CRISPR/Cas9 gene editing demonstrates metabolic importance of GPR55 in the modulation of GIP release and pancreatic beta cell function.

G-protein coupled receptor-55 (GPR55), an endocannabinoid receptor, is a novel anti-diabetic target. This study aimed to assess the metabolic functionality of GPR55 ligands using CRISPR/Cas9 gene editing to determine their regulatory role in beta cell function and incretin-secreting enteroendocrine cells. A clonal Gpr55 knockout beta cell line was generated by CRISPR/Cas9 gene editing to investigate insulin secretion and Gpr55 signalling. Acute effects of GPR55 agonists were investigated in high fat fed (HFD) diabetic HsdOla:TO (Swiss TO) mice. Atypical and endogenous endocannabinoid ligands (10-7-10-4M) stimulated insulin secretion (p < 0.05-0.001) in rodent (BRIN-BD11) and human (1.1B4) beta cells, with 2-2.7-fold (p < 0.001) increase demonstrated in BRIN-BD11 cells (10-4M). The insulinotropic effect of Abn-CBD (42 %), AM251 (30 %) and PEA (53 %) were impaired (p < 0.05) in Gpr55 knockout BRIN-BD11 cells, with the secretory effect of O-1602 completely abolished (p < 0.001). Gpr55 ablation abolished the release of intracellular Ca2+ upon treatment with O-1602, Abn-CBD and PEA. Upregulation of insulin mRNA by Abn-CBD and AM251 (1.7-3-fold; p < 0.01) was greatly diminished (p < 0.001) in Gpr55 null cells. Orally administered Abn-CBD and AM251 (0.1 µmol/kgBW) improved GIP (p < 0.05-p < 0.01), GLP-1 (p < 0.05-p < 0.001), glucose tolerance (p < 0.001) and circulating insulin (p < 0.05-p < 0.001) in HFD diabetic mice. Abn-CBD in combination therapy with DPP-IV inhibitor (Sitagliptin) resulted in greater improvement in glucose tolerance (p < 0.05) and insulin release (p < 0.05). Antagonism of Gpr55 in-vivo attenuated the glucoregulatory effects of Abn-CBD (p < 0.05). Conclusively, GPR55 agonists enhance insulin, GIP and GLP-1 release, thereby promoting GPR55 agonist monotherapy and combinational therapy as a novel approach for the treatment of type-2-diabetes.

Gene editing in the context of an increasingly complex genome.

The reporting of the first draft of the human genome in 2000 brought with it much hope for the future in what was felt as a paradigm shift toward improved health outcomes. Indeed, we have now mapped the majority of variation across human populations with landmark projects such as 1000 Genomes; in cancer, we have catalogued mutations across the primary carcinomas; whilst, for other diseases, we have identified the genetic variants with strongest association. Despite this, we are still awaiting the genetic revolution in healthcare to materialise and translate itself into the health benefits for which we had hoped. A major problem we face relates to our underestimation of the complexity of the genome, and that of biological mechanisms, generally. Fixation on DNA sequence alone and a 'rigid' mode of thinking about the genome has meant that the folding and structure of the DNA molecule -and how these relate to regulation- have been underappreciated. Projects like ENCODE have additionally taught us that regulation at the level of RNA is just as important as that at the spatiotemporal level of chromatin.In this review, we chart the course of the major advances in the biomedical sciences in the era pre- and post the release of the first draft sequence of the human genome, taking a focus on technology and how its development has influenced these. We additionally focus on gene editing via CRISPR/Cas9 as a key technique, in particular its use in the context of complex biological mechanisms. Our aim is to shift the mode of thinking about the genome to that which encompasses a greater appreciation of the folding of the DNA molecule, DNA- RNA/protein interactions, and how these regulate expression and elaborate disease mechanisms.Through the composition of our work, we recognise that technological improvement is conducive to a greater understanding of biological processes and life within the cell. We believe we now have the technology at our disposal that permits a better understanding of disease mechanisms, achievable through integrative data analyses. Finally, only with greater understanding of disease mechanisms can techniques such as gene editing be faithfully conducted.

Progesterone profiles in postpartum Holstein dairy cows as an aid in the study of retained fetal membranes, pyometra and anestrus.

This study was carried out to investigate the utility of enzymeimmunoassay (EIA) - derived progesterone profiles in the investigation of postpartum reproductive abnormalities and the effect of their use on reproductive performance in Holstein cows. Whole milk samples, collected twice weekly from parturition until confirmation of pregnancy or removal from the herd, were assayed for progesterone (P4) concentration using a commercially available microtitre plate EIA. The sensitivity, specificity, precision and accuracy were satisfactory to distinguish cyclic changes of P4 reflecting ovarian activity. The P4 profiles indicated that retained fetal membranes and anestrus were associated with increased mean intervals to first ovulation and first estrus (P < 0.05). The first luteal phase of cows with pyometra was prolonged compared with that of normal herdmates (P < 0.05). Anestrus cows had an increased number of ovulations before first service and before pregnancy (3.9 and 5.9) in comparison with normal herdmates (2.1 and 3.5; P < 0.05). Calving to pregnancy interval was increased above normal (74.8) for pyometra (125.0) and anestrus (152.7) (P < 0.05). Pregnancy rate for anestrus cows (82%) was lower than for normal cows (100%) and more anestrous cows were culled (27%) than normal cows (5%) (P < 0.5). The P4 profiles indicated that the major problem in the herd studied, anestrus (32% incidence rate), was most likely due to the failure to observe estrus rather than acyclicity.

Epidemiologic evidence for early onset of mental disorders and higher risk of drug abuse in young adults.

Data from the National Institute of Mental Health (NIMH) Epidemiologic Catchment Area Program, an epidemiologic survey of five communities, showed that four major disorders commonly begin in late adolescence or young adulthood. The median age at onset for anxiety disorders is 15 years; for major depressive episode, 24 years; for drug abuse or dependence, 19 years; and for alcohol abuse or dependence, 21 years. Findings also suggest that for respondents 18-30 years old, having a major depressive episode or anxiety disorder doubles the risk for later drug abuse or dependence.