A mixed-method survey to understand the role of dog welfare organisations in Ireland, including reported challenges and potential solutions.

BACKGROUND: This novel study forms part of a larger research programme seeking an improved understanding of aspects of the owned dog population in Ireland. Dog welfare organisations (DWOs) in Ireland are recognised as an instrumental pillar of the animal welfare sector with some receiving substantial public funding. We conducted a survey of DWOs in Ireland (n = 39) to gain a better understanding of their role and function, including their policies and procedures and the rehoming of dogs to other regions. In addition, we wanted to get a better understanding of the challenges experienced by DWOs in fulfilling their role and their perspectives on potential solutions to these challenges. The survey questions consisted of closed and open-ended items. Closed items were analysed quantitively; open-ended items were analysed thematically. RESULTS: Most DWOs (> 80%) had written protocols for important welfare actions including rehoming procedures, assessment of owner suitability and euthanasia. DWOs sent dogs to Northern Ireland (13%), Great Britain (38.5%) and to other countries outside the United Kingdom (36%, including Germany, Sweden, Italy, the Netherlands and Czechia). Reported challenges included a general lack of funding, limited public awareness of the importance of dog welfare and insufficient capacity to handle dog numbers. To address these challenges, the DWOs highlighted the potential contribution of subsidised programmes and access to resources to educate potential owners. In a further qualitative evaluation to capture perceptions of appropriate solutions by DWOs, several themes emerged, relating to legislation, education, an overwhelmed workforce, and funding. CONCLUSIONS: This study provides important insights into the roles and functions of DWOs and challenges they experience in Ireland. It is hoped that the findings from this research will inform future research investigating potential solutions to these challenges as well as the development of policy in Ireland.

Understanding the dog population in the Republic of Ireland: insight from existing data sources?

BACKGROUND: Reliable information about national pet dog populations is an important contributor to informed decision-making, both by governments and national dog welfare organisations. In some countries, there is an improved understanding of aspects of the national pet dog population, but as yet limited published information is available in Ireland. The current study reviews the utility of existing data to inform our understanding of recent changes to the pet dog population in Ireland, including both biological and organisational processes. RESULTS: Based on national data on dog licencing and microchipping registration, pet dog numbers have remained relatively stable in recent years (ie prior to the COVID-19 pandemic). Since 2015, there has been a substantial decrease in the number of dogs managed through dog control centres. Although the completeness of the data are likely variable, there appears to be substantial, and increasing, number of dogs moving from Ireland to other countries, including UK, Sweden, Italy, Germany and Singapore. We also note an increase (albeit much smaller) in the number of dogs being moved into Ireland. CONCLUSIONS: This study highlights the challenges faced when using existing national data to gain insights into the dog population of Ireland. The linking of existing national databases (individual dog identification, dog licencing, dog control statistics) has the potential to improve both the representativeness and accuracy of information about the Irish pet dog population. In the next phases of our work, we will focus on the work of dog welfare organisations, given both the increased role played by these organisations and the substantial public funding that has been committed in this sector.

Acyl chain selection couples the consumption and synthesis of phosphoinositides.

Phosphoinositides (PIPn) in mammalian tissues are enriched in the stearoyl/arachidonoyl acyl chain species ("C38:4"), but its functional significance is unclear. We have used metabolic tracers (isotopologues of inositol, glucose and water) to study PIPn synthesis in cell lines in which this enrichment is preserved to differing relative extents. We show that PIs synthesised from glucose are initially enriched in shorter/more saturated acyl chains, but then rapidly remodelled towards the C38:4 species. PIs are also synthesised by a distinct 're-cycling pathway', which utilises existing precursors and exhibits substantial selectivity for the synthesis of C38:4-PA and -PI. This re-cycling pathway is rapidly stimulated during receptor activation of phospholipase-C, both allowing the retention of the C38:4 backbone and the close coupling of PIPn consumption to its resynthesis, thus maintaining pool sizes. These results suggest that one property of the specific acyl chain composition of PIPn is that of a molecular code, to facilitate 'metabolic channelling' from PIP2 to PI via pools of intermediates (DG, PA and CDP-DG) common to other lipid metabolic pathways.

Gßγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils.

The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gßγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gßγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gßγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gßγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP3 production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to fMLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gßγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gßγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.

The History of In Vivo Tuberculin Testing in Bovines: Tuberculosis, a "One Health" Issue.

Tuberculosis (TB) is more than 3 million years old thriving in multiple species. Ancestral Mycobacterium tuberculosis gave rise to multiple strains including Mycobacterium bovis now distributed worldwide with zoonotic transmission happening in both directions between animals and humans. M. bovis in milk caused problems with a significant number of deaths in children under 5 years of age due largely to extrapulmonary TB. This risk was effectively mitigated with widespread milk pasteurization during the twentieth century, and fewer young children were lost to TB. Koch developed tuberculin in 1890 and recognizing the possibility of using tuberculin to detect infected animals the first tests were quickly developed. Bovine TB (bTB) control/eradication programmes followed in the late nineteenth century/early twentieth century. Many scientists collaborated and contributed to the development of tuberculin tests, to refining and optimizing the production and standardization of tuberculin and to determining test sensitivity and specificity using various methodologies and injection sites. The WHO, OIE, and EU have set legal standards for tuberculin production, potency assay performance, and intradermal tests for bovines. Now, those using tuberculin tests for bTB control/eradication programmes rarely, see TB as a disease. Notwithstanding the launch of the first-ever roadmap to combat zoonotic TB, many wonder if bTB is actually a problem? Is there a better way of dealing with bTB? Might alternative skin test sites make the test "better" and easier to perform? Are all tuberculins used for testing equally good? Why have alternative "better" tests not been developed? This review was prompted by these types of questions. This article attempts to succinctly summarize the data in the literature from the late nineteenth century to date to show why TB, and zoonotic TB specifically, was and still is important as a "One Health" concern, and that the necessity to reduce the burden of zoonotic TB, to save lives and secure livelihoods is far too important to await the possible future development of novel diagnostic assays for livestock before renewing efforts to eliminate it. Consequently, it is highly probable that the tuberculin skin test will remain the screening test of choice for farmed livestock for the considerable future.

Cadmium and other heavy metal concentrations in bovine kidneys in the Republic of Ireland.

In Ireland, an estimated 15% of Irish soils exceed the EU threshold limit for soil Cd of 1mg/kg. The aim was to determine the concentrations of Cd and other heavy metals (As, Hg and Pb) in kidneys collected from cattle at slaughter. Systematic sampling of eligible animals (animals that were born and reared until slaughter in the same Irish county) at the time of slaughter was conducted, until a threshold number of animals from all 26 counties and 6 age categories was reached. A predictive surface of soil Cd was generated, by kriging the Cd values of 1310 previously reported soil samples. A linear regression weighted model was developed to model kidney Cd concentration, using the risk factors of age, sex, breed, province and estimated soil Cd concentration. Kidney Cd (n=393) concentrations varied between 0.040 and 8.630 mg/kg wet weight; while concentrations of As, Hg and Pb were low. The estimated weighted proportion of animals with a high (≥1 mg/kg) kidney Cd concentration was 11.25% (95% CI: 8.63-14.53%). Key predictors for high kidney Cd concentration were soil Cd, animal age and province. At a soil Cd concentration of 1.5 mg/kg, it was predicted that an age threshold to avoid exceeding a kidney Cd concentration of 1 mg/kg in most animals would be ~3 y in Connacht, >4 y in Ulster, and >5 y in Leinster and Munster. In naturally occurring areas of high Cd levels in soils in Ireland, the Cd level in bovine kidneys can exceed the current EU ML of 1mg/kg in older animals. Kidneys of most cattle under three years of age will conform with EU requirements.

Seroprevalence of Louping Ill virus (LIV) antibodies in sheep submitted for post mortem examination in the North West of Ireland in 2011.

Blood samples were collected opportunistically at routine post mortem examination from 199 sheep which came from 152 flocks. The location of each submitting flock was mapped. Sera were tested using a goose blood haemagglutination inhibition assay for louping ill virus. There was an animal level prevalence of 8.5%, and a flock level prevalence of 9.8%. The greatest proportion of seropositive animals was identified among the animals older than 24 months of age. The elevation of the land associated with positive flocks was greater than that of negative flocks. Lesions of non-suppurative meningoencephalitis were observed in three of the 199 animals.

The spatial distribution of pet dogs and pet cats on the island of Ireland.

BACKGROUND: There is considerable international research regarding the link between human demographics and pet ownership. In several international studies, pet ownership was associated with household demographics including: the presence of children in the household, urban/rural location, level of education and age/family structure. What is lacking across all these studies, however, is an understanding of how these pets are spatially distributed throughout the regions under study. This paper describes the spatial distribution of pet dog and pet cat owning households on the island of Ireland. RESULTS: In 2006, there were an estimated 640,620 pet dog owning households and 215,542 pet cat owning households in Ireland. These estimates are derived from logistic regression modelling, based on household composition to determine pet dog ownership and the type of house to determine pet cat ownership. Results are presented using chloropleth maps. There is a higher density of pet dog owning households in the east of Ireland and in the cities than the west of Ireland and rural areas. However, in urban districts there are a lower proportion of households owning pet dogs than in rural districts. There are more households with cats in the urban areas, but the proportion of households with cats is greater in rural areas. CONCLUSIONS: The difference in spatial distribution of dog ownership is a reflection of a generally higher density of households in the east of Ireland and in major cities. The higher proportion of ownership in the west is understandable given the higher proportion of farmers and rural dwellings in this area. Spatial representation allows us to visualise the impact of human household distribution on the density of both pet dogs and pet cats on the island of Ireland. This information can be used when analysing risk of disease spread, for market research and for instigating veterinary care.

Clinically failed eggs as a source of normal human embryo stem cells.

The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.

Ndel1 alters its conformation by sequestering cAMP-specific phosphodiesterase-4D3 (PDE4D3) in a manner that is dynamically regulated through Protein Kinase A (PKA).

The involvement of the Nuclear distribution element-like (Ndel1; Nudel) protein in the recruitment of the dynein complex is critical for neurodevelopment and potentially important for neuronal disease states. The PDE4 family of phosphodiesterases specifically degrades cAMP, an important second messenger implicated in learning and memory functions. Here we show for the first time that Ndel1 can interact directly with PDE4 family members and that the interaction of Ndel1 with the PDE4D3 isoform is uniquely disrupted by elevation of intracellular cAMP levels. While all long PDE4 isoforms are subject to stimulatory PKA phosphorylation within their conserved regulatory UCR1 domain, specificity for release of PDE4D3 is conferred due to the PKA-dependent phosphorylation of Ser13 within the isoform-specific, unique amino-terminal domain of PDE4D3. Scanning peptide array analyses identify a common region on Ndel1 for PDE4 binding and an additional region that is unique to PDE4D3. The common site lies within the stutter region that links the second coiled-coil region to the unstable third coiled-coil regions of Ndel1. The additional binding region unique to PDE4D3 penetrates into the start of the third coiled-coil region that can undergo tail-to-tail interactions between Ndel1 dimers to form a 4 helix bundle. We demonstrate Ndel1 self-interaction in living cells using a BRET approach with luciferase- and GFP-tagged forms of Ndel1. BRET assessed Ndel1-Ndel1 self-interaction is amplified through the binding of PDE4 isoforms. For PDE4D3 this effect is ablated upon elevation of intracellular cAMP due to PKA-mediated phosphorylation at Ser13, while the potentiating effects of PDE4B1 and PDE4D5 are resistant to cAMP elevation. PDE4D long isoforms and Ndel1 show a similar sub-cellular distribution in hippocampus and cortex and locate to post-synaptic densities. We show that Ndel1 sequesters EPAC, but not PKA, in order to form a cAMP signalling complex. We propose that a key function of the Ndel1 signalling scaffold is to signal through cAMP by sequestering EPAC, whose activity may thus be specifically regulated by sequestered PDE4 that also stabilizes Ndel1-Ndel1 self-interaction. In the case of PDE4D3, its association with Ndel1 is dynamically regulated by PKA input through its ability to phosphorylate Ser13 in the unique N-terminal region of this isoform, triggering the specific release of PDE4D3 from Ndel1 when cAMP levels are elevated. We propose that Ser13 may act as a redistribution trigger in PDE4D3, allowing it to dynamically re-shape cAMP gradients in distinct intracellular locales upon its phosphorylation by PKA.

