Protein Kinase C-ß Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis.

PKCß-null (Prkcb-/-) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCß failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb-/- B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCß as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate.

Opposing roles for ADAMTS2 and ADAMTS14 in myofibroblast differentiation and function.

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor ß availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The mediating role of reflective functioning and general psychopathology in the relationship between childhood conduct disorder and adult aggression among offenders.

BACKGROUND: The nature of the pathway from conduct disorder (CD) in adolescence to antisocial behavior in adulthood has been debated and the role of certain mediators remains unclear. One perspective is that CD forms part of a general psychopathology dimension, playing a central role in the developmental trajectory. Impairment in reflective functioning (RF), i.e., the capacity to understand one's own and others' mental states, may relate to CD, psychopathology, and aggression. Here, we characterized the structure of psychopathology in adult male-offenders and its role, along with RF, in mediating the relationship between CD in their adolescence and current aggression. METHODS: A secondary analysis of pre-treatment data from 313 probation-supervised offenders was conducted, and measures of CD symptoms, general and specific psychopathology factors, RF, and aggression were evaluated through clinical interviews and questionnaires. RESULTS: Confirmatory factor analyses indicated that a bifactor model best fitted the sample's psychopathology structure, including a general psychopathology factor (p factor) and five specific factors: internalizing, disinhibition, detachment, antagonism, and psychoticism. The structure of RF was fitted to the data using a one-factor model. According to our mediation model, CD significantly predicted the p factor, which was positively linked to RF impairments, resulting in increased aggression. CONCLUSIONS: These findings highlight the critical role of a transdiagnostic approach provided by RF and general psychopathology in explaining the link between CD and aggression. Furthermore, they underscore the potential utility of treatments focusing on RF, such as mentalization-based treatment, in mitigating aggression in offenders with diverse psychopathologies.

Cancer-associated fibroblasts in pancreatic cancer: new subtypes, new markers, new targets.

Cancer-associated fibroblasts (CAFs) have conflicting roles in the suppression and promotion of cancer. Current research focuses on targeting the undesirable properties of CAFs, while attempting to maintain tumour-suppressive roles. CAFs have been widely associated with primary or secondary therapeutic resistance, and strategies to modify CAF function have therefore largely focussed on their combination with existing therapies. Despite significant progress in preclinical studies, clinical translation of CAF targeted therapies has achieved limited success. Here we will review our emerging understanding of heterogeneous CAF populations in tumour biology and use examples from pancreatic ductal adenocarcinoma to explore why successful clinical targeting of protumourigenic CAF functions remains elusive. Single-cell technologies have allowed the identification of CAF subtypes with a differential impact on prognosis and response to therapy, but currently without clear consensus. Identification and pharmacological targeting of CAF subtypes associated with immunotherapy response offers new hope to expand clinical options for pancreatic cancer. Various CAF subtype markers may represent biomarkers for patient stratification, to obtain enhanced response with existing and emerging combinatorial therapeutic strategies. Thus, CAF subtyping is the next frontier in understanding and exploiting the tumour microenvironment for therapeutic benefit. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

PKN2 deficiency leads both to prenatal 'congenital' cardiomyopathy and defective angiotensin II stress responses.

The protein kinase PKN2 is required for embryonic development and PKN2 knockout mice die as a result of failure in the expansion of mesoderm, cardiac development and neural tube closure. In the adult, cardiomyocyte PKN2 and PKN1 (in combination) are required for cardiac adaptation to pressure-overload. The specific role of PKN2 in contractile cardiomyocytes during development and its role in the adult heart remain to be fully established. We used mice with cardiomyocyte-directed knockout of PKN2 or global PKN2 haploinsufficiency to assess cardiac development and function using high resolution episcopic microscopy, MRI, micro-CT and echocardiography. Biochemical and histological changes were also assessed. Cardiomyocyte-directed PKN2 knockout embryos displayed striking abnormalities in the compact myocardium, with frequent myocardial clefts and diverticula, ventricular septal defects and abnormal heart shape. The sub-Mendelian homozygous knockout survivors developed cardiac failure. RNASeq data showed up-regulation of PKN2 in patients with dilated cardiomyopathy, suggesting an involvement in adult heart disease. Given the rarity of homozygous survivors with cardiomyocyte-specific deletion of PKN2, the requirement for PKN2 in adult mice was explored using the constitutive heterozygous PKN2 knockout. Cardiac hypertrophy resulting from hypertension induced by angiotensin II was reduced in these haploinsufficient PKN2 mice relative to wild-type littermates, with suppression of cardiomyocyte hypertrophy and cardiac fibrosis. It is concluded that cardiomyocyte PKN2 is essential for heart development and the formation of compact myocardium and is also required for cardiac hypertrophy in hypertension. Thus, PKN signalling may offer therapeutic options for managing congenital and adult heart diseases.

