Exploring Greater Flexibility for Chronic Toxicity Study Designs to Support Human Safety Assessment While Balancing 3Rs Considerations.

Chronic repeated-dose toxicity studies are required to support long-term dosing in late-stage clinical trials, providing data to adequately characterize adverse effects of potential concern for human safety. Different regulatory guidances for the design and duration of chronic toxicity studies are available, with flexibility in approaches often adopted for specific drug modalities. These guidances may provide opportunities to reduce time, cost, compound requirement and animal use within drug development programs if applied more broadly and considered outside their current scopes of use. This article summarizes presentations from a workshop at the 43rd Annual Meeting of the American College of Toxicology (ACT) in November 2022, discussing different approaches for chronic toxicity studies. A recent industry collaboration between the Netherlands Medicines Evaluation Board (MEB) and UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) illustrated current practices and the value of chronic toxicity studies for monoclonal antibodies (mAbs) and evaluated a weight of evidence (WOE) model where a 3-month study rather than a 6-month study might be adequate. Other topics included potential opportunities for single-species chronic toxicity studies for small molecules, peptides and oligonucleotides and whether a 6-month duration non-rodent study can be used more routinely than a 9-month study (similar to ICH S6(R1) for biological products). Also addressed were opportunities to optimize recovery animal use if warranted and whether restriction to one study only (if at all) can be applied more widely within and outside ICH S6(R1).

Identification and expression of MarCE, a marine carboxylesterase with synthetic ester-degrading activity.

Carboxylic ester hydrolases with the capacity to degrade polyesters are currently highly sought after for their potential use in the biological degradation of PET and other chemically synthesized polymers. Here, we describe MarCE, a carboxylesterase family protein identified via genome mining of a Maribacter sp. isolate from the marine sponge Stelligera stuposa. Based on phylogenetic analysis, MarCE and its closest relatives belong to marine-associated genera from the Cytophaga-Flavobacterium-Bacteroides taxonomic group and appear evolutionarily distinct to any homologous carboxylesterases that have been studied to date in terms of structure or function. Molecular docking revealed putative binding of BHET, a short-chain PET derivative, onto the predicted MarCE three-dimensional structure. The synthetic ester-degrading activity of MarCE was subsequently confirmed by MarCE-mediated hydrolysis of 2 mM BHET substrate, indicated by the release of its breakdown products MHET and TPA, which were measured, respectively, as 1.28 and 0.12 mM following 2-h incubation at 30°C. The findings of this study provide further insight into marine carboxylic ester hydrolases, which have the potential to display unique functional plasticity resulting from their adaptation to complex and fluctuating marine environmentsw.

Alcohol is a risk factor for helmet non-use and fatalities in off-road vehicle and motorcycle crashes.

OBJECTIVES: Off-road vehicle (ORV) and motorcycle use is common in Canada; however, risk of serious injury is heightened when these vehicles are operated without helmets and under the influence of alcohol. This study evaluated the impact of alcohol intoxication on helmet non-use and mortality among ORV and motorcycle crashes. METHODS: Using data collected from the Nova Scotia Trauma Registry, a retrospective analysis (2002-2017) of ORV and motorcycle crashes resulting in major traumatic brain injury was performed. Patients were grouped by blood alcohol concentration (BAC) as negative (< 2 mmol/L), legally intoxicated (2-17.3 mmol/L) or criminally intoxicated (> 17.3 mmol/L). Logistic regression models were constructed to test for helmet non-use and mortality. RESULTS: A total of 424 trauma patients were included in the analysis (220 ORV, 204 motorcycle). Less than half (45%) of patients involved in ORV crashes were wearing helmets and 65% were criminally intoxicated. Most patients involved in motorcycle crashes were helmeted at time of injury (88.7%) and 18% were criminally intoxicated. Those with criminal levels of intoxication had 3.7 times the odds of being unhelmeted and were 3 times more likely to die prehospital compared to BAC negative patients. There were significantly increased odds of in-hospital mortality among those with both legal (OR = 5.63), and criminal intoxication levels (OR = 4.97) compared to patients who were BAC negative. CONCLUSION: Alcohol intoxication is more frequently observed in ORV versus motorcycle crashes. Criminal intoxication is associated with helmet non-use. Any level of intoxication is a predictor of increased in-hospital mortality.

Fidelity, pragmatism and the "grey line" in between-exploring the delivery of a pragmatic physical activity randomised controlled trial-a secondary analysis.

