Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry

Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, and N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction by xenobiotics. Ratios of different caffeine metabolites in urine or other body fluids are frequently used to characterise the individual/actual activity of these enzymes. The common analytical method involves extensive sample preparation, followed by HPLC-UV. The presence of numerous other UV-absorbing chemicals in body fluids affects the sensitivity and selectivity of this method. We have developed an HPLC-electrospray-MS-MS method for the determination of 11 caffeine metabolites and two internal standards after a simple, extractionless preparation. Blank urine, obtained after 5 days on a methylxanthine-free diet, contained small amounts of some caffeine metabolites, but no other components producing any confounding signals. Eleven metabolites and internal standards were recovered at 90 to 110% after addition to the blank urine (0.1 to 2.5 micro M in the final sample involving a 20-fold dilution of urine) in the 0.1-2.5 micro M concentration range. Other metabolites, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU), were detected with similar recovery and precision, but required higher concentrations (3 to 30 micro M). AFMU was completely converted into AAMU by a short alkalisation of urine. The method was explored in six healthy individuals after consuming coffee (4 mg caffeine per kg body mass). These experiments demonstrated the simplicity, high sensitivity and selectivity of the method under conditions used for phenotyping.     

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies

 A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid. Received: 9 August 1999 / Accepted: 28 September 1999     

Investigation of Biophysical Mechanisms in Gold Nanoparticle Mediated Laser Manipulation of Cells Using a Multimodal Holographic and Fluorescence Imaging Setup

Laser based cell manipulation has proven to be a versatile tool in biomedical applications. In this context, combining weakly focused laser pulses and nanostructures, e.g. gold nanoparticles, promises to be useful for high throughput cell manipulation, such as transfection and photothermal therapy. Interactions between laser pulses and gold nanoparticles are well understood. However, it is still necessary to study cell behavior in gold nanoparticle mediated laser manipulation. While parameters like cell viability or perforation efficiency are commonly addressed, the influence of the manipulation process on other essential cell parameters is not sufficiently investigated yet. Thus, we set out to study four relevant cell properties: cell volume and area, ion exchange and cytoskeleton structure after gold nanoparticle based laser manipulation. For this, we designed a multimodal imaging and manipulation setup. 200 nm gold nanoparticles were attached unspecifically to canine cells and irradiated by weakly focused 850 ps laser pulses. Volume and area change in the first minute post laser manipulation was monitored using digital holography. Calcium imaging and cells expressing a marker for filamentous actin (F-actin) served to analyze the ion exchange and the cytoskeleton, respectively. High radiant exposures led to cells exhibiting a tendency to shrink in volume and area, possibly due to outflow of cytoplasm. An intracellular raise in calcium was observed and accompanied by an intercellular calcium wave. This multimodal approach enabled for the first time a comprehensive analysis of the cell behavior in gold nanoparticle mediated cell manipulation. Additionally, this work can pave the way for a better understanding and the evaluation of new applications in the context of cell transfection or photothermal therapy.     

Stable individual differences in separation calls during early development in cats and mice

Background

The development of ethologically meaningful test paradigms in young animals is an essential step in the study of the ontogeny of animal personality. Here we explore the possibility to integrate offspring separation (distress) calls into the study of consistent individual differences in behaviour in two species of mammals, the domestic cat (Felis silvestris catus) and the mound-building mouse (Mus spicilegus). Such vocal responses in young mammals are a potentially useful test option as they represent an important element of mother-offspring communication with strong implications for offspring survival. In addition, the neural control of vocalisation is closely associated with emotional state.

Results

We found marked similarities in the pattern of individual responses of the young of both species to separation from their mother and littermates. In the domestic cat as well as in the mound-building mouse, individual differences in the frequency of calls and to a lesser extent in locomotor activity were repeatable across age, indicating the existence of personality types. Such consistencies across age were also apparent when only considering relative individual differences among litter siblings. In both species, however, individual patterns of vocalisation and locomotor activity were unrelated. This suggests that these two forms of behavioural responses to isolation represent different domains of personality, presumably based on different underlying neurophysiological mechanisms.

Conclusions

Brief separation experiments in young mammals, and particularly the measurement of separation calls, provide a promising approach to study the ontogeny of personality traits. Future long-term studies are needed to investigate the association of these traits with biologically meaningful and potentially repeatable elements of behaviour during later life.

     

Inverse Agonistic Action of 3-Iodothyronamine at the Human Trace Amine-Associated Receptor 5

ObjectiveApplication of 3-iodothyronamine (3-T₁AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T₁AM. However, 3-T₁AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T₁AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct G_s-, G_i/o-, G_12/13-, G_q/11- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T₁AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the G_q/11 pathway but show differences in the basal activity in G_s and MAP kinase signaling. In contrast to mTaar5, 3-T₁AM application at hTAAR5 resulted in significant reduction in basal IP₃ formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T₁AM, exhibiting inhibitory effects on IP₃ formation and MAP kinase signaling pathways, but does not mediate G_s signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T₁AM-mediated effects may differ between rodents and humans.     

