Prevention of aminoglycoside antibiotic-induced ototoxicity of auditory hair cells via block of mechano-electrical transducer channels or intracellular mechanisms

This thesis addresses the pressing concern of clinical drug-induced hearing loss (ototoxicity). Described herein is the mechanism by which ototoxicity emerges following drug administration both in a clinical setting and in an in vitro model assay system used for its investigation, through use of mouse cochlear cultures. The predominant nature of this research concerns the identification of novel otoprotectants - compounds that when co-administered alongside clinical drug treatments can prevent the unfortunate ototoxic side-effect from occurring. Here I present my research, focussing on the identification of a number of novel compounds that have the potential to be taken forward to in vivo screening and, ultimately, clinical trials. Moreover, for each identified compound I present my investigation of their mechanism of protection – which could arise either by preventing the entry of the ototoxicity-inducing drugs into the sensory hair cells of the inner ear, or prevent their induction of apoptosis once inside the cell. To investigate their protective mechanism I employed a variety of methods, including: electrophysiology, fluorescent imaging and mitochondrial respirometry. Conclusively, I have identified at least five novel otoprotectants with the potential for clinical use. I show that three of these compounds likely block the entry of the damaging drugs into sensory hair cells, whereas the remaining two are thought to work intracellularly. Moreover, the two most effective compounds that I have identified seemingly work intracellularly, suggesting this to be the most viable mechanism of otoprotection for further investigation. I also show the potential for compound modification based on their mechanism of protection as a way of improving a compound’s otoprotective profile. Lastly, I devised an assay for the screening of clinical drug effects on mitochondria and employ this as a new avenue of screening for otoprotection

Building and Restructuring Social Media Platforms for Non-Binary Users : Pilot Survey of Non-Binary Social Media Users

The past ten years has seen a positive trend in people openly identifying outside of the gender binary, as knowledge of Non-Binary identities becomes more widespread and accepted. In an increasingly online world, there is pressure on social media platforms to accommodate these diverse gender identities. In online spaces driven by self-expression, Non-Binary users feel frustrated and forgotten by the lack of diversity in the options they are presented with. There is also an absence of standardisation across platforms: it seems every social media site has its own idea of how to tackle the problem. In this work in progress, I present the findings of a pilot study carried out in March 2022, wherein I asked Non-Binary social media users about their experiences across various social media platforms. The results of this study create the foundation of a larger user study that will explore these questions in more depth, and inform proposed guidelines and standardisation for social media platforms in accommodating diverse gender options

d-Tubocurarine and Berbamine: Alkaloids That Are Permeant Blockers of the Hair Cell's Mechano-Electrical Transducer Channel and Protect from Aminoglycoside Toxicity

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell’s stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 µM) or berbamine (≥1.55 µM) protect zebrafish hair cells in vivo from neomycin (6.25 µM, 1 h). Protection of zebrafish hair cells against gentamicin (10 µM, 6 h) was provided by ≥25 µM dTC or ≥12.5 µM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 µM, 48 h) by 20 µM berbamine or 25 µM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 µM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 µM (dTC) and 2.8 µM (berbamine) in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell’s basolateral K + current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants

Design, synthesis and biological evaluation of a new series of carvedilol derivatives that protect sensory hair cells from aminoglycoside-induced damage by blocking the mechanoelectrical transducer channel

Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious bacterial infections but have use-limiting side effects including irreversible hearing loss. Here, we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo zebrafish assays and investigated its mechanism of protection which, we found, may be mediated by a block of the hair cell’s mechanoelectrical transducer (MET) channel, the major entry route for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues were prepared and evaluated for their effect against AG-induced damage as well as their affinity for the MET channel. One derivative was found to confer greater protection than carvedilol itself in cochlear cultures and also to bind more tightly to the MET channel. At higher concentrations, both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting a good therapeutic window under in vivo conditions

Familial atrial fibrillation mutation M1875T-SCN5A increases early sodium current and dampens the effect of flecainide