BACKGROUND: Intervention fidelity in health services research has been poor with a reported lack of understanding about what constitutes pragmatic adaptation of interventions and what constitutes failure to maintain intervention fidelity. However, the challenges facing those delivering such interventions have not been thoroughly explored. The aims of this study were to critically explore the challenges in maintaining fidelity experienced by physiotherapy staff and support workers when delivering a complex intervention for older people living with frailty. METHODS: This study is a secondary analysis of data from a process evaluation of a large randomised controlled trial (RCT). The process evaluation employed qualitative methodologies with mixed methods including a variety of data collection methods, including participant observation, semi-structured interviews and documentary analysis. Thematic analysis was used to make sense of the data. RESULTS: Many therapy staff felt ongoing confusion about what was acceptable to adapt and what needed to follow the protocol exactly. We found that some therapy staff were able to embrace the challenges of pragmatically adapting interventions while maintaining intervention fidelity, others stuck rigidly to the protocol and failed to adapt interventions where it was necessary. CONCLUSION: It was clear that the understanding of fidelity and pragmatism was poor. While pragmatic trials are vital to replicate real world clinical practice, further guidance may need to be developed in order to guide the level of adaptation that is acceptable before fidelity is undermined.

Microbial metabolites as modulators of host physiology.

The gut microbiota is increasingly recognised as a key player in influencing human health and changes in the gut microbiota have been strongly linked with many non-communicable conditions in humans such as type 2 diabetes, obesity and cardiovascular disease. However, characterising the molecular mechanisms that underpin these associations remains an important challenge for researchers. The gut microbiota is a complex microbial community that acts as a metabolic interface to transform ingested food (and other xenobiotics) into metabolites that are detected in the host faeces, urine and blood. Many of these metabolites are only produced by microbes and there is accumulating evidence to suggest that these microbe-specific metabolites do act as effectors to influence human physiology. For example, the gut microbiota can digest dietary complex polysaccharides (such as fibre) into short-chain fatty acids (SCFA) such as acetate, propionate and butyrate that have a pervasive role in host physiology from nutrition to immune function. In this review we will outline our current understanding of the role of some key microbial metabolites, such as SCFA, indole and bile acids, in human health. Whilst many studies linking microbial metabolites with human health are correlative we will try to highlight examples where genetic evidence is available to support a specific role for a microbial metabolite in host health and well-being.

Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models.

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered "Crab"-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. "Crab"-like configuration of two catalytic peptides ("pincers") flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for "single-pincer" conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled "Crabs" to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5'-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models.

A native mass spectrometry approach to qualitatively elucidate interfacial epitopes of transient protein-protein interactions.

Native mass spectrometric analysis of TPR2A and GrpE with unpurified peptides derived from limited proteolysis of their respective PPI partners (HSP90 C-terminus and DnaK) facilitated efficient, qualitative identification of interfacial epitopes involved in transient PPI formation. Application of this approach can assist in elucidating interfaces of currently uncharacterised transient PPIs.

What influences provision of information about recovery on stroke units? A focused ethnographic case study.

OBJECTIVE: Patients and carers frequently report dissatisfaction with post-stroke information provision. This study aimed to develop an in-depth understanding of the factors influencing provision of information about recovery in stroke units. METHODS: Focused ethnographic case-studies in two UK stroke units, including non-participant observations and semi-structured interviews with professionals, patients and carers, and documentary analysis. A Framework approach to analysis was undertaken. RESULTS: Twenty patients, 17 carers and 47 professionals participated. The unpredictable recovery trajectory led professionals to present prognostic estimates as uncertain possibilities. The need to maintain patients' motivation limited sharing of negative predictions, and generic information over-emphasised the importance of therapy in recovery. A structured multidisciplinary team approach to delivering information improved consistency. Complex clinical reasoning was required to identify and meet patients' needs. Hospital environments and routines restricted opportunities for dialogue, particularly with carers. CONCLUSIONS: The process of providing information about post-stroke recovery is complex, requiring enhanced clinical reasoning and communication. The challenges faced by professionals are numerous and if not addressed can result in suboptimal provision. PRACTICE IMPLICATIONS: Professionals should develop a co-ordinated multidisciplinary approach to information provision; and engage in dialogue to ensure a tailored approach to identifying and meeting patients' and carers' information needs.

Global indicators of the environmental impacts of invasive alien species and their information adequacy.

