Prescription drugs with potential for misuse: protocol for a multi-indicator analysis of supply, detection and the associated health burden in Ireland between 2010 and 2020.

INTRODUCTION: There is an increasing concern about the misuse of prescription drugs. Misuse refers to the intentional repurposing of prescribed drugs and/or the use of illicitly sourced prescription drugs, which may be counterfeit or contaminated. Drugs with the greatest potential for misuse are prescription opioids, gabapentinoids, benzodiazepines, Z-drugs and stimulants. OBJECTIVE: The aim of this study is to provide a comprehensive analysis of the supply, patterns of use and health burden associated with prescription drugs with potential for misuse (PDPM) in Ireland between 2010 and 2020. Three inter-related studies will be carried out. The first study will describe trends in supply of PDPM using law enforcement drug seizures data and national prescription records from national community and prison settings. The second study aims to estimate trends in the detection of PDPM across multiple early warning systems using national forensic toxicology data. The third study aims to quantify the health burden associated with PDPM nationally, using epidemiological indicators of drug-poisoning deaths, non-fatal intentional drug overdose presentations to hospitals and drug treatment demand. METHODS AND ANALYSIS: A retrospective observational study design, with repeated cross-sectional analyses, using negative binomial regression models or, where appropriate, joinpoint regression. ETHICS AND DISSEMINATION: The study has received approval from the RCSI Ethics Committee (REC202202020). Results will be disseminated in peer-reviewed journals, scientific and drug policy meetings and with key stakeholders via research briefs.

Population Pharmacokinetics of Periarticular Ketorolac in Adult Patients Undergoing Total Hip or Total Knee Replacement Surgery.

BACKGROUND: Ketorolac tromethamine has been used for joint infiltration by the orthopedic surgeons as a part of postoperative multimodal analgesia. The objective of this study is to investigate the pharmacokinetic properties of S (-) and R (+) enantiomers of ketorolac in adult patients undergoing total hip (THA) and knee arthroplasty (TKA). METHODS: Adult patients with normal preoperative renal function received a periarticular infiltration of 30 mg of ketorolac tromethamine along with 100 mL of 0.2% ropivacaine and 1 mg of epinephrine at the end of their THA or TKA surgery. Blood samples were taken from a venous cannula at various time points after infiltration. Pharmacokinetic modeling was performed using PMetrics 1.5.0. RESULTS: From 18 participants, 104 samples were analyzed. The peak plasma concentration for S (-) ketorolac was found to be lower than that of R (+) ketorolac, for both THA (0.19-1.22 mg/L vs 0.39-1.63 mg/L, respectively) and TKA (0.28-0.60 mg/L vs 0.48-0.88 mg/L, respectively). The clearance of the S (-) ketorolac enantiomer was higher than R (+) ketorolac (4.50 ± 2.27 vs 1.40 ± 0.694 L/h, respectively). CONCLUSIONS: Our study demonstrates that with periarticular infiltration, S (-) ketorolac was observed to have increased clearance rate and highly variable volume of distribution and lower peak plasma concentration compared to R (+) ketorolac.

TWIST1 Integrates Endothelial Responses to Flow in Vascular Dysfunction and Atherosclerosis.

RATIONALE: Blood flow-induced shear stress controls endothelial cell (EC) physiology during atherosclerosis via transcriptional mechanisms that are incompletely understood. The mechanosensitive transcription factor TWIST is expressed during embryogenesis, but its role in EC responses to shear stress and focal atherosclerosis is unknown. OBJECTIVE: To investigate whether TWIST regulates endothelial responses to shear stress during vascular dysfunction and atherosclerosis and compare TWIST function in vascular development and disease. METHODS AND RESULTS: The expression and function of TWIST1 was studied in EC in both developing vasculature and during the initiation of atherosclerosis. In zebrafish, twist was expressed in early embryonic vasculature where it promoted angiogenesis by inducing EC proliferation and migration. In adult porcine and murine arteries, TWIST1 was expressed preferentially at low shear stress regions as evidenced by quantitative polymerase chain reaction and en face staining. Moreover, studies of experimental murine carotid arteries and cultured EC revealed that TWIST1 was induced by low shear stress via a GATA4-dependent transcriptional mechanism. Gene silencing in cultured EC and EC-specific genetic deletion in mice demonstrated that TWIST1 promoted atherosclerosis by inducing inflammation and enhancing EC proliferation associated with vascular leakiness. CONCLUSIONS: TWIST expression promotes developmental angiogenesis by inducing EC proliferation and migration. In addition to its role in development, TWIST is expressed preferentially at low shear stress regions of adult arteries where it promotes atherosclerosis by inducing EC proliferation and inflammation. Thus, pleiotropic functions of TWIST control vascular disease and development.