EPAC and PKA allow cAMP dual control over DNA-PK nuclear translocation.

We identify a compartmentalized signaling system that identifies a functional role for the GTP exchange factor, exchange protein activated by cAMP (EPAC) coupled to Rap2 in the nucleus. In this system, cAMP regulates the nuclear/cytoplasmic trafficking of DNA-dependent protein kinase (DNA-PK), a critical kinase that acts to repair double-stranded breaks (DSBs) in damaged DNA and to phosphorylate the cell survival kinase, PKB/Akt. Intersecting regulatory inputs for cAMP employ EPAC to transduce positive effects, namely the Rap2-dependent nuclear exit and activation of DNA-PK, whereas protein kinase A (PKA) provides the negative input by antagonizing these actions. We identify this as a compartmentalized regulatory system where modulation of cAMP input into the stimulatory, EPAC and inhibitory, PKA intersecting arms is provided by spatially discrete, cAMP degradation systems. The distribution of DNA-PK between nuclear and cytoplasmic compartments can thus potentially be influenced by relative inputs of cAMP signaling through the EPAC and PKA pathways. Through this signaling system EPAC activation can thereby impact on the Ser-473 phosphorylation status of PKB/Akt and the repair of etoposide-induced DSBs.

Isoform-selective susceptibility of DISC1/phosphodiesterase-4 complexes to dissociation by elevated intracellular cAMP levels.

Disrupted-in-schizophrenia 1 (DISC1) is a genetic susceptibility factor for schizophrenia and related severe psychiatric conditions. DISC1 is a multifunctional scaffold protein that is able to interact with several proteins, including the independently identified schizophrenia risk factor phosphodiesterase-4B (PDE4B). Here we report that the 100 kDa full-length DISC1 isoform (fl-DISC1) can bind members of each of the four gene, cAMP-specific PDE4 family. Elevation of intracellular cAMP levels, so as to activate protein kinase A, caused the release of PDE4D3 and PDE4C2 isoforms from fl-DISC1 while not affecting binding of PDE4B1 and PDE4A5 isoforms. Using a peptide array strategy, we show that PDE4D3 binds fl-DISC1 through two regions found in common with PDE4B isoforms, the interaction of which is supplemented because of the presence of additional PDE4B-specific binding sites. We propose that the additional binding sites found in PDE4B1 underpin its resistance to release during cAMP elevation. We identify, for the first time, a functional distinction between the 100 kDa long DISC1 isoform and the short 71 kDa isoform. Thus, changes in the expression pattern of DISC1 and PDE4 isoforms offers a means to reprogram their interaction and to determine whether the PDE4 sequestered by DISC1 is released after cAMP elevation. The PDE4B-specific binding sites encompass point mutations in mouse Disc1 that confer phenotypes related to schizophrenia and depression and that affect binding to PDE4B. Thus, genetic variation in DISC1 and PDE4 that influence either isoform expression or docking site functioning may directly affect psychopathology.