Monitoring the extent to which invasive alien species (IAS) negatively impact the environment is crucial for understanding and mitigating biological invasions. Indeed, such information is vital for achieving Target 6 of the Kunming-Montreal Global Biodiversity Framework. However, to-date indicators for tracking the environmental impacts of IAS have been either lacking or insufficient. Capitalizing on advances in data availability and impact assessment protocols, we developed environmental impact indicators to track realized and potential impacts of IAS. We also developed an information status indicator to assess the adequacy of the data underlying the impact indicators. We used data on 75 naturalized amphibians from 82 countries to demonstrate the indicators at a global scale. The information status indicator shows variation in the reliability of the data and highlights areas where absence of impact should be interpreted with caution. Impact indicators show that growth in potential impacts are dominated by predatory species, while potential impacts from both predation and disease transmission are distributed worldwide. Using open access data, the indicators are reproducible and adaptable across scales and taxa and can be used to assess global trends and distributions of IAS, assisting authorities in prioritizing control efforts and identifying areas at risk of future invasions. This article is part of the theme issue 'Ecological novelty and planetary stewardship: biodiversity dynamics in a transforming biosphere'.

A nature-positive future with biological invasions: theory, decision support and research needs.

In 2050, most areas of biodiversity significance will be heavily influenced by multiple drivers of environmental change. This includes overlap with the introduced ranges of many alien species that negatively impact biodiversity. With the decline in biodiversity and increase in all forms of global change, the need to envision the desired qualities of natural systems in the Anthropocene is growing, as is the need to actively maintain their natural values. Here, we draw on community ecology and invasion biology to (i) better understand trajectories of change in communities with a mix of native and alien populations, and (ii) to frame approaches to the stewardship of these mixed-species communities. We provide a set of premises and actions upon which a nature-positive future with biological invasions (NPF-BI) could be based, and a decision framework for dealing with uncertain species movements under climate change. A series of alternative management approaches become apparent when framed by scale-sensitive, spatially explicit, context relevant and risk-consequence considerations. Evidence of the properties of mixed-species communities together with predictive frameworks for the relative importance of the ecological processes at play provide actionable pathways to a NPF in which the reality of mixed-species communities are accommodated and managed. This article is part of the theme issue 'Ecological novelty and planetary stewardship: biodiversity dynamics in a transforming biosphere'.

FIVE YEARS OF PARTULA SNAIL PRE-EXPORT HEALTH SCREENING.

Between 2015 and 2019, a health screening was carried out annually on captive-bred Partula snails prior to export for reintroduction as part of an international effort to repopulate areas of French Polynesia, where the snails were extinct or critically endangered. In total, 129 separate tank populations of 12 different species were screened at ZSL London Zoo. Wet mounts and smears stained with modified Ziehl-Neelsen (MZN) of 535 fecal samples were examined, and 45% contained flagellated protozoa, and 35.5% had MZN-positive oocysts, measuring 3-5 µm in diameter. Smaller (2 µm) presumptive spores, MZN-positive bacilli, ciliated protozoa and nematodes were recorded less frequently. Fecal bacterial culture yielded mixed species, with a clear predominance of Myroides species (88.9% of samples). The MZN-positive oocysts (3-5 µm) were present in 6.5% of impression smears from the apices of 432 snails examined postmortem, plus acid-fast bacilli in a few cases, but no 2 µm spores. Mixed bacteria were cultured from coelomic swabs, with Myroides species again the most common (63.5%). Histologic examination was carried out on 292 snails. Autolysis affected almost 90% of those found dead but only 3.4% of euthanized snails. Histology commonly identified microsporidial sporocysts in the digestive gland and midgut epithelium of all but two species. Intracellular, extracytoplasmic Cryptosporidium-like organisms were also common in the midgut but were only observed when snails were fixed in 10% formalin (2017-2019), not ethanol. There were no clear pathologic changes associated with either organism. Pigmented hemocytic nodules were commonly observed, most frequently in the foot process; these were either age related or evidence of prior chronic inflammatory reaction and of low clinical significance. With no evidence of poor health and no significant organisms found, a total of 4,978 individuals representing 12 species were exported for reintroduction.

Utilising Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to track the oxidation of lignin by an alkaliphilic laccase.

Lignin is a complex heteroaromatic polymer which is one of the most abundant and diverse biopolymers on the planet. It comprises approximately one third of all woody plant matter, making it an attractive candidate as an alternative, renewable feedstock to petrochemicals to produce fine chemicals. However, the inherent complexity of lignin makes it difficult to analyse and characterise using common analytical techniques, proving a hindrance to the utilisation of lignin as a green chemical feedstock. Herein we outline the tracking of lignin degradation by an alkaliphilic laccase in a semi-quantitative manner using a combined chemical analysis approach using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to characterise shifts in chemical diversity and relative abundance of ions, and NMR to highlight changes in the structure of lignin. Specifically, an alkaliphilic laccase was used to degrade an industrially relevant lignin, with compounds such as syringaresinol being almost wholly removed (95%) after 24 hours of treatment. Structural analyses reinforced these findings, indicating a >50% loss of NMR signal relating to ß-ß linkages, of which syringaresinol is representative. Ultimately, this work underlines a combined analytical approach that can be used to gain a broader semi-quantitative understanding of the enzymatic activity of laccases within a complex, non-model mixture.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.