Dermatofibrosarcoma Protuberans: The role of tissue expansion in reconstructive surgery of the scalp.

INTRODUCTION: Dermatofibrosarcoma Protuberans is an uncommon tumour, making up less than 0.1% of all malignancies. With regards to soft tissue tumours; this pathology is thought to make up less than 2% of the sum total. Traditionally treatment has been wide local excision, with or without adjuvant radiotherapy. PRESENTATION OF CASE: We present a case of a 42 year old man referred by his GP with a lump on the right parietal region of the scalp. An USS done by his GP revealed a complex hypoechoic cystic mass, some 2cm×1cm×2cm. Excision biopsy was performed and on review of the pathology it was noted that the lesion was a Dermatofibrosarcoma Protuberans. Due to the relatively low grade of this sarcoma, it was decided to treat with wide local excision with 2-4cm margins. The expected residual scalp defect would be difficult to close with local flaps. To facilitate closure tissue expansion was undertaken for 6 weeks prior to definitive surgery. DISCUSSION: With regards to tumours of the head and neck, use of a tissue expander has been recommended to improve cosmetic outcomes following respective surgery with wide margins. Ultimately the timing of tissue expansion i.e. before/after resection of the tumour, must weight the risk of delayed resective surgery on prognosis against the benefits of this reconstructive technique. CONCLUSION: Head and neck tumours requiring careful reconstruction may benefit from tissue expansion to provide adequate volumes of matching soft tissue, as shown in this case.

A case of suboccipital lump with a rare and unexpected diagnosis of apocrine cystadenoma.

INTRODUCTION: Cystadenoma of apocrine origin is a tumour of the sweat gland that is benign in nature. Classification of this pathology is based upon histological characteristics plus histochemical analysis. Prevalence of cystadenoma has been suggested to be quite rare, in the region of 1 in 1000 of subcutaneous biopsies observed. PRESENTATION OF CASE: We present a case of a 40 year old man referred by his GP with a suboccipital lump, present for some years. On examination the lump was approximately 4-5cm in diameter and an unusual punctum was present. The patient proceeded to an excision of the lesion and the gross specimen showed characteristics of a multiloculated cyst, measuring some 5cm×3.5cm. Histopathology of the tumour revealed an apocrine cystadenoma; there were no features suggestive of malignancy. DISCUSSION: Previous classification of cystadenoma via histological and immunohistochemical method; has revealed only two distinct entities and the term hydrocystoma was often used in place of cystadenoma. More recent studies have suggested that a third type can be identified via immunohistochemical analysis. This third type; apocrine hydrocystoma, reveals that those previously defined as eccrine in origin may also be related to the apocrine ducts. CONCLUSION: Apocrine cystadenoma remains a benign pathology and treatment should be focussed on excision, without need for further intervention. Apocrine cystadenoma remains a relatively rare pathology, though one which should not recur if adequate treatment is given.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Computational approaches for understanding the diagnosis and treatment of Parkinson's disease.

This study describes how the application of evolutionary algorithms (EAs) can be used to study motor function in humans with Parkinson's disease (PD) and in animal models of PD. Human data is obtained using commercially available sensors via a range of non-invasive procedures that follow conventional clinical practice. EAs can then be used to classify human data for a range of uses, including diagnosis and disease monitoring. New results are presented that demonstrate how EAs can also be used to classify fruit flies with and without genetic mutations that cause Parkinson's by using measurements of the proboscis extension reflex. The case is made for a computational approach that can be applied across human and animal studies of PD and lays the way for evaluation of existing and new drug therapies in a truly objective way.

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

INTRODUCTION AND OBJECTIVES: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development. METHODS AND RESULTS: Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants. CONCLUSIONS: The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

Sulf1 has ligand-dependent effects on canonical and non-canonical Wnt signalling.

Wnt signalling plays essential roles during embryonic development and is known to be mis-regulated in human disease. There are many molecular mechanisms that ensure tight regulation of Wnt activity. One such regulator is the heparan-sulfate-specific 6-O-endosulfatase Sulf1. Sulf1 acts extracellularly to modify the structure of heparan sulfate chains to affect the bio-availability of Wnt ligands. Sulf1 could, therefore, influence the formation of Wnt signalling complexes to modulate the activation of both canonical and non-canonical pathways. In this study, we use well-established assays in Xenopus to investigate the ability of Sulf1 to modify canonical and non-canonical Wnt signalling. In addition, we model the ability of Sulf1 to influence morphogen gradients using fluorescently tagged Wnt ligands in ectodermal explants. We show that Sulf1 overexpression has ligand-specific effects on Wnt signalling: it affects membrane accumulation and extracellular levels of tagged Wnt8a and Wnt11b ligands differently, and inhibits the activity of canonical Wnt8a but enhances the activity of non-canonical Wnt11b.

Sulf1 influences the Shh morphogen gradient during the dorsal ventral patterning of the neural tube in Xenopus tropicalis.

Genetic studies have established that heparan sulphate proteoglycans (HSPGs) are required for signalling by key developmental regulators, including Hedgehog, Wnt/Wg, FGF, and BMP/Dpp. Post-synthetic remodelling of heparan sulphate (HS) by Sulf1 has been shown to modulate these same signalling pathways. Sulf1 codes for an N-acetylglucosamine 6-O-endosulfatase, an enzyme that specifically removes the 6-O sulphate group from glucosamine in highly sulfated regions of HS chains. One striking aspect of Sulf1 expression in all vertebrates is its co-localisation with that of Sonic hedgehog in the floor plate of the neural tube. We show here that Sulf1 is required for normal specification of neural progenitors in the ventral neural tube, a process known to require a gradient of Shh activity. We use single-cell injection of mRNA coding for GFP-tagged Shh in early Xenopus embryos and find that Sulf1 restricts ligand diffusion. Moreover, we find that the endogenous distribution of Shh protein in Sulf1 knockdown embryos is altered, where a less steep ventral to dorsal gradient forms in the absence of Sulf1, resulting in more a diffuse distribution of Shh. These data point to an important role for Sulf1 in the ventral neural tube, and suggests a mechanism whereby Sulf1 activity shapes the Shh morphogen gradient by promoting ventral accumulation of high levels of Shh protein.

Lin28 proteins are required for germ layer specification in Xenopus.

Lin28 family proteins share a unique structure, with both zinc knuckle and cold shock RNA-binding domains, and were originally identified as regulators of developmental timing in Caenorhabditis elegans. They have since been implicated as regulators of pluripotency in mammalian stem cells in culture. Using Xenopus tropicalis, we have undertaken the first analysis of the effects on the early development of a vertebrate embryo resulting from global inhibition of the Lin28 family. The Xenopus genome contains two Lin28-related genes, lin28a and lin28b. lin28a is expressed zygotically, whereas lin28b is expressed both zygotically and maternally. Both lin28a and lin28b are expressed in pluripotent cells of the Xenopus embryo and are enriched in cells that respond to mesoderm-inducing signals. The development of axial and paraxial mesoderm is severely abnormal in lin28 knockdown (morphant) embryos. In culture, the ability of pluripotent cells from the embryo to respond to the FGF and activin/nodal-like mesoderm-inducing pathways is compromised following inhibition of lin28 function. Furthermore, there are complex effects on the temporal regulation of, and the responses to, mesoderm-inducing signals in lin28 morphant embryos. We provide evidence that Xenopus lin28 proteins play a key role in choreographing the responses of pluripotent cells in the early embryo to the signals that regulate germ layer specification, and that this early function is probably independent of the recognised role of Lin28 proteins in negatively regulating let-7 miRNA biogenesis.

Early transcriptional targets of MyoD link myogenesis and somitogenesis.

In order to identify early transcriptional targets of MyoD prior to skeletal muscle differentiation, we have undertaken a transcriptomic analysis on gastrula stage Xenopus embryos in which MyoD has been knocked-down. Our validated list of genes transcriptionally regulated by MyoD includes Esr1 and Esr2, which are known targets of Notch signalling, and Tbx6, mesogenin, and FoxC1; these genes are all are known to be essential for normal somitogenesis but are expressed surprisingly early in the mesoderm. In addition we found that MyoD is required for the expression of myf5 in the early mesoderm, in contrast to the reverse relationship of these two regulators in amniote somites. These data highlight a role for MyoD in the early mesoderm in regulating a set of genes that are essential for both myogenesis and somitogenesis.