Metabolism and toxicological detection of the designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) in rat urine using gas chromatography-mass spectrometry

The amphetamine-derived designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) is an upcoming substance on the illicit drug market. In the current study, the identification of its metabolites in rat urine and their toxicological detection in the authors' systematic toxicological analysis (STA) procedure were examined. DOI is extensively metabolized by O-demethylation and beside small amounts of parent compound it was found to be excreted mainly in form of metabolites. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug users' dose of DOI by detection of the two O-demethyl metabolite isomers in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOI in human urine.     

Transplacental Zika virus transmission in ex vivo perfused human placentas

A Zika virus (ZIKV) infection during pregnancy can result in severe birth defects such as microcephaly. To date, it is incompletely understood how ZIKV can cross the human placenta. Furthermore, results from studies in pregnant mice and non-human primates are conflicting regarding the role of cross-reactive dengue virus (DENV) antibodies on transplacental ZIKV transmission. Elucidating how ZIKV can cross the placenta and which risk factors contribute to this is important for risk assessment and for potential intervention strategies for transplacental ZIKV transmission. In this study we use an ex vivo human placental perfusion model to study transplacental ZIKV transmission and the effect that cross-reactive DENV antibodies have on this transmission. By using this model, we demonstrate that DENV antibodies significantly increase ZIKV uptake in perfused human placentas and that this increased uptake is neonatal Fc-receptor-dependent. Furthermore, we show that cross-reactive DENV antibodies enhance ZIKV infection in term human placental explants and in primary fetal macrophages but not in primary trophoblasts. Our data supports the hypothesis that presence of cross-reactive DENV antibodies could be an important risk factor for transplacental ZIKV transmission. Furthermore, we demonstrate that the ex vivo placental perfusion model is a relevant and animal friendly model to study transplacental pathogen transmission.     

Single-molecule fluorescence observed with mercury lamp illumination

We demonstrate that it is possible to observe single fluorescent molecules using a standard fluorescence microscope with mercury lamp excitation and an inexpensive cooled charge-coupled device (CCD) camera. With this equipment, we have been able to observe single molecules of tetramethyl-rhodamine, rhodamine 6G, fluorescein isothiocyanate and green fluorescent protein. Immobilized molecules were observed both in air and in aqueous solution.     

Computational and experimental investigation of flow and particle settling in a roller bottle bioreactor

It is shown that cell settling is a key factor affecting the performance of roller bottle bioreactors. The two-dimensional cross-sectional flow at the center of a roller bottle is simulated using a finite difference method, and the settling behavior of cells is simulated using particle dynamics algorithms and validated experimentally using fluorescent particles. The settling behavior of particles in the roller bottle flow is studied using both steady and time dependent rotation rates. Under steady flow conditions the flow is divided into two regions: one where the particles settle to the wall and one where the particles remain suspended indefinitely. The relative size of these two regions depends on the ratio of the settling velocity to the rotation rate of the bottle. For unsteady flows generated by periodic changes of the bottle rotation direction, the settling of cells is accelerated significantly, leading to complete deposition in just a few turns of the bottle.     

Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination

Site-specific mutagenesis provides the ability to alter DNA with precision so that the function of any given gene can be more fully understood. Several methods of in vitro mutagenesis are time-consuming and imprecise, requiring the subcloning and sequencing of products. Here we describe a rapid, high fidelity method of in situ mutagenesis in bacteriophage lambda using transplacement. Using this method, mutations are transferred from oligonucleotides to target phages using a plasmid interface. A small (50 bp) homology region bearing a centred point mutation is generated from oligonucleotides and subcloned into a transplacement plasmid bearing positive and negative phage selectable markers. Following a positive/negative selection cycle of integrative recombination and excision, the point mutation is transferred precisely from plasmid to phage in a subset ( approximately 25-50%) of recombinants. As the fidelity of both oligonucleotide synthesis and phage-plasmid recombination is great, this approach is extremely reliable. Using transplacement, point mutations can be accurately deposited within large phage clones and we demonstrate the utility of this technique in the construction of gene targeting vectors in bacteriophages.     

Integrated data acquisition system for medical device testing and physiology research in compliance with good laboratory practices

In seeking approval from the US Food and Drug Administration (FDA) for clinical trial evaluation of an experimental medical device, a sponsor is required to submit experimental findings and support documentation to demonstrate device safety and efficacy that are in compliance with Good Laboratory Practices (GLP). The objective of this project was to develop an integrated data acquisition (DAQ) system and documentation strategy for monitoring and recording physiological data when testing medical devices in accordance with GLP guidelines mandated by the FDA. Data aquisition systems were developed as stand-alone instrumentation racks containing transducer amplifiers and signal processors, analog-to-digital converters for data storage, visual display and graphical user-interfaces, power conditioners, and test measurement devices. Engineering standard operating procedures (SOP) were developed to provide a written step-by-step process for calibrating, validating, and certifying each individual instrumentation unit and the integrated DAQ system. Engineering staff received GLP and SOP training and then completed the calibration, validation, and certification process for the individual instrumentation components and integrated DAQ system. Eight integrated DAQ systems have been successfully developed that were inspected by regulatory affairs consultants and determined to meet GLP guidelines. Two of these DAQ systems were used to support 40 of the pre-clinical animal studies evaluating the AbiCor artificial heart (ABIOMED, Danvers, MA). Based in part on these pre-clinical animal data, the AbioCor clinical trials began in July 2001. The process of developing integrated DAQ systems, SOP, and the validation and certification methods used to ensure GLP compliance are presented in this article.     

Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins

Membrane proteins destined for the vacuolar or lysosomal lumen are typically ubiquitinated, the ubiquitin serving as a targeting signal for the multivesicular body pathway. The RING-domain ubiquitin ligase Tul1 is an integral membrane protein that modifies the yeast vacuolar enzyme carboxypeptidase S (Cps1), the polyphosphatase Ppn1/Phm5 and other proteins containing exposed hydrophilic residues within their transmembrane domains (TMDs). Here we show that Bsd2 provides an alternative ubiquitination mechanism for Cps1, Phm5 and other proteins. Bsd2 is a three-TMD protein with a PPXY motif that binds the HECT domain ubiquitin ligase Rsp5. It can thus act as a specific adaptor linking Rsp5 to its substrates. Like Tul1, the Bsd2 system recognises polar TMDs. Bsd2 also controls the vacuolar targeting of a manganese transporter and a mutant plasma membrane ATPase, and together with the ER retrieval receptor Rer1, it protects cells from stress. We suggest that Bsd2 has a wide role in the quality control of membrane proteins. Bsd2 is the yeast homologue of human NEDD4 binding protein N4WBP5, which may therefore have similar functions.     

Thymosin beta 4 and its N-terminal tetrapeptide, AcSDKP, inhibit proliferation, and induce dysplastic, non-apoptotic nuclei and degranulation of mast cells

Thymosin beta4 (Tbeta4), a 5 kDa polypeptide, is a member of the beta-thymosin family. It acts as the principal intracellular G-actin sequestering peptide and exhibits extracellular functions in angiogenesis and wound healing. The N-terminus of Tbeta4 contains a bioactive tetrapeptide, acSDKP, a negative regulator of hematopoietic stem-cell proliferation. Here, we show that both peptides inhibit mast-cell proliferation over the concentration range of 10(-6) to 10(-17) M with the maximum effect of both at 10(-14) M. Both Tbeta4 and acSDKP caused dysplastic mast-cell nuclei that were confirmed by DAPI fluorescent staining. Flow-cytometric analysis of ploidy revealed that the dysplastic nuclei were not multinucleated, but fragmented nuclei in G2 growth arrest. We could further demonstrate that 10(-8) or 10(-14) M Tbeta4 or acSDKP induce mast-cell degranulation. A concentration of 10(-8) M Tbeta4 or acSDKP caused 57 or 89% degranulation, respectively. A number of tryptic fragments of Tbeta4 were assayed beside intact Tbeta4 and the tetrapeptide, and found to be inactive.     

Sleep assessment in a population-based study of chronic fatigue syndrome

Background

Chronic fatigue syndrome (CFS) is a disabling condition that affects approximately 800,000 adult Americans. The pathophysiology remains unknown and there are no diagnostic markers or characteristic physical signs or laboratory abnormalities. Most CFS patients complain of unrefreshing sleep and many of the postulated etiologies of CFS affect sleep. Conversely, many sleep disorders present similarly to CFS. Few studies characterizing sleep in unselected CFS subjects have been published and none have been performed in cases identified from population-based studies.

Methods

The study included 339 subjects (mean age 45.8 years, 77% female, 94.1% white) identified through telephone screen in a previously described population-based study of CFS in Wichita, Kansas. They completed questionnaires to assess fatigue and wellness and 2 self-administered sleep questionnaires. Scores for five of the six sleep factors (insomnia/hypersomnia, non-restorative sleep, excessive daytime somnolence, sleep apnea, and restlessness) in the Centre for Sleep and Chronobiology's Sleep Assessment Questionnaire^© (SAQ^©) were dichotomized based on threshold. The Epworth Sleepiness Scale score was used as a continuous variable.

Results

81.4% of subjects had an abnormality in at least one SAQ^© sleep factor. Subjects with sleep factor abnormalities had significantly lower wellness scores but statistically unchanged fatigue severity scores compared to those without SAQ^© abnormality. CFS subjects had significantly increased risk of abnormal scores in the non-restorative (adjusted odds ratio [OR] = 28.1; 95% confidence interval [CI]= 7.4–107.0) and restlessness (OR = 16.0; 95% CI = 4.2–61.6) SAQ^© factors compared to non-fatigued, but not for factors of sleep apnea or excessive daytime somnolence. This is consistent with studies finding that, while fatigued, CFS subjects are not sleepy. A strong correlation (0.78) of Epworth score was found only for the excessive daytime somnolence factor.

Conclusions

SAQ^© factors describe sleep abnormalities associated with CFS and provide more information than the Epworth score. Validation of these promising results will require formal polysomnographic sleep studies.