A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest.

Multipotent neural crest (NC) progenitors generate an astonishing array of derivatives, including neuronal, skeletal components and pigment cells (chromatophores), but the molecular mechanisms allowing balanced selection of each fate remain unknown. In zebrafish, melanocytes, iridophores and xanthophores, the three chromatophore lineages, are thought to share progenitors and so lend themselves to investigating the complex gene regulatory networks (GRNs) underlying fate segregation of NC progenitors. Although the core GRN governing melanocyte specification has been previously established, those guiding iridophore and xanthophore development remain elusive. Here we focus on the iridophore GRN, where mutant phenotypes identify the transcription factors Sox10, Tfec and Mitfa and the receptor tyrosine kinase, Ltk, as key players. Here we present expression data, as well as loss and gain of function results, guiding the derivation of an initial iridophore specification GRN. Moreover, we use an iterative process of mathematical modelling, supplemented with a Monte Carlo screening algorithm suited to the qualitative nature of the experimental data, to allow for rigorous predictive exploration of the GRN dynamics. Predictions were experimentally evaluated and testable hypotheses were derived to construct an improved version of the GRN, which we showed produced outputs consistent with experimentally observed gene expression dynamics. Our study reveals multiple important regulatory features, notably a sox10-dependent positive feedback loop between tfec and ltk driving iridophore specification; the molecular basis of sox10 maintenance throughout iridophore development; and the cooperation between sox10 and tfec in driving expression of pnp4a, a key differentiation gene. We also assess a candidate repressor of mitfa, a melanocyte-specific target of sox10. Surprisingly, our data challenge the reported role of Foxd3, an established mitfa repressor, in iridophore regulation. Our study builds upon our previous systems biology approach, by incorporating physiologically-relevant parameter values and rigorous evaluation of parameter values within a qualitative data framework, to establish for the first time the core GRN guiding specification of the iridophore lineage

Binocular summation improves performance to defocus-induced blur

PURPOSE. To assess whether there are any advantages of binocular over monocular vision under blur conditions.\ud \ud METHODS. We measured the effect of defocus, induced by positive lenses, on the pattern reversal Visual Evoked Potential (VEP) and on visual acuity (VA). Monocular (dominant eye) and binocular VEPs were recorded from thirteen volunteers (average age: 28±5 years, average spherical equivalent: -0.25±0.73 D) for defocus up to 2.00 D using positive powered lenses. VEPs were elicited using reversing 10 arcmin checks at a rate of 4 reversals/second. The stimulus subtended a circular field of 7 degrees with 100% contrast and mean luminance 30 cd/m2. VA was measured under the same conditions using ETDRS charts. All measurements were performed at 1m viewing distance with best spectacle sphero-cylindrical correction and natural pupils. \ud \ud RESULTS. With binocular stimulation, amplitudes and implicit times of the P100 component of the VEPs were greater and shorter, respectively, in all cases than for monocular stimulation. Mean binocular enhancement ratio in the P100 amplitude was 2.1 in-focus, increasing linearly with defocus to be 3.1 at +2.00 D defocus. Mean peak latency was 2.9 ms shorter in-focus with binocular than for monocular stimulation, with the difference increasing with defocus to 8.8 ms at +2.00 D. As for the VEP amplitude, VA was always better with binocular than with monocular vision, with the difference being greater for higher retinal blur.\ud \ud CONCLUSIONS. Both subjective and electrophysiological results show that binocular vision ameliorates the effect of defocus. The increased binocular facilitation observed with retinal blur may be due to the activation of a larger population of neurons at close-to-threshold detection under binocular stimulation

A porous graphitized carbon LC-ESI/MS method for the quantitation of metronidazole and fluconazole in breast milk and human plasma

Information on drug transfer into the breast milk is essential to protect the infant from undesirable adverse effects of maternal consumption of drugs and to allow effective pharmacological treatment of breastfeeding mothers. Metronidazole and fluconazole are two drugs frequently used in nursing women to treat various infections, thus questioning infant's safety due to drug exposure through breast milk. In this article a porous graphitized carbon LC/ESI-MS assay was developed for the quantitation of metronidazole and fluconazole in breast milk and human plasma. The assay was based on the use of 150 μL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the LC/ESI-MS system. All analytes and the internal standard, ropinirole, were separated by using a porous graphitized carbon analytical column (150 × 2.1 mm i.d., particle size 5 μm) with isocratic elution. The mobile phase consists of 55% acetonitrile in water acidified with 0.1% concentrated formic acid and pumped at a flow rate of 0.25 mL min−1. The assay was linear over a concentration range of 0.1 to 15 μg mL−1 for all analytes in both biological samples. Intermediate precision was found to be <8.4% over the tested concentration ranges. A run time of <5 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application for the analysis of metronidazole and fluconazole in both breast milk and human plasma and it can be used to support a wide range of clinical studies. © 2018 Elsevier B.V

Quantification of three beta-lactam antibiotics in breast milk and human plasma by hydrophilic interaction liquid chromatography/positive-ion electrospray ionization mass spectrometry

The use of cephalosporins during breast feeding raises several issues, including the risk of drug exposure through breast milk for the infant. In this paper, a hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed for the quantitation of cefuroxime, cefoxitin, and cefazolin in breast milk and human plasma. The assay was based on the use of small sample size, 25 μL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the HILIC/ESI-MS system. All analytes and the internal standard, alfuzosin, were separated by using a ZIC®-HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 µm, 200 Å) with isocratic elution. The mobile phase was composed of a 6% 12.5 mM ammonium acetate water solution in acetonitrile and pumped at a flow rate of 0.25 mL min-1. The assay was linear over a concentration range of 0.2 to 5 µg mL-1 and 0.4 to 20 µg mL-1 for all the analytes in breast milk and in human plasma, respectively. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 12 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application of HILIC in the analysis of antibiotics in breast milk and human plasma and it can be used to support a wide range of clinical studies. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd