Genetic services survey-experience of people with rare diseases and their families accessing genetic services in the Irish Republic.

Irish Health Service objectives state that patients with rare diseases should have timely access to genomic diagnostics with appropriate pre and post-test counselling. However, waiting times for clinical genetics outpatient appointments, during the study period, were up to two years as staffing levels remain low. A targeted public online survey was conducted in January 2022 to capture the experiences of Rare Disease families trying to access genetic testing and clinical genetic clinics in the Irish Republic. Irish patients experience significant waiting times to access clinical genetic services and self-report anxiety and stress, related to delayed access to diagnosis, clarity around recurrence risk and follow-up management. This negatively impacts personal decisions around family planning, education and employment and has a significant impact on family members seeking clarity on their own risk. Mainstream genetic testing activity is significant. Families report concern over the competency of health care professionals arranging and delivering genetic results and delays in accessing clinical genetics expertise to take them through the clinical implications. Timely access to clinical genetics expertise is important to ensure families with rare diseases have an appropriate understanding of the medical and reproductive implications of a genetic diagnosis and access to relevant care pathways. A national framework to develop competency in genomic literacy for health-care professionals including a national genetic test directory may be beneficial. Clinical genetics teams require ongoing support and investment to ensure the delivery of a safe and effective service for Irish families with rare diseases.

Ancient population genomics and the study of evolution.

Recently, the study of ancient DNA (aDNA) has been greatly enhanced by the development of second-generation DNA sequencing technologies and targeted enrichment strategies. These developments have allowed the recovery of several complete ancient genomes, a result that would have been considered virtually impossible only a decade ago. Prior to these developments, aDNA research was largely focused on the recovery of short DNA sequences and their use in the study of phylogenetic relationships, molecular rates, species identification and population structure. However, it is now possible to sequence a large number of modern and ancient complete genomes from a single species and thereby study the genomic patterns of evolutionary change over time. Such a study would herald the beginnings of ancient population genomics and its use in the study of evolution. Species that are amenable to such large-scale studies warrant increased research effort. We report here progress on a population genomic study of the Adélie penguin (Pygoscelis adeliae). This species is ideally suited to ancient population genomic research because both modern and ancient samples are abundant in the permafrost conditions of Antarctica. This species will enable us to directly address many of the fundamental questions in ecology and evolution.

Insight into the medicinal chemistry of the endocannabinoid hydrolase inhibitors.

Endocannabinoid hydrolases are nowadays increasingly considered as potential therapeutic targets for treating several pathological states. So far, numerous classes of endocannabinoid hydrolase inhibitors have been described. We herein review the medicinal chemistry of these inhibitors with a particular emphasis on the basis of their design, chemical structure, structure-activity relationships, and inhibition mechanisms.

Ancient DNA recovers the origins of Maori feather cloaks.

Feather cloaks ("kakahu"), particularly those adorned with kiwi feathers, are treasured items or "taonga" to the Maori people of "Aotearoa"/New Zealand. They are considered iconic expression of Maori culture. Despite their status, much of our knowledge of the materials used to construct cloaks, the provenance of cloaks, and the origins of cloak making itself, has been lost. We used ancient DNA methods to recover mitochondrial DNA sequences from 849 feather samples taken from 109 cloaks. We show that almost all (>99%) of the cloaks were constructed using feathers from North Island brown kiwi. Molecular sexing of nuclear DNA recovered from 92 feather cloak samples also revealed that the sex ratio of birds deviated from a ratio of 1:1 observed in reference populations. Additionally, we constructed a database of 185 mitochondrial control region DNA sequences of kiwi feathers comprising samples collected from 26 North Island locations together with data available from the literature. Genetic subdivision (G(ST)), nucleotide subdivision (N(ST)) and Spatial Analysis of Molecular Variants (SAMOVA) analyses revealed high levels of genetic structuring in North Island brown kiwi. Together with sequence data from previously studied ancient and modern kiwi samples, we were able to determine the geographic provenance of 847 cloak feathers from 108 cloaks. A surprising proportion (15%) of cloaks were found to contain feathers from different geographic locations, providing evidence of kiwi trading among Maori tribes or organized hunting trips into other tribal areas. Our data also suggest that the east of the North Island of New Zealand was the most prolific of all kiwi cloak making areas, with over 50% of all cloaks analyzed originating from this region. Similar molecular approaches have the potential to discover a wealth of lost information from artifacts of endemic cultures worldwide.

A review on the monoacylglycerol lipase: at the interface between fat and endocannabinoid signalling.

Together with anandamide, 2-arachidonoylglycerol (2-AG) constitutes one of the main representatives of a family of endogenous lipids known as endocannabinoids. These act by binding to CB(1) and CB(2) cannabinoid receptors, the molecular target of the psychoactive compound Delta(9)-THC, both in the periphery and in the central nervous system, where they behave as retrograde messengers to modulate synaptic transmission. These last years, evidence has accumulated to demonstrate the lead role played by the monoacylglycerol lipase (MAGL) in the regulation of 2-arachidonoylglycerol (2-AG) levels. Considering the numerous physiological functions played by this endocannabinoid, MAGL is now considered a promising target for therapeutics, as inhibitors of this enzyme could reveal useful for the treatment of pain and inflammatory disorders, as well as in cancer research, among others. Here we review the milestones that punctuated MAGL history, from its discovery to recent advances in the field of inhibitors development. An emphasis is given on the recent elucidation of the tridimensional structure of the enzyme, which could offer new opportunities for rational drug design.

Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability.

BACKGROUND AND AIMS: Obese and diabetic mice display enhanced intestinal permeability and metabolic endotoxaemia that participate in the occurrence of metabolic disorders. Our recent data support the idea that a selective increase of Bifidobacterium spp. reduces the impact of high-fat diet-induced metabolic endotoxaemia and inflammatory disorders. Here, we hypothesised that prebiotic modulation of gut microbiota lowers intestinal permeability, by a mechanism involving glucagon-like peptide-2 (GLP-2) thereby improving inflammation and metabolic disorders during obesity and diabetes. METHODS: Study 1: ob/ob mice (Ob-CT) were treated with either prebiotic (Ob-Pre) or non-prebiotic carbohydrates as control (Ob-Cell). Study 2: Ob-CT and Ob-Pre mice were treated with GLP-2 antagonist or saline. Study 3: Ob-CT mice were treated with a GLP-2 agonist or saline. We assessed changes in the gut microbiota, intestinal permeability, gut peptides, intestinal epithelial tight-junction proteins ZO-1 and occludin (qPCR and immunohistochemistry), hepatic and systemic inflammation. RESULTS: Prebiotic-treated mice exhibited a lower plasma lipopolysaccharide (LPS) and cytokines, and a decreased hepatic expression of inflammatory and oxidative stress markers. This decreased inflammatory tone was associated with a lower intestinal permeability and improved tight-junction integrity compared to controls. Prebiotic increased the endogenous intestinotrophic proglucagon-derived peptide (GLP-2) production whereas the GLP-2 antagonist abolished most of the prebiotic effects. Finally, pharmacological GLP-2 treatment decreased gut permeability, systemic and hepatic inflammatory phenotype associated with obesity to a similar extent as that observed following prebiotic-induced changes in gut microbiota. CONCLUSION: We found that a selective gut microbiota change controls and increases endogenous GLP-2 production, and consequently improves gut barrier functions by a GLP-2-dependent mechanism, contributing to the improvement of gut barrier functions during obesity and diabetes.

Ancient genetic variation in one of the world's rarest seabirds.

The Chatham Island Taiko (Tchaik, Pterodroma magentae) is one of the world's rarest seabirds. In the past there were millions of breeding pairs of Taiko and it was the most abundant burrowing petrel on Chatham Island. The present population consists of just 120-150 birds, including only 8-15 breeding pairs. Surprisingly high genetic variation was revealed by DNA sequencing of almost every known adult Taiko (N=90). Given the massive population decline, genetic variation may have been even larger in the past. Therefore, we investigated past genetic diversity by sequencing regions of the mitochondrial cytochrome b gene in 44 ancient Taiko bones. We identified a total of 12 haplotypes in Taiko. Eight haplotypes were revealed in the ancient DNA: four were unique to the bones and four corresponded to those found in the modern Taiko population. Surprisingly, despite the critically endangered status of the Taiko, no significant reduction in mitochondrial DNA haplotype diversity was observed between ancient samples (N=44) and modern adult Taiko (N=90). The modern population may have however lost four haplotypes present in the ancient populations.

Ancient DNA and conservation: lessons from the endangered kiwi of New Zealand.

Conservation genetics typically seeks to map the distribution of contemporary genetic variation across space, and to use the resulting genetic parameters to infer any likely short-term evolutionary consequences for rare and endangered species. Recent developments in the study of ancient DNA now enable the extension of genetic variation studies backwards in time and provide a context by which to interpret contemporary levels of genetic variation, in addition to any patterns of genetic change over time. Ancient DNA research can also help to determine past levels of genetic diversity, identify species' boundaries and reveal former ranges among morphologically cryptic taxa. Ancient DNA sequence data for the New Zealand kiwi (Apteryx spp.) are presented and we show that most ancient populations of rowi and tokoeka exhibited private mitochondrial DNA haplotypes. Moreover, we illustrate that the extinction of these populations has led to the loss of considerable genetic variation. We also use ancient DNA methodology to determine past species distributions for brown kiwi and great spotted kiwi whose bones are morphologically indistinguishable.

A MBP-FAAH fusion protein as a tool to produce human and rat fatty acid amide hydrolase: expression and pharmacological comparison.

Fatty acid amide hydrolase (FAAH), a membrane-anchored enzyme responsible for the termination of endocannabinoid signalling, is an attractive target for treating conditions such as pain and anxiety. Inhibitors of the enzyme, optimized using rodent FAAH, are known but their pharmacology and medicinal chemistry properties on the human FAAH are missing. Therefore recombinant human enzyme would represent a powerful tool to evaluate new drug candidates. However, the production of high amounts of enzyme is hampered by the known refractiveness of FAAH to overexpression. Here, we report the successful overexpression of rat and human FAAH as a fusion to the E. coli maltose-binding protein, retaining catalytic properties of native FAAH. Several known FAAH inhibitors were tested and differences in their potencies toward the human and rat FAAH were found, underscoring the importance of using a human FAAH in the development of inhibitors.

The effect of the palmitoylethanolamide analogue, palmitoylallylamide (L-29) on pain behaviour in rodent models of neuropathy.

BACKGROUND AND PURPOSE: Cannabinoids are associated with analgesia in acute and chronic pain states. A spectrum of central cannabinoid (CB(1)) receptor-mediated motor and psychotropic side effects limit their therapeutic potential. Here, we investigate the analgesic effect of the palmitoylethanolamide (PEA) analogue, palmitoylallylamide (L-29), which via inhibition of fatty acid amide hydrolase (FAAH) may potentiate endocannabinoids thereby avoiding psychotropic side effects. EXPERIMENTAL APPROACH: The in vivo analysis of the effect of L-29 on measures of pain behaviour in three rat models of neuropathic pain. KEY RESULTS: Systemically administered L-29 (10 mg kg(-1)) reduced hypersensitivity to mechanical and thermal stimuli in the partial sciatic nerve injury (PSNI) model of neuropathic pain; and mechanical hypersensitivity in a model of antiretroviral (ddC)-associated hypersensitivity and a model of varicella zoster virus (VZV)-associated hypersensitivity. The effects of L-29 were comparable to those of gabapentin (50 mg kg(-1)). The CB(1) receptor antagonist SR141716a (1 mg kg(-1)) and the CB(2) receptor antagonist SR144528 (1 mg kg(-1)) reduced the effect of L-29 on hypersensitivity in the PSNI and ddC models, but not in the VZV model. The peroxisome proliferator-activated receptor-alpha antagonist, MK-886 (1 mg kg(-1)), partially attenuated the effect of L-29 on hypersensitivity in the PSNI model. L-29 (10 mg kg(-1)) significantly attenuated thigmotactic behaviour in the open field arena without effect on locomotor activity. CONCLUSIONS AND IMPLICATIONS: L-29 produces analgesia in a range of neuropathic pain models. This presents L-29 as a novel analgesic compound that may target the endogenous cannabinoid system while avoiding undesirable side effects associated with direct cannabinoid receptor activation.

Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro.

BACKGROUND AND PURPOSE: Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2-arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated. EXPERIMENTAL APPROACH: Homogenates and cell lines were incubated with test compounds and, thereafter, with either [(3)H]-2-oleoylglycerol or [(3)H]-anandamide. Labelled reaction products were separated from substrate using chloroform: methanol extraction. KEY RESULTS: In cytosolic fractions from rat brain, URB602 and URB754 inhibited the hydrolysis of 2-oleoylglycerol with IC(50) values of 25 and 48 microM, respectively. Anandamide hydrolysis by brain membranes was not sensitive to URB754, but was inhibited by URB602 (IC(50) value 17 microM). Hydrolysis of 2-oleoylglycerol by human recombinant monoacylglycerol lipase was sensitive to URB602, but not URB754. The lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis was also observed for intact RBL2H3 basophilic leukaemia cells. C6 glioma expressed mRNA for monoacylglycerol lipase, and hydrolyzed 2-oleoylglycerol in a manner sensitive to inhibition by methyl arachidonoyl fluorophosphonate but not URB754 or URB597. MC3T3-E1 mouse osteoblastic cells, which did not express mRNA for monoacylglycerol lipase, hydrolyzed 2-oleoylglycerol in the presence of URB597, but the hydrolysis was less sensitive to methyl arachidonoyl fluorophosphonate than for C6 cells. CONCLUSIONS AND IMPLICATIONS: The data demonstrate that the compounds URB602 and URB754 do not behave as selective and/or potent inhibitors of monoacylglycerol lipase.