Off-road vehicle crashes: dangers of alcohol and drug impairment.

BACKGROUND: Impaired driving is a public health issue, yet little is known concerning the prevalence of substance use in drivers involved in off-road vehicle crashes. The goal of the present study was to describe the demographics and prevalence of alcohol and drug use in drivers of off-road vehicle crashes. METHODS: In this observational substudy, we collected clinical and toxicological data on all moderately or severely injured off-road vehicle drivers who had blood samples obtained within 6 h of the crash. Clinical data were extracted from patients' medical charts and toxicology analyses were performed for blood alcohol, cannabinoids, recreational drugs, and impairing medications. RESULTS: Thirty-three injured drivers met the inclusion criteria. The mean age was 37.6 ± 13.4 years and 79% were male. Blood alcohol was detected in 58% of drivers and 42% of these were above the legal limit. Tetrahydrocannabinol was positive in 12% of drivers, and 18% were positive for recreational drugs. Opiates were detected in 21% of drivers. Overall, 85% were positive for at least one substance and 39% tested positive for multiple substances. CONCLUSION: This study presents the first evidence of alcohol and drug use in off-road vehicle drivers after cannabis legalization in Canada. Our results show that over half of drivers in off-road vehicle crashes test positive for alcohol and 30% tested positive for THC, cocaine, or amphetamines. Opiates are also commonly detected in off-road vehicle drivers. Emergency department (ED) visits resulting from drug driving of off-road vehicles serve as an opportunity for screening, initiating treatment, and connecting patients to interventions for substance use disorders.

ABSTRAIT: CONTEXTE: La conduite avec facultés affaiblies est un problème de santé publique, mais on sait peu de choses sur la prévalence de la toxicomanie chez les conducteurs impliqués dans des accidents de la route. L'objectif de la présente étude était de décrire la démographie et la prévalence de la consommation d'alcool et de drogues chez les conducteurs de véhicules hors route. MéTHODES: Dans le cadre de cette sous-étude observationnelle, nous avons recueilli des données cliniques et toxicologiques sur tous les conducteurs de véhicules hors route ayant subi des blessures modérées ou graves qui avaient reçu des échantillons de sang dans les 6 heures suivant l'accident. Les données cliniques ont été extraites des dossiers médicaux des patients et des analyses toxicologiques ont été effectuées pour l'alcool dans le sang, les cannabinoïdes, les drogues récréatives et les médicaments pour les facultés affaiblies. RéSULTATS: Trente-trois conducteurs blessés répondaient aux critères d'inclusion. L'âge moyen était de 37,6 13,4 ans et 79 % étaient des hommes. L'alcool dans le sang a été détecté chez 58 % des conducteurs et 42 % d'entre eux dépassaient la limite légale. Le tétrahydrocannabinol était positif chez 12 % des conducteurs et 18 % étaient positifs aux drogues récréatives. Des opiacés ont été détectés chez 21 % des conducteurs. Dans l'ensemble, 85 % étaient positifs pour au moins une substance et 39 % étaient positifs pour plusieurs substances. CONCLUSION: Cette étude présente les premières preuves de la consommation d'alcool et de drogues chez les conducteurs de véhicules hors route après la légalisation du cannabis au Canada. Nos résultats montrent que plus de la moitié des conducteurs de véhicules hors route ont un résultat positif au test de dépistage de l'alcool et 30 % ont un résultat positif au test de dépistage du THC, de la cocaïne ou des amphétamines. Les opiacés sont également couramment détectés chez les conducteurs de véhicules hors route. Les visites aux services d'urgence (SU) découlant de la conduite de véhicules hors route avec facultés affaiblies par la drogue constituent une occasion de dépistage, d'amorcer un traitement et de mettre les patients en contact avec des interventions pour les troubles liés à la consommation de substances.

Kinetic modeling of leucine-mediated signaling and protein metabolism in human skeletal muscle.

