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Epicardial pulsed field ablation of ganglionated plexi: Computational and pre-clinical evaluation of a bipolar sub-xiphoid catheter for the treatment of atrial fibrillation
No full textEpicardial pulsed field ablation (PFA) of ganglionated plexi (GPs) is being explored as a potential treatment for atrial fibrillation. Initial work using open-chest access with a monopolar ablation device has been completed. This study describes the early development work for a device that can be used with subxiphoid access and deliver bipolar ablation pulses. Electric field computational models have been used for the initial guidance on pulse parameters. An in vivo assessment of these ablation parameters has been performed in an open-chest canine study, while subxiphoid access and navigation of the device has been demonstrated in a porcine model. Results from this acute study have demonstrated the promising potential of this approach.This research was funded by AtriAN Medical Ltd., with additional grant funding as follows: Government of Ireland, Disruptive Technologies Innovation Fund (grant No. DT20180123); Science Foundation Ireland (grant No. 22/PATH-S/10719); Spanish Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación, Fondo Europeo de Desarrollo Regional (grant PID2022-136273OA-C33 funded by MCIN/AEI/10.13039/501100011033/FEDER, UE). The APC was funded by AtriAN Medical Ltd.peer-reviewe
Development of 3D breast cancer model to study extracellular vesicle crosstalk
No full text3D model systems are crucial in establishing novel therapeutics and understanding cell interactions in the tumor microenvironment. Extracellular vesicles (EVs) are promising carriers of therapeutic cargo for tumor-targeted delivery, and biocompatible hydrogels offer controlled EV delivery. The three core elements of this study included generation and scale up of GFP-EVs in a FiberCell bioreactor, followed by assessment of EV encapsulation and release from hyaluronic acid hydrogels and finally establishing a dynamic 3D breast cancer model that mimics the tumor niche. MDA-MB-231 cells were genetically modified to produce CD63-GFP-labeled EVs. 1x109 of these cells were cultured in a 20kD bioreactor with the introduction of serum free media once stable culture was established. Over five weeks, multiple collections of GFP-labeled EVs were isolated by size exclusion chromatography and characterized by Nanoparticle Tracking Analysis (NTA), western blot, and Transmission Electron Microscopy (TEM). Furthermore, tyramine modified hyaluronic acid (HA-TA) hydrogels were established with hydrogen peroxide and horse radish peroxidase crosslinkers to investigate EV release patterns in static and dynamic conditions. To establish a dynamic 3D multicellular model of breast cancer, isolation and characterization of patient-derived tumor and lymph node stromal cells was performed. Mixed stromal/epithelial tumor spheroids and lymph node spheroids encapsulated in alginate hydrogels were formed. Spheroid viability was assessed and these were introduced into a dynamic multi in vitro organ (MIVO®) system with their respective endothelial barriers to establish crosstalk and support secretome analysis using angiogenesis arrays and ELISA. GFP expression was demonstrated in transduced cells with longitudinal expression confirmed in cells throughout bioreactor culture. NTA and TEM revealed both plasma-EVs and GFP-EVs in the size range of 30-200nm with an intact lipid bilayer were successfully isolated. Initial harvests of GFP-EVs contained subpopulations in a higher size range which disappeared within a few days of serum withdrawal, highlighting initial serum contamination. Western blot confirmed the expression of EV markers CD63, TSG101, CD81, CD 82. Successful incorporation and release of plasma-derived EVs from the hydrogels was demonstrated, with release patterns dependent on loaded EV concentrations and hydrogel formulations. Further investigation into EV release patterns using GFP-EVs under static and dynamic conditions highlighted a significant increase in EV release under fluidic flow conditions. Characterization of tumor and LN stromal populations confirmed presence of stromal markers and absence of hematopoietic markers. Spheroid growth within the alginate gel was monitored maintenance of an intact structure, cell viability and metastatic potential shown. Secretome analysis of the spheroid culture in the dynamic fluidic system supported the tumor-mimicking characteristics of the 3D breast cancer model system. These preliminary findings demonstrate the potential of the fibercell bioreactor system that supported efficient, serum free and reproducible scale up of GFP-EV production which will facilitate tracking EV transfer in the cancer setting. HA-TA hydrogels also showed promise for incorporation and sustained release of EVs. Finally, the 3D organoids in the dynamic system mimicked the tumor niche, demonstrating compelling potential for future study of intercellular EV trafficking and therapeutic potential prior to clinical translation.2025-01-2
Learning from what goes well: Improving the quality of general practice