The Future of Precision Oncology.

Our understanding of the molecular mechanisms underlying cancer development and evolution have evolved rapidly over recent years, and the variation from one patient to another is now widely recognized. Consequently, one-size-fits-all approaches to the treatment of cancer have been superseded by precision medicines that target specific disease characteristics, promising maximum clinical efficacy, minimal safety concerns, and reduced economic burden. While precision oncology has been very successful in the treatment of some tumors with specific characteristics, a large number of patients do not yet have access to precision medicines for their disease. The success of next-generation precision oncology depends on the discovery of new actionable disease characteristics, rapid, accurate, and comprehensive diagnosis of complex phenotypes within each patient, novel clinical trial designs with improved response rates, and worldwide access to novel targeted anticancer therapies for all patients. This review outlines some of the current technological trends, and highlights some of the complex multidisciplinary efforts that are underway to ensure that many more patients with cancer will be able to benefit from precision oncology in the near future.

INVESTIGATING BLOOD LACTATE CONCENTRATION AS A PROGNOSTIC INDICATOR FOR BIRDS PRESENTING WITH BREVETOXICOSIS: 2020-2021.

Large blooms of the dinoflagellate Karenia brevis cause annual harmful algal bloom events, or "red tides" on Florida's Gulf Coast. Each year, the Clinic for the Rehabilitation of Wildlife (CROW) is presented with hundreds of cases of aquatic birds that exhibit neurologic clinical signs due to brevetoxicosis. Double-crested cormorants (Phalacrocorax auratus) are the most common species seen, and typically present with a combination of ataxia, head tremors, knuckling, and/or lagophthalmos. Blood lactate levels are known to increase in mammals for a variety of reasons, including stress, hypoxia, sepsis, and trauma, but there is limited literature on blood lactate values in avian species. The objective of this study was to determine the prognostic value of blood lactate concentration on successful rehabilitation and release of birds presenting with clinical signs consistent with brevetoxicosis. Blood lactate levels were collected on intake, the morning after presentation and initial therapy, and prior to disposition (release or euthanasia) from 194 birds (including 98 cormorants) representing 17 species during the 2020-2021 red tide season. Overall, mean blood lactate at intake, the morning after intake, and predisposition was 2.9, 2.8, and 3.2 mmol/L, respectively, for released birds across all species (2.9, 2.9, and 3.2 mmol/L for released cormorants); 3.4, 3.4, and 6.5 mmol/L for birds that died (4.0, 3.5, and 7.9 mmol/L for cormorants that died); and 3.1, 3.5, and 4.7 mmol/L for birds that were euthanized (3.5, 4.7, and 4.9 mmol/L for cormorants that were euthanized). On average, birds that died or were euthanized had an elevated lactate at all time points as compared to those that were released, but these results were not statistically significant (P = 0.13). These results indicate that blood lactate levels do not appear to be useful as a prognostic indicator for successful release of birds, including double-crested cormorants, affected by brevetoxicosis.

PKC and the control of localized signal dynamics.

Networks of signal transducers determine the conversion of environmental cues into cellular actions. Among the main players in these networks are protein kinases, which can acutely and reversibly modify protein functions to influence cellular events. One group of kinases, the protein kinase C (PKC) family, have been increasingly implicated in the organization of signal propagation, particularly in the spatial distribution of signals. Examples of where and how various PKC isoforms direct this tier of signal organization are becoming more evident.

Systematic review of the status of veterinary epidemiological research in two species regarding the FAIR guiding principles.

BACKGROUND: The FAIR (Findable, Accessible, Interoperable, Reusable) principles were proposed in 2016 to set a path towards reusability of research datasets. In this systematic review, we assessed the FAIRness of datasets associated with peer-reviewed articles in veterinary epidemiology research published since 2017, specifically looking at salmonids and dairy cattle. We considered the differences in practices between molecular epidemiology, the branch of epidemiology using genetic sequences of pathogens and hosts to describe disease patterns, and non-molecular epidemiology. RESULTS: A total of 152 articles were included in the assessment. Consistent with previous assessments conducted in other disciplines, our results showed that most datasets used in non-molecular epidemiological studies were not available (i.e., neither findable nor accessible). Data availability was much higher for molecular epidemiology papers, in line with a strong repository base available to scientists in this discipline. The available data objects generally scored favourably for Findable, Accessible and Reusable indicators, but Interoperability was more problematic. CONCLUSIONS: None of the datasets assessed in this study met all the requirements set by the FAIR principles. Interoperability, in particular, requires specific skills in data management which may not yet be broadly available in the epidemiology community. In the discussion, we present recommendations on how veterinary research could move towards greater reusability according to FAIR principles. Overall, although many initiatives to improve data access have been started in the research community, their impact on the availability of datasets underlying published articles remains unclear to date.

Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids.

Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.

Functional implications of assigned, assumed and assembled PKC structures.

The empirical derivation of PKC (protein kinase C) domain structures and those modelled by homology or imputed from protein behaviour have been extraordinarily valuable both in the elucidation of PKC pathway mechanisms and in the general lessons that extrapolate to other signalling pathways. For PKC family members, there are many domain/subdomain structures and models, covering all of the known domains, variably present in this family of protein serine/threonine kinases (C1, C2, PB1, HR1, kinase domains). In addition to these structures, there are a limited number of complexes defined, including the structure of the PKCε V3-14-3-3 complex. In the context of structure-driven insights into PKC pathways, there are several broadly applicable principles and mechanisms relevant to the operation of and intervention in signalling pathways. These principles have an impact in unexpected ways, from the regulation of membrane targeting, through strategies for pharmacological intervention, to biomarkers.

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin.

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.

Targets in the Tumour Matrisome to Promote Cancer Therapy Response.

The extracellular matrix (ECM) is composed of complex fibrillar proteins, proteoglycans, and macromolecules, generated by stromal, immune, and cancer cells. The components and organisation of the matrix evolves as tumours progress to invasive disease and metastasis. In many solid tumours, dense fibrotic ECM has been hypothesised to impede therapy response by limiting drug and immune cell access. Interventions to target individual components of the ECM, collectively termed the matrisome, have, however, revealed complex tumour-suppressor, tumour-promoter, and immune-modulatory functions, which have complicated clinical translation. The degree to which distinct components of the matrisome can dictate tumour phenotypes and response to therapy is the subject of intense study. A primary aim is to identify therapeutic opportunities within the matrisome, which might support a better response to existing therapies. Many matrix signatures have been developed which can predict prognosis, immune cell content, and immunotherapy responses. In this review, we will examine key components of the matrisome which have been associated with advanced tumours and therapy resistance. We have primarily focussed here on targeting matrisome components, rather than specific cell types, although several examples are described where cells of origin can dramatically affect tumour roles for matrix components. As we unravel the complex biochemical, biophysical, and intracellular transduction mechanisms associated with the ECM, numerous therapeutic opportunities will be identified to modify tumour progression and therapy response.

Expanding access to veterinary clinical decision support in resource-limited settings: a scoping review of clinical decision support tools in medicine and antimicrobial stewardship.

Introduction: Digital clinical decision support (CDS) tools are of growing importance in supporting healthcare professionals in understanding complex clinical problems and arriving at decisions that improve patient outcomes. CDS tools are also increasingly used to improve antimicrobial stewardship (AMS) practices in healthcare settings. However, far fewer CDS tools are available in lowerand middle-income countries (LMICs) and in animal health settings, where their use in improving diagnostic and treatment decision-making is likely to have the greatest impact. The aim of this study was to evaluate digital CDS tools designed as a direct aid to support diagnosis and/or treatment decisionmaking, by reviewing their scope, functions, methodologies, and quality. Recommendations for the development of veterinary CDS tools in LMICs are then provided. Methods: The review considered studies and reports published between January 2017 and October 2023 in the English language in peer-reviewed and gray literature. Results: A total of 41 studies and reports detailing CDS tools were included in the final review, with 35 CDS tools designed for human healthcare settings and six tools for animal healthcare settings. Of the tools reviewed, the majority were deployed in high-income countries (80.5%). Support for AMS programs was a feature in 12 (29.3%) of the tools, with 10 tools in human healthcare settings. The capabilities of the CDS tools varied when reviewed against the GUIDES checklist. Discussion: We recommend a methodological approach for the development of veterinary CDS tools in LMICs predicated on securing sufficient and sustainable funding. Employing a multidisciplinary development team is an important first step. Developing standalone CDS tools using Bayesian algorithms based on local expert knowledge will provide users with rapid and reliable access to quality guidance on diagnoses and treatments. Such tools are likely to contribute to improved disease management on farms and reduce inappropriate antimicrobial use, thus supporting AMS practices in areas of high need.

Needs and capabilities for improving poultry production and health management in Indonesia.

In Indonesia, the development of the poultry industry is facing numerous challenges. Major constraints include high disease burdens, large fluctuations in farm input and output prices, and inadequate biosecurity. Timely and reliable information about animal production and health can help stakeholders at all levels of the value chain make appropriate management decisions to optimize their profitability and productivity while reducing risks to public health. This study aimed to describe the challenges in the Indonesian poultry industry, assess stakeholders' needs and capabilities in terms of generating and using poultry information for making production and health management decisions, and identify levers for improvement. Interviews were conducted with a diversity of key informants and value chain actors in five Indonesian provinces. Thematic analysis was applied with an interpretivist approach to gain an in-depth understanding of the lived experiences of various stakeholders and their opinions as to what might constitute appropriate solutions. Our findings indicate that market and political instability, ineffective management of poultry data, and limited inter-sectoral collaboration are limiting the development of the sector. Increased intersectoral cooperation is needed to implement standards for data collection and sharing across the industry, provide education and practical training on the use of information technologies for farm management, and accelerate research and innovation. Our study can contribute to the development of data-driven tools to support evidence-based decision-making at all levels of the poultry system.

Protein kinase N1 deficiency results in upregulation of cerebral energy metabolism and is highly protective in in vivo and in vitro stroke models.

BACKGROUND AND AIM: We recently identified protein kinase N1 (PKN1) as a master regulator of brain development. However, its function in the adult brain has not been clearly established. In this study, we assessed the cerebral energetic phenotype of wildtype (WT) and global Pkn1 knockout (Pkn1-/-) animals under physiological and pathophysiological conditions. METHODS: Cerebral energy metabolism was analyzed by 13C6-glucose tracing in vivo and real time seahorse analysis of extracellular acidification rates as well as mitochondrial oxygen consumption rates (OCR) of brain slice punches in vitro. Isolated WT and Pkn1-/- brain mitochondria were tested for differences in OCR with different substrates. Metabolite levels were determined by mass spectrometric analysis in brain slices under control and energetic stress conditions, induced by oxygen-glucose deprivation and reperfusion, an in vitro model of ischemic stroke. Differences in enzyme activities were assessed by enzymatic assays, western blotting and bulk RNA sequencing. A middle cerebral artery occlusion stroke model was used to analyze lesion volumes and functional recovery in WT and Pkn1-/- mice. RESULTS: Pkn1 deficiency resulted in a remarkable upregulation of cerebral energy metabolism, in vivo and in vitro. This was due to two separate mechanisms involving an enhanced glycolytic flux and higher pyruvate-induced mitochondrial OCR. Mechanistically we show that Pkn1-/- brain tissue exhibits an increased activity of the glycolysis rate-limiting enzyme phosphofructokinase. Additionally, glucose-1,6-bisphosphate levels, a metabolite that increases mitochondrial pyruvate uptake, were elevated upon Pkn1 deficiency. Consequently, Pkn1-/- brain slices had more ATP and a greater accumulation of ATP degradation metabolites during energetic stress. This translated into increased phosphorylation and activity of adenosine monophosphate (AMP)-activated protein kinase (AMPK) during in vitro stroke. Accordingly, Pkn1-/- brain slices showed a post-ischemic transcriptional upregulation of energy metabolism pathways and Pkn1 deficiency was strongly protective in in vitro and in vivo stroke models. While inhibition of mitochondrial pyruvate uptake only moderately affected the protective phenotype, inhibition of AMPK in Pkn1-/- slices increased post-ischemic cell death in vitro. CONCLUSION: This is the first study to comprehensively demonstrate an essential and unique role of PKN1 in cerebral energy metabolism, regulating glycolysis and mitochondrial pyruvate-induced respiration. We further uncovered a highly protective phenotype of Pkn1 deficiency in both, in vitro and in vivo stroke models, validating inhibition of PKN1 as a promising new therapeutic target for the development of novel stroke therapies.

Rinderpest virus sequestration and use in posteradication era.

After the 2011 declaration of rinderpest disease eradication, we surveyed 150 countries about rinderpest virus stocks. Forty-four laboratories in 35 countries held laboratory-attenuated strains, field strains, or diagnostic samples. Vaccine and reagent production and laboratory experiments continued. Rigorous standards are necessary to ensure that stocks are kept under safe conditions.

Pseudokinase drug intervention: a potentially poisoned chalice.

Pseudokinases, the catalytically impaired component of the kinome, have recently been found to share more properties with active kinases than previously thought. In many pseudokinases, ATP binding and even some activity is preserved, highlighting these proteins as potential drug targets. In both active kinases and pseudokinases, binding of ATP or drugs in the nucleotide-binding pocket can stabilize specific conformations required for activity and protein-protein interactions. We discuss the implications of locking particular conformations in a selection of (pseudo)kinases and the dual potential impact on the druggability of these proteins.

A comparison of medetomidine and its active enantiomer dexmedetomidine when administered with ketamine in mice.

BACKGROUND: Medetomidine-ketamine (MK) and dexmedetomidine-ketamine (DK) are widely used to provide general anaesthesia in laboratory animals, but have not been compared directly in many of these species, including rodents. This study aimed to compare the onset and depth of anaesthesia, and changes in vital signs, after intraperitoneal (IP) or subcutaneous (SC) administration of ketamine (75 mg kg(-1)) combined with medetomidine (1 mg kg(-1)) or dexmedetomidine (0.5 mg kg(-1)) using a randomised semi-crossover design with ≥ 48 hours between treatments in 10 male and 10 female mice. Each mouse was anaesthetised twice using the same administration route (IP or SC): once with each drug-ketamine combination. Anaesthetised mice were monitored on a heating pad without supplemental oxygen for 89 minutes; atipamezole was administered for reversal. The times that the righting reflex was lost post-injection and returned post-reversal were analysed using general linear models. Tail-pinch and pedal reflexes were examined using binomial generalized linear models. Pulse rate (PR), respiratory rate (fr), and arterial haemoglobin saturation (S(p)O2) were compared using generalized additive mixed models. RESULTS: There were no significant differences among treatments for the times taken for loss and return of the righting reflex, or response of the tail-pinch reflex. The pedal withdrawal reflex was abolished more frequently with MK than DK over time (P = 0.021). The response of PR and S(p)O2 were similar among treatments, but fr was significantly higher with MK than DK (P ≤ 0.0005). Markedly low S(p)O2 concentrations occurred within 5 minutes post-injection (83.8 ± 6.7%) in all treatment groups and were most severe after 89 minutes lapsed (66.7 ± 7.5%). No statistical differences were detected in regards to administration route (P ≤ 0.94). CONCLUSIONS: This study failed to demonstrate clinical advantages of the enantiomer dexmedetomidine over medetomidine when combined with ketamine to produce general anaesthesia in mice. At the doses administered, deep surgical anaesthesia was not consistently produced with either combination; therefore, anaesthetic depth must be assessed before performing surgical procedures. Supplemental oxygen should always be provided during anaesthesia to prevent hypoxaemia.

"It's a habit. They've been doing it for decades and they feel good and safe.": A qualitative study of barriers and opportunities to changing antimicrobial use in the Indonesian poultry sector.

Interventions to change antimicrobial use (AMU) practices can help mitigate the risk of antimicrobial resistance (AMR) development. However, changing AMU practices can be challenging due to the complex nature of the factors influencing AMU-related behaviours. This study used a qualitative approach to explore the factors that influenced decision-making on AMU by farmers and other actors in the Indonesian poultry sector. Thirty-five semi-structured interviews were conducted with farmers, technical services staff from the private sector, and representatives of associations, universities, and international organisations in Central Java, West Java, and East Java. Thematic analysis identified three patterns of influence on AMU: how farmers used information to make AMU-related decisions, the importance of farmers' social and advisory networks, and the motivations driving changes in AMU behaviours. Key barriers identified included a lack of shared understanding around when to use antibiotics, financial pressures in the poultry sector, and a lack of engagement with government veterinary services. Potential opportunities identified included high farmer awareness of AMU, identification of private sector actors and peer networks as the stakeholders with established relationships of trust with farmers, and the importance of farmers' conceptions of good farming practices, which could be engaged with to improve AMU practices.