Evolution of genome sequencing techniques

The quality and the speed for genome sequencing has advanced at the same time that technology boundaries are stretched. This advancement has been divided so far in three generations. The first-generation methods enabled sequencing of clonal DNA populations. The second-generation massively increased throughput by parallelizing many reactions while the third-generation methods allow direct sequencing of single DNA molecules. The first techniques to sequence DNA were not developed until the mid-1970s, when two distinct sequencing methods were developed almost simultaneously, one by Alan Maxam and Walter Gilbert, and the other one by Frederick Sanger. The first one is a chemical method to cleave DNA at specific points and the second one uses ddNTPs, which synthesizes a copy from the DNA chain template. Nevertheless, both methods generate fragments of varying lengths that are further electrophoresed. Moreover, it is important to say that until the 1990s, the sequencing of DNA was relatively expensive and it was seen as a long process. Besides, using radiolabeled nucleotides also compounded the problem through safety concerns and prevented the automation. Some advancements within the first generation include the replacement of radioactive labels by fluorescent labeled ddNTPs and cycle sequencing with thermostable DNA polymerase, which allows automation and signal amplification, making the process cheaper, safer and faster. Another method is Pyrosequencing, which is based on the “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation. By the end of the last millennia, parallelization of this method started the Next Generation Sequencing (NGS) with 454 as the first of many methods that can process multiple samples, calling it the 2º generation sequencing. Here electrophoresis was completely eliminated. One of the methods that is sometimes used is SOLiD, based on sequencing by ligation of fluorescently dye-labeled di-base probes which competes to ligate to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. The widely used Solexa/Illumina method uses modified dNTPs containing so called “reversible terminators” which blocks further polymerization. The terminator also contains a fluorescent label, which can be detected by a camera. Now, the previous step towards the third generation was in charge of Ion Torrent, who developed a technique that is based in a method of “sequencing-by-synthesis”. Its main feature is the detection of hydrogen ions that are released during base incorporation. Likewise, the third generation takes into account nanotechnology advancements for the processing of unique DNA molecules to a real time synthesis sequencing system like PacBio; and finally, the NANOPORE, projected since 1995, also uses Nano-sensors forming channels obtained from bacteria that conducts the sample to a sensor that allows the detection of each nucleotide residue in the DNA strand. The advancements in terms of technology that we have nowadays have been so quick, that it makes wonder: ¿How do we imagine the next generation?

Galanin interacts with Neuropeptide Y Y1 receptor in the dentate gyrus of the hippocampus through GALR2/NPYY1R heterodimers

We have shown Galanin(GAL) and Neuropeptide Y Y1(NPYY1) interactions at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers in the amygdala. The aim of this work was to analyze GAL/NPYY1R interactions in the Dentate Gyrus(DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay(PLA). Rats(n=6) were sacrificed 15 minutes or 5 hours after icv injections of GAL(3nmol) and DG sections were incubated with NPYY1R agonist [I125]-[Leu31,Pro34]PYY(25 pM) or NPYY1R-33PdATP specific probe, for autoradiography and in situ hybridization respectively. Autoradiograms were analyzed using NIH image analysis system and Student’s unpaired t-test was used. For PLA, DG sections were incubated with anti-GALR2 Rabbit(1:100) and anti-NPYY1R Goat(1:200). PLA signals were detected with PLA PLUS or MINUS probes for rabbit or goat/mouse antibodies. PLA signals were visualized by using a confocal microscope Leica TCS-SL confocal microscope(Leica). We observed that GAL significant increased the NPYY1R agonist [I125]-[Leu31,Pro34]PYY binding in the DG by 20% (p<0,05) and the NPYY1R mRNA expression in the granular layer of DG by 31% (p<0,001). Moreover, PLA-positive red clusters were found specifically in the polymorphic layer and subgranular zone of the DG. No PLA clusters were observed neither in the molecular layer of the DG nor in the corpus callosum, an area that seems to lack of GALR2 receptor. These results demonstrate a novel mechanism of interaction between GAL and NPY1R in the DG at receptor level, probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. Study supported by Junta de Andalucia CVI6476.

Galanin and galanin fragment 1-15 modulate antidepressant responses by targeting 5-HT1AR-GALR heteroreceptor complexes of the ascending midbrain serotonin pathways.