No full textImproving the quality of primary care is a global imperative, one only increasing in importance as health systems internationally move towards community-based models of care, and patients, tasks, technologies, practice environments and expectations get increasingly complex. Contemporary approaches to improvement, focused on learning from instances of poor performance, have not generated sustained improvement in quality and are likely insufficient to address the increasing complexities of delivering care in the general practice setting. Recently, new approaches to understanding quality and safety have been advocated, that emphasise learning from when things go well, or even exceptionally well. One such approach, the ‘positive deviance’ approach focuses learning on exceptionally high-performing individuals, teams or practices, and applications of the approach in primary care thus far have been promising. Given the increasingly complex healthcare landscape, applying strengths-based approaches like PD is an attractive strategy as it allows us to engage patients and staff to learn from positive deviations in care and attend to key systems and contextualising factors that impact the success of improvement efforts. The current thesis applied the principles of positive deviance and strengths-based learning to identify and disseminate the key factors and strategies that support the delivery of exceptional care in general practice to stimulate learning and improvement in this domain of healthcare.Irish Research Council (GOIPG/2020/977
Investigating the roles of MARCKS and MARCKS-like 1 proteins in Xenopus laevis spinal cord development and regeneration
No full textThe identification and characterisation of key molecules that can promote or suppress regeneration has been an elusive, long-sought objective for researchers in spinal cord injury. The myristoylated alanine-rich C-kinase substrate (MARCKS) and MARCKS-like 1 (MARCKSL1) are two homologous proteins that are highly expressed in early developmental nervous tissue, with implications in gastrulation, embryogenesis, brain development, and myogenesis. Sharing conserved domains, both proteins act as primary substrates for protein kinase C (PKC), which enables them to dynamically regulate the actin cytoskeleton, membrane phosphoinositides, and other sub-cellular components. Previously, MARCKSL1 has been shown to promote appendage regeneration in other vertebrate models. Proteomic data from a former study in our lab also indicates that MARCKS and MARCKSL1 are differentially expressed after spinal cord injury (SCI) in regenerative and non-regenerative systems. Yet, there have been no functional studies exploring the roles of MARCKS and MARCKSL1 in spinal cord development and regeneration. This study reveals that marcks and marcksl1 are expressed in various tissues of Xenopus laevis throughout embryonic development, including the spinal cord. Genetic disruption of MARCKS and MARCKSL1 using both CRISPR/Cas9 and morpholino approaches results in a significant reduction in neurite outgrowth and mitotic and neural stem cell activity during spinal cord development, indicating that these proteins have essential and redundant functions during normal spinal cord development. Alternatively, mRNA overexpression of MARCKS and MARCKSL1 further enhances neurite outgrowth and cell proliferation. Pharmacological activation and inhibition of PKC, PIP2 and other signaling pathways in MARCKS and MARCKSL1 CRISPR mutants suggests that the proteins may modulate neurite outgrowth and cell proliferation by two different mechanisms. First, MARCKS and MARCKSL1 can promote cell proliferation and neurite outgrowth through a PIP2-dependent mechanism that is inhibited by PKC and, thus, probably involves unphosphorylated forms of MARCKS and MARCKSL1. Second, targets of PKC phosphorylation, which may include phosphorylated MARCKS and MARCKSL1 can promote cell proliferation and neurite outgrowth through additional mechanisms. This study also indicates that MARCKS and MARCKSL1 are upregulated at 5 days post spinal cord transection (DPT) in Xenopus laevis tadpoles. Here, higher levels of MARCKS and MARCKSL1 can be observed in the ependymal layer, white matter area, and meninges. These findings also demonstrate a general increase in cell proliferation at 2 DPT, followed by a gradual increase of Sox2+ neural progenitor cells along the ependyma, gradually filling the injury gap and posterior injury stump. Following CRISPR mediated knockdown of MARCKS and MARCKSL1, tadpoles show significant delays and deficiencies in behavioural recovery, injury gap closure, proliferative response, and stem cell activation, indicating that MARCKS and MARCKSL1 are required for these processes during spinal cord regeneration. A pharmacological study after spinal cord injury in MARCKS/MARCKSL1-mutant tadpoles indicates that Phospholipase D (PLD) activation significantly rescues regenerative outcomes in the absence of MARCKS and MARCKSL1, suggesting that these proteins may promote spinal cord regeneration via a PLD-dependent mechanism. Taken together, this study provides evidence for novel roles of MARCKS and MARCKSL1 for axon outgrowth and proliferation of neural progenitor cells during spinal cord development and suggests that these proteins are redeployed after spinal cord injury to recapitulate similar functions during spinal cord regeneration.2025-02-1
Designing necks and wrinkles in inflated auxetic membranes
