A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos

Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)^1Hri, to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates some previous observations on Hes1 expression and suggests new, hitherto unrecognised expression domains including expression in the definitive endoderm at early somite stages before gut tube closure and thus preceding organogenesis. This mouse line will be a valuable tool for studies addressing the role of Hes1 in a number of different research areas including organ specification, development and regeneration.     

Schizosaccharomyces pombe encodes a mutated AP endonuclease 1

Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis.     

A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions

Macroautophagy is a catabolic process that maintains cellular homeostasis and protects cells against various external stresses including starvation. Except for the identification of the Akt-mTORC1 pathway as a major negative regulator, little is known about signaling networks that control macroautophagy under optimal growth conditions. Therefore, we screened a human kinome siRNA library for siRNAs that increase the number of autophagosomes in normally growing MCF-7 human breast carcinoma cells, and identified 10 kinases as regulators of constitutive macroautophagy. Further analysis of these kinases with respect to the autophagic flux, kinase signaling and endolysosomal function identified WNK2 as a positive regulator of autophagosome maturation and nine others as macroautophagy inhibitors. The depletion of MK2, PACSIN1, DAPK2, CDKL3 and SCYL1 functioned upstream of Akt-mTORC1 pathway, whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation of macroautophagy and open new possibilities for its pharmacological manipulation.     

Development and evaluation of a liquid chromatography tandem mass spectrometry method for simultaneous determination of salivary melatonin, cortisol and testosterone

Circadian disruption can have several possible health consequences, but is not well studied. In order to measure circadian disruption, in relation to shift or night work, we developed a simple and sensitive method for the simultaneous determination of melatonin, cortisol and testosterone in human saliva. We used liquid-liquid extraction (LLE) followed by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) recorded in positive ion mode. Saliva samples were collected by spitting directly into tubes and 250 μL were used for analysis. The limits of detection were 4.1 pmol/L, 0.27 nmol/L and 10.8 pmol/L for melatonin, cortisol, and testosterone, respectively. The developed method was sensitive enough to measure circadian rhythms of all 3 hormones in a pilot study among four healthy volunteers. It can therefor be used to study the impact of night work and working in artificial light on the workers circadian rhythms. To our knowledge this is the first LC-ESI-MS/MS method for simultaneous determination of salivary melatonin, cortisol and testosterone.     

Quantification of antidepressants and antipsychotics in human serum by precipitation and ultra high pressure liquid chromatography-tandem mass spectrometry

The present article describes the quantification of mirtazapine, O-desmethylvenlafaxine, quetiapine, venlafaxine, and ziprasidone (group 1), and amitriptyline, citalopram, clomipramine, clozapine, desmethylclomipramine, desipramine, imipramine, and nortriptyline (group 2) in human serum for therapeutic drug monitoring. The method was developed to replace old techniques which applied solid phase extraction and ultra-violet detection. The old methods had reached their limit of capacity regarding the number of samples and co-medicated drugs interfering with the detection. Serum samples were precipitated with zinc sulphate and methanol containing a stable isotope labelled analog for each analyte. Quantitative analysis was performed by ultra high pressure liquid chromatography combined with a tandem mass spectrometer using a Zorbax SB-C8 column (2.0 × 50 mm; 1.8 μm) with a mobile phase consisting of 0.1% formic acid in water and methanol, respectively. The total run time of the chromatography was 4 min. Precision and trueness varied from 2.6% to 14.9% and 87.6% to 103.5%, respectively. At the lower limit of quantification, precision was up to 17.9% and trueness varied from 89.5% to 111.5%. A five point standard curve covering the clinically relevant ranges with a power function fit was applied for calibration. Ion suppression from matrix effects and internal standards were thoroughly investigated and are discussed. Process efficiency rates varied from 42% to 99%. The method has shortened the response time, reduced interference from other drugs, avoided acetonitrile usage, and reduced the amount of serum needed for analysis 50-fold.     

Bipolar disorder risk alleles in adult ADHD patients

Attention-deficit/hyperactivity disorder (ADHD) has an estimated prevalence of 3-5% in adults. Genome-wide association (GWA) studies have not been performed in adults with ADHD and studies in children have so far been inconclusive, possibly because of the small sample sizes. Larger GWA studies have been performed on bipolar disorder (BD) and BD symptoms, and several potential risk genes have been reported. ADHD and BD share many clinical features and comorbidity between these two disorders is common. We therefore wanted to examine whether the reported BD genetic variants in CACNA1C, ANK3, MYO5B, TSPAN8 and ZNF804A loci are associated with ADHD or with scores on the Mood Disorder Questionnaire (MDQ), a commonly used screening instrument for bipolar spectrum disorders. We studied 561 adult Norwegian ADHD patients and 711 controls from the general population. No significant associations or trends were found between any of the single nucleotide polymorphisms (SNPs) studied and ADHD [odds ratios (ORs) ≤ 1.05]. However, a weak association was found between rs1344706 in ZNF804A (OR = 1.25; P = 0.05) and MDQ. In conclusion, it seems unlikely that these six SNPs with strong evidence of association in BD GWA studies are shared risk variants between ADHD and BD.     

Preclinical safety evaluations supporting pediatric drug development with biopharmaceuticals: strategy,challenges, current practices

Evaluation of pharmaceutical agents in children is now conducted earlier in the drug development process. An important consideration for this pediatric use is how to assess and support its safety. This article is a collaborative effort of industry toxicologists to review strategies, challenges, and current practice regarding preclinical safety evaluations supporting pediatric drug development with biopharmaceuticals. Biopharmaceuticals include a diverse group of molecular, cell‐based or gene therapeutics derived from biological sources or complex biotechnological processes. The principles of preclinical support of pediatric drug development for biopharmaceuticals are similar to those for small molecule pharmaceuticals and in general follow the same regulatory guidances outlined by the Food and Drug Administration and European Medicines Agency. However, many biopharmaceuticals are also inherently different, with limited species specificity or immunogenic potential which may impact the approach taken. This article discusses several key areas to aid in the support of pediatric clinical use, study design considerations for juvenile toxicity studies when they are needed, and current practices to support pediatric drug development based on surveys specifically targeting biopharmaceutical development. Birth Defects Res (Part B) 92:359–380, 2011. © 2011 Wiley‐Liss, Inc.