Mood disorders, including depression and anxiety, are among the most prevalent mental illnesses with high socioeconomic impact. Although the underlying mechanisms have not yet been clearly defined in the last decade the importance of the role of neuropeptides, including Galanin (GAL), and/or their receptors in the treatment of stress-related mood disorders is becoming increasingly apparent. GAL is involved in mood regulation, including depression-related and anxiety-like behaviors. Activation of GALR1 and GALR3 receptors results in a depression like behavior while stimulation of GALR2 receptor leads to anti-depressant-like effects. Moreover, GAL modulates 5-HT1A receptors (5-HT1AR), a key receptor in depression at autoreceptor and postsynaptic level in the brain. This interaction can in part be due to the existence of GALR1-5-HT1AR heteroreceptor complexes in discrete brain regions [1]. Not only GAL but also the N-terminal fragments like GAL(1-15) are active in the Central Nervous System [2, 3]. Recently, we described that GAL(1-15) induces strong depression-related and anxiogenic-like effects in rats, and these effects were significantly stronger than the ones induced by GAL [4]. The GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe (DR), areas rich in GAL(1-15) binding sites [5] were involved in these effects [4, 6] and demonstrated also in cellular models. In the present study, we have analyzed the ability of GAL(1-15) to modulate 5-HT1AR located at postjunctional sites and at the soma-dendritic level in rats. We have analyzed the effect of GAL(1-15) on the 5-HT1AR-mediated response in a behavioral test of depression and the involvement of the GALR2 in these effects. GAL(1-15) enhanced the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT in the forced swimming test [7]. These effects were stronger than the ones induced by GAL. The mechanism of this action involved interactions at the receptor level in the plasma membrane with changes also at the transcriptional level. Thus, GAL(1-15) affected the binding characteristics as well as the mRNA level of 5-HT1AR in the dorsal hippocampus and DR. GALR2 was involved in these effects, since the specific GALR2 antagonist M871 blocked GAL(1-15) mediated actions at the behavioral and receptor level [7]. Furthermore, the results on the proximity ligation assay (PLA) in this work suggest the existence of GALR1-GALR2-5-HT1AR heteroreceptor complexes since positive PLA were obtained for both GALR1-5-HT1AR and GALR2-5-HT1AR complexes in the DR and hippocampus. Moreover the studies on RN33B cells, where GALR1, GALR2 and 5-HT1AR exist [4], also showed PLA-positive clusters indicating the existence of GALR1-5-HT1AR and GALR2-5-HT1AR complexes in these cells [7]. In conclusion, our results indicate that GAL(1–15) enhances the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT probably acting on GALR1-GALR2-5-HT1AR heteroreceptor located at postjunctional sites and at the soma-dendritic level. The development of new drugs specifically targeting these heteroreceptor complexes may offer a novel strategy for treatment of depression. This work has been supported by Junta de Andalucia CVI646 1. Borroto-Escuela, D.O., et al., Galanin receptor-1 modulates 5-hydroxtryptamine-1A signaling via heterodimerization. Biochem Biophys Res Commun, 2010. 393(4): p. 767-72. 2. Hedlund, P.B. and K. Fuxe, Galanin and 5-HT1A receptor interactions as an integrative mechanism in 5-HT neurotransmission in the brain. Ann N Y Acad Sci, 1996. 780: p. 193-212. 3. Diaz-Cabiale, Z., et al., Neurochemical modulation of central cardiovascular control: the integrative role of galanin. EXS, 2010. 102: p. 113-31. 4. Millon, C., et al., A role for galanin N-terminal fragment (1-15) in anxiety- and depression-related behaviors in rats. Int J Neuropsychopharmacol, 2015. 18(3). 5. Hedlund, P.B., N. Yanaihara, and K. Fuxe, Evidence for specific N-terminal galanin fragment binding sites in the rat brain. Eur J Pharmacol, 1992. 224(2-3): p. 203-5. 6. Borroto-Escuela, D.O., et al., Preferential activation by galanin 1-15 fragment of the GalR1 protomer of a GalR1-GalR2 heteroreceptor complex. Biochem Biophys Res Commun, 2014. 452(3): p. 347-53. 7. Millon, C., et al., Galanin (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT: involvement of the raphe-hippocampal 5-HT neuron system. Brain Struct Funct, 2016.