher variation of temperature (SD of 0.89) than both passive cooling in combination with gel packs (SD of 0.79) and active cooling (SD of 0.76). aEEG before TH was obtained in 35.8% of the infants and 86.5% had full EEG. One cUS was performed in 95.3% and MRI in 62.2% of the infants. Conclusion: Target temperature can be achieved with all three cooling methods. Passive cooling has the highest variation of temperature. Neuromonitoring should be improved in Swiss neonatal and paediatric intensive care units. Our results stress the importance of national registries.

Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated in cancer 1

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), located on human chromosome 17p13.3, is frequently silenced in cancer by epigenetic mechanisms. Hypermethylated in cancer 1 belongs to the bric à brac/poxviruses and zinc-finger family of transcription factors and acts by repressing target gene expression. It has been shown that enforced p53 expression leads to increased HIC1 mRNA, and recent data suggest that p53 and Hic1 cooperate in tumorigenesis. In order to elucidate the regulation of HIC1 expression, we have analysed the HIC1 promoter region for p53-dependent induction of gene expression. Using progressively truncated luciferase reporter gene constructs, we have identified a p53-responsive element (PRE) 500 bp upstream of the TATA-box containing promoter P0 of HIC1, which is sequence specifically bound by p53 in vitro as assessed by electrophoretic mobility shift assays. We demonstrate that this HIC1 p53-responsive element (HIC1.PRE) is necessary and sufficient to mediate induction of transcription by p53. This result is supported by the observation that abolishing endogenous wild-type p53 function prevents HIC1 mRNA induction in response to UV-induced DNA damage. Other members of the p53 family, notably TAp73beta and DeltaNp63alpha, can also act through this HIC1.PRE to induce transcription of HIC1, and finally, hypermethylation of the HIC1 promoter attenuates inducibility by p53.

Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents

The CYP17A1 gene is the qualitative regulator of steroidogenesis. Depending on the presence or absence of CYP17 activities mineralocorticoids, glucocorticoids or adrenal androgens are produced. The expression of the CYP17A1 gene is tissue as well as species-specific. In contrast to humans, adrenals of rodents do not express the CYP17A1 gene and have therefore no P450c17 enzyme for cortisol production, but produce corticosterone. DNA methylation is involved in the tissue-specific silencing of the CYP17A1 gene in human placental JEG-3 cells. We investigated the role of DNA methylation for the tissue-specific expression of the CYP17A1 gene in rodents. Rats treated with the methyltransferase inhibitor 5-aza-deoxycytidine excreted the cortisol metabolite tetrahydrocortisol in their urine suggesting that treatment induced CYP17 expression and 17alpha-hydroxylase activity through demethylation. Accordingly, bisulfite modification experiments identified a methylated CpG island in the CYP17 promoter in DNA extracted from rat adrenals but not from testes. Both methyltransferase and histone deacetylase inhibitors induced the expression of the CYP17A1 gene in mouse adrenocortical Y1 cells which normally do not express CYP17, indicating that the expression of the mouse CYP17A1 gene is epigenetically controlled. The role of DNA methylation for CYP17 expression was further underlined by the finding that a reporter construct driven by the mouse -1041 bp CYP17 promoter was active in Y1 cells, thus excluding the lack of essential transcription factors for CYP17 expression in these adrenal cells.

Who Counts? Demography of Swidden Cultivators in Southeast Asia

Swidden cultivators are often found as a distinct category of farmers in the literature, but rarely appear in population censuses or other national and regional classifications. This has led to a worldwide confusion on how many people are dependent on this form of agriculture. The most often cited number of 200–300 million dates back to the early 1970s, but the source is obscure. We assess available, published data from nine countries in Southeast Asia and conclude that on this basis it is not possible to provide a firm estimate of the number of swidden cultivators in the region. A conservative range of 14–34 million people engaged in swidden cultivation in the region is suggested, however. We argue that along with improved knowledge of swidden livelihoods, there is an urgent need to develop techniques that will allow for better estimates of swidden populations in order to secure appropriate rural development and poverty reduction in swidden areas.

