A novel tetrabranched neurotensin(8-13) cyclam derivative: synthesis, 64Cu-labeling and biological evaluation

New macrocyclic 1,4,8,11-tetraazacyclotetradecane (cyclam) derivatives with 1, 2 and 4 neurotensin(8-13) units 4, 5 and 7 have been synthesized. Compounds 4 and 5 were prepared by the reaction of non-stabilized neurotensin(8-13) and cyclamtetrapropionic acid 2 using 1-ethyl-3-(3-dimethylaminocarbonyl)carbodiimide-hydrochloride and N-hydroxysulfosuccinimide. The tetrameric compound 7 was synthesized by Michael addition of neurotensin(8-13) acrylamide 6 and cyclam 1. The copper(II) complexation behavior of 4, 5 and 7 was investigated by UV/visible spectrophotometry and shows that the metal center resides inside the N4 chromophore with additional apical interactions established with pendant arms. The novel tetrabranched NT(8-13) cyclam 7 with nanomolar neurotensin receptor 1 binding affinity was efficiently radiolabeled with ⁶⁴Cu under mild conditions. ⁶⁴Cu ⊂ 7 showed slow transchelation in the presence of a large amount of cyclam as competing ligand, while it completely remains intact in the presence of EDTA. The in vivo behavior of ⁶⁴Cu ⊂ 7 was studied in rats and mice. The metabolic stability in rodent models was high with a half-life of intact ⁶⁴Cu ⊂ 7 in plasma of 34 min in rats and 60 min in the mice, respectively. The binding affinity was high enough to demonstrate in vivo binding of ⁶⁴Cu ⊂ 7 to NTR1 overexpressing HT-29 tumor xenotransplants in nude mice. Regarding elimination, ⁶⁴Cu ⊂ 7 showed a substantial renal and reticuloendothelial accumulation. On the other hand, metabolization of the compound in vivo with a resulting metabolite—postulated to be the ⁶⁴Cu-cyclam-tetraarginine complex—also showed long retention in the circulating blood, preventing a better contrast of tumor imaging.     

Regulatory T cells in patients with Whipple's disease

Classical Whipple's disease (CWD) is caused by chronic infection with Tropheryma whipplei that seems to be associated with an underlying immune defect. The pathognomonic hallmark of CWD is a massive infiltration of the duodenal mucosa with T. whipplei-infected macrophages that disperse systemically to many other organ systems. An alleviated inflammatory reaction and the absence of T. whipplei-specific Th1 reactivity support persistence and systemic spread of the pathogen. In this article, we hypothesized that regulatory T cells (T(reg)) are involved in immunomodulation in CWD, and we asked for the distribution, activation, and regulatory capacity of T(reg) in CWD patients. Whereas in the lamina propria of CWD patients before treatment numbers of T(reg) were increased, percentages in the peripheral blood were similar in CWD patients and healthy controls. However, peripheral T(reg) of CWD patients were more activated than those of controls. Elevated secretion of IL-10 and TGF-β in the duodenal mucosa of CWD patients indicated locally enhanced T(reg) activity. Enhanced CD95 expression on peripheral memory CD4(+) T cells combined with reduced expression of IFN-γ and IL-17A upon polyclonal stimulation by CD4(+) cells from untreated CWD patients further hinted to T(reg) activity-related exhaustion of effector CD4(+) T cells. In conclusion, increased numbers of T(reg) can be detected within the duodenal mucosa in untreated CWD, where huge numbers of T. whipplei-infected macrophages are present. Thus, T(reg) might contribute to the chronic infection and systemic spread of T. whipplei in CWD but in contrast prevent mucosal barrier defect by reducing local inflammation.     

Does sex make a difference? Genetic diversity and spatial genetic structure in two co-occurring species of Gagea (Liliaceae) with contrasting reproductive strategies

Gagea lutea and G. spathacea are spring geophytes naturally co-occurring in woodlands, characterised by contrasting reproductive strategies probably caused by divergent ploidy levels. The hexaploid G. lutea relies on vegetative reproduction by subterranean bulbils in young stages but completely switches to sexual reproduction once a certain bulb size is attained. The nonaploid G. spathacea seems to be sterile and reproduces only vegetatively; the plants continue to form bulbils even in the rare event of flowering. This study used AFLP genotyping to investigate the consequences of these reproductive strategies for genetic diversity. For 150 and 100 samples from three Western Pomeranian populations of G. lutea and G. spathacea, respectively, AFLP fingerprints were analysed for three different spatial scales, the patch, the transect, and the region. Applying a threshold for genotypic identity of <0.05 simple matching distance, 22?C30 genets were detected in the three G. lutea populations, with all genets confined to single populations. Clonal genets consisted of 2?C9 samples and extended over up to 28?m, but never occupied the whole length of a transect; 67?C75% of all patches had different genets. Genetic distances between genets within populations were similar to those recorded between populations. Genotyping of G. spathacea revealed a single clonal genet for all three populations sampled within a distance of 30?km. The absent genetic diversity confirms the suspected sexual sterility. Gagea spathacea seems to be one of the few non-apomictic, fully clonal vascular plants able to occupy a significant range solely by dispersal of vegetative diaspores.     