Microevolution and mega-icebergs in the Antarctic.

Microevolution is regarded as changes in the frequencies of genes in populations over time. Ancient DNA technology now provides an opportunity to demonstrate evolution over a geological time frame and to possibly identify the causal factors in any such evolutionary event. Using nine nuclear microsatellite DNA loci, we genotyped an ancient population of Adélie penguins (Pygoscelis adeliae) aged approximately 6,000 years B.P. Subfossil bones from this population were excavated by using an accurate stratigraphic method that allowed the identification of individuals even within the same layer. We compared the allele frequencies in the ancient population with those recorded from the modern population at the same site in Antarctica. We report significant changes in the frequencies of alleles between these two time points, hence demonstrating microevolutionary change. This study demonstrates a nuclear gene-frequency change over such a geological time frame. We discuss the possible causes of such a change, including the role of mutation, genetic drift, and the effects of gene mixing among different penguin populations. The latter is likely to be precipitated by mega-icebergs that act to promote migration among penguin colonies that typically show strong natal return.

Mutational bias in penguin microsatellite DNA.

Analysis of nucleotide sequence variation at a microsatellite DNA locus revealed extensive size homoplasy of alleles in Adélie penguins (Pygoscelis adeliae). Variation in the flanking regions at this locus allowed discrimination between mechanisms proposed for length changes in microsatellite DNA alleles. We further examined the structure of alleles for the same microsatellite DNA locus across 11 additional species of penguin (Spheniscidae) by mapping allele sequences onto an independent penguin phylogeny. Our analysis indicated that the repeat motifs appear to have evolved independently on several occasions. We observed sequence instability in the region bordering the repeat tract with a transversional bias predominating. We propose that this bias results from inaccurate DNA replication owing to the sequence context of this repeat tract. Because we show that regions flanking repeat sequences exhibit this mutational bias, this cautions against the use of such regions for phylogeny reconstruction.

Current knowledge on the antagonists and inverse agonists of cannabinoid receptors.

Ten years elapsed since the discovery by Sanofi of SR141716A the first selective CB(1) cannabinoid receptor antagonist. Shortly after, Sanofi also reported the synthesis of the first selective CB(2) cannabinoid receptor antagonist, SR144528. Since these two milestones in the cannabinoid field, many other compounds, more or less related to the Sanofi compounds, or based on a completely different scaffold appeared. Several of these compounds are currently involved in clinical trials for diseases such as obesity, nicotine and alcohol addictions, or allergies. Further, the cannabinoid receptors knock-out mice production strengthened the hypothesis of the existence of several other "cannabinoid" receptors for which the first antagonists begin to appear. The large amount of patents taken by many different pharmaceutical companies prove, if necessary, the great therapeutic potential expected for the cannabinoid receptors antagonists.

Is a large-scale DNA-based inventory of ancient life possible?

A complete DNA-based inventory of the Earth's present biota using large-scale high-throughput DNA sequencing of signature region(s) (DNA barcoding) is an ambitious proposal rivaling the Human Genome Project. We examine whether this approach will also enable us to assess the past diversity of the earth's biota. To test this, we sequenced the 5' terminus of the mitochondrial cytochrome c oxidase I (COI) gene of individuals belonging to a group of extinct ratite birds, the moa of New Zealand. Moa comprised a large number of taxa that radiated in isolation on this oceanic landmass. Using a phylogenetic approach based on a large data set including protein coding and 12S DNA sequences as well as morphology, we now have precise information about the number of moa species that once existed. We show that each of the moa species detected using this extensive data set has a unique COI barcode(s) and that they all show low levels of within-species COI variation. Consequently, we conclude that COI sequences accurately identify the species discovered using the larger data set. Hence, more generally, this study suggests that DNA barcoding might also help us detect other extinct animal species and that a large-scale inventory of ancient life is possible.

Nuclear DNA sequences detect species limits in ancient moa.

Ancient DNA studies have typically used multi-copy mitochondrial DNA sequences. This is largely because single-locus nuclear genes have been difficult to recover from sub-fossil material, restricting the scope of ancient DNA research. Here, we have isolated single-locus nuclear DNA markers to assign the sex of 115 extinct moa and, in combination with a mitochondrial DNA phylogeny, tested competing hypotheses about the specific status of moa taxa. Moa were large ratite birds that showed extreme size variation both within and among species. For some taxa, this large variation was hypothesized to represent sexual dimorphism, while for others it was argued to reflect the existence of different species. Our results show that moa were characterized by extreme reverse sexual dimorphism and as a result we have been able to clarify the number of moa species. For example, we show that the three recognized 'species' of Dinornis comprised only two monophyletic groups and that two of these 'species' comprised individuals of one sex only. This study also illustrates that single-locus nuclear DNA sequences can be consistently recovered from ancient material.

Polymorphism of N-stearoylethanolamine: differential scanning calorimetric, vibrational spectroscopic (FTIR), and crystallographic studies.

Based on a series of physicochemical properties (differential scanning calorimetry, powder X-ray crystallographic studies and Fourier-transform infra red spectroscopic analysis) determined for N-stearoylethanolamine (NSEA) (C18:0) at different temperatures, evidence has been given that this compound can exist in (at least) three polymorphic forms. Powder X-ray crystallography clearly demonstrates the presence of three distinct molecular packings at distinct temperatures while spectral changes in the vibrational spectra reveal that the geometry of the CH(2)z.sbnd;CO functional group of the molecule is affected during the polymorphic transitions. Rationalization of the thermal physicochemical behavior of NSEA in terms of molecular packing is also proposed. It supposes rearrangement of the hydrocarbon chains upon heating of the molecule.

C-Terminal gp40 peptide analogs inhibit feline immunodeficiency virus: cell fusion and virus spread.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1), gp160, is synthesized as a protein precursor that when proteolytically cleaved yields two subunits, gp120 and gp41. gp120 is the surface glycoprotein on HIV-1 responsible for binding to CD4, and gp41 is the transmembrane glycoprotein involved in the membrane fusion process. gp41 is divided into the N-terminal fusion peptide, the heptad repeat 1 (HR1) and HR2 regions, and the C-terminal transmembrane region, which are collectively responsible for virus fusion and entry into the cell. Synthetic peptides derived from the HR2 and HR1 regions of HIV-1(LAI) have been shown to prevent virus-cell fusion and infection in vitro. In phase II clinical trials in HIV patients, data revealed that T20 has antiviral efficacy and is well tolerated. Similar results were obtained in vitro with HIV-2 and simian immunodeficiency virus, supporting the conservation of the gp41 ectodomain among lentiviruses. Feline immunodeficiency virus (FIV) infection in the cat has been used as a model to develop potential antivirals for HIV. To determine if synthetic gp40 analogs capable of inhibiting FIV infection could be identified, 15 overlapping 35-amino-acid peptides derived from the C-terminal HR2 domain of FIV gp40 were synthesized. These peptides were tested for efficacy against FIV in a syncytium-forming assay with FIV-infected CrFK cells and HeLa cells expressing the FIV receptor CXCR4. Several peptides exhibited activity at the nanogram level. Antiviral activity was confirmed by suppression of reverse transcriptase in a FIV feline CD4(+)-T-cell (FCD4-E) acute-infection assay. These data demonstrate that synthetic peptides derived from the HR2 domain of the FIV gp41 protein are effective inhibitors of FIV infection.

Rates of evolution in ancient DNA from Adélie penguins.

Well-preserved subfossil bones of Adélie penguins, Pygoscelis adeliae, underlie existing and abandoned nesting colonies in Antarctica. These bones, dating back to more than 7000 years before the present, harbor some of the best-preserved ancient DNA yet discovered. From 96 radiocarbon-aged bones, we report large numbers of mitochondrial haplotypes, some of which appear to be extinct, given the 380 living birds sampled. We demonstrate DNA sequence evolution through time and estimate the rate of evolution of the hypervariable region I using a Markov chain Monte Carlo integration and a least-squares regression analysis. Our calculated rates of evolution are approximately two to seven times higher than previous indirect phylogenetic estimates.