Transcription pattern of reproduction relevant genes along the brain-pituitary-gonad axis of female, male and intersex thicklip grey mullets, Chelon labrosus, from a polluted harbor.

The reproductive cycle of teleost fishes is regulated by the brain-pituitary-gonad (BPG) axis. The transcription profile of genes involved in the reproduction signalling in the BPG-axis differs in females and males during the gametogenic cycle. Impacts of endocrine disrupting chemicals on these signalling pathways in fish are known, but the participation of the BPG-axis in the development of the intersex condition is not well understood. Intersex thicklip grey mullets (Chelon labrosus) have been identified in several estuaries from the SE Bay of Biscay, revealing the presence of feminizing contaminants in the area. In previous studies, transcription patterns of genes related with steroidogenesis and gamete growth have been shown to differ among female, male and intersex mullets. However, many components of the reproduction control have not been studied yet. The aim of this study was to assess the transcription levels of target BPG-axis genes in female, male and intersex mullets captured in the polluted harbour of Pasaia, during their gametogenic cycle. After histologically examining the gonads, the transcription levels of previously sequenced target genes were measured by qPCR: kiss2, gpr54 and gnrh1 in brain, fshß and lhß in pituitary and fshr and lhr in gonads. In both females and males, brain genes were most transcribed in early gametogenesis, proving their relevance in the onset of both oogenesis and spermatogenesis. Pituitary gonadotropins in females showed upregulation as oogenesis progressed, reaching the highest transcription levels at vitellogenic stage, while in males transcript levels were constant during spermatogenesis. Transcription levels of gonadotropin receptors showed different patterns in ovaries and testes, suggesting differing function in relation to gametogenesis and maturation. Intersex mullets showed transcription levels of brain target genes similar to those observed in females at cortical alveoli stage and to those in mid spermatogenic males. In intersex testes the transcription pattern of gonadotropin receptor fshr was downregulated in comparison to non-intersex testes.

Phage-displayed peptides targeting specific tissues and organs.

Phage display is a powerful and widely used technique to find novel peptide ligands. A massive amount of peptide sequences have been identified for all kinds of materials, and peptides that may have targeting capabilities towards specific cells and tissues have received special attention in biomedical sciences. As a result, it is increasingly harder to follow all the work that has been done, which sometimes leads to many promising ligands receiving little attention, together with the publication of false positives that have already been found. The aim of this review is to provide an updated and comprehensive list of phage-displayed peptides targeting different tissues and organs. The limitations of the technique are carefully analysed and the future perspectives envisaged.

Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET.

Quantification of the intracellular equilibrium dissociation constant of the interaction, Kd , is challenging due to the variability of the relative concentrations of the interacting proteins in the cell. Fluorescence lifetime imaging microscopy (FLIM) of the donor provides an accurate measurement of the molecular fraction of donor involved in FRET, but the fraction of bound acceptor is also needed to reliably estimate Kd . We present a method that exploits the spectroscopic properties of the widely used eGFP - mCherry FRET pair to rigorously determine the intracellular Kd based on imaging the fluorescence lifetime of only the donor (single-channel FLIM). We have assessed the effect of incomplete labelling and determined its range of application for different Kd using Monte Carlo simulations. We have demonstrated this method estimating the intracellular Kd for the homodimerisaton of the oncogenic protein 3-phosphoinositide-dependent kinase 1 (PDK1) in different cell lines and conditions, revealing a competitive mechanism for its regulation. The measured intracellular Kd was validated against in-vitro data. This method provides an accurate and generic tool to quantify protein interactions in situ.