No full textThis article presents the potentiality of inflatable, functionally-graded auxetic membranes to produce wrinkles and necks. We obtain elastic instabilities at desired locations in axisymmetric membranes and with prescribed patterns in square membranes. First, we use an analytical approach to obtain a series of universal results providing insights into the formation of wrinkles and necks in inflated, axisymmetric membranes. For example, we prove analytically that necks and wrinkles may never overlap in pressurized, axially symmetric membranes. Second, we implement the relaxed strain energy of tension field theory into a Finite Element solver (COMSOL). By tuning spatial inhomogeneities of the material moduli, we corroborate our universal results, describe the onset of wrinkling in an averaged way, and also generate non-trivial instabilities at desired locations. This study on membranes with morphing or corrugation on demand has potential applications in Braille reading and haptics.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 956401. GZ gratefully acknowledges the support of GNFM (Gruppo Nazionale di Fisica Matematica) of the INdAM F. Severi, Italy . The authors thank the anonymous referees for their valuable comments
Deciphering anaerobic ethanol oxidation for better recovery of renewable energy from wastewater
No full textAnaerobic digestion is a promising technology relying on complex microbial interactions to convert organics into methane, achieving sustainable energy recovery. Ethanol is an important low-molecular intermediate during anaerobic digestion, and ethanol-type fermentation is a major fermentation type in mixed cultures of acidogenesis, alongside butyric acid-type and propionic acid-type fermentations. Furthermore, anaerobic ethanol degradation has been identified through various metabolic pathways: (i) conventional ethanol degradation to acetate and hydrogen via interspecies hydrogen transfer (IHT), (ii) ethanol consumption leading to propionate production, and (iii) a newly suggested mechanism wherein ethanol releases electrons for electron acceptors via direct extracellular electron transfer (EET) or direct interspecies electron transfer (DIET). These metabolic pathways illustrate the intricacy and diverse possibilities of reactions when ethanol serves as a substrate during anaerobic digestion. Therefore, it is imperative to elucidate the regulatory strategies governing ethanol metabolism, encompassing aspects such as the degradation rate and the specific metabolic pathways, within anaerobic digestion ecosystems. The objectives of this study were: (i) to interpret and regulate the participation of EET pathway in ethanol metabolism to favour the overall ethanol oxidation; (ii) to enrich syntrophic bacteria and modulate ethanol metabolic pathways through the manipulation of operational parameters, including solids retention times (SRT) and operational modes; and (iii) to regulate syntrophic relationships among microorganisms and microbial activities by adjusting operational parameters and introducing powdered activated carbon (PAC). A thermodynamic approach was employed to analyse IHT and EET pathways in ethanol consumption. The effects of the fraction of EET pathway in ethanol degradation, product feedback, and the redox potential of redox-active mediator on biomass yields and biogas production were evaluated. The involvement of EET makes it thermodynamically favourable for ethanol oxidation. It was found that the EET fraction played a crucial role in maintaining biomass yields, and ethanol oxidation occurred when the redox potential was above -0.408 V through EET or when the product concentration was below the threshold value. Moreover, strategies for the application of one-reactor and zone-separation systems were proposed to optimize system performance and bioenergy recovery with the appropriate redox potential range. Different operational modes (sequencing batch reactors, SBRs, and continuous-flow reactors, CFRs) and SRTs (25 days and 10 days) were employed to regulate syntrophic interactions in four reactors. Microorganisms with high half-saturation constants were enriched in reactors with a 25-day SRT. SBRs favoured the acclimation of ethanol oxidizing bacteria and acetotrophic methanogens with high half-saturation constants. In SBRs, Syner-01 and Methanothrix dominated, and a low SRT of 10 days increased the relative abundance of Geobacter to 38.0% for possible performing DIET. In CFRs, a low SRT of 10 days increased the relative abundance of Desulfovibrio among syntrophic bacteria in mediating IHT. Two operational modes (SBRs and CFRs) with or without the addition of PAC were adopted as the regulatory approach to modulate microbial activities and drive metabolic pathways towards acetate or propionate. The operational mode of SBR and the presence of CO2 facilitated ethanol metabolism towards propionate production, while CFRs with an extended SRT enriched high relative abundances of Geobacter, reaching 71.7% and 70.4% under conditions with and without the addition of PAC, respectively. Although both long-term and short-term PAC additions increased sludge conductivity and reduced the methanogenic lag phase, only the long-term PAC addition resulted in enhanced rates of ethanol degradation and propionate production/degradation. This study advances anaerobic digestion technology by unravelling the complex ethanol metabolic pathways. The study could offer insights into the EET pathway with a novel approach to favouring ethanol oxidation, further affecting overall system performance. Manipulating operational parameters, especially operational modes and SRT, and introducing PAC emerge as effective strategies for regulating microbial activities, enrich functional microorganisms, and directing metabolic pathways. Proposed strategies for system optimization, including one-reactor and zone separation systems, present practical solutions for practical applications. The findings not only contribute to the improvement of bioenergy recovery and wastewater treatment but also provide insights for guiding reactor design based on different operational modes and SRT.2026-03-2
Computational approaches to identify and explain sources of error in cancer somatic mutation data
No full textErrors in the identification of somatic mutations in cancer samples can have critical implications in both research and clinical applications. Failure to detect potential variants of interest can lead to missed opportunities in patient treatment or sci entific research. Incorrectly identifying a somatic variant may result in inaccurate prognosis, unsuitable treatment selection, or misleading research. By understanding the sources of error in somatic mutation calling, we are better placed to mitigate these risks. The reevaluation of variants that have been excluded from analysis by mutation calling methodologies can provide valuable insights in this regard. By considering the allele frequency, nucleotide context, and potential impact on pro tein of a mutation that has been discarded from analysis, we can incorporate the overall biological context into our assessment of the variant call. This approach enables us to identify putative somatic variants that were overlooked by the caller and, importantly, investigate the reason for their omission. In Chapter 2, we outline vcfView, an interactive R Shiny tool designed to support the evaluation and exploratory analysis of somatic mutation records from cancer se quencing data. We use vcfView to reevaluate the TCGA acute myeloid leukaemia data and identify clinically actionable mutation records in patients that were incor rectly excluded from analysis due to the presence of tumour sample DNA in the matched normal sample. The validation of somatic mutation calling pipelines is a critical step in ensur ing the accuracy and reliability of the results obtained from the analysis of cancer genomic sequencing data. However, the trustworthiness of the validation results is directly linked to the quality of the truth set used for validation. In Chapter 3, we introduce a simulation framework designed to generate comprehensive and realistic tumour genomic sequencing data. This framework takes into account the inherent randomness of genomic sequencing, providing an accurate representation of the fre quency profile as it is observed in real sequencing data. It generates a corresponding truth set alongside the simulated sequencing data, documenting the true source of each non-reference base in the data. Unlike existing validation methods, this truth set not only identifies variant caller errors but, crucially, enables us to understand the reasons behind the erroneous calls. Using the GATK Mutect2 variant calling pipeline, we apply this framework to highlight and explain sources of error in somatic mutation data and biases in the estimation of somatic allele frequency. Finally in Chapter 4, we analyse tumour-only sequencing and somatic variant data from an unpublished dataset comprising 60 individuals diagnosed with early onset and aggressive pancreatic ductal adenocarcinoma. We apply the tools and methods we have developed previously to recover somatic variant information from sequence data obtained from heavily damaged FFPE samples. We provide an im proved estimate of the true incidence of pathogenic KRAS variants within the cohort that accounts for the sequencing strategy and sample preparation methods used. We also highlight recurrent mutations in several other cancer associated genes that may have played a role in disease progression in these patients
Assessing ISO 18404 standard applicability in the service sector: a qualitative study
No full textThis research aims to establish the applicability of the International Organisation for Standardisation (ISO) 18404 standard to the service sector, identify any required amendments and identify the critical success factors and barriers to deploying the standard within the service sector. Design/methodology/approach The study used a qualitative approach by interviewing operational excellence (OPEX) professionals who work in the service sector. Findings The findings indicate a significant lack of knowledge about the existence of the standard and a general scepticism regarding the applicability of the current ISO 18404 standard to the service sector. Research limitations/implications Limited examples of the application of ISO 18404 in organisations exist, as only a few organisations have adopted the standard. Therefore, the research focussed on the challenges and obstacles that experienced OPEX professionals perceived could be an issue. Originality/value The study will aid service sector organisations in understanding the standard and, subsequently, determine whether to pursue it as part of an OPEX programme. This research is the first study on the application of ISO 18404 to the service sector.peer-reviewe
Using genomics to aid in the conservation of the native Irish honey bee
No full textApis mellifera mellifera, or the European dark bee, is a sub-lineage of the European honey bee that is native to North Western Europe including Ireland. Recent studies have shown that the free-living population of honey bees in Ireland is widespread and has a high level of genetic purity. This is threatened by consistent and increasing importation of foreign sub-lineages, namely C-lineage queens from either A. m. ligustica and A. m. carnica, with numbers of importations rising from just over 100 queens per year in 2012 to over 1200 per year in 2021. A survey of Irish beekeepers was undertaken to identify attitudes of Irish beekeepers towards issues such as conservation and scientific research. The results indicated that the overwhelming majority are currently supporting the conservation of the native Irish honey bee through their beekeeping practices with only a small group of respondents keeping a non-native sub-lineage in their apiaries. A whole genome pooled sequencing analysis was undertaken to identify hybrid colonies and model the current population of Irish honey bees. Over 150 Irish honey bee colonies were sampled alongside three UK and five German colonies. The use of the f3-statistics method found an increase in hybridisation rates compared with previous studies and confirmed the reports of increased importations, however, the Irish population was found to contain a large percentage of extremely pure M-lineage honey bee colonies that are an important resource in the conservation of this sub-lineage. The use of pool-seq allowed the data generated in this study to be combined with two pan-European pool-seq datasets to create a network model of European honey bee population. Groups from this model were then extracted for admixture analysis that helped identify the best course of action in regards to the future methods of conservation of the native Irish honey bee. The reliance on abdominal colour alone to detect hybridised honey bees has been challenged in recent studies and pure-black hybrids were also detected in this study. A Cochran-Mantel-Haenszel test followed by a gene ontology analysis discovered genes and processes related to melanin production and cuticle development in honey bees. A microbiome analysis using shotgun metagenomics methods was carried out to investigate if management, and treatment, results in a different microbiome profile in comparison with free-living honey bee colonies. Relative abundance analysis and differential absolute abundance analysis methods both identified differences between managed and free-living honey bee microbiomes.SFI CRT grant number: 18/CRT/62142025-02-1
The application of continuous flow chemistry in the synthesis of iminosugars and c-glycosyl compounds
No full textFlow chemistry is a field of chemical synthesis which involves carrying out reactions in continuous streams of solvents. Despite the numerous potential advantages, flow chemistry has been applied in a limited number of areas in synthetic glycochemistry, such as oligosaccharide synthesis and deprotection steps. The main aim of this thesis is to apply continuous flow techniques in other areas of synthetic glycochemistry, and to provide a comparison with batch techniques for the reactions studied. Chapter 1 introduces carbohydrates and iminosugars, which are the main synthetic focus of this work. The bioactivity of iminosugars and major synthetic routes towards these substances are described. An overview of flow chemistry, its advantages over batch techniques and its application to date in glycochemistry is also included. Chapter 2 focuses on flow syntheses of iminosugars adapted from existing batch methods. This chapter includes a flow synthesis of 1-deoxynojirimycin (1-DNJ) and miglustat from an L-sorbose derivative, along with the synthesis of a C-glycosyl mannonojirimycin compound via cascade azide-alkene cycloaddition/aziridine formation/ring opening. Different protecting groups were also investigated for the synthesis of the mannonojirimycin structure. Chapter 3 describes the synthesis of new iminosugars in batch and flow, including protected deoxymannonojirimycin (DMJ) and L-DNJ structures via tandem intramolecular azide-alkene cycloaddition/aziridine formation/ring opening reactions. A Design of Experiments (DoE) study to improve the yield of the L-DNJ compound is discussed, and further attempts to improve yield via photosynthetic methods in batch and thermolysis in flow are also described. Additional synthetic modifications to the azide-alkene precursor with an aim to improve yield are also discussed. The main achievement was the development of a route to l-DNJ from D-xylose, which is also a new sequence. Chapter 4 details the application of cascade Wittig/intramolecular oxa-Michael additions to synthesise 2-(C-glycosyl)acetates under continuous flow conditions, from commercially available pyranoses. Access to higher temperatures in flow enabled the synthesis of these C-glycosyl compounds, while batch attempts are mostly unsuccessful using this method. A modified flow approach involving delayed addition of base (DBU), which further increased yields, is also described in this chapter. The flow synthesis of a precursor to b-D-galactopyranosyl-2-methylpropane, an inducer of protein synthesis in E. coli, is also described