Genome-wide Association Study and Meta-Analysis Identify ISL1 as Genome-wide Significant Susceptibility Gene for Bladder Exstrophy

The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the uro-rectal malformation spectrum, and is thought to result from aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). To identify susceptibility loci for CBE, we performed a genome-wide association study (GWAS) of 110 CBE patients and 1,177 controls of European origin. Here, an association was found with a region of approximately 220kb on chromosome 5q11.1. This region harbors the ISL1 (ISL LIM homeobox 1) gene. Multiple markers in this region showed evidence for association with CBE, including 84 markers with genome-wide significance. We then performed a meta-analysis using data from a previous GWAS by our group of 98 CBE patients and 526 controls of European origin. This meta-analysis also implicated the 5q11.1 locus in CBE risk. A total of 138 markers at this locus reached genome-wide significance in the meta-analysis, and the most significant marker (rs9291768) achieved a P value of 2.13 × 10⁻¹². No other locus in the meta-analysis achieved genome-wide significance. We then performed murine expression analyses to follow up this finding. Here, Isl1 expression was detected in the genital region within the critical time frame for human CBE development. Genital regions with Isl1 expression included the peri-cloacal mesenchyme and the urorectal septum. The present study identified the first genome-wide significant locus for CBE at chromosomal region 5q11.1, and provides strong evidence for the hypothesis that ISL1 is the responsible candidate gene in this region.     

Systematic Cell-Based Phenotyping of Missense Alleles Empowers Rare Variant Association Studies: A Case for LDLR and Myocardial Infarction

A fundamental challenge to contemporary genetics is to distinguish rare missense alleles that disrupt protein functions from the majority of alleles neutral on protein activities. High-throughput experimental tools to securely discriminate between disruptive and non-disruptive missense alleles are currently missing. Here we establish a scalable cell-based strategy to profile the biological effects and likely disease relevance of rare missense variants in vitro. We apply this strategy to systematically characterize missense alleles in the low-density lipoprotein receptor (LDLR) gene identified through exome sequencing of 3,235 individuals and exome-chip profiling of 39,186 individuals. Our strategy reliably identifies disruptive missense alleles, and disruptive-allele carriers have higher plasma LDL-cholesterol (LDL-C). Importantly, considering experimental data refined the risk of rare LDLR allele carriers from 4.5- to 25.3-fold for high LDL-C, and from 2.1- to 20-fold for early-onset myocardial infarction. Our study generates proof-of-concept that systematic functional variant profiling may empower rare variant-association studies by orders of magnitude.     

Only-Child Status in Relation to Perceived Stress and Studying-Related Life Satisfaction among University Students in China: A Comparison with International Students

Objectives

University students in general face multiple challenges, which may affect their levels of perceived stress and life satisfaction. Chinese students currently face specific strains due to the One-Child Policy (OCP). The aim of this study was to assess (1) whether the levels of perceived stress and studying-related life satisfaction are associated with only-child (OC) status after controlling for demographic and socio-economic characteristics and (2) whether these associations differ between Chinese and international students.

Materials and Methods

A cross-sectional health survey based on a self-administrated standardised questionnaire was conducted among 1,843 (1,543 Chinese, 300 international) students at two Chinese universities in 2010–2011. Cohen’s Perceived Stress Scale (PSS-14) and Stock and Kraemer’s Studying-related Life Satisfaction Scale were used to measure perceived stress and studying-related life satisfaction respectively. Multivariable logistic regression analyses were used to examine the associations of OC status with perceived stress and studying-related life satisfaction by sex for Chinese students and international students separately.

Results

The Chinese non-only-children (NOCs) were more likely to come from small cities. Multivariable regression models indicate that the Chinese NOCs were more stressed than OCs (OR = 1.39, 1.11–1.74) with a stronger association in men (OR = 1.48, 1.08–2.02) than women (OR = 1.26, 0.89–1.77). NOCs were also more dissatisfied than their OC fellows in the Chinese subsample (OR = 1.37, 1.09–1.73). Among international students, no associations between OC status and perceived stress or studying-related life satisfaction were found.

Conclusions

To promote equality between OCs and NOCs at Chinese universities, the causes of more stress and less studying-related life satisfaction among NOCs compared to OCs need further exploration.     

Genome-Based Identification of Active Prophage Regions by Next Generation Sequencing in Bacillus licheniformis DSM13

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).