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF in patients. Methods and results We designed a new murine model carrying the Scn5a-M1875T mutation enabling us to study the effects of the Nav1.5 mutation in detail in vivo and in vitro using patch clamp and microelectrode recording of atrial cardiomyocytes, optical mapping, electrocardiogram, echocardiography, gravimetry, histology, and biochemistry. Atrial cardiomyocytes from newly generated adult Scn5a-M1875T+/− mice showed a selective increase in the early (peak) cardiac sodium current, larger action potential amplitude, and a faster peak upstroke velocity. Conduction slowing caused by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/− compared to wildtype atria. Overt hypertrophy or heart failure in Scn5a-M1875T+/− mice could be excluded. Conclusion The Scn5a-M1875T point mutation causes gain-of-function of the cardiac sodium channel. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability

The application of antimicrobial stewardship knowledge to nursing practice : A national survey of United Kingdom pre-registration nursing students

© 2024 The Authors. Journal of Advanced Nursing published by John Wiley & Sons Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/AIM: To assess student nurses understanding and skills in the application of antimicrobial stewardship knowledge to practice. DESIGN: Quantitative. METHODS: Cross-sectional survey. RESULTS: Five hundred and twenty three student nurses responded across 23 UK universities. Although students felt prepared in competencies in infection prevention and control, patient-centred care and interprofessional collaborative practice, they felt less prepared in competencies in which microbiological knowledge, prescribing and its effect on antimicrobial stewardship is required. Problem-based learning, activities in the clinical setting and face-to-face teaching were identified as the preferred modes of education delivery. Those who had shared antimicrobial stewardship teaching with students from other professions reported the benefits to include a broader understanding of antimicrobial stewardship, an understanding of the roles of others in antimicrobial stewardship and improved interprofessional working. CONCLUSION: There are gaps in student nurses' knowledge of the basic sciences associated with the antimicrobial stewardship activities in which nurses are involved, and a need to strengthen knowledge in pre-registration nurse education programmes pertaining to antimicrobial management, specifically microbiology and antimicrobial regimes and effects on antimicrobial stewardship. Infection prevention and control, patient-centred care and interprofessional collaborative practice are areas of antimicrobial stewardship in which student nurses feel prepared. Interprofessional education would help nurses and other members of the antimicrobial stewardship team clarify the role nurses can play in antimicrobial stewardship and therefore maximize their contribution to antimicrobial stewardship and antimicrobial management. IMPLICATIONS FOR THE PROFESSION: There is a need to strengthen knowledge from the basic sciences, specifically pertaining to antimicrobial management, in pre-registration nurse education programmes. PATIENT OR PUBLIC CONTRIBUTION: No patient or public contribution. IMPACT: What Problem Did the Study Address? Nurses must protect health through understanding and applying antimicrobial stewardship knowledge and skills (Nursing and Midwifery Council 2018); however, there is no research available that has investigated nurses understanding and skills of the basic sciences associated with the antimicrobial stewardship activities in which they are involved. What Were the Main Findings? There are gaps in student nurses' knowledge of the basic sciences (specifically microbiology and prescribing) associated with the antimicrobial stewardship activities in which nurses are involved. Problem-based learning, and activities in the clinical setting, were reported as useful teaching methods, whereas online learning, was seen as less useful. Where and on Whom Will the Research Have an Impact? Pre-registration nurse education programmes. REPORTING METHOD: The relevant reporting method has been adhered to, that is, STROBE.Peer reviewe

Long-term prognosis for 1-year relapse-free survivors of CD34 cell-selected allogeneic hematopoietic stem cell transplantation : a landmark analysis