Skeletal muscle protein levels are governed by the relative rates of muscle protein synthesis (MPS) and breakdown (MPB). The mechanisms controlling these rates are complex, and their integrated behaviors are challenging to study through experiments alone. The purpose of this study was to develop and analyze a kinetic model of leucine-mediated mTOR signaling and protein metabolism in the skeletal muscle of young adults. Our model amalgamates published cellular-level models of the IRS1-PI3K-Akt-mTORC1 signaling system and of skeletal-muscle leucine kinetics with physiological-level models of leucine digestion and transport and insulin dynamics. The model satisfactorily predicts experimental data from diverse leucine feeding protocols. Model analysis revealed that total levels of p70S6K are a primary determinant of MPS, insulin signaling substantially affects muscle net protein balance via its effects on MPB, and p70S6K-mediated feedback of mTORC1 signaling reduces MPS in a dose-dependent manner.

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Blood-Brain Barrier Dysfunction and Exposure to Head Impacts in University Football Players.

OBJECTIVE: To investigate the link between dysfunction of the blood-brain barrier (BBB) and exposure to head impacts in concussed football athletes. DESIGN: This was a prospective, observational pilot study. SETTING: Canadian university football. PARTICIPANTS: The study population consisted of 60 university football players, aged 18 to 25. Athletes who sustained a clinically diagnosed concussion over the course of a single football season were invited to undergo an assessment of BBB leakage. INDEPENDENT VARIABLES: Head impacts detected using impact-sensing helmets were the measured variables. MAIN OUTCOME MEASURES: Clinical diagnosis of concussion and BBB leakage assessed using dynamic contrast-enhanced MRI (DCE-MRI) within 1 week of concussion were the outcome measures. RESULTS: Eight athletes were diagnosed with a concussion throughout the season. These athletes sustained a significantly higher number of head impacts than nonconcussed athletes. Athletes playing in the defensive back position were significantly more likely to sustain a concussion than remain concussion free. Five of the concussed athletes underwent an assessment of BBB leakage. Logistic regression analysis indicated that region-specific BBB leakage in these 5 athletes was best predicted by impacts sustained in all games and practices leading up to the concussion-as opposed to the last preconcussion impact or the impacts sustained during the game when concussion occurred. CONCLUSIONS: These preliminary findings raise the potential for the hypothesis that repeated exposure to head impacts may contribute to the development of BBB pathology. Further research is needed to validate this hypothesis and to test whether BBB pathology plays a role in the sequela of repeated head trauma.

Spatial Hotspots and Sociodemographic Profiles Associated With Traumatic Brain Injury in Nova Scotia.

Traumatic brain injury (TBI) is a leading cause of death and disability, primarily caused by falls and motor vehicle collisions (MVCs). Although many TBIs are preventable, there is a notable lack of studies exploring the association of geographically defined TBI hotspots with social deprivation. Geographic information systems (GIS) can be used to identify at-risk neighborhoods (hotspots) for targeted interventions. This study aims to determine the spatial distribution of TBI by major causes and to explore the sociodemographic and economic characteristics of TBI hotspots and cold spots in Nova Scotia. Patient data for TBIs from 2003 to 2019 were obtained from the Nova Scotia Trauma Registry. Residential postal codes were geocoded and assigned to dissemination areas (DA). Area-based risk factors and deprivation status (residential instability [RI], economic dependency [ED], ethnocultural composition [EC], and situational vulnerability [SV]) from the national census data were linked to DAs. Spatial autocorrelation was assessed using Moran's I, and hotspot analysis was performed using Getis-Ord Gi* statistic. Differences in risk factors between hot and cold spots were evaluated using the Mann-Whitney U test for numerical variables and the χ2 test or Fisher's exact test for categorical variables. A total of 5394 TBI patients were eligible for inclusion in the study. The distribution of hotspots for falls exhibited no significant difference between urban and rural areas (p = 0.71). Conversely, hotspots related to violence were predominantly urban (p = 0.001), whereas hotspots for MVCs were mostly rural (p < 0.001). Distinct dimensions of deprivation were associated with falls, MVCs, and violent hotspots. Fall hotspots were significantly associated with areas characterized by higher RI (p < 0.001) and greater ethnocultural diversity (p < 0.001). Conversely, the same domains exhibited an inverse relationship with MVC hotspots; areas with low RI and ethnic homogeneity displayed a higher proportion of MVC hotspots. ED and SV exhibited a strong gradient with MVC hotspots; the most deprived quintiles displayed the highest proportion of MVC hotspots compared with cold spots (ED; p = 0.002, SV; p < 0.001). Areas with the highest levels of ethnocultural diversity were found to have a significantly higher proportion of violence-related hotspots than cold spots (p = 0.005). This study offers two significant contributions to spatial epidemiology. First, it demonstrates the distribution of TBI hotspots by major injury causes using the smallest available geographical unit. Second, we disentangle the various pathways through which deprivation impacts the risk of main mechanisms of TBI. These findings provide valuable insights for public health officials to design targeted injury prevention strategies in high-risk areas.