Finding Homogeneity in Heterogeneity - A New Approach to Quantifying Landscape Mosaics Developed for the Lao PDR

A key challenge for land change science in general and research on swidden agriculture in particular, is linking land cover information to human–environment interactions over larger spatial areas. In Lao PDR, a country facing rapid and multi-level land change processes, this hinders informed policy- and decision-making. Crucial information on land use types and people involved is still lacking. This article proposes an alternative approach for the description of landscape mosaics. Instead of analyzing local land use combinations, we studied land cover mosaics at a meso-level of spatial scale and interpreted these in terms of human–environmental interactions. These landscape mosaics were then overlaid with population census data. Results showed that swidden agricultural landscapes, involving 17% of the population, dominate 29% of the country, while permanent agricultural landscapes involve 74% of the population in 29% of the territory. Forests still form an important component of these landscape mosaics.

An Assessment of Trends in the Extent of Swidden in Southeast Asia

Swidden systems consisting of temporarily cultivated land and associated fallows often do not appear on land use maps or in statistical records. This is partly due to the fact that swidden is a diverse and dynamic land use system that is difficult to map and partly because of the practice of grouping land covers associated with swidden systems into land use or land cover categories that are not self-evidently linked to swiddening. Additionally, in many parts of Southeast Asia swidden systems have changed or are in the process of changing into other land use systems. This paper assesses the extent of swidden on the basis of regional and national sources for nine countries, and determines the pattern of changes of swidden on the basis of 151 cases culled from 67 articles. Findings include (1) a majority of the cases document swidden being replaced by other forms of agriculture or by other livelihood systems; (2) in cases where swiddening is still practiced, fallow lengths are usually, but not always, shorter; and (3) shortened fallow length does not necessarily indicate a trend away from swidden since it is observed that short fallow swidden is sometimes maintained along with other more intensive farming practices and not completely abandoned. The paper concludes that there is a surprising lack of conclusive data on the extent of swidden in Southeast Asia. In order to remedy this, methods are reviewed that may lead to more precise future assessments.

Controversies of fetal cardiac intervention

Remarkable advances in ultrasound imaging technology have made it possible to diagnose fetal cardiovascular lesions as early as 12-14 weeks of gestation and to assess their physiological relevance by echocardiography. Moreover, invasive techniques have been developed and refined to relieve significant congenital heart disease (CHD), such as critical aortic and pulmonary stenoses in the pediatric population including neonates. Recognition of the fact that certain CHDs can evolve in utero, and early intervention may improve the outcome by altering the natural history of such conditions has led to the evolution of a new fetal therapy, i.e. fetal cardiac intervention. Two entities, pulmonary valvar atresia and intact ventricular septum (PA/IVS) and hypoplastic left heart syndrome (HLHS), are associated with significant morbidity and mortality even with postnatal surgical therapy. These cases are believed to occur due to restricted blood flow, leading to impaired growth and function of the right or left ventricle. Therefore, several centers started the approach of antenatal intervention with the primary goal of improving the blood flow through the stenotic/atretic valve orifices to allow growth of cardiac structures. Even though centers with a reasonable number of cases seem to have improved the technique and the immediate outcome of fetal interventions, the field is challenged by ethical issues as the intervention puts both the mother and the fetus at risk. Moreover, the perceived benefits of prenatal treatment have to be weighed against steadily improving postnatal surgical and hybrid procedures, which have been shown to reduce morbidity and mortality for these complex heart defects. This review is an attempt to provide a balanced opinion and an update on fetal cardiac intervention.

The unstable (CTG)$\sb{\rm n}$ trinucleotide repeat associated with myotonic dystrophy: What it does and where it came from

Myotonic dystrophy (DM), an autosomal dominant disorder mapping to human chromosome 19q13.3, is the most common neuromuscular disease in human adults.^ Following the identification of the mutation underlying the DM phenotype, an unstable (CTG)$\sb{n}$ trinucleotide repeat in the 3$\prime$ untranslated region (UTR) of a gene encoding a ser/thr protein kinase named DM protein kinase (DMPK), the study was targeted at two questions: (1) the identification of the disease-causing mechanism(s) of the unstable repeat, and at a more basic level, (2) the identification of the origin and the mechanism(s) involved in repeat instability. The first goal was to identify the pathophysiological mechanisms of the (CTG)$\sb{n}$ repeat.^ The normal repeat is transcribed but not translated; therefore, initial studies centered on the effect on RNA transcript levels. The vast majority of DM affecteds are heterozygous for the mutant expansion, so that the normal allele interferes with the analysis of the mutant allele. A quantitative allele-specific RT-PCR procedure was developed and applied to a spectrum of patient tissue samples and cell lines. Equal levels of unprocessed pre-mRNA were determined for the wild type (+) and disease (DM) alleles in skeletal muscle and cell lines of heterozygous DM patients, indicating that any nucleosome binding has no effect at the level of transcriptional initiation and transcription of the mutant DMPK locus. In contrast, processed mRNA levels from the DM allele were reduced relative to the + allele as the size of the expansion increased. The unstable repeat, therefore, impairs post-transcriptional processing of DM allele transcripts. This phenomenon has profound effects on overall DMPK locus steady-state transcript levels in cells missing a wild type allele and does not appear to be mediated by imprinting, decreased mRNA stability, generation of aberrant splice forms, or absence of polyadenylation of the mutant allele.^ In Caucasian DM subjects, the unstable repeat is in complete linkage disequlibrium with a single haplotype composed of nine alleles within and flanking DMPK over a physical distance of 30 kb. A detailed haplotype analysis of the DM region was conducted on a Nigerian (Yoruba) DM family, the only indigenous sub-Saharan DM case reported to date. Each affected member of this family had an expanded (CTG)$\sb{n}$ repeat in one of their DMPK alleles. However, unlike all other DM populations studied thus far, disassociation of the (CTG)$\sb{n}$ repeat expansion from other alleles of the putative predisposing haplotype was found. Thus, the expanded (CTG)$\sb{n}$ repeat in this family was the result of an independent mutational event. Consequently, the origin of DM is unlikely the result of a single mutational event, and the hypothesis that a single ancestral haplotype predisposes to repeat expansion is not compelling. (Abstract shortened by UMI.) ^

Intergroup bias in third-party punishment stems from both ingroup favoritism and outgroup discrimination

Social norms pervade almost every aspect of social interaction. If they are violated, not only legal institutions, but other members of society as well, punish, i.e., inflict costs on the wrongdoer. Sanctioning occurs even when the punishers themselves were not harmed directly and even when it is costly for them. There is evidence for intergroup bias in this third-party punishment: third-parties, who share group membership with victims, punish outgroup perpetrators more harshly than ingroup perpetrators. However, it is unknown whether a discriminatory treatment of outgroup perpetrators (outgroup discrimination) or a preferential treatment of ingroup perpetrators (ingroup favoritism) drives this bias. To answer this question, the punishment of outgroup and ingroup perpetrators must be compared to a baseline, i.e., unaffiliated perpetrators. By applying a costly punishment game, we found stronger punishment of outgroup versus unaffiliated perpetrators and weaker punishment of ingroup versus unaffiliated perpetrators. This demonstrates that both ingroup favoritism and outgroup discrimination drive intergroup bias in third-party punishment of perpetrators that belong to distinct social groups.

Do we need antiplatelet therapy in thrombocytosis? Pro. Diagnostic and pathophysiologic considerations for a treatment choice.

Thrombocytosis (defined as platelets >450 x 10(9)/l) has several aetiologies. After having excluded spurious thrombocytosis (e. g., due to microspherocytes, schistocytes, cryoglobulins, or bacteria), the differential diagnosis of true thrombocytosis encompasses secondary causes (as diverse as inflammation, infection, malignancy, iron deficiency, or asplenia), primary hereditary (rare forms of familial thrombocytosis) and primary acquired entities (either in the context of a myelodysplastic syndrome or more frequently a myeloproliferative neoplasia). This manuscript addresses the following aspects: 1) diagnostic approach to thrombocytosis; 2) various mechanisms leading to a high platelet count; 3) potential of some of these mechanisms to modulate platelet function, producing hyper-reactive platelets and thus exerting a direct impact on the thrombotic risk; 4) indication of anti-thrombotic treatment in patients with thrombocytosis. There is a single prospective randomized clinical trial showing the benefit of acetyl-salicylic acid in polycythaemia vera. For other types of primary thrombocytosis and for secondary forms, treatment decisions have to be individualized according to the patient thrombotic and bleeding risks, taking into account the mechanism causing thrombocytosis. This manuscript discusses experimental and clinical data suggesting that besides patients with essential thrombocythaemia and other forms of primary thrombocytosis also those with thrombocytosis in the context of chronic inflammation, malignancy, or exposure to high altitude might benefit from anti-platelet treatment.