Altres ajuts: This research was supported in part by National Institutes of Health award number P01 CA23766 and NIH/NCI Cancer Center Support Grant P30 CA008748. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.CD34 selection significantly improves GVHD-free survival in allogeneic hematopoietic cell transplantation (allo-HSCT). Specific information regarding long-term prognosis and risk factors for late mortality after CD34-selected allo-HSCT is lacking, however. We conducted a single-center landmark analysis in 276 patients alive without relapse 1 year after CD34-selected allo-HSCT for AML (n=164), ALL (n=33), or MDS (n=79). At 5 years' follow-up after the 1-year landmark (range 0.03-13 years), estimated RFS was 73% and OS 76%. The 5-year cumulative incidence of relapse and NRM were 11% and 16%, respectively. In multivariate analysis, HCT-CI score ≥ 3 correlated with marginally worse RFS (HR 1.78, 95% CI 0.97-3.28, p=0.06) and significantly worse OS (HR 2.53, 95% CI 1.26-5.08, p=0.004). Despite only 24% of patients with acute GVHD within 1 year, this also significantly correlated with worse RFS and OS, with increasing grades of acute GVHD associating with increasingly poorer survival on multivariate analysis (p<0.0001). Of 63 deaths after the landmark, GVHD accounted for 27% of deaths and was the most common cause of late mortality, followed by relapse and infection. While prognosis is excellent for patients alive without relapse 1 year after CD34-selected allo-HSCT, risks of late relapse and NRM persist, particularly due to GVHD

Reduced plakoglobin increases the risk of sodium current defects and atrial conduction abnormalities in response to androgenic anabolic steroid abuse

Androgenic anabolic steroids (AAS) are commonly abused by young men. Male sex and increased AAS levels are associated with earlier and more severe manifestation of common cardiac conditions, such as atrial fibrillation, and rare ones, such as arrhythmogenic right ventricular cardiomyopathy (ARVC). Clinical observations suggest a potential atrial involvement in ARVC. Arrhythmogenic right ventricular cardiomyopathy is caused by desmosomal gene defects, including reduced plakoglobin expression. Here, we analysed clinical records from 146 ARVC patients to identify that ARVC is more common in males than females. Patients with ARVC also had an increased incidence of atrial arrhythmias and P wave changes. To study desmosomal vulnerability and the effects of AAS on the atria, young adult male mice, heterozygously deficient for plakoglobin (Plako+/−), and wild type (WT) littermates were chronically exposed to 5α-dihydrotestosterone (DHT) or placebo. The DHT increased atrial expression of pro-hypertrophic, fibrotic and inflammatory transcripts. In mice with reduced plakoglobin, DHT exaggerated P wave abnormalities, atrial conduction slowing, sodium current depletion, action potential amplitude reduction and the fall in action potential depolarization rate. Super-resolution microscopy revealed a decrease in NaV1.5 membrane clustering in Plako+/− atrial cardiomyocytes after DHT exposure. In summary, AAS combined with plakoglobin deficiency cause pathological atrial electrical remodelling in young male hearts. Male sex is likely to increase the risk of atrial arrhythmia, particularly in those with desmosomal gene variants. This risk is likely to be exaggerated further by AAS use

Human and Non-Human Primate Genomes Share Hotspots of Positive Selection

Among primates, genome-wide analysis of recent positive selection is currently limited to the human species because it requires extensive sampling of genotypic data from many individuals. The extent to which genes positively selected in human also present adaptive changes in other primates therefore remains unknown. This question is important because a gene that has been positively selected independently in the human and in other primate lineages may be less likely to be involved in human specific phenotypic changes such as dietary habits or cognitive abilities. To answer this question, we analysed heterozygous Single Nucleotide Polymorphisms (SNPs) in the genomes of single human, chimpanzee, orangutan, and macaque individuals using a new method aiming to identify selective sweeps genome-wide. We found an unexpectedly high number of orthologous genes exhibiting signatures of a selective sweep simultaneously in several primate species, suggesting the presence of hotspots of positive selection. A similar significant excess is evident when comparing genes positively selected during recent human evolution with genes subjected to positive selection in their coding sequence in other primate lineages and identified using a different test. These findings are further supported by comparing several published human genome scans for positive selection with our findings in non-human primate genomes. We thus provide extensive evidence that the co-occurrence of positive selection in humans and in other primates at the same genetic loci can be measured with only four species, an indication that it may be a widespread phenomenon. The identification of positive selection in humans alongside other primates is a powerful tool to outline those genes that were selected uniquely during recent human evolution

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles