Why Are We Still Cloning Melatonin Receptors? A Commentary.

Cloning may seem to be a view from the past. The time before software, computers and AI were invented. It seems to us worth discussing these points in view of our favorite target: the melatoninergic system. In a few stances, it might be important to point out that even in the new era of dry science, there is still a need to experiment and to prove at the bench that our in silico assertions are right. Most of the living animals express to some extend the melatonin receptors. Some of these animal genomes were completely or partially sequenced, and it is tempting to extract from this huge information the sequence(s) of our favorite genes (MLT receptors). Then, why bother cloning, as opposed to simply built the gene and express it in a host cell? Because the genetic boundaries of the expressed sequence(s) are not 100% sure. Because the melatonin receptor gene(s) comprise a first exon 25,000 base pair far from the second one and the limits between this Ex1 and In1-as between In1 and Ex2-are subject to changes that might have a huge impact on the biochemical properties of the receptor, once expressed. Because a receptor is a biochemical entity with characteristics that are important for the functioning of this particular pathway, and more generally, for the functioning of life.

A Randomized Controlled Clinical Trial in Healthy Older Adults to Determine Efficacy of Glycine and N-Acetylcysteine Supplementation on Glutathione Redox Status and Oxidative Damage.

Glycine and cysteine are non-essential amino acids that are required to generate glutathione, an intracellular tripeptide that neutralizes reactive oxygen species and prevents tissue damage. During aging glutathione demand is thought to increase, but whether additional dietary intake of glycine and cysteine contributes towards the generation of glutathione in healthy older adults is not well understood. We investigated supplementation with glycine and n-acetylcysteine (GlyNAC) at three different daily doses for 2 weeks (low dose: 2.4 g, medium dose: 4.8 g, or high dose: 7.2 g/day, 1:1 ratio) in a randomized, controlled clinical trial in 114 healthy volunteers. Despite representing a cohort of healthy older adults (age mean = 65 years), we found significantly higher baseline levels of markers of oxidative stress, including that of malondialdehyde (MDA, 0.158 vs. 0.136 µmol/L, p < 0.0001), total cysteine (Cysteine-T, 314.8 vs. 276 µM, p < 0.0001), oxidized glutathione (GSSG, 174.5 vs. 132.3 µmol/L, p < 0.0001), and a lower ratio of reduced to oxidized glutathione (GSH-F:GSSG) (11.78 vs. 15.26, p = 0.0018) compared to a young reference group (age mean = 31.7 years, n = 20). GlyNAC supplementation was safe and well tolerated by the subjects, but did not increase levels of GSH-F:GSSG (end of study, placebo = 12.49 vs. 7.2 g = 12.65, p-value = 0.739) or that of total glutathione (GSH-T) (end of study, placebo = 903.5 vs. 7.2 g = 959.6 mg/L, p-value = 0.278), the primary endpoint of the study. Post-hoc analyses revealed that a subset of subjects characterized by high oxidative stress (above the median for MDA) and low baseline GSH-T status (below the median), who received the medium and high doses of GlyNAC, presented increased glutathione generation (end of study, placebo = 819.7 vs. 4.8g/7.2 g = 905.4 mg/L, p-value = 0.016). In summary GlyNAC supplementation is safe, well tolerated, and may increase glutathione levels in older adults with high glutathione demand. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT05041179, NCT05041179.

Generation of a zebrafish SWATH-MS spectral library to quantify 10,000 proteins.

Sequential window acquisition of all theoretical mass spectra (SWATH-MS) requires a spectral library to extract quantitative measurements from the mass spectrometry data acquired in data-independent acquisition mode (DIA). Large combined spectral libraries containing SWATH assays have been generated for humans and several other organisms, but so far no publicly available library exists for measuring the proteome of zebrafish, a rapidly emerging model system in biomedical research. Here, we present a large zebrafish SWATH spectral library to measure the abundance of 104,185 proteotypic peptides from 10,405 proteins. The library includes proteins expressed in 9 different zebrafish tissues (brain, eye, heart, intestine, liver, muscle, ovary, spleen, and testis) and provides an important new resource to quantify 40% of the protein-coding zebrafish genes. We employ this resource to quantify the proteome across brain, muscle, and liver and characterize divergent expression levels of paralogous proteins in different tissues. Data are available via ProteomeXchange (PXD010876, PXD010869) and SWATHAtlas (PASS01237).

Gene expression profiling during hibernation in the European hamster.

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body's clocks at the beginning of the active period.

Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2.

For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT1 and MT2) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT1, MT2, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT1 receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (Phodopus sungorus) expresses a non-functional MT2. We observed that unlike this hamster, the European hamster (Cricetus cricetus) does not have a stop codon in the MT2 sequence. Thus, we undertook the tedious task of cloning the MT2 receptor. We partially succeeded, sequencing the complete exon 2 and a fragment of exon 1 (from putative amino acids 12 to 38 and 77 to 323), after several years of efforts. In order to show that the protein parts we cloned were capable to sustain some binding capacities, we designed a chimeric MT2 receptor using a consensus sequence to replace the unknown amino acids, based on other small rodent MT2 sequences. This chimeric construct could bind melatonin in the nanomolar range. This work is meant to be the basis for attempts from other laboratories of the community to determine the complete natural sequence of the European hamster MT2 receptor. The present work is the first to show that, among the hamsters, if the Siberian is a natural knockout for MT2, the European one is not.

Library construction for ancient genomics: single strand or double strand?

A novel method of library construction that takes advantage of a single-stranded DNA ligase has been recently described and used to generate high-resolution genomes from ancient DNA samples. While this method is effective and appears to recover a greater fraction of endogenous ancient material, there has been no direct comparison of results from different library construction methods on a diversity of ancient DNA samples. In addition, the single-stranded method is limited by high cost and lengthy preparation time and is restricted to the Illumina sequencing platform. Here we present in-depth comparisons of the different available library construction methods for DNA purified from 16 ancient and modern faunal and human remains, covering a range of different taphonomic and climatic conditions. We further present a DNA purification method for ancient samples that permits the concentration of a large volume of dissolved extract with minimal manipulation and methodological improvements to the single-stranded method to render it more economical and versatile, in particular to expand its use to both the Illumina and the Ion Torrent sequencing platforms. We show that the single-stranded library construction method improves the relative recovery of endogenous to exogenous DNA for most, but not all, of our ancient extracts.

Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery.

AIM: To develop an in vitro model based on neural stem cells derived from transgenic animals, to be used in the study of pathological mechanisms of Alzheimer's disease and for testing new molecules. METHODS: Neural stem cells (NSCs) were isolated from the subventricular zone of Wild type (Wt) and Tg2576 mice. Primary and secondary neurosphere generation was studied, analysing population doubling and the cell yield per animal. Secondary neurospheres were dissociated and plated on MCM Gel Cultrex 2D and after 6 d in vitro (DIVs) in mitogen withdrawal conditions, spontaneous differentiation was studied using specific neural markers (MAP2 and TuJ-1 for neurons, GFAP for astroglial cells and CNPase for oligodendrocytes). Gene expression pathways were analysed in secondary neurospheres, using the QIAGEN PCR array for neurogenesis, comparing the Tg2576 derived cell expression with the Wt cells. Proteins encoded by the altered genes were clustered using STRING web software. RESULTS: As revealed by 6E10 positive staining, all Tg2576 derived cells retain the expression of the human transgenic Amyloid Precursor Protein. Tg2576 derived primary neurospheres show a decrease in population doubling. Morphological analysis of differentiated NSCs reveals a decrease in MAP2- and an increase in GFAP-positive cells in Tg2576 derived cells. Analysing the branching of TuJ-1 positive cells, a clear decrease in neurite number and length is observed in Tg2576 cells. The gene expression neurogenesis pathway revealed 11 altered genes in Tg2576 NSCs compared to Wt. CONCLUSION: Tg2576 NSCs represent an appropriate AD in vitro model resembling some cellular alterations observed in vivo, both as stem and differentiated cells.