Centuries-Old Viable Fruit of Sacred Lotus Nelumbo nucifera Gaertn var. China Antique

During the Sino-Japanese conflict of the 1920s, Japanese botanist Ichiro Ohga was presented single-seeded fruit of Nelumbo nucifera var. China Antique collected by a local farmer from a dry lakebed in Northeast China (then, “Manchuria”). Ohga studied the fruit and published his findings. Years later, we tested the germination of Nelumbo fruit from the same locality. The oldest seed sprouted, having a germination time of ~3 days, was radiocarbon dated to be ~1300 years old. These cold- and drought-tolerant seeds exhibited shoot-before-root emergence and a primary green plumule capable of “dim-light” photosynthesis. Such traits and the notable long-term viability of the fruit spurred the interest of Ray Ming, University of Illinois that has now led to the sequencing of the Nelumbo genome. Analyses of this genome may provide insight into the biochemistry of Nelumbo on wax-biosynthesis genes, and application of aging-related thermostable proteins to the extension of seed-life and improvement of food quality of economic crops. Here, we review the history of these long-lived Nelumbo fruit, and their occurrence, discovery, collection, propagation, and methods of seedling care. The robust impermeable wax- and suberin-covered pericarp is a major factor contributing to their remarkable longevity. New findings are presented on the modern and 459- and 464-year-old pericarp anatomy, impermeability to water, and whole fruit and pericarp mechanical properties, and comparison of the mode of fruit weight-gain during imbibition and germination time relative to fruit maturity.     

A common layer of interoperability for biomedical ontologies based on OWL EL

MOTIVATION: Ontologies are essential in biomedical research due to their ability to semantically integrate content from different scientific databases and resources. Their application improves capabilities for querying and mining biological knowledge. An increasing number of ontologies is being developed for this purpose, and considerable effort is invested into formally defining them in order to represent their semantics explicitly. However, current biomedical ontologies do not facilitate data integration and interoperability yet, since reasoning over these ontologies is very complex and cannot be performed efficiently or is even impossible. We propose the use of less expressive subsets of ontology representation languages to enable efficient reasoning and achieve the goal of genuine interoperability between ontologies. RESULTS: We present and evaluate EL Vira, a framework that transforms OWL ontologies into the OWL EL subset, thereby enabling the use of tractable reasoning. We illustrate which OWL constructs and inferences are kept and lost following the conversion and demonstrate the performance gain of reasoning indicated by the significant reduction of processing time. We applied EL Vira to the open biomedical ontologies and provide a repository of ontologies resulting from this conversion. EL Vira creates a common layer of ontological interoperability that, for the first time, enables the creation of software solutions that can employ biomedical ontologies to perform inferences and answer complex queries to support scientific analyses. Availability and implementation: The EL Vira software is available from http://el-vira.googlecode.com and converted OBO ontologies and their mappings are available from http://bioonto.gen.cam.ac.uk/el-ont.     

Analysis of the free energy in a stochastic RNA secondary structure model

There are two custom ways for predicting RNA secondary structures: minimizing the free energy of a conformation according to a thermodynamic model and maximizing the probability of a folding according to a stochastic model. In most cases, stochastic grammars are used for the latter alternative applying the maximum likelihood principle for determining a grammar's probabilities. In this paper, building on such a stochastic model, we will analyze the expected minimum free energy of an RNA molecule according to Turner's energy rules. Even if the parameters of our grammar are chosen with respect to structural properties of native molecules only (and therefore, independent of molecules' free energy), we prove formulae for the expected minimum free energy and the corresponding variance as functions of the molecule's size which perfectly fit the native behavior of free energies. This gives proof for a high quality of our stochastic model making it a handy tool for further investigations. In fact, the stochastic model for RNA secondary structures presented in this work has, for example, been used as the basis of a new algorithm for the (nonuniform) generation of random RNA secondary structures.     

Intrauterine growth retarded progeny of pregnant sows fed high protein:low carbohydrate diet is related to metabolic energy deficit

High and low protein diets fed to pregnant adolescent sows led to intrauterine growth retardation (IUGR). To explore underlying mechanisms, sow plasma metabolite and hormone concentrations were analyzed during different pregnancy stages and correlated with litter weight (LW) at birth, sow body weight and back fat thickness. Sows were fed diets with low (6.5%, LP), adequate (12.1%, AP), and high (30%, HP) protein levels, made isoenergetic by adjusted carbohydrate content. At -5, 24, 66, and 108 days post coitum (dpc) fasted blood was collected. At 92 dpc, diurnal metabolic profiles were determined. Fasted serum urea and plasma glucagon were higher due to the HP diet. High density lipoprotein cholesterol (HDLC), %HDLC and cortisol were reduced in HP compared with AP sows. Lowest concentrations were observed for serum urea and protein, plasma insulin-like growth factor-I, low density lipoprotein cholesterol, and progesterone in LP compared with AP and HP sows. Fasted plasma glucose, insulin and leptin concentrations were unchanged. Diurnal metabolic profiles showed lower glucose in HP sows whereas non-esterified fatty acids (NEFA) concentrations were higher in HP compared with AP and LP sows. In HP and LP sows, urea concentrations were 300% and 60% of AP sows, respectively. Plasma total cholesterol was higher in LP than in AP and HP sows. In AP sows, LW correlated positively with insulin and insulin/glucose and negatively with glucagon/insulin at 66 dpc, whereas in HP sows LW associated positively with NEFA. In conclusion, IUGR in sows fed high protein:low carbohydrate diet was probably due to glucose and energy deficit whereas in sows with low protein:high carbohydrate diet it was possibly a response to a deficit of indispensable amino acids which impaired lipoprotein metabolism and favored maternal lipid disposal.     

Serine-71 phosphorylation of rac1 modulates downstream signaling

The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.     

Induced Tauopathy in a Novel 3D-Culture Model Mediates Neurodegenerative Processes: A Real-Time Study on Biochips

Tauopathies including Alzheimer’s disease represent one of the major health problems of aging population worldwide. Therefore, a better understanding of tau-dependent pathologies and consequently, tau-related intervention strategies is highly demanded. In recent years, several tau-focused therapies have been proposed with the aim to stop disease progression. However, to develop efficient active pharmaceutical ingredients for the broad treatment of Alzheimer’s disease patients, further improvements are necessary for understanding the detailed neurodegenerative processes as well as the mechanism and side effects of potential active pharmaceutical ingredients (API) in the neuronal system. In this context, there is a lack of suitable complex in vitro cell culture models recapitulating major aspects of taupathological degenerative processes in sufficient time and reproducible manner.Herewith, we describe a novel 3D SH-SY5Y cell-based, tauopathy model that shows advanced characteristics of matured neurons in comparison to monolayer cultures without the need of artificial differentiation promoting agents. Moreover, the recombinant expression of a novel highly pathologic fourfold mutated human tau variant lead to a fast and emphasized degeneration of neuritic processes. The neurodegenerative effects could be analyzed in real time and with high sensitivity using our unique microcavity array-based impedance spectroscopy measurement system. We were able to quantify a time- and concentration-dependent relative impedance decrease when Alzheimer’s disease-like tau pathology was induced in the neuronal 3D cell culture model. In combination with the collected optical information, the degenerative processes within each 3D-culture could be monitored and analyzed. More strikingly, tau-specific regenerative effects caused by tau-focused active pharmaceutical ingredients could be quantitatively monitored by impedance spectroscopy.Bringing together our novel complex 3D cell culture taupathology model and our microcavity array-based impedimetric measurement system, we provide a powerful tool for the label-free investigation of tau-related pathology processes as well as the high content analysis of potential active pharmaceutical ingredient candidates.     

Personalized medicine using DNA biomarkers: a review

Biomarkers are of increasing importance for personalized medicine, with applications including diagnosis, prognosis, and selection of targeted therapies. Their use is extremely diverse, ranging from pharmacodynamics to treatment monitoring. Following a concise review of terminology, we provide examples and current applications of three broad categories of biomarkers-DNA biomarkers, DNA tumor biomarkers, and other general biomarkers. We outline clinical trial phases for identifying and validating diagnostic and prognostic biomarkers. Predictive biomarkers, more generally termed companion diagnostic tests predict treatment response in terms of efficacy and/or safety. We consider suitability of clinical trial designs for predictive biomarkers, including a detailed discussion of validation study designs, with emphasis on interpretation of study results. We specifically discuss the interpretability of treatment effects if a large set of DNA biomarker profiles is available and the number of therapies is identical to